调节性T细胞在人乳头瘤病毒16整合状态的宫颈癌组织中的表达及意义

目的 探讨调节性T细胞（Treg）活化与人乳头瘤病毒（HPV）16存在状态的关系及其与HPV16整合状态的宫颈鳞癌患者临床病理资料之间的相关性。 方法 2012年1-10月在中国医科大学附属盛京医院妇产科,采用多重实时PCR技术对HPV16阳性的17例正常宫颈组织、65例宫颈上皮内瘤变（CIN）、60例宫颈鳞癌（CC）的宫颈脱落细胞标本检测E2和E6基因,通过E2/E6比值法评估HPV16 DNA的体内存在状态。采用免疫组化方法检测相应宫颈组织中叉头状／翅膀状螺旋转录因子（Foxp3）的表达。结果 Foxp3阳性积分光密度值在HPV16游离状态、整合状态宫颈组织中,均与宫颈病变严重程度呈正比,在CC与CINⅢ组比较差异有统计学意义（P<0.05）。在同一病变中,HPV16整合型组Foxp3阳性表达均高于游离型组,在CC组比较差异有统计学意义（t=-2.685,P<0.05）。Foxp3阳性在HPV16整合状态宫颈鳞癌中的表达与FIGO分期、组织学分级及淋巴结转移相关（P<0.05）,而与年龄无关（P>0.05）。结论 Treg与HPV存在状态密切相关,Treg在HPV持续感染所致宫颈癌的发生及发展中可能起着重要作用。     

HPV16/18 prevalence in cervical lesions/cancers and p53 genotypes in cervical cancer patients from India

OBJECTIVES: The HPV16/18 code for an oncoprotein-E6, which binds to p53 tumor suppressor protein and degrades the protein via ubiquitination. A common polymorphism of p53 in exon 4 codon 72, resulting in either proline (Pro) or arginine (Arg), affects HPV16/18 E6-mediated degradation of p53 protein in vivo. Hence, in the current study we investigated the prevalence of HPV16/18 in cervical lesions and the distribution of p53 genotypes in cervical cancers and normal healthy women. METHODS: DNA from 337 Indian women with invasive cervical cancers, 164 women with clinically normal cervix, 64 women with low-grade squamous intraepithelial lesions (LSIL), and 5 women with high-grade squamous intraepithelial lesions (HSIL) was examined for the presence of HPV16/18 using consensus primers in a polymerase chain reaction (PCR), and the specific HPV type was identified by Southern hybridization of the PCR product using HPV16/18 type-specific nucleotide sequences as probes. Further, 134 women with cervical cancers and 131 healthy women were used to determine the frequency of p53 genotypes, Pro/Pro, Arg/Arg, and Pro/Arg, using peripheral blood cell DNA to indicate the constitutional genotypes and allele-specific primers, in a PCR-based assay. RESULTS: We observed a prevalence of HPV16/18 in 77% (258/337) of cervical cancer patients, 38% (24/64) of LSILs, 4 of 5 HSILs, and 15.2% (25/164) of normal healthy women. The frequency of distribution of the three genotypes of p53 codon 72 in a subgroup of the HPV16/18-positive cervical cancer patients was Pro/Pro 0.18 and Arg/Arg 0.26, with the heterozygous Pro/Arg 0.56, differing significantly from the genotype frequency in the normal healthy women (chi(2) = 6.928, df = 2, P < 0.05). CONCLUSIONS: A high prevalence of HPV16/18 was observed in the cervical cancers. The prevalence in LSILs confirms HPV16/18 infection as an early event and further indicates a role in progression of lesions. The p53 genotype distribution indicated that women homozygous for Arg genotype were at a 2.4-fold higher risk for developing HPV16/18-associated cervical carcinomas, compared to those showing heterozygous Pro/Arg genotype (odds ratio 2.4, 95% confidence interval 1.89 to 3.04).     

