Abnormal expression and mutation of p53 in cervical cancer--a study at protein, RNA and DNA levels.

OBJECTIVES: The objectives of this study are to document the status of p53 expression and mutation in cervical cancer at protein, RNA and DNA levels and to relate this to the presence of HPV. MATERIALS AND METHODS: Biopsy specimens from one hundred and three squamous cell carcinoma of the cervix and histologically normal ectocervix were analysed. Fresh tissues were extracted for protein, RNA and DNA and flash frozen tissue cryostat sectioned for immunohistochemical staining. HPV DNA status was determined by PCR using L1 consensus primers and typed for HPV 16 and 18 with E6 specific primers. p53 expression was determined at the protein level by Western blotting on protein extracts and at RNA level by Northern blotting. RESULTS: There was no p53 overexpression or mutation detectable in the protein extracts. Three of 65 (4.6%) of the carcinomas were positive for p53 by immunostaining with the polyclonal antibody CM1. Overexpression at the RNA level was detected in 2 of 32 (6.3%) carcinomas. p53 mutation was screened for by PCR/SSCP (single strand conformation polymorphism) followed by sequencing to define the site of mutation. Two of the cervical cancers (2.0%) showed mutation in p53 in exons 7 or 8. The mutation rate in HPV positive tumours was 1.2% (1/81) and in HPV negative tumours was 5.2% (1/19). CONCLUSION: p53 overexpression or mutation does not seem to play a significant role in cervical carcinomas.     

Human papillomavirus infection in Bowen disease: Negative p53 expression,not p16INK4a overexpression,is correlated with human papillomavirus‐associated Bowen disease

Genital Bowen disease (BD) has been linked to the high‐risk types of human papillomavirus (HPV) infection. Recently, it has been recognized that HPV also can be associated with extragenital BD. HPV oncoproteins E6 and E7 interfere with the function of p53 and pRb, respectively, leading carcinogenesis. p16^INK4a overexpression induced by inactivation of pRb is recognized as a surrogate marker for HPV‐associated cervical cancer. In this study, we examined the presence of HPV DNA in 142 BD lesions by polymerase chain reaction (PCR), and determined the type of HPV by PCR restriction fragment length polymorphism or direct DNA sequencing. HPV DNA was detected in 66.7% of genital BD and 8.3% of extragenital BD. The types of HPV detected were HPV types 6, 16, 33, 52, 56, 58 and 59. We also investigated the expression of p16^INK4a, pRb and p53 by immunohistochemistry. Positive expression was detected in 88.6% for p16^INK4a, 25.2% for pRb, and 63.8% for p53. There was no significant difference in p16^INK4a and pRb expression between HPV‐positive and ‐negative BD. However, a strong correlation of HPV positivity with p53 negativity was found. A total of 66.7% of HPV‐positive BD showed no p53 expression, whereas the corresponding rate was 32.8% of HPV‐negative BD. This study demonstrated that HPV can participate in the development of BD, not only in the genital lesion, but also in extragenital lesion. p16^INK4a overexpression is not a marker for HPV infection in BD. Instead, negative p53 expression is correlated with HPV‐associated BD.     

Simultaneously detected aberrant p53 tumor suppressor protein and HPV-DNA localize mostly in separate keratinocytes in anogenital and common warts

