The p53 codon 72 genotypes in HPV infection and cervical disease

OBJETIVE: An experimental study has indicated that individuals homozygous for the Arg-encoding allele of p53 gene may have an increased susceptibility to HPV-related cervical cancer but many epidemiological studies have failed to repeat this result. Many epidemiological studies have failed in the attempt to repeat this results. The aim of the present work was to investigate whether the p53 arginine allele confers a risk factor for cervical carcinogenesis. STUDY DESIGN: Using PCR based technology, DNAs from 90 normal cervical samples and 205 abnormal cervical tissue scrapes were analyzed for the type of HPV present and p53 codon 72 polymorphism. RESULTS: Non statistically significant differences were found for the frequencies of p53 genotypes in the different cytological/histological groups (chi2=1.4; P=0.97) nor for the risk for HPV infection (chi2=1; P=0.9). CONCLUSION: This study showed that polymorphism at codon 72 of TP53 gene is not associated with an increased susceptibility to cervical disease and/or HPV infection in the Argentine women population.     

HPV oligonucleotide microarray-based detection of HPV genotypes in cervical neoplastic lesions

BACKGROUND: In this study we examined the use of a new-human papillomavirus (HPV) detection method, the HPV oligonucleotide microarray system (Biomedlab Co., Korea), which we compared with the well-established HPV DNA detection system (Hybrid Capture II; HC-II, Digene Co.). This new method prompted us to develop a new HPV genotyping technique, using the oligonucleotide microarray, to detect the generic and type-specific sequence of HPV types. In particular, we undertook the evaluation of the clinical efficacy of the HPV oligonucleotide microarray for detecting HPV in cervical neoplastic lesions. METHODS: One hundred forty patients were involved and classified into three groups according to their histopathologic diagnoses: Group I (nonspecific chronic cervicitis; n = 61), Group II (low-grade squamous intraepithelial lesion (SIL); koilocytosis, and mild dysplasia; n = 39), and Group III (high-grade SIL; moderate, severe dysplasia and in situ carcinoma; n = 40). Cytological diagnoses were based on the Bethesda System and cervical samples were analyzed by the two methods. The HPV oligonucleotide microarray detected 15 types of high-risk HPV (HPV-16/-18/-31/-33/-35/-39/-45/-51/-52/-56/-58/-59/-66/-68/-69) and 7 types of low-risk HPV (HPV-6/-11/-34/-40/-42/-43/-44). RESULTS: In 105 of the 140 cervical samples (75%), HPV DNAs were examined using the HC-II method. HPV detection rates using the HPV microarray agreed with those of HC-II. One HC-II-positive, but HPV microarray-negative, case occurred in the low-grade SIL (Group II) and was later confirmed negative for HPV. The other HPV microarray-positive but HC-II-negative case was found to be HPV-18 by PCR. Low-risk types of HPV were detected in 3 of 39 low-grade SIL cases (Group II) using the HPV microarray. HPV-16 was the most frequent type (32.1%) in all specimens tested, and was significantly more frequent in low-grade or high-grade intraepithelial lesions (Groups II or III) than in normal controls (Group I) (P < 0.05). HPV-58 was the second most common type (17.5%) in Group III. The HPV microarray was found to have advantages in terms of identifying the HPV genotypes and cases of multiple HPV infection. Double HPV infections were detected in 12 cases and triple HPV infections in 7 cases. Two cases were positive for four types of HPV (HPV-16/18/33/35, HPV-16/18/58/68). The sensitivity of HPV testing (HC-II; 94.9%, HPV microarray; 93.7%) for identifying patients with squamous intraepithelial lesion was significantly better than the sensitivity of cytology (77.1%, P < 0.05). On using multiple logistic regression analysis to estimate the relative risk of SIL versus HPV type, HPV-16-positive cases were found to have a 7.5-fold risk of SIL (95% CI = 3.28-16.51; P < 0.01). HPV-33 and HPV-58 were found to be significantly related to high-grade SILs (P < 0.01). CONCLUSIONS: Our results suggest that the HPV oligonucleotide microarray is highly comparable to HC-II for detecting HPV in cervical specimens. The HPV oligonucleotide microarray provides useful information on viral genotype and multiple HPV infections in HPV-related cervical lesions. Genetic information on HPV in cervical specimens might be a particular benefit of the new procedure in the management of cervical neoplastic lesions     

