Human papillomavirus,smoking status and outcomes in tonsillar squamous cell carcinoma

It is now clear that the two separate entitles of tonsillar cancer, HPV induced and non‐HPV induced (smoking induced), have significantly different presenting stage and outcomes. A significant proportion of patients with human papillomavirus positive tonsillar cancer have had exposure to smoking. We examined the combined effect of human papillomavirus and smoking on the outcomes and determined whether smoking can modify the beneficial effect of human papillomavirus. A total of 403 patients from nine centers were followed up for recurrence or death for a median of 38 months. Determinants of the rate of loco‐regional recurrence, death from tonsillar cancer and overall survival were modeled using Cox regression. Smoking status was a significant predictor of overall survival (p = 0.04). There were nonstatistically significant trends favoring never smokers for loco‐regional recurrence and disease specific survival. In addition, there was no statistically significant interactions between smoking and human papillomavirus (p‐values for the interaction were 0.26 for loco‐regional recurrence, 0.97 for disease specific survival and 0.73 for overall survival). The effect of smoking on loco‐regional recurrence and disease specific survival outcomes was not statistically significant, nor was there significant evidence that the effect of smoking status on these outcomes was modified by HPV status. Irrespective of HPV status, however, smokers did have poorer overall survival than never‐smokers, presumably due to effects of smoking that are unrelated to the primary cancer.     

Marked differences in survival rate between smokers and nonsmokers with HPV 16-associated tonsillar carcinomas

Oncogenic human papillomavirus (HPV) is a causative agent in a subgroup of head and neck carcinomas, particularly tonsillar squamous cell carcinomas (TSCC). This study was undertaken because controversial data exist on the physical status of HPV-DNA and the use of p16(INK4A) overexpression as surrogate HPV marker, and to examine the impact of HPV and tobacco consumption on the clinical course of TSCC. Tissue sections of 81 TSCC were analyzed by HPV 16-specific fluorescence in situ hybridization (FISH) and p16(INK4A)-specific immunohistochemistry. Results were correlated with clinical and demographic data. HPV 16 integration was detected by FISH as punctate signals in 33 out of 81 (41%) TSCC, 32 of which showed p16(INK4A) accumulation. Only 5 out of 48 HPV-negative tumors showed p16(INK4A) immunostaining (p < 0.0001). The presence of HPV furthermore correlates significantly with low tobacco (p = 0.002) and alcohol intake (p = 0.011), poor differentiation grade (p = 0.019), small tumor size (p = 0.024), presence of a local metastasis (p = 0.001) and a decreased (loco)regional recurrence rate (p = 0.039). Statistical analysis revealed that smoking significantly increases the risk of cancer death from TSCC and that non-smoking patients with HPV-containing TSCC show a remarkably better disease-specific survival rate. HPV 16 is integrated in 41% of TSCC and strongly correlates with p16(INK4A) overexpression, implicating the latter to be a reliable HPV biomarker. Patients with HPV-positive tumors show a favorable prognosis as compared to those with HPV-negative tumors, but tobacco use is the strongest prognostic indicator. These findings indicate that oncogenic processes in the tonsils of non-smokers differ from those occurring in smokers, the former being related to HPV 16 infection.     

乳腺癌组织中HPV16 18E6及p53 MCM7蛋白的表达及意义

目的: 探讨人乳腺癌及乳腺良性病变中高危型HPV16、18感染和细胞周期复制调控蛋白p53、MCM7异常表达与人乳腺癌发生、发展的关系。 方法: 采用免疫组织化学SP法,检测30例正常乳腺、30例乳腺腺病,55例乳腺癌、5例导管原位癌及其癌旁组织中HPV16、18E6,p53和MCM7蛋白的表达。 结果: 55例乳腺癌组织中HPV16、18E6,p53和MCM7蛋白的阳性表达率分别为58.18%、38.18%和96.36%,均显著高于正常组和腺病组(P<0.01、P<0.001、P<0.05)。浸润性导管癌HPV16、18E6阳性组中p53蛋白的阳性表达率显著低于HPV16、18E6阴性组(P<0.01)。HPV16、18E6与p53蛋白的表达呈负相关(r=-0.5769;P<0.001),而与MCM7蛋白表达呈正相关(r=0.5442;P<0.001)。MCM7蛋白的阳性表达率与浸润性导管癌组织学分级、淋巴结转移和肿块直径有关(P<0.01、P<0.03、P<0.01)。 结论: 高危型HPV16、18E6感染后p53功能的失活和MCM7蛋白的高表达导致细胞周期复制调控异常,可能涉及了HPV感染后乳腺癌的发生、发展过程。MCM7的高表达与乳腺癌细胞的增殖、侵袭和转移有关。二者联合检测可作为评价HPV感染后乳腺上皮细胞的增殖状态、早期诊断和预测肿瘤生物学行为的重要指标。     