Abstract The E6 oncoprotein of human papillomavirus (HPV) is known to inactivate the control function on cell cycle exerted by p53 tumor suppressor protein in vitro by binding to p53 and thus facilitating the degradation of p53. We have applied a simultaneous in situ demonstration method for detecting p53 protein and HPV-DNA on formalin-fixed tissue sections, and investigated the in vivo interrelationship of p53 protein and HPV-DNA. Immunohistochemical staining for p53 protein with polyclonal and monoclonal antibodies, recognizing both wild-type (wt) and mutated p53 protein, was performed first and in situ DNA hybridization (ISH) for HPV types 6/11 or 16/18 with digoxigenin-labelled probes thereafter. 47% (25/53) of 48 histologically confirmed primary or recurrent condylomata acuminata (CA), 2 Bowenoid papulosis (BP) and 3 common wart (CW) biopsies, positive for HPV 6/11 or HPV 16/18 DNA, showed keratinocytes immunopositive for p53 protein. Of these. 11 lesions with abundant numbers of p53-positive cells were further analyzed with the double method. Signals for abnormal p53 protein and HPV-DNA were detected in separate cell nuclei in all biopsies and, additionally, in the same cell nuclei in 3 biopsies (1 BP, 1 CA, 1 CW). Usually the p53 positivity localized more basally in the epidermis than HPV-DNA, although p53- and HPV-positive keratinocytes were always located closely. The findings were similar for HPV-types 6/11 and 16/18. Our finding of both p53 and HPV-6/11 signals in the same cell nuclei may indicate complexing of p53 and low-risk HPV's without degradation of p53. Our results show abnormal p53 expression in HPV-infected skin lesions, and suggest that p53 protein is susceptible to aberrations even in the cells in the vicinity of productive HPV infection. However, it is not yet fully understood how HPV interferes with p53 protein in these cells.     

Molecular mechanism of carcinogenesis by human papillomavirus-16

Human papillomaviruses (HPVs) are common DNA viruses in humans. Recently, epithelial cancers associated with HPV infection have been used as models of virus-induced carcinogenesis. HPVs can be divided into two groups, mucosal and cutaneous. HPV-16 is the most frequent mucosal type associated with cervical cancer. Although the molecular mechanisms of carcinogenesis by HPV-16 have not been completely elucidated, it is apparent that HPV infection is the major risk factor in cervical carcinogenesis. Two viral early genes, E6 and E7, and an upstream regulatory region (URR) are preserved in cervical carcinoma cell lines as well as in clinical samples of cervical cancer, indicating that these regions are important in cancer development. E6 and E7 function as transforming genes. E6 protein binds to and promotes degradation of the tumor suppressor protein, p53, while E7 protein complexes and inactivates the Rb protein; together, they disrupt cell cycle regulation. E6 and E7 are transcribed from a promoter, P97. P97 is regulated by complex interactions between multiple, positive and negative, cellular factors and the viral E2 product. E2 disruption caused by the integration into the cellular genome may induce overexpression of E6 and E7. The E6 and E7 proteins are thought to act as critical factors in cervical carcinogenesis by inactivating the two tumor suppressor proteins, p53 and Rb, which are commonly mutated in other human cancers.     

鲍温样丘疹病中hTERT蛋白表达与高危型HPV感染的相关性研究

目的研究hTERT蛋白在鲍温样丘疹病中的表达及与高危型HPV存在的相关性。方法通过原位杂交法研究HPV的存在和免疫组化法检测hTERT蛋白在鲍温样丘疹病中的表达,并探讨hTERT蛋白表达与高危型HPV存在的相关性。结果在26例鲍温样丘疹病标本中,18例(69.2%)为HPV16/18阳性,其中1例HPV16/18阳性标本也有HPV6/11阳性。3例(11.5%)为HPV31/33/35阳性,2例(7.7%)为未定型HPV阳性,21例有高危型HPV存在的标本是hTERT阳性,16例高危型HPV感染的标本呈现了强而弥漫性hTERT蛋白表达。hTERT蛋白表达与高危型HPV的存在显著相关。结论高危型HPV可能诱导了hTERT蛋白的高表达。     

