Detection of High-Risk Human Papillomaviruses in the Prevention of Cervical Cancer in India

Human papillomaviruses (HPVs) are small, non-enveloped, double-stranded DNA viruses that infect epithelial tissues. Specific genotypes of human papillomavirus are the single most common etiological agents of cervical intraepithelial lesions and cervical cancer. Cervical cancer usually arises at squamous metaplastic epithelium of transformation zone (TZ) of the cervix featuring infection with one or more oncogenic or high-risk HPV (HRHPV) types. A hospital- based study in a rural set up was carried out to understand the association of HR-HPV with squamous intraepithelial lesions (SILs) and cervical cancer. In the present study, HR-HPV was detected in 65.7% of low-grade squamous intraepithelial lesions (LSILs), 84.6% of high grade squamous intraepithelial lesions (HSILs) and 94% of cervical cancer as compared to 10.7% of controls. The association of HPV infection with SIL and cervical cancer was analyzed with Chi square test (p<0.001). The significant association found confirmed that detection of HR-HPV is a suitable candidate for early identification of cervical precancerous lesions and in the prevention of cervical cancer in India.     

Human Papillomavirus Infection and Anogenital Neoplasia in Human Immunodeficiency Virus-Positive Men and Women

Human immunodeficiency virus (HIV)-positive women have a higher prevalenceof human papillomavirus (HPV) infection in the cervix and anus,as well as squamous intraepithelial lesions (SILs) at thesesites, than do HIV-negative women matched for age and HIV riskfactors. Similarly, HIV-positive homosexual or bisexual men havea higher prevalence of anal HPV infection and anal SIL than doHIV-negative homosexual or bisexual men. In HIV-positive individuals,the prevalence of HPV infection, the proportion infected withmultiple HPV types, and the prevalence of anogenital SILs increasewith decreasing CD4 count. This situation may reflect loss ofsystemic immune response to HPV antigens or local HPV-HIV interactionsat the tissue or cellular level. Despite the high levels ofanogenital SILs, to date, there has not been a significant increasein reported cases of invasive anogenital cancer in HIV-positiveindividuals. However, several years may be required for SILto progress to invasive cancer, and the advent of newer therapiesfor HIV that are expected to prolong survival may paradoxicallyincrease the risk of progression to cancer in individuals withSILs if these lesions do not regress spontaneously and remainuntreated.     

Cervicovaginal microbiota composition correlates with the acquisition of high‐risk human papillomavirus types

High‐risk (hr) human papillomavirus (HPV) infection is closely associated with the clinical conditions of both squamous intraepithelial lesions (SILs) and cervical carcinoma. However, it remains unclear what factors determine the type of hrHPV infection. Here, we have comprehensively investigated the bacterial composition of the cervicovaginal microbiota of 280 women infected with one type of hrHPV (HPV 16, 52 or 58) by the pyrosequencing of barcoded 16S rRNA genes. Differential microbiota composition was observed among various SIL groups and within the subgroups of each group. This result showed that it is not the microbiota diversity or the common microbiota, but rather agents that are specific to each SIL that might have a positive influence on the acquisition of hrHPV types, independent of abundance. Specifically, a composition of Oribacterium, Lachnobacterium and Thermus in the cervicovaginal microbiota is more likely to be associated with HPV 16, while a composition of Motilibacter in the cervicovaginal microbiota is more likely to be associated with HPV 52, and a composition of Litorilinea and Paludibaculum with a concomitant paucity of L. iners in the cervicovaginal microbiota is more likely to be associated with HPV 58. Furthermore, functional predictions regarding infectious diseases and cancer‐related genes disclosed significant differences (p < 0.01) among the different (sub)groups. Our study provides an elucidation of the relationship between the composition of the cervicovaginal microbiota and the type of hrHPV acquired.     

Deregulation of the G1/S phase transition in cancer and squamous intraepithelial lesions of the uterine cervix: a case control study

High-risk types of HPV express the oncoproteins, E6 and E7, that can inactivate TP53 and RB1, respectively, and thus take control of both cell cycle and apoptosis. Herein, the mRNA expression profiles of 24 G1/S checkpoint genes were analysed in cancer and squamous intraepithelial lesions (SIL) of the uterine cervix. In total 35 squamous cervical carcinomas, 26 high-grade SIL (HSIL), 33 low-grade SIL (LSIL) tissues, and 28 normal uterine cervix specimens as controls were assessed by RT-PCR. Five genes were found to be upregulated only in tumours, RBL2, E2F2, CDK6, CCNE1 and MYC; eight in tumours and HSILs, E2F1, E2F3, E2F5, CCND1, CDK2, CDKN1B, PCNA and POLA, and five in tumours, HSILs and LSILs, TP53, E2F4, CDKN1A, CDKN2A and DHFR. MDM2 was found to be upregulated in SIL, while RBL1 was found to be downregulated in all three groups of cases. TP73 exhibited lower levels in carcinomas; however, its exon 13-containing isoforms were increased and exon 2-containing isoforms were reduced in both cancer and HSIL. Three genes, RB1, CDK4 and CDKN2D, did not exhibit any significant alteration in gene expression. Hierarchical clustering revealed that this set of G1/S checkpoint genes was able to discriminate the total 122 samples into groups of disease and non-disease with only 8 exceptions (6.6%). Our data suggest that deregulation of G1/S phase transition in cervical carcinogenesis is a progressive process. Certain clusters of genes are activated very early in pre-cancerous SILs while others are activated later, during malignant transformation. The ability of this array of markers to identify disease status suggests that it could be used for diagnostic purposes.     