HPV16/18E6蛋白和PKR/p-PKR在宫颈病变的表达及意义

目的:探讨宫颈病变组织HPV16/18感染对蛋白激酶R(PKR)激活的影响及两者在宫颈癌形成、演进过程中的作用和对宫颈癌患者预后的影响。方法:用免疫组化SP法检测HPV16/18型E6蛋白(E6)、PKR、磷酸化型PKR(p-PKR)在63例宫颈癌、114例宫颈上皮内瘤样病变(CINⅠ~Ⅲ)、15例正常宫颈组织的表达。结果:E6蛋白与PKR在各组的阳性表达率与宫颈病变级别呈正相关(P<0.05,P<0.05),E6蛋白与PKR在各组的阳性表达率呈正相关(P<0.05);宫颈癌组PKR阳性表达率明显高于p-PKR(P<0.01);E6、PKR阳性表达率与肿瘤大小有关(P<0.05,P<0.05);宫颈癌患者病情进展与临床分期显著相关(P<0.01),病情进展与E6、p-PKR阳性表达率相关(P<0.05,P<0.05)。结论:HPV16/18感染可阻碍PKR激活,突破机体防御HPV16/18感染的机制,在宫颈癌的发生及演进过程中可能起了重要作用,对宫颈癌患者预后不利;p-PKR能抑制宫颈癌患者病情进展,可能改善其预后。     

hWAPL基因多态性增加HPV16/18阳性宫颈癌患者的发病危险性

目的:检测宫颈癌患者外周血人半翼基因(hWAPL)的单核苷酸多态性(SNP)及宫颈癌组织中的HPV DNA感染型别,探讨宫颈癌的发病易感性。方法:应用基质辅助激光解吸附电离飞行时间质谱检测技术检测150例宫颈癌患者(病例组)和150健康妇女(对照组)外周血hWAPL基因的12个标签SNP(tagSNP)位点的多态性;采用基因芯片方法对宫颈脱落细胞进行HPV DNA分型检测。结果:与对照组比较,病例组hWAPL基因的SNP位点rs11595882 T(P=0.001,OR=2.481)、rs10887621 C(P=0.040,OR=1.610)、rs11202058 G(P=0.043,OR=1.479)的危险等位基因频率明显高于对照组(P0.05)。病例组hWAPL基因SNP rs7083506(CC+CT)(P=0.011,OR=3.273)、rs11595882(TT+TC)(P=0.002,OR=2.510)、rs7918136(TT+TA)(P=0.011,OR=3.273)、rs11202058的(GG+GA)(P=0.011,OR=3.273)危险等位基因型的频率显著高于对照组(P0.05)。HPV16/18阳性的宫颈癌患者的rs11595882和rs11202058位点的危险等位基因频率和等位基因型频率显著高于HPV16/18阳性的正常人群(P值均小于0.01)。结论:hWAPL基因的多态性改变是宫颈癌发生的危险因素,尤其对合并高危型HPV16/18阳性的患者尤为显著。     

HPV prevalence, E6 sequence variation and physical state of HPV16 isolates from patients with cervical cancer in Sichuan, China