外阴HPV相关疾病中p~(53),cyclinD1和ki67蛋白表达及其与HPV感染的关系

目的 探讨人乳头瘤病毒(HPV)在外阴HPV相关疾病—尖锐湿疣(CA)、外阴上皮内瘤变(VIN)和外阴鳞状细胞癌(VSCC)中的感染情况及与p53,cyclinD1,ki67蛋白表达的关系。方法 在110例外阴HPV相关疾病中应用核酸分子快速导流杂交基因分型技术检测HPV16/18,HPV6/11的表达,同时用免疫组化SP法检测p53,cyclinD1,ki67蛋白的表达,并与10例正常组织进行对照。结果 HPV16/18在CA组,VIN组及VSCC组的阳性表达率均高于正常对照组,而且其阳性表达率随外阴上皮恶性程度增加而增加;HPV6/11在CA组及VINⅠ组的阳性表达率均高于VINⅡ组及VINⅢ组和正常对照组;以上差异均有统计学意义(P均<0.05)。HPV6/11在VSCC组无阳性表达,HPV16/18感染与p53,ki67表达呈正相关,与cyclinD1表达无相关性。HPV6/11感染与p53,cyclinD1,ki67表达无相关性。结论 HPV16/18感染与重度外阴上皮内瘤变及外阴鳞状细胞癌密切相关;HPV6/11感染是尖锐湿疣及轻度外阴上皮内瘤变的重要病因。HPV16/18感染与p53,ki67蛋白表达呈正相关,它们在外阴鳞状上皮恶变过程中有着协同的作用。     

尖锐湿疣皮损中HPV类型及p53、bcl—2分子表达研究

目的 探讨尖锐湿疣(CA)中HPV（人乳头瘤病毒）感染类型及其p53和bcl－2（B细胞淋巴瘤样因子2）分子在HPV6／11阳性尖锐湿疣发病中的可能作用。方法 用PCR法检测了22例CA皮损中HPV6／11和HPV16／18,用免疫组化方法检测了尖锐湿疣组织中p53和bcl－2分子的表达。结果 （1）所有标本均为HPV6／11阳性,未检测出HPV16／18;（2）40．9％的尖锐疣皮损在表皮角质形成细胞中有p53蛋白的表达,但阳性细胞数量较少,主要散在分布于基底层;（3）bcl－2蛋白在CA及鳞状细胞癌中未见表达。结论 （1）HPV6／11是CA的主要致病病毒;（2）CA中部分出现p53分子表达提示这部分细胞可能已出现恶性转化倾向;（3）bcl-2可能与角质形成细胞来源的良性增生性疾病以及恶性肿瘤关系不大。     

Human papillomavirus (HPV) detection among human immunodeficiency virus-infected pregnant Thai women: implications for future HPV immunization

BACKGROUND: Human immunodeficiency virus (HIV)-infected women are at increased risk for developing cervical cancer and for infection with human papillomavirus (HPV). Prophylactic vaccines targeting HPV types 16 and 18 are being evaluated for efficacy among young women. GOAL: The goal was to assess the prevalence of HPV among HIV-infected pregnant women in Bangkok and to evaluate the need for prophylactic HPV vaccines studies in this population. STUDY DESIGN: The study population consisted of 256 HIV-infected pregnant women who participated in a mother-to-child HIV transmission trial. Stored cervicovaginal lavage samples were tested for the presence of HPV DNA by polymerase chain reaction with PGMY09/11 primers and reverse line-blot hybridization for determination of anogenital HPV types. RESULTS: HPV prevalence was 35.5% (91/256); high-risk HPV prevalence was 23.4% (60/256). HPV type 16 or 18 was present in 8.2% (21/256). Almost half of all infections were multiple. Furthermore, overall HPV detection was associated with abnormal cervical cytology (P<0.001) and higher HIV-plasma viral load (P=0.007). CONCLUSIONS: Only one-quarter of HIV-infected pregnant women in Bangkok had high-risk HPV types; less than 10% had HPV types 16 or 18. As the HPV prevalence is expected to increase during HIV disease, prophylactic vaccines targeting HPV types 16 and 18 should be studied among HIV-infected women not yet infected with these HPV types and not previously exposed.     