Detection of human papillomavirus infection of the cervix in very elderly women using PCR.

PURPOSE: To determine the incidence of human papillomavirus (HPV) infection and the morphologic lesions induced by HPV infection of the uterine cervix in elderly Japanese women (including very elderly women) and to clarify the natural history and outcome of HPV infection. EXPERIMENTAL DESIGN: We detected squamous intraepithelial lesions (SIL) by cytology and the presence of HPV infection by PCR on cervical smears obtained at autopsy from 335 women ages 60 to 105 years (mean, 82.7 years). Two primers were used for PCR, one for low-risk HPV subtypes and one for high-risk HPV subtypes. RESULTS: SILs were observed in 20 (6.0%) of the women autopsied; 18 (5.4%) had low-grade SIL and 2 (0.6%) had high-grade SIL. HPV-DNA was not detected in any of the women with normal cervical cytology but was found in 9 (45.0%) of the 20 with SIL (2.7% of all subjects). Of these 9 women, 2 (22%) were positive for low-risk types of HPV and 7 (78%) were positive for high-risk types. All 9 had spent their last days in hospitals or nursing homes, the duration of institutional care ranging from 17 days to 10 years 2 months. CONCLUSIONS: Our results suggest that HPV infection of the cervix may occasionally persist for long periods of time (the maximum duration noted in this study being 10 years 2 months). It is also possible that HPV infection in young women with normal cervical cytology will not persist into advanced age.     

Serum levels of insulin-like growth factor I and risk of squamous intraepithelial lesions of the cervix.

Squamous intraepithelial lesions (SILs) are areas of precancerous growth in the cervix that can be indicative of future cervical cancer. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) have been implicated in cancer development. Recent studies have demonstrated that elevated plasma IGF-I levels are associated with increased risk of prostate, lung, colon, and breast cancers. In this case-control study, we analyzed the relationship between serum levels of IGF-I and IGFBP-3, and SILs of the cervix. The case patients were comprised of 267 women treated at The University of Texas M. D. Anderson Cancer Center Colposcopy Clinic in Houston, Texas for abnormal Pap smears. The clinic serves minority and economically disadvantaged women referred from the County Health Department clinics of Harris County, Texas. The control subjects were 238 healthy women receiving family planning and screening services at two Harris County Health Department clinics. Case patients with either high-grade or low-grade SILs had significantly higher serum levels of IGF-I, IGFBP-3, and molar ratios of IGF-I:IGFBP-3 than the control subjects did. IGF-I levels in the highest quartile were associated with significantly higher risk of SILs compared with the lowest quartile, independent of IGFBP-3 levels. The odds ratio for the fourth quartile of IGF-I level, relative to the first quartile, was 8.54 (95% confidence interval, 4.15-17.60; P < 0.0001) after adjustment for age, ethnicity, smoking status, and IGFBP-3 level. There was a dose-response relationship between risk of SILs and the level of IGF-I: as the level of IGF-I increased, so did the risk of SILs. In addition, the serum level of IGFBP-3 was significantly higher in case patients than in control subjects. However, after adjustment for IGF-I, no relationship was evident between IGFBP-3 level and risk of SILs. Serum levels of IGF-I may be a useful biomarker for assessing risk of SIL development.     

Analysis of aberrant DNA methylation and human papillomavirus DNA in cervicovaginal specimens to detect invasive cervical cancer and its precursors.