OBJECTIVES: Infection with high-risk human papillomavirus (hr-HPV) is an important factor associated with cervical cancer. The genetic mutation of HPV16 E6 and integration of HPV16 DNA in the cervical carcinoma tissues are considered important genetic changes in cervical lesion progression. But the studies of hr-HPV epidemiology are relatively less in the area of Sichuan, China. Therefore, we investigated the prevalence of 9 high-risk subtypes and analyzed the genetic mutation characteristic of HPV16 E6 and physical state of HPV16 DNA. METHODS: The fragments of L1 and E6 genes were amplified by PCR or nested PCR and then directly sequenced. Further, the multiplex PCR for HPV16 E2 and E6 genes was performed for detection of integration. RESULTS: HPV16, 58 and 18 were prominent, accounting for 78.6%, 20.0% and 9.7%, respectively in 145 isolates. E6 variants revealed that the European (EP) prototype and East Asia (EA) strain were 26 (23.0%) and 34 (30.1%), respectively. Furthermore, there were 14 base substitutions in E6 regions of the study group, of which 12 resulted in amino acid changes and the rest was silent mutation. Significantly, the 240G substitution exactly located the P53 degradation site. Overall, 8 of 114 (7.0%) isolates only contained integrated HPV16 DNA, 43 (37.7%) only contained episomal DNA and 63 (55.3%) contained both integrated and episomal DNA. The proportion of disruption of an intact E2 gene in the patients with cervical cancer is much lower than that in the previous studies. CONCLUSIONS: HPV16, 58 and 18 were mainly prevailing subtypes in patients with cervical cancer from Sichuan areas, China and EP/EA strains were predominant in these areas. Some mutations of E6 gene, which lead to the amino acid changes, may be more potentially carcinogenic and the proportion of disruption of an intact E2 gene is much lower.     

Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray

OBJECTIVES: This study was conducted to evaluate a clinical efficacy of human papillomavirus (HPV) oligonucleotide microarray (Biomedlab Co., Seoul, South Korea) for the detection of HPVs in various cervical lesions. RESULTS: HPV DNAs from 234 patients were analysed by two methods. Among those, 212 patients were classified into 5 groups according to the histologic diagnosis: chronic cervicitis, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III, and invasive cervical carcinoma. PCR-RFLP could detect 7 types of high-risk HPVs (HPV 16/18/31/33/35/52/58) and HPV microarray could detect 15 types of high-risk HPVs (HPV 16/18/31/33/35/39/45/51/52/56/58/59/66/68/69) and 7 types of low-risk HPVs (HPV 6/11/34/40/42/43/44).HPV genotyping by HPV oligonucleotide microarray revealed that HPV16 was the most frequent type (30.2%) in all specimens tested and was significantly more frequent in CIN III and invasive carcinomas than other lesions. METHODS: HPV DNAs were detected in 158 and 174 of the 234 cervical samples by PCR-RFLP and HPV microarray, respectively. The correlation between the two methods was good in detecting HPVs in general (kappa index = 0.69) and HPVs 31 and 52 (kappa index = 0.70 and 0.70, respectively) and excellent in detecting HPVs 16, 18, 33, 35, and 58 (kappa index = 0.90, 0.88, 0.92, 0.77, and 0.84, respectively). Double HPV infection was detected in 10 cases and one triple infection was detected. By combining cytology and HPV testing, the sensitivity was improved to 87.5, 95.5, 96.1, and 97.2% in CIN I, CIN II, CIN III, and carcinoma, respectively. CONCLUSIONS: This results suggest that HPV oligonucleotide microarray is a highly comparable method to the previously used PCR-RFLP method for the detection of HPVs in cervical specimens. Genetic informations for HPV infection in cervical specimens may offer new strategies in manipulating the patients harboring cervical intraepithelial neoplasia and cervical carcinoma.     

Detection of HPV genotypes in cervical lesions by the HPV DNA Chip and sequencing

OBJECTIVE: A newly introduced HPV detection technique in cervical lesion, the HPV DNA Chip test, contains 24 HPV probes and has the advantage of being able to detect 24 HPV types at once. We performed HPV DNA sequencing and compared the results with that of the HPV DNA Chip for evaluation of the accuracy of the DNA Chip test. METHODS: The HPV DNA sequencing was performed in samples of 282 patients, where specific HPV type had been shown in HPV DNA Chip test. The sixteen cases where multiple HPV types had been found in HPV DNA Chip test were included in 282 cases. The sequencing was also performed in HPV-other type samples of 95 patients, where positive in HPV-PCR, but specific HPV type had not been found. RESULTS: In 257 cases (91.1%) of 282 cases, the HPV types of the HPV DNA sequencing test were in agreement with types of the HPV DNA Chip. In 16 cases (5.7%), the sequencing types were different from the types of HPV DNA Chip. But, in 9 of 16 cases, types in HPV DNA sequencing were absent types in HPV DNA Chip test. The interpretation of HPV DNA sequencing was impossible in nine cases (3.2%). The HPV DNA sequencing test of 95 cases of HPV-other type showed that the sequencing types from 94 cases (98.9%) were absent types in HPV DNA Chip test. In sequencing test of HPV-other type, HPV-81 (20.0%), HPV-62 (14.7%), HPV-84 (13.7%), and HPV-61 (13.7%) were frequently detected. CONCLUSION: HPV DNA Chip is an accurate method for detecting the 24 HPV genotypes.     