Correlation of p16 and pRb expression with HPV detection in Bowen's disease

BACKGROUND: Bowen's disease is a form of cutaneous squamous cell carcinoma which may be caused by ultraviolet radiation, human papilloma virus (HPV) infection, or other causes. Although p16 over-expression is a surrogate marker of HPV E7-mediated catabolism of pRb in premalignant and malignant lesions of the cervical mucosa, the correlation of p16 and pRb expression with HPV detection in Bowen's disease has not been well characterized. METHODS: A retrospective study on formalin-fixed tissues was performed. Immunohistochemistry for p16 and pRb was performed on 32 cases. DNA was successfully extracted from 20 cases, and polymerase chain reaction was performed to amplify a highly conserved region of the HPV L1 open reading frame. RESULTS: Twenty-eight of 32 (88%) cases showed strong diffuse staining for p16 but were negative for pRb; two of 32 cases (6%) were negative for p16 but were diffusely positive for pRb; one case was strongly positive for both p16 and pRb, and one case was negative for both p16 and pRb. Three of 20 cases (15%) contained HPV DNA. All three of these cases showed strong p16 expression and lack of pRb staining. CONCLUSIONS: Most cases of Bowen's disease strongly express p16 but not pRb. In contrast to HPV-associated lesions of the cervical mucosa, p16 overexpression in cutaneous Bowen's disease appears to be unrelated to HPV status. The p16 overexpression in Bowen's disease may reflect disruption of the G1/S checkpoint, resulting in unregulated cell cycle progression.     

Presence of human papilloma virus type 16 DNA sequences in human nonmelanoma skin cancers

The presence of human papillomaviruses (HPV) has been shown to be associated with the development and progression of invasive cancers of the genital tract, skin, and head and neck. In this study we analyzed 37 human nonmelanoma skin cancers (21 squamous cell carcinomas and 16 basal cell carcinomas) for the presence of HPV sequences. The polymerase chain reaction (PCR) was employed using primers designed to amplify DNA encoding the E6-E7 region of HPV types 6b/11, 16, and 18. HPV type 6b/11 and 18 sequences were absent from the DNA of all 37 tumors examined. However, HPV type 16 sequences were detected in four of 21 squamous cell carcinomas (SCC) (19%) and three of 16 basal cell carcinomas (BCC) (19%) as indicated on agarose gel electrophoresis by the presence of a single specific DNA band of predicted length. Furthermore, HPV type 16 sequences were absent in the DNA of normal skin from these seven skin cancer patients. The presence of HPV type 16 sequences in the seven skin tumors was confirmed by dot blot hybridization of PCR-amplified material to 32P-labeled HPV type 16, but not to HPV type 6/11 or 18-specific probes. These data indicate that HPV type 16, but not 6b/11 or 18, is associated with development of some human nonmelanoma skin cancers.     

P53,Fas,Bcl-2蛋白在尖锐湿疣组织中的检出及其与HPV感染的相关性

目的　探讨P5 3 ,Fas ,Bcl 2蛋白在尖锐湿疣 (CA)组织中的检出情况及其与人乳头瘤病毒 (HPV)感染的关系。方法　采用标准免疫组化法检测了 2 4例CA、46例宫颈癌和 2 8例正常宫颈组织P5 3 ,Fas ,Bcl 2蛋白 ,用PCR技术对各组织标本的HPV DNA进行检测和 6,11,16,18分型。结果　P5 3 ,Fas ,Bcl 2蛋白在CA组的阳性率显著高于正常对照组 (P <0 .0 0 1) ;CA组和宫颈癌组之间差异无显著性 (P >0 .0 5 )。综合分析 ,蛋白阳性率与HPV感染率密切相关 (P <0 .0 5 ) ,但CA组或宫颈癌组的蛋白阳性率与HPV 6/11型或 16/18型的检出与否无统计学意义 (P >0 .0 5 )。结论　P5 3 ,Fas ,Bcl 2蛋白与尖锐湿疣的发病有密切关系 ,凋亡基因的表达可能抑制HPV的增殖     

Detection of human papillomavirus DNA in anogenital condylomata in men using in situ DNA hybridisation applied to paraffin sections.