RNA干扰在宫颈癌HPV16E6/E7中的研究进展

RNA干扰(RNAi)是一种有效的基因沉默过程,宫颈癌的重要基因型HPV16的关键致癌基因是E6和E7,目前RNAi在HPV16 E6/E7中的基因沉默作用已取得一些突破性进展.在体外研究方面,正在进一步确定稳定干扰目的 基因的载体形式和筛选更好的干扰序列,在体内研究方面,已建立相应的动物模型,为RNAi的临床研究打下...     

Double positivity for HPV DNA/p16 in tonsillar and base of tongue cancer improves prognostication: Insights from a large population‐based study

The aim was to explore the overall survival (OS) for palatine tonsillar squamous cell carcinoma (TSCC), subdivided, according to certainty of tonsillar tumour origin, into specified tonsillar squamous cell carcinomas (STSCCs) and nonspecified tonsillar squamous cell carcinomas (NSTSCCs), and base of tongue squamous cell carcinoma (BSCC) when stratifying for HPV DNA status, p16 expression and combined HPV/p16 status. We included all patients (n = 797) diagnosed with TSCCs and BSCCs in Eastern Denmark as registered in the Danish Head and Neck Cancer Group (DAHANCA) database and the Danish Pathology Databank, 2000–2010. Patients were treated according to national guidelines (radiotherapy +/? concomitant cisplatin). All specimens were analysed using HPV DNA PCR and p16 immunohistochemistry. Clinical information was retrieved from the DAHANCA database and the Danish National Patient Registry. Information on vital status was obtained from the Danish Civil Registration System. We observed improved OS for HPV+/p16+ BSCCs compared to HPV?/p16? (hazard ratio for death [HR], 0.15; 95% CI, 0.09–0.24). Among STSCCs, HPV+/p16+ showed the lowest HR (0.19, 95% CI, 0.13–0.29); whereas, HPV?/p16+ showed an intermediate HR (0.39; 95% CI, 0.22–0.70). For NSTSCCs, HPV+/p16+ and HPV?/p16+ showed similar OS (HRs, 0.39; 95% CI, 0.26–0.59; and 0.48; 95% CI, 0.24–0.95, respectively). Combined HPV+/p16+ was a significantly better prognostic marker in BSCCs and STSCCs than HPV DNA and p16, alone (all p‐values < 0.05). Whereas, combined testing in NSTSCC was not better than p16 (p = 0.53), alone. In conclusion, double positivity for HPV/p16 in conjunction with the certainty of tumour site improved prognosis.     

人乳头瘤病毒16型E6、E7反义RNA对人宫颈癌SiHa细胞的恶性逆转作用

背景与目的:人乳头瘤病毒16型(human papillomavirus 16,HPV16)的早期基因E6、E7可分别诱导p53的泛素化降解和pRb的高磷酸化失活,与宫颈癌的发生发展关系密切.本研究通过真核表达载体介导HPV16型E6、E7基因反义RNA在SiHa细胞中的表达,探讨反义RNA能否降低E6、E7基因的表达和诱导细胞凋亡.方法:利用pEGFP构建HPV16型E6、E7基因反义RNA的真核表达载体并转染SiHa细胞,利用RT-PCR和Western blot技术检测转染后E6、E7基因的mRNA和蛋白的变化,利用MTT法检测SiHa细胞转染后细胞增殖活性,利用流式细胞仪和激光共聚焦显微镜检测转染后细胞的凋亡情况.结果:转染携带HPV16型E6、E7反义基因的质粒后,SiHa细胞的E6、E7基因的mRNA和蛋白表达均明显下调;MTT结果显示转染后细胞的增殖活性(0.50±0.05)与转染空载体细胞(1.01±0.06)和未转染细胞(1.28±0.06)比较明显降低(P＜0.05);流式细胞仪检测结果显示转染后细胞凋亡率(59.3±11.3)%与转染空载体细胞(9.4±1.8)%和未转染细胞(2.1±0.4)%比较有显著性差异(P＜0.05).激光共聚焦显微镜结果示转染后细胞凋亡明显增多.结论:HPV16型E6、E7基因反义RNA可降低宫颈癌细胞中E6、E7癌基因的表达,诱导宫颈癌细胞凋亡.     