Immune response to p53 and HPV-16 E6 proteins in patients with cervical cancer

Park JS, Kim CJ, Um SJ, Hwang ES, Kim HS, Park SN, Namkoong SE, Kim SJ. Immune response to p53 and HPV-16 E6 proteins in patients with cervical cancer. Int J Gynecol Cancer 1998; 8 : 328–335.
To investigate whether p53 autoantibodies could be found in the sera of patients with cervical cancers, we have therefore studied by radioimmunoprecipitation assay, using in vitro translated p53 protein, sera from such patients. The sero-positive patients for p53 were also evaluated in relation to immunoreactivity to p53 antigens by immunohistochemistry, for genomic alterations of p53 by PCR-SSCP, and for the presence of HPV-16/18 DNAs in the cervical cancer cells. In immunohistochemistry, expression of p53 protein was seen in 47% (14/30) of HPV-16 or -18 positive cervical cancers and 13 % (2/15) of HPV-16/18 negative cervical cancers ( P < 0.01). Eight out of 12 control ovarian cancers (67%) showed positive p53 staining in most tumor cells. Cases of cervical cancer and ovarian cancer, which were positively expressed p53 protein in the tissue or the sera, were studied for genomic alterations in exons 5–8 of the p53 by PCR-SSCP. Serum antibodies to in vitro translated p53 protein were found in two cases from 63 cervical cancers; one patient was stage IIA, having HPV-16 DNA in a tumor of squamous cell type, and another patient was stage IIIB, having HPV-16 and -18 DNAs in an adenocarcinoma. The cervical cancer tissues from the two sero-positive patients were also positive for p53 immunostaining. None of the cervical cancer samples showed aberrant bands, but three of eight cases of ovarian cancer which were positive for p53 protein by immunostaining were shown to have aberrant bands by PCR-SSCP. In contrast to ovarian cancers, alteration of p53 tumor suppressor gene and positive antibody response to p53 protein seem to be rare events in patients with cervical cancer.     

HPV16-specific cytotoxic T lymphocyte responses are detected in all HPV16-positive cervical cancer patients

OBJECTIVES: The specific CTL response against human papillomavirus (HPV) antigens in women with cervical cancer has been poorly studied. Immunological monitoring of this response is central for understanding the principles that underlie successful immunotherapeutic strategies. The aim of the study was to investigate the HPV16 E6/E7-specific CTL immune response in a group of untreated HPV16-positive cervical cancer patients. METHODS: Peripheral blood mononuclear cells from 21 untreated cervical cancer patients and 4 healthy controls were isolated prior to any therapy. Autologous monocyte-derived dendritic cells (MDDCs) were transiently transfected with HPV16 E6 or E7 expression vectors and used for one round of in vitro restimulation and as target cells in chromium release assays with restimulated peripheral blood lymphocytes. RESULTS: Transfected monocyte-derived dendritic cells were differentiated to exhibit a fully mature phenotype. HPV16 E6 and E7 transgenes were expressed and translated as measured by RT-PCR and intracellular flow cytometry, respectively. All HPV16-associated cervical cancer patients showed evidence of specific CTLs. Lytic activity for HPV16 E6 (11/12) and/or E7 (8/9) was above 30% at the 100:1 effector to target ratio. None of the HPV16-negative cervical cancer patients or healthy controls were above 15% of lysis. CONCLUSIONS: These data suggest that HPV-specific cytolytic immune responses can be detected in all untreated cervical cancer patients. Our approach, using dendritic cells for restimulation and as target cells, may enhance immunomonitoring of cervical cancer patients.     