An in situ DNA hybridisation method was used to detect human papillomavirus (HPV) DNA (HPV types 6, 11, 16, and 18), and an immunoperoxidase (IP-PAP) method to detect HPV structural protein expression in paraffin sections of biopsy specimens from 133 men treated for penile (in 114 cases) and anal (in 19 cases) warts. The anatomical distribution on the penis of classic condyloma acuminatum and of papular and flat condylomata was practically identical. The gross appearance of the warts did not correlate with their morphology on light microscopy. The detection rate of dysplasia was very different in the three types of lesions (25% in flat, 50% in acuminatum, and 75% in papular warts). Of 133 lesions, 59 (44.4%) contained HPV antigens, their expression being inversely related to the grade of dysplasia; only 17% of HPV 16 lesions had detectable HPV antigen compared with 50% to 67% in lesions of the other three HPV types. HPV 16 and HPV 18 DNA were most commonly (11%) detectable in Bowenoid lesions; however, most of the HPV 16 and 18 positive cases were found among the flat and acuminatum type of lesions. Though the overall detection rate of HPV DNA (76%) did not correlate with the grade of dysplasia, a clear cut association of HPV 16 and HPV 18 with dysplastic lesions was found, none of the HPV 16 and 25% of the HPV 18 positive cases being devoid of concomitant dysplasia. The corresponding figures for HPV 6 and HPV 11 were 59.2% and 68.8%, respectively. The implications of these findings are discussed in terms of epidemiologically established connections between penile and cervical cancer, with special emphasis of the high risk HPV types 16 and 18. The applicability of in situ DNA hybridisation as a powerful tool in the analysis of specific HPV DNA sequences in routinely processed biopsy specimens from these lesions is emphasised.     

高危型人乳头状瘤病毒分型检测在宫颈癌及癌前病变中的价值研究

目的:评价高危型人乳头状瘤病毒HPV16,HPV18检测在宫颈癌及癌前病变筛查中的应用价值。方法:以宫颈组织病理学诊断为金标准,采用荧光PCR技术检测筛查360例病例的HPV16,HPV18分型情况,并分别通过传统细胞涂片和液基细胞学法进行验证,比较筛查方法的灵敏性和特异性指标,同时了解永川地区HPV16和HPV18的感染率,评价检测意义。结果:HPV16或HPV18总感染率为41.4%(149/360)。其中HPV16,HPV18阳性率分别为62.4%(93/149)和32.2%(48/149),混合感染率为6%(9/149)。HPV16和HPV18分型的检测方法的灵敏度,特异度,阴性预测值和阳性预测值均高于传统细胞涂片和液基细胞学法,且HPV检测联合液基细胞筛查效能高于联合传统细胞学涂片法。结论:高危型HPV16,HPV18的感染与宫颈癌及宫颈癌前病变密切相关,HPV16,HPV18的检测对筛查、预防宫颈癌,降低宫颈癌发病率有重要作用。     

The impact of a quadrivalent human papillomavirus (types 6, 11, 16, 18) virus-like particle vaccine in European women aged 16 to 24

Background  Quadrivalent human papillomavirus (HPV types 6/11/16/18) L1 VLP vaccine is highly effective in preventing HPV 6/11/16/18-related cervical and external genital disease. Herein, we evaluated the impact of the quadrivalent HPV 6/11/16/18 L1 VLP vaccine on prevention of HPV-associated cervico-genital lesions in a broad population of sexually active European women.
Methods  Female subjects ( N = 9265) aged 16–24 with four or fewer lifetime sexual partners were enrolled and randomized to quadrivalent HPV vaccine or placebo. Subjects underwent cervicovaginal sampling for HPV DNA detection. Papanicolaou testing and anti-HPV 6/11/16/18 serology testing was also performed.
Results  Vaccine efficacy against lesions representing immediate cervical cancer precursors (cervical intraepithelial neoplasia grade 2/3 or adenocarcinoma in situ ) related to HPV 6/11/16/18 in the per-protocol population was 100.0%[95% confidence interval (95% CI), 89.8–100.0]. Efficacy against external genital lesions (vulvar or vaginal intraepithelial neoplasia, condyloma, vulvar or vaginal cancer) related to vaccine HPV types in the per-protocol European population was 99.0% (95% CI, 94.4–100.0).
Conclusion  These data demonstrate that quadrivalent HPV 6/11/16/18 vaccination programs in 16- to 24-year-old European women can be beneficial.
NCT0009252, NCT00092534, NCT00092495     