HPV16 E6 E7 siRNA作用于人宫颈癌细胞株的实验研究

目的: 使用HPV16 E6、E7 siRNA处理表达HPV16阳性的人宫颈鳞癌细胞株CaSki和SiHa,观察其对HPV16 E6、E7原癌基因的特异性抑制作用,为探讨使用siRNA治疗宫颈癌的可行性提供实验基础. 方法: 分别设计并合成靶向于HPV16 E6、E7的siRNA各3条,经脂质体包裹后转染两株细胞,分别采用RT-PCR和Western Blot方法,通过对转染前后各时间段两基因mRNA及蛋白的表达情况的检测,验证RNAi作用的特异性及时效性. 结果: 同对照组相比,各E6、E7 siRNA均可引起靶基因表达水平的显著性下降(P<0.05);而空脂质体组及阴性对照siRNA组的靶基因表达水平则无明显变化.其中E6-1 siRNA和E7-2 siRNA对靶基因抑制作用较强.E6-1 siRNA和E7-2 siRNA转染两株细胞后24h、48h、72h及96h均可观察到靶基因表达的显著性降低(P<0.05),而对相应的非靶基因无明显抑制作用. 结论: 不同序列的E6、E7 siRNA,对靶基因的干扰效率不同;E6、E7 siRNA分别作用于宫颈鳞癌细胞株,均引起了靶基因的特异、高效性的抑制,而时非靶基因的表达无明显变化,且抑制作用至少维持到转染后96h.为进一步研究采用RAN干扰技术进行宫颈癌的生物治疗奠定了基础.     

靶向切割HPV16E6 mRNA的核酶诱导宫颈癌细胞CaSKi凋亡的研究(英)

背景与目的:人乳头瘤病毒与宫颈癌的发生相关,核酶是一种能切割靶RNA的特殊RNA。本研究旨在探讨转染了靶向切割HPV16E6mRNA核酶的宫颈癌细胞株的表型,以及核酶对宫颈癌细胞增殖与调亡的作用。方法:计算机设计特异性切割HPV16E6mRNA的核酶,构建抗HPV16E6核酶(HRz)的真核表达质粒。以脂质体法将抗HPV16E6核酶、空载体质粒分别导入CaSKi细胞,命名为CaSKi-R和CaSKi-P细胞。Northern杂交检测3种细胞中E6基因的表达。流式细胞仪检测3种细胞的凋亡率,将3种细胞在相差显微镜和荧光显微镜下观察,采用荧光(Hoechst33342)染色和TUNEL(TDT-mediateddUTPnickendlabeling)两种方法测定细胞凋亡。流式细胞术测定HPV16E6、c-myc、bcl-2、p53、Fas等蛋白的表达。结果:RNA点杂交证实核酶mRNA能稳定表达于CaSKi-R细胞中。Northern杂交证实CaSKi-R中E6mRNA表达水平较CaSKi细胞明显降低,而CaSKi-P和CaSKi细胞的E6mRNA表达水平无差异。CaSKi-R细胞凋亡率明显高于CaSKi和CaSKi-P细胞,细胞周期阻滞于G2期,S期细胞百分率下降。抗HPV16E6核酶能明显减少CaSKi-R细胞中HPV16E6、c-myc、bcl-2蛋白的表达,增高p53蛋白的表达;这种现象并不发生于CaSKi-P细胞中。Fas蛋白的表达在3种细胞中都相近。结论:抗HPV16E6核酶的导入能诱导宫颈癌细     

HPV16型E6、E7变异与宫颈癌发生发展的研究进展

宫颈癌是全球15 ～44岁女性中第二常见的恶性肿瘤,每年的死亡人数约为265 653人,在中国,宫颈癌的发生率及死亡率仍较高.高危型人乳头状瘤病毒(HPV)持续感染是宫颈癌前病变及宫颈癌发生的必要条件,HPV16是最常见的高危人乳头瘤病毒.HPV16编码的E6和E7蛋白在HPV相关的肿瘤中起关键作用.近年来的研究揭示了HPV16 E6、E7基因的变异引起氨基酸变化可影响E6、E7蛋白与p53、pRb 的结合,进而与宿主细胞恶性转化相关.本文将对近年来HPV16 E6、E7变异在宫颈癌发生发展中的作用作一综述.     