HPV 11, 16 and 18 DNA sequences in cervical swabs from women with cervical dysplasia: prevalence and associated risk of progression.

Cervical swabs obtained from 164 women with histologically proven preinvasive and invasive cervical neoplasia were analysed for HPV type 11, 16 and 18 DNA by filter in situ hybridisation. HPV 16 or 18 was detected in 8 of 24 swabs from patients with invasive squamous cell carcinoma (33%), in 59 of 100 patients with carcinoma in situ or severe dysplasia (59%) and in 16 of 40 patients with mild or moderate dysplasia (40%). HPV 6 or 11 was found in only 2% of all swabs. Thirty-eight of the patients participated in a prospective follow-up study and were monitored non-invasively by cytology and colposcopy for 8 to 36 months. 25 patients had persisting or progressive lesions, 13 of which harboured HPV 16 or 18. Of 13 patients who had complete resolution of the dysplasia, only 2 were HPV-positive. The study indicates a significantly higher risk of malignant progression when the cervical dysplasia is associated with HPV 16 or 18 infection.     

Distribution of HPV16 and 18 intratypic variants in normal cytology, intraepithelial lesions, and cervical cancer in a Mexican population

OBJECTIVE: Several intratype variants of HPV16 and 18 have been identified. These variants are associated with populations from different geographic regions, and show a differential distribution among the severity of the cervical lesion, most likely due to different pathogenic potential. The objective of this study was to investigate the variant distribution of HPV16 and 18 in a Mexican population and its association with the severity of the cervical lesion and the histological lineage of cervical cancer. METHODS: HPV types 16 and 18 detection was performed in 412 samples of preinvasive and invasive specimens from patients attending a Primary Health-Care Center, an Early Cervical Lesion Clinic, or a Cancer Center. Distribution of HPV variants was correlated with the cytological findings and tumor cell types using contingency tables. Statistical difference was tested with the Fisher's Exact Test or its Fisher-Freeman-Halton extension for RXC tables. Alpha value was set at the P < 0.05. RESULTS: Among the 277 women included in this study without cancer, 63.5% (176 cases) had a normal cytology; from the remaining 101 women, 53.5% were LSIL (54 cases), and 46.5% HSIL (47 cases). From a total of 135 invasive carcinomas, 78.5% were squamous (106 cases); 6.6% adenocarcinoma (9 cases); 9.6% adenosquamous (ADSC) (13 cases); and 5.1% were undifferentiated carcinoma (7 cases). HPV16 E and AA-a were evenly distributed among preinvasive and invasive lesions. However, the isolate AA-c was exclusively found in cervical cancer. HPV18 Var-1(E) was almost exclusively found in invasive lesions, while the HPV18 Var-2(Af) predominated in normal or preinvasive lesions. In invasive cancer, this variant was found only in squamous tumors. CONCLUSIONS: The differential distribution of HPV16 and 18 variants in cervical lesions we found further supports experimental data on the different pathogenic potential of HPV16 and 18 variants for cervical cancer development.     

G2 checkpoint in uterine cervical cancer with HPV 16 E6 according to p53 polymorphism and its screening value