PURPOSE: Cancer of the uterine cervix is an important cause of death in women worldwide. Pap smears as a tool for screening decreased the incidence and mortality of cervical cancer dramatically. This proof of principle study aimed to develop a potential tool for cervical screening using a test that can be applied by patients without visiting a physician and to increase the coverage rate, especially of the high-risk population with low socioeconomic status. EXPERIMENTAL DESIGN: Human papillomavirus (HPV) DNA testing and methylation analysis of DNA obtained from cervicovaginal specimens of 13, 31, and 11 patients with no dysplasia/low-grade squamous intraepithelial lesion (SIL), high-grade SIL, and invasive cervical cancer, respectively, collected on a tampon, was performed using PCR-based methods to detect invasive cervical cancer and study whether these changes are already present in the precursor lesions. RESULTS: High-risk HPV DNA was present in 68 and 82% of patients with high-grade SIL and invasive cervical cancer. DNA methylation of the 11 genes tested increased with severity of the cervical lesion. Unsupervised hierarchical cluster analysis using solely information on DNA methylation of the 11 genes was able to predict the presence of invasive cervical cancers: one of the two clusters formed contained 9 of 11 invasive cervical cancers, as well as two high-grade SILs. CONCLUSIONS: HPV DNA and DNA methylation analyzed in cervicovaginal specimens are able to predict invasive cervical cancers. To detect all high-grade SILs when applying this test, genes that become methylated earlier throughout cervical carcinogenesis have to be defined.     

Frequent aberration of the transforming growth factor‐β receptor II gene in cell lines but no apparent mutation in pre‐invasive and invasive carcinomas of the uterine cervix

The type II transforming growth factor‐β (TGF‐β) receptor (RII) gene located at 3p22 plays an important role in regulating growth and differentiation of epithelium, including that of the uterine cervix. Loss‐of‐function mutations of RII have frequently been found in gastrointestinal cancers, with a replication‐error (RER) phenotype characterized by the presence of microsatellite instability (MI). In this study, genomic PCR, SSCP and DNA sequencing were conducted to investigate the coding sequences of the RII gene in cell lines (n = 5) and tissues (n = 15) of squamous carcinomas of the uterine cervix. Intragenic deletions were noted in 2 of 5 cervical‐cancer cell lines (ME180 and HeLa cells). However, no mutation, other than DNA polymorphisms, was found in 15 cervical cancers with either alleleic loss at 3p22 (n = 11) or MI (n = 4). Further analysis of squamous intraepithelial lesions (SIL) with (n = 12) or without (n = 4) MI for the (A)₁₀ change, a prototypic mutation found in over 90% of RER‐positive colon cancers, also showed no aberration. Our study concludes that the RII gene is frequently disrupted in cervical‐cancer cell lines, but is rarely mutated in CC and SIL tissues, including those showing MI or alleleic loss at 3p22. The underlined mechanism of genomic instability in CC and SIL may thus differ from that of colorectal cancer. The allelic loss at 3p22‐24 in CC does not involve the coding sequence of the RII gene. The non‐coding sequence of RII or an unidentified gene may be responsible for it. Int. J. Cancer 80:506–510, 1999. © 1999 Wiley‐Liss, Inc.     

Telomerase activity in cervical cancer is quantitatively distinct from that in its precursor lesions

Studies using the telomeric repeat amplification protocol (TRAP) assay have demonstrated telomerase activity not only in cancers but also in non-cancerous lesions. However, quantitative differences in activity between both lesions have not been examined. In the present study, using a stretch PCR assay, telomerase activity was analyzed quantitatively in cervical cancer, its precursor squamous intra-epithelial lesions (SILs) and normal cervix. In stretch PCR assay, telomerase activity was expressed in relative units vs. control activity from C33A cells (100 units). Mean telomerase activities in cervical cancer, SIL and normal cervix were 72 ± 35 units, 18 ± 17 units and 7 ± 4 units, respectively, suggesting that telomerase activity in cancer lesions was quantitatively distinct from that in pre-malignant lesions, which may mean a much more pronounced activation of telomerase in cancers than in SIL. Our findings also suggest that stretch PCR assay can distinguish telomerase activity in cancer from that in non-cancerous lesions and may be useful for the differential diagnosis of cancer lesions. Int. J. Cancer (Pred. Oncol.) 79:66–70, 1998. © 1998 Wiley-Liss, Inc.     

Prognosis and recurrence risk for patients with cervical squamous intraepithelial lesions diagnosed during pregnancy

BACKGROUND: In the current study, the authors sought to examine the prognosis and recurrence risk for patients with cervical squamous intraepithelial lesions (SILs) diagnosed during pregnancy. METHODS: A retrospective review of all women who gave birth at Walter Reed Army Medical Center (Washington, DC) or the National Naval Medical Center (Bethesda, MD) between 1986 and 1997 was performed. One hundred fifty-seven patients with SILs who underwent antepartum and postpartum evaluation were identified from a total of 6248 records of birth at these two institutions. Patient demographics and cervical cytology and histology were reviewed. RESULTS: One-hundred twenty-nine patients were diagnosed with low-grade squamous intraepithelial lesions (LSILs) antepartum. Of these patients, 49 (38%) had a previous history of abnormal cervical cytology (30 LSILs and 19 high-grade squamous intraepithelial lesions [HSILs]). Twenty-eight patients were diagnosed with HSIL antepartum. Of these patients, 24 (86%) had a history of abnormal cervical cytology