HPV16E6蛋白及抑癌基因p53在口腔癌中的表达研究

目的 研究HPV16E6蛋白及抑癌基因p53在口腔癌中的表达.方法 回顾性分析100例口腔癌患者的临床资料和病理切片,采用免疫组织化学方法检测口腔癌组织中p53和HPV16E6蛋白的阳性表达情况,分析其与患者临床分期和病理分级的关系.结果 男性口腔癌患者的p53阳性表达率低于女性患者,HPV16E6阳性表达率高于女性患者;年龄﹥50岁口腔癌患者的p53阳性表达率高于年龄≤50岁的患者,HPV16E6阳性表达率低于年龄≤50岁的患者,但差异均无统计学意义(P﹥0.05).口腔癌患者的组织切片显示,p53阳性表达细胞较少,而HPV16E6阳性表达细胞较多.随着患者临床分期的升高(Ⅰ～Ⅳ期),p53阳性表达率呈下降趋势,HPV16E6阳性表达率呈上升趋势,Ⅱ、Ⅲ、Ⅳ期患者与Ⅰ期患者的p53和HPV16E6阳性表达率比较,差异均有统计学意义(P﹤0.05);随着患者病理分级的升高(Ⅰ～Ⅲ级),p53阳性表达率呈下降趋势,HPV16E6阳性表达率呈上升趋势,Ⅱ、Ⅲ级患者与Ⅰ级患者的p53和HPV16E6阳性表达率比较,差异均有统计学意义(P﹤0.05).结论口腔癌患者的p53阳性表达率随患者病理分级与临床分期的升高而降低,HPV16E6阳性表达率随患者病理分级与临床分期的升高而升高,p53阳性表达率的降低和HPV16E6阳性表达率的升高可能与口腔癌的发生和发展有关.     

宫颈癌及其癌前病变组织中HPV16 E6、HPV16 E7蛋白的表达及其意义

目的 探讨宫颈癌及其癌前病变组织中HPV16E6、E7蛋白的表达及其意义。方法 采用免疫组化SP法对15例正常宫颈、25例宫颈上皮内瘤变（C1N）以及61例浸润性宫颈癌组织进行了HPVl6E6、HPVl6E7蛋白表达的检测。结果在正常宫颈、CINI～Ⅱ、CINⅢ及浸润性宫颈癌中，HPVl6E6蛋白的阳性表达率分别为0（0／15）、7．14％（1／14）、36．36％（4／11）、59．02％（36／61）；CINⅢ和浸润性宫颈癌中HPVl6E6蛋白阳性表达率明显高于正常宫颈组织和CINI～Ⅱ（P〈0．05）；在高、中、低分化宫颈癌中，HPVl6E6蛋白阳性表达率分别为45．45％（5／11）、77．78％（14／18）、53．13％（17／32）；HPVl6E6蛋白在不同分化程度的宫颈癌组织中的阳性表达率有显著性差异（P〈0．05），但HPVl6E6蛋白表达与官颈癌组织分化程度无明显相关性（ys=0．123），HPVl6E6蛋白阳性表达率与宫颈癌临床分期和淋巴结转移无关（P〉0．05）。HPVl6E7蛋白在正常宫颈上皮、CINI-Ⅱ、CIN Ⅲ及浸润性富颈癌组织中的阳性表达率分别为20．00％（3／15）、42．86％（6／14）、63．64％（7／11）、57．38％（35／61），HPVl6E7蛋白在不同宫颈组织中的阳性表达率无明显差异（P=0．05）；HPVl6E7蛋白的表达与富颈癌临床分期、淋巴结转移和组织分化均无关（P〉0．05）。结论 HPVl6E6蛋白的检测有可能作为宫颈癌前病变转归的指标。     