INTRODUCTION: We aimed to verify not only whether homozygous Arg at codon 72 of the p53 apoptotic domain is a possible risk factor for cervical human papillomavirus (HPV)-related cancer, but whether degraded p53 may have an effect on a G2 checkpoint of the cell cycle. The implication of the codon 72 polymorphism of p53 in cervical tumor remains controversial. Furthermore, G2 checkpoint alteration and its relationship with p53, the codon 72 allotype, according to HPV infection in cervical tumors, has not been studied. MATERIALS AND METHODS: The purified genomic DNA from 252 archival cervical tissues [102 cervical intraepithelial neoplasias (CINs) and 46 squamous cell carcinomas of the uterine cervix (SCCs), and 104 normal] were amplified by nested polymerase chain reaction (PCR) for HPV-16/HPV-18. In addition, all of them were amplified by PCR for exon 4 of p53, where the codon 72 resides. The amplified PCR products were then sequenced using the forward primer. A polymorphism analysis was done by SnaPshot ddNTP primer extension and following direct sequencing. The reaction mixture was treated with 0.25 unit of shrimp alkaline phosphatase (Amersham) at 37 degrees C for 1 h, subsequently performed in an ABI Prism 310 Genetic Analyzer (Perkin-Elmer). The archival slides were incubated overnight at 4 degrees C using mouse anti-human recombinant cyclin B1 polyclonal antibody or mouse anti-Xenopus p34(cdc2) monoclonal antibody for immunohistochemistry (Santa Cruz Biotech, Santa Cruz, CA). RESULTS: The frequency of Arg allelic homozygosity was high in both cases (89.1%) and the control (80.8%) group (P = 0.4703). All groups except CIN were in Hardy-Weinberg equilibrium. There was no significant difference in the frequency of p53 polymorphism between the HPV-positive (Arg, 88.0%) and the negative (Arg, 88.8%) groups, or between CIN (Arg, 88.2%) and SCC (Arg, 89.1%). Both immunoreactivities to cyclin B and p34(cdc2) were strongly correlated with the HPV infection (P = 0.0001) and the histological types (P = 0.0001) between CIN and SCC, being strongly correlated with each other (alpha:0.62954, P = 0.0001). CONCLUSION: The particular type of the p53 polymorphism does not bear relation to the progression of cervical cancer, HPV infection, or to the p53 codon 72 polymorphism. However, the G2 checkpoint appears to be altered in the case of a HPV-positive SCC.     

Analysis of p53 codon 72 polymorphism and its association with human papillomavirus 16 and 18 E6 in Chinese cervical lesions

The aim of this study was to analysis the relationship between p53 codon 72 polymorphism with human papillomavirus (HPV) 16 and 18 E6 in Chinese cervical cancer. A total of 81 cervical squamous cancer (specimens of G1, G2, and G3 are 13, 24, and 44, respectively; and of stage IB, IIA, IIB, and IIIA are 15, 37, 24, and 5, respectively), 18 cervical adenocarcinoma, 88 cervical intraepithelial neoplasm (CIN) (specimens of CIN II and III are 30 and 58), and 60 normal cervical specimens were included in this study. Polymerase chain reaction was used to examine p53 genotypes and HPV 16 and 18 E6. The frequencies of p53 Arg homozygosity in cervical squamous cancer, cervical adenocarcinoma, and CIN (II-III) were 58.02%, 55.55%, and 59.09%, respectively, that was much higher than that of p53 Arg/Pro heterozygosity (30.86%, 27.78%, and 21.59%) and of p53 Pro homozygosity (11.12%, 16.67%, and 19.32%) in each groups and higher than the frequency of p53 Arg homozygosity in normal samples (23.33%). There is no statistic difference in the normal samples for the frequency of p53 Arg homozygosity, p53 Arg/Pro heterozygosity, and p53 Pro homozygosity (23.33%, 40.00%, and 36.67%, respectively). The frequency of p53 Arg homozygosity in high risk (HR)-HPV E6-positive cervical squamous cancer samples (64.06%) is much higher than that in (HR)-HPV E6-negative cervical squamous cancer samples (35.29%) and in HR-HPV E6-positive normal samples (33.33%). No difference of p53 codon 72 polymorphism was found according to FIGO staging and grades. In conclusion, based on the findings of this study, it is suggested that p53 Arg homozygosity could act as a potential risk factor for the tumorigenesis of the cervix. p53 codon 72 polymorphism has no relation with the FIGO staging and grades of cervical cancer. p53 Arg homozygosity and HR-HPV E6 positive simultaneously can predict the fate of cervical lesions.     