HPV-related cancer susceptibility and p53 codon 72 polymorphism

Conflicting results regarding the association of a polymorphism at codon 72 of the p53 tumour suppressor gene and susceptibility to develop human papilloma virus (HPV)-associated cervical cancer have been published over the last year, implicating differences in ethnic background, sample origin, sample size and/or detection assay. The material for this study was collected in the identical geographical region as for 2 previous reports with contradictory results regarding the association of codon 72 genotype with squamous cell cancer (SCC). We have used an alternative detection assay, based on pyrosequencing technology, that interrogates the variable position by the accuracy of DNA polymerase. In addition to cervical clinical specimens from SCC, HPV16- and HPV18-infected adenocarcinoma cases as well as cervical intraepithelial neoplasia (CIN) were investigated. No significant association was found between p53 codon 72 genotype and the risk to develop adenocarcinoma, SCC or CIN in the Swedish population.     

宫颈癌标本中HPV16的E6、E7和LCR的变异研究

目的:探究分析E6、E7和LCR(long control region,LCR)在宫颈癌标本中HPV16中的变异情况。方法:随机选取我院病理实验室于2015年1月至2016年1月期间留存的HPV16阳性宫颈癌标本100例为研究对象,分别采用PCR技术进行E6、E7和LCR片段的扩增处理,并采用DNA序列进行PCR扩增产物的序列测定,分析E6、E7和LCR的变异表现。结果:E6基因中最常见变异为T350G(67.27%),E7基因中最常见变异为T789C(72.67%),LCR最常见变异为G7521A(90.90%),LCR区中出现G7799A、A7636C、C13T、C7678T新变异,E7区高度保守,YY1转录因子结合点是LCR变异的主要集中点。结论:宫颈癌标本中HPV16存在E6、E7和LCR变异情况,分析高危型HPV变异有助于宫颈癌HPV的早期诊断,可将其应用于宫颈癌防治的疫苗设计中,具有广泛的临床应用前景。     

热休克蛋白在尖锐湿疣组织中的检测及其与HPV感染相关性

目的　探讨热休克蛋白 70(HSP70)在尖锐湿疣(CA)组织中的检测及其与人乳头瘤病毒 (HPV)感染的关系。方法　采用免疫组化法检测了 24例CA、46例宫颈癌和 28例正常宫颈组织HSP70,用PCR技术对各组织标本的HPV-DNA进行检测和 6, 1 1, 1 6, 1 8分型。结果　HSP7 0在CA和宫颈癌组的阳性率显著高于正常宫颈对照组 (P<0. 01)。CA组HPV6, 11型DNA阳性者HSP70的检出率显著高于阴性者(P<0. 05),宫颈癌组HPV16, 18型DNA阳性者与阴性者HSP70的检出率差异无显著性(P>0. 05)。结论　HSP70的表达与尖锐湿疣的发病有关,HSP70在尖锐湿疣组织中的表达与HPV的感染明显相关。     

Mucosal human papillomavirus types in squamous cell carcinomas of the uterine cervix and subsequently on fingers

Human papillomavirus (HPV), especially type 16, is causally involved in the pathogenesis of anogenital cancer. There is an increasing number of reports of HPV infections in squamous cell carcinoma (SCC) of the fingers. A search of the Swedish cancer register covering the period 1958-94 inclusive for women with a history of genital and upper extremity SCC revealed 63 cases. Archival material from both cervical and cutaneous lesions was traced and analysed for the presence of HPV DNA in 32 of these patients. A newly developed 'neighbour primer' polymerase chain reaction (PCR) for HPV 16 DNA, aimed at overcoming the obstacle of cross-linked target DNA, was shown to be superior to conventional general and type-specific HPV PCR tests. HPV DNA was significantly more frequently found in digital tumours than in tumours at other cutaneous sites of the upper extremities [67% (10 of 15) vs. 7% (three of 43); P < 0.001]. Among 13 patients with a history of both cervical and finger SCC, HPV 16 was found in cervical samples from seven patients. From five of these seven patients, HPV 16 was also present in the corresponding finger lesions. The results support the hypothesis of a possible transmission of patients' genital HPV infections to fingers.