新疆哈萨克族食管癌组织中HPV16 E6、E7基因的检测与p53的关系

目的探讨人乳头状瘤病毒(HPV)16型感染在新疆哈萨克族(哈族)食管癌发病中的作用,并分析HPV16感染与p53过表达之间的关系.方法采用聚合酶链(PCR)技术,检测41例食管鳞状细胞癌组织中HPV16 E6与E7基因表达差异;用LSAB免疫组织化学方法分析p53蛋白的表达.结果癌组织中HPV16 E6与E7基因阳性检出率分别为34.15%(14/41)和63.41%(26/41);用免疫组化检出p53蛋白阳性率分别在HPV16 E6阳性组(57.1%)与HPV16 E6阴性组(14.8%)间、HPV16 E7阳性组(42.3%)与HPV16 E7阴性组(6.7%)间均存在显著性差异(P＜0.05);用PCR检出HPV16 E6、E7基因在食管癌的高分化组、中低分化组中的检出率分别为7.69%(1/13)、46.43%(13/28)和38.46%(5/13)、75.00%(21/28),差别均有统计学意义(P＜0.05).结论提示p53基因突变与HPV16感染在哈族食管癌的发病过程中存在相互促进作用;另外,HPV16 E6与E7基因和哈萨克族食管癌病理组织学分级的生物学行为密切相关.     

Characterization of humoral immune responses against p16, p53, HPV16 E6 and HPV16 E7 in patients with HPV-associated cancers

The cellular tumor suppressor p16 is strongly overexpressed in cervical cancers and precancers. We have previously demonstrated that infiltrating T lymphocytes reactive against p16 can be found in cervical cancer patients. Here, we analyzed whether p16 induces humoral immune responses. Sera of patients with cervical cancer, oropharyngeal cancer, colorectal cancer and autoimmune disease were included. A total of 919 sera were analyzed, including 486 matched sera from a cervical cancer case control study. p16 antibodies were analyzed in Western blot and a newly developed peptide ELISA covering the complete p16 protein. In addition, a Luminex-based multiplex assay was used for simultaneous detection of antibodies directed against p16, p53, HPV16 E6 and HPV16 E7. In all entities, only low p16 antibody reactivity was observed. Epitope mapping revealed 2 predominant epitope regions of the p16 protein. No significant difference in p16 antibody frequency (OR = 0.9; 95% CI = 0.6-1.3) and p53 antibody frequency (OR = 0.6; 95% CI = 0.3-1.2) was found between patients and healthy controls in the cervical cancer case control study. Antibodies against the HPV16 oncoproteins E6 and E7 were detected more frequently in cervical cancer patients when compared with healthy controls (E6 OR = 27.8; 95% CI = 11.1-69.7, E7 OR = 5.7; 95% CI = 2.9-11.1). In conclusion, despite the strong expression of p16 and the observed induction of cellular immune responses, antibody reactivity against p16 was observed only at very low levels independent of the disease background.     

A high and increasing HPV prevalence in tonsillar cancers in Eastern Denmark, 2000–2010: The largest registry‐based study to date

The aim was to explore whether the incidence of tonsillar squamous cell carcinomas (TSCCs) increased in Eastern Denmark, 2000–2010, and whether human papillomavirus (HPV) could explain the increase, and to assess the association of HPV prevalence with gender, age, and origin (i.e., the certainty of tonsillar tumor origin). We applied HPV DNA PCR and p16 immunohistochemistry to all TSCCs registered in the Danish Head and Neck Cancer Group (DAHANCA) and in the Danish Pathology Data Bank (n = 632). Pathologists reviewed and subdivided the tumors into two groups: specified and nonspecified TSCCs. Approximately 10% of HPV‐positive tumors was genotyped by amplicon next‐generation sequencing. The overall crude incidence of TSCCs increased significantly (2.7% per year) and was explained by an increasing incidence of HPV‐positive TSCCs (4.9% per year). The overall HPV prevalence was 58%, with HPV16 being the predominant HPV type. In multivariate analysis, the HPV prevalence was associated with age (<55 vs. >60 years) (OR, 1.72; 95% CI 1.13–2.63) and origin (nonspecified vs. specified TSCCs) (OR, 0.15; 95% CI 0.11–0.22). The association of HPV prevalence with origin increased over time in specified TSCCs (OR per year, 1.10; 95% CI 1.01–1.19), whereas no change over time was observed among nonspecified TSCCs (OR per year, 0.99; 95% CI 0.90–1.08). In conclusion, the observed increase in the number of HPV‐positive TSCCs can explain the increasing number of TSCCs in Eastern Denmark, 2000–2010. HPV prevalence was associated with younger age (<55 years) and a high certainty of tonsillar tumor origin.     