The presence of HPV 16, 18 and p53 immunohistochemical staining in tumor tissue of Israeli Jewish women with cervical and vulvar neoplasia

The incidence of cervical neoplasia in Israeli Jewish women is persistently lower, while that of vulvar carcinoma is comparable to that in other populations. The aim of the present investigation was to assess the prevalence of HPV and of immunohistochemically detected mutant p53 in Israeli Jewish women with cervical and vulvar neoplasia compared with other populations. Tissue sections from formalin-fixed paraffin-embedded blocks of ten patients with CIN III, 29 with invasive squamous cell carcinoma, three with adenocarcinoma and 14 with invasive vulvar carcinoma, were examined for the presence of HPV 16 and HPV 18 DNA by PCR amplification, and for mutant p53 protein by immunohistochemical staining. HPV negative cases were re-examined with a sensitive primer. HPV DNA was detected in eight patients with CIN III and in 23 patients with invasive squamous carcinoma. In the remaining cervical squamous neoplasia tissue analysis with the sensitive primer could not be done. HPV DNA was also detected in two patients with adenocarcinoma and in nine (64.2%) patients with vulvar carcinoma. Positive p53 immunohistochemical staining was found only in one CIN III patient, in six (20.7%) squamous carcinoma and in 11 (78.6%) vulvar carcinoma patients. Of the p53 immunohistochemical staining positive tissues, two with cervical carcinoma and six with vulvar carcinoma were also HPV-positive. The prevalence of HPV and of positive p53 immunohistochemical staining in our series of Israeli Jewish women with cervical and vulvar neoplasia is similar to that in other populations, suggesting that the etiological factors are probably also alike.     

HPV16/18 E7-pulsed dendritic cell vaccination in cervical cancer patients with recurrent disease refractory to standard treatment modalities

OBJECTIVE: To evaluate the potential of human papillomavirus (HPV) type 16 and 18 E7 antigen-loaded autologous dendritic cells (DC) as a therapeutic cellular vaccine in a case series of cervical cancer patients harboring recurrent/metastatic disease refractory to standard treatment modalities. METHODS: Autologous monocyte-derived DC were pulsed with recombinant HPV16 E7 or HPV18 E7 oncoproteins and administered to 4 cervical cancer patients. Vaccinations were followed by subcutaneous administration twice daily of low doses of human recombinant interleukin-2 (1 x 10(6) IU/m2) from day 3 to day 7. Safety, toxicity, delayed type hypersensitivity reactions (DTH), clinical responses, and induction of serological and cellular immunity against HPV16/18 E7 were monitored. RESULTS: The vaccine was well-tolerated in all patients and no local or systemic side effects or toxicity were recorded. Three out of four patients were found to be significantly immunocompromised before starting the vaccination treatment, as assessed by DTH with a panel of recall antigens. Specific humoral and cellular CD4+ T cell responses to the E7 vaccine were detected in 2 patients, as detected by ELISA and by IFN-gamma ELISpot assays, respectively. Increased numbers of E7-specific IFN-gamma secreting CD8+ T cells were detected in all patients after vaccination. Swelling and induration (i.e., a positive DTH response) to the intradermal injection of HPV E7 oncoprotein and/or irradiated autologous tumor cells were detected in two patients after six vaccinations. No objective clinical responses were observed. However, both patients who developed a positive DTH to the vaccine experienced a slow tumor progression (i.e., 13 months survival) while DTH unresponsive patients died within 5 months from the beginning of therapy. CONCLUSIONS: Autologous DC pulsed with HPV16/18 E7 proteins can induce systemic B and T cell responses in patients unresponsive to standard treatment modalities. However, treatment-induced immunosuppression may impose severe limitations on the efficacy of active vaccination strategies in late stage cervical cancer patients. DC-based vaccination trials are warranted in immunocompetent cervical cancer patients with early stage disease and/or limited tumor burden, and at significant risk for tumor recurrence or disease progression.     