T and B lymphocyte counts and blast transformation in patients with Stage I cervical cancer

In 57 patients with Stage I cervical cancer and in 25 healthy women, a determination was made of leukocytosis, counts of lymphocytes and monocytes per 1 mm³ of blood, counts of T and B lymphocytes, and PHA-induced lymphocyte ability of in vitro blast transformation. Patients with cervical cancer, as compared with controls, exhibited a decrease in lymphocytosis and in the counts and percentages of T and B lymphocytes, as well as an increase of lymphocyte reactivity to PHA. An increase in the PHA-induced lymphocyte ability of in vitro blast transformation occurred in patients with a clinical picture characterized by the presence of a large tumor of the cervix or of metastases to the lymph nodes of the pelvis. In the group of patients in Stage I with a small cervix the mean indices of blast transformation did not differ from those observed for the healthy control group; on the other hand, in the group of patients with a large cervix and metastases to the lymph nodes of the pelvis, these indices exceeded on the average by about 88% those in the control group.     

HPV 16 DNA occurrence in lymph nodes of patients with cervical carcinoma

OBJECTIVES: We estimated the occurrence of DNA HPV 16 presence in lymph nodes of 25 patients undergoing abdominal operation for cervical carcinoma. MATERIALS AND METHODS: The presence of HPV 16 DNA was detected during the preoperative diagnosis procedure by the PCR method. RESULTS: According to the histopathological examination, metastases in the lymph nodes were present in material from two patients. It was confirmed HPV 16 DNA was detected with PCP. We found also 6 patients with HPV 16 DNA in their lymph nodes without histological confirmation. CONCLUSIONS: We consider PCR detection of HPV DNA as a simple and useful support of pathology diagnosis.     

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.     

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。     

The level of antibody against E6 HPV 16 oncoprotein in blood sera of women with chronic HPV 16 infection and cervical cancer

The aim of this study was to estimate of the role of chronic HPV 16 infection and the presence of anti E6 HPV 16 in the initiation of the cancerogenesis process of cervical cancer. MATERIAL AND METHODS: The study included two groups of patients. The first group comprised 323 women observed for three consecutive years (1998-2000), in whom the presence of HPV 16 viruses was estimated by PCR, and the level of anti E6 HPV 16 antibodies was estimated in the plasma with ELISA. A similar test was performed in a group of 46 patients with cervical intraepithelial neoplasia (CIN), 91 patients with invasive cervical cancer and 22 women after hysterectomy and RTG-therapy. RESULTS: In 32 patients, chronic HPV 16 infection showed a steady rise in the mean absorbance level of anti E6 HPV 16 antibodies from 0.04 in 1998 to 0.06 in 2000, while in HPV-negative women the mean absorbance value was 0.03-0.04. Mean absorbance value in patients with CIN III and invasive cancer rose with advancing stage of the cancer process and lowered after completion of oncological treatment. The values were 0.14, 0.33 and 0.13, respectively. CONCLUSION: The persistence of chronic HPV 16 infection and accompanying steady rise in absorbance index caused by an increase in the level of antiviral antibodies are a clear warning signal preceding in time the histological process of cancerogenesis.     

Low incidence of HPV DNA in sera of pretreatment cervical cancer patients

OBJECTIVE: The aim of this study was to investigate the feasibility of using DNA in the circulation as a diagnostic tool for cervical cancer. METHODS: We used PCR followed by Southern hybridization to detect human papillomavirus (HPV) DNA in serum samples taken from patients of cervical cancer before treatment. RESULTS: A total of 60 samples were analyzed. In a set of 40 samples, without knowledge of the HPV DNA status in the corresponding cervical carcinomas, we could detect 8 (20%) positive samples, of which 7 (17.5%) were HPV 16 and 1 (2.5%) was HPV 18. In another set of 20 samples, known to be HPV 16 infected in the corresponding cervical carcinomas, we detected only 4 (20%) HPV-16-positive samples. The occurrence of HPV DNA in sera of cervical cancer patients seems sporadic. CONCLUSION: The low incidence indicates that serum HPV DNA has limited application in the diagnosis of cervical cancer.     