MHC class I expression in HPV positive and negative tonsillar squamous cell carcinoma in correlation to clinical outcome

Human papillomavirus (HPV) is an important factor for the development of tonsillar squamous cell carcinoma (TSCC). In addition, patients with HPV‐positive TSCC have a better clinical outcome than patients with HPV‐negative TSCC. Although, HPV is an important prognostic marker, additional biomarkers are needed to better predict clinical outcome to individualize treatment. Hence, we examined if classical HLA HLA‐A,B,C and nonclassical HLA‐E,G could serve as such marker. Formalin‐fixed paraffin‐embedded TSCC from 150 patients diagnosed 2000–2006, earlier analyzed for HPV DNA and p16^INK4a, and treated with intention to cure were evaluated for the expression of HLA‐A,B,C and HLA‐E,G by immunohistochemistry. For HPV‐positive TSCC a low expression of HLA‐A,B,C, whereas for HPV‐negative TSCC, a normal expression of HLA‐A,B,C was significantly correlated to a favorable clinical outcome. These correlations were more pronounced for membrane staining of HLA‐A,B,C when compared with cytoplasmatic staining. No significant correlation was found between HLA‐E,G and HPV status or clinical outcome. The unexpected contrasting correlation between HLA‐A,B,C expression, and clinical outcome depending on HPV, indicates essential differences between HPV‐positive and HPV‐negative TSCC. Furthermore, our data demonstrate that for both HPV‐positive and HPV‐negative TSCC, the expression of HLA‐A,B,C together with HPV may serve as a useful biomarker for predicting clinical outcome.     

HPV integration begins in the tonsillar crypt and leads to the alteration of p16, EGFR and c-myc during tumor formation

The prevalence of human papillomavirus (HPV) infection is high in the oropharyngeal mucosal regions, of which the tonsil is the most commonly affected. There may be a link between HPV and the pathogenesis of tonsillar cancer (TC), because of common anatomical characteristics between cervical and tonsillar cancer. We aimed to clarify whether HPV directly affects the oncogenesis and biologic behavior of TC by making a comparison between infection prevalence, physical status and viral loading numbers, and clinicopathologic prognostic factors. To compare HPV-related molecules between TC and tonsillitis (CFT), p16, survivin, HIF-1alpha, skp-1, cyclin A, cyclin B1, c-myc and EGFR were investigated. We observed a significant difference in HPV prevalence between 52 TCs and 69 CFTs (73.1% vs. 11.6%), and most of the HPVs were type 16 (87.2%) and nonepisomal (94.1%). Most TCs associated with HPV arose from the tonsillar crypts, and tended to be inverted and poorly differentiated. Compared with HPV-negative TC, HPV-positive TC showed a strong association with p16 overexpression (p<0.0001), and an inverse association with EGFR amplification (p=0.0478). HPV-16 integration status was strongly associated with c-myc amplification (p=0.034) and HIF-1alpha overexpression (p=0.022). HPV-16 integration could be directly related to tonsillar carcinogenesis initially in tonsillar crypts, followed by cell cycle aberration such as p16 overexpression related to the G1-S phase.     

乳腺浸润性导管癌组织中HPV16DNA与p53蛋白表达的研究

 目的 检测人乳腺浸润性导管癌组织中HPV16DNA和p53蛋白，探讨HPV16型感染和p53蛋白表达与人乳腺癌之间的关系。方法 采用分子原位杂交和免疫组化技术检测和分析50例乳腺浸润性导管癌、30例正常乳腺组织中HPV16DNA和p53蛋白二者之间的表达关系。结果 癌组中HPV16DNA阳性和HPV16DNA阴性两组阃p53蛋白的表达均有显著性差异（P〈0．01），HPV16DNA阳性组显示与p53蛋白表达呈负相关（P〈0．02）。结论 HPV16感染后导致野生型p53的降解可能涉及HPV16感染后人乳腺上皮细胞癌变的病理发生过程。