人宫颈鳞癌和宫颈上皮内瘤变组织内HPV16 DNA及p53基因变异的研究

目的 本文采用常规PCR技术对40例宫颈鳞癌组织学标本和200例宫颈上皮内瘤变（CIN）Ⅰ～Ⅱ级的宫颈分泌物标本的HPV16 DNA及P53基因5～6，7～8外显子变异进行了研究，方法 采用常规PCR技术检测HPV16 DNA和p53基因及免疫组织化学方法检测P53基因。结果 在40例宫颈鳞癌标本中HPV16 DNA阳性检出率有20例（50％）；200例CIN标本中有76例（38％）。P53基因在40例宫颈鳞癌标本中有20例出现变异（50％），其中外显子5缺失10例（50％），外显子6缺失1例（5％），外显子7缺失3例（15％），外显子8缺失6例（30％）。200例CIN标本均未检出p53外显子5～6，7～8的变异，宫颈鳞癌HPVl6DNA阳性与p53基因变异的符合率为80％。结论HPVl6I）NA与宫颈鳞癌的发生有密切的相关性，p53基因在宫颈鳞癌组织中确实存在着变异。本研究认为常规PCR技术作为辅助诊断方法是一种快速、简单、适用于临床的方法。     

The role of p53, Bcl-2 and Ki-67 in premalignant cervical lesions and cervical cancer

PURPOSE: The aim of this study was to determine the role of p53, Bcl-2 and Ki-67 expression in the carcinogenesis of cervical carcinoma and aggressiveness of cervical intraepithelial neoplasia (CIN). METHODS: The pathology specimens of 63 patients with a diagnosis of normal squamous epithelium (22 cases), CIN I (14), CIN II (5), CIN III (8) and squamous cell carcinoma (14) were evaluated immunohistochemically for the expression of p53, Bcl-2 and Ki-67 in paraffin sections. RESULTS: The expression of p53 and Ki-67 increased proportionally to the grade of CIN and cervical cancer, but only the increase of p53 expression was statistically significant (p = 0.002). There was no significant correlation between Bcl-2 expression and premalignant and malignant cervical lesions. CONCLUSION: p53 expression may have a role in the carcinogenesis of squamous cell cervical carcinoma whereas Bcl-2 expression has no role. Ki-67 expression can not be used in determining the aggressiveness of CIN lesions.     

Methylation marker analysis and HPV16/18 genotyping in high-risk HPV positive self-sampled specimens to identify women with high grade CIN or cervical cancer

Objectives

Methylation marker analysis using bi-marker panel MAL/miR-124-2 is a promising triage test for identifying cervical (pre)cancer in high-risk human papillomavirus (hrHPV) positive women. Bi-marker panel MAL/miR-124-2 can be applied directly on self-sampled cervico-vaginal material and its sensitivity is non-inferior to that of cytology, yet at the cost of more colposcopy referrals. Our objective was to increase specificity of MAL/miR-124-2 methylation analysis by varying the assay thresholds and adding HPV16/18 genotyping.

Methods

1019 hrHPV-positive women were selected from a randomized controlled self-sampling trial (PROHTECT-3; 33–63 years, n = 46,001) and nine triage strategies with methylation testing of MAL/miR-124-2 and HPV16/18 genotyping were evaluated. The methylation assay threshold was set at four different predefined levels which correspond with clinical specificities for end-point cervical intra-epithelial grade 3 or worse (CIN3 +) of 50%, 60%, 70%, and 80%.

Results

The CIN3 + sensitivity of methylation analysis decreased (73.5 to 44.9%) while specificity increased (47.2 to 83.4%) when increasing the assay threshold. CIN3 + sensitivity and specificity of HPV16/18 genotyping were 68.0% and 65.6%, respectively. Combined methylation analysis at threshold-80 and HPV16/18 genotyping yielded similar CIN3 + sensitivity as that of methylation only at threshold-50 (77.6%) with an increased specificity (54.8%).

Conclusions

Combined triage by MAL/miR-124-2 methylation analysis with threshold-80 and HPV16/18 genotyping reaches high CIN3 + sensitivity with increased specificity to identify women with cervical (pre)cancer among HPV self-sample positive women. The combined strategy is attractive as it is fully molecular and identifies women at the highest risk of cervical (pre)cancer because of strongly elevated methylation levels and/or HPV16/18 positivity.