人免疫重建荷人HPV16阳性宫颈癌SCID鼠模型的建立及其生物学特征研究

目的 :建立人免疫重建荷人HPV16阳性宫颈癌 -严重联合免疫缺陷小鼠模型并研究其生物学特征。方法 :向严重联合免疫缺陷 (SCID)鼠腹腔注射人外周血淋巴细胞 (PBL)后 2 4h ,皮下接种HPV16阳性的人宫颈癌细胞株SiHa细胞建立人免疫重建荷人HPV16阳性宫颈癌SCID鼠模型 ,观察荷瘤鼠成瘤、一般特征和移植瘤生长、转移情况及组织学特征 ,检测外周血、肿瘤组织和肺脾等其它组织中HPV16DNA、血清中人IgG含量和移植物抗宿主情况。结果 :SCID小鼠成瘤率为 10 0 % ,移植瘤生长以局部浸润为主 ,未见转移瘤。免疫重建荷瘤组生存期 (136 .2± 6 .3d)显著长于未重建荷瘤组 (97.8± 3.7d) ;组织学检查示移植前后肿瘤细胞形态相似 ;所有肿瘤组织中HPV16DNA呈阳性 ,而外周血和肺脾等其它组织均为阴性。未发现移植物抗宿主情况。结论 :成功建立的人免疫重建荷人HPV16阳性宫颈癌SCID小鼠模型能较好地模拟人自发宫颈癌的生物学特征     

Anti-embryoglycan antibodies detected in sera of patients with uterine cervical cancer]

Embryoglycan is a high molecular weight glycopeptide, found abundantly in the cell surface of F9, stem cell clone derived from mouse teratocarcinoma. Many differentiation-associated antigens and receptors for lectins have been shown to be carried by embryoglycan. Furthermore, antibodies reacting with embryoglycan have been detected in the sera of patients with ovarian germ cell tumor. We investigated anti-embryoglycan antibodies in the sera of patients with uterine cervical cancer, considering the possibility of the appearance of embryoglycan-like antigen accompanied by malignant changes in the uterine cervix. Reaction between the patients' sera and embryoglycan (or F9 cells) was assayed by Farr's assay and an indirect immunofluorescence method. Among the patients with uterine cervical cancer (squamous cell carcinoma), 16 of 61 cases (26%) were positive in invasive cases, whereas early cases (14 cases) were all negative. Cases of benign ovarian tumor (23 cases), uterine myoma (25 cases) and 50 normal volunteers were all negative. The antigenic determinant of embryoglycan was found to be alpha-galactosyl residue since alpha-galactosidase-treated embryoglycan lost the activity to bind these antisera. However, the antigenic structure of alpha-galactosyl residue was shown to be distinct from the antigenic determinant of erythrocytes blood type B, but to some degree cross reacted with it, in an absorption test. These results indicate that an unusual alpha-galactosyl residue carried by embryoglycan is expressed on at least some uterine cervical cancer cells. Furthermore the immune system of patients produces antibodies reacting with the alpha-galactosyl residue of embryoglycan. It would be possible to use this method in monitoring patients' specific immune response.     

HPV genotyping from invasive cervical cancer in Chile

Objective

To determine the prevalence rates of the different HPV types in cervical cancer lesions in Chile to facilitate the development of prophylactic human papillomavirus (HPV) vaccines effective for that country.

Method

Biopsy samples of 312 cervical cancer lesions were assessed for HPV type by reverse-line blotting assay.

Results

HPV DNA was found in 94.2% of the lesions, 67.2% harboring 1 viral type and the remainder harboring more than 1 type. HPV-16 was the most frequent type in single infections (50.5%), followed by HPV-18 (7.8%), HPV-31 (2.4%), and HPV-45 (2.0%). HPV-16 was also present in 98.7% of dual and multiple infections, its most frequent association being with HPV-18.

Conclusions

HPV types 16, 18, 31, and 45, alone or combined with other types, were observed in the biopsy samples of up to 80.5% of cervical cancer lesions.     

宫颈癌及其癌前病变组织中FHIT蛋白异常表达与HPV16E6、HPV16E7蛋白表达的相关性

目的 探讨宫颈癌中三联脆组(FHIT)蛋白表达与HPV16 E6、E7蛋白表达的相关性.方法 采用免疫组化SP法对四川大学华西第二医院1999年1月至2003年2月的15例正常宫颈、25例宫颈上皮内瘤变(CIN)以及61例浸润性宫颈鳞癌组织标本进行FHIT蛋白、HPV16E6、HPV16 E7蛋白表达的检测.结果 (1)在正常宫颈上皮、CINI～II、CINⅢ及浸润性宫颈鳞癌中,FHIT 蛋白阳性表达率分别为100%(15/15)、71.43%(10/14)、36.36%(4/11)、14.75%(9/61),P＜0.05;HPV16E6蛋白阳性表达率分剐为0(0/15)、7.14%(1/14)、36.36%(4/11)、59.02%(36/61),P＜0.05;HPV16E7蛋白阳性表达率分别为20.00%(3/15)、42.86%(6/14)、63.64%(7/11)、57.38%(35/61),P＞0.05.(2) 宫颈病变组织中FHIT蛋白的阳性表达与HPV16E6蛋白阳性表达呈负相关(P＜0.0l,r=-0.449),与HPV16E7蛋白表达无相关性(P＞0.05).结论 宫颈癌中FHIT 蛋白的异常表达与HPV16 E6蛋白表达有关,FHIT蛋白和HPV16E6蛋白的联合检测可能可作为宫颈癌前病变转归的指标.     

HPV 16 DNA in autopsy material of a metastatic cervical carcinoma

Summary Human papillomavirus (HPV) DNA has been regularly detected in primary cervical carcinomas and in some metastatic lesions. Using Southern blot hybridization on autopsy material we found HPV 16 DNA in a primary cervical carcinoma and in multiple metastases therefrom.     

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.     

E2 sequence variations of HPV 16 among patients with cervical neoplasia seen in the Indian subcontinent

OBJECTIVES: Specific nucleotide variations in the E2 DNA sequence were looked for in samples with an intact human papillomavirus (HPV) 16 episomal E2 DNA. METHODS: Ninety-two women, 76 with invasive cervical carcinoma and 16 with cervical intraepithelial neoplasia (CIN) were recruited. HPV DNA typing was performed by polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP). Intact episomal E2 DNA of HPV 16 was detected by PCR. Important nucleotide variations in samples with amplifiable E2 DNA were detected by RFLP. Nucleotide sequencing was performed on representative samples to confirm RFLP findings. RESULTS: A total of 89 (96.7%) women were positive for HPV DNA. Of these, 56 (63%) were positive for HPV 16, and of these, 38 (68%) were positive for intact episomal HPV 16 E2 DNA while 18 (32%) were negative. Samples with intact episomal HPV 16 E2 DNA sequences were grouped into four different digestion profiles I to IV based on RFLP patterns. Digestion patterns revealed absence of any sequence variations in samples with digestion profile I and presence of a 2983 A-G variation in those with profile II. Samples with digestion profiles III and IV revealed three variations in the hinge region (3516 C-A, 3538 A-C, 3566 T-G) and two in the DNA binding domain (3684 C-A, 3694 T-A) of the E2 sequence. Sequencing performed on representative samples confirmed RFLP findings. CONCLUSIONS: PCR-RFLP helped in the identification of important HPV 16 E2 sequence variations, circumventing the need for sequencing. The presence of the nucleotide variations in positions that could alter the biological and immunological functions of the E2 protein combined with its increased occurrence in this study bring out the importance of these variations.     

宫颈癌中抑癌基因FHIT表达与HPV16基因状态

目的:探讨抑癌基因FHIT表达及人乳头瘤病毒16(HPV16)的基因型整合状态在宫颈癌发生发展中的作用及相关性。方法:选取柳州市人民医院2013年6月至2014年12月收治的42例宫颈癌、55例宫颈上皮内瘤样病变(CIN)和20例宫颈正常组织的患者,免疫组化法检测宫颈组织中FHIT蛋白表达;多重PCR法检测HPV16 E2/E7表达。结果:FHIT蛋白的总阳性表达率为57.26%(67/117),正常宫颈组织、CINⅠ、CINⅡ、CINⅢ和宫颈癌中FHIT蛋白阳性率分别为85.00%、80.00%、75.00%、60.00%和26.19%。随着宫颈病变加重,FHIT蛋白阳性表达率下降,组间差异有统计学意义(χ~2=7.335;P=0.003)。117例单纯HPV16阳性标本HPV16总整合率为81.20%,正常宫颈组织、CINⅠ、CINⅡ、CINⅢ和宫颈癌中整合率分别为60.00%、66.67%、75.00%、95.00%和92.86%;随病变加重,整合率增强,组间差异有统计学意义(χ~2=5.713,P=0.003);FHIT蛋白阳性表达在不同HPV16整合时不同,差异有统计学意义(χ~2=11.989,P=0.000)。结论:HPV16基因整合可能通过诱导FHIT基因低表达从而促使宫颈癌发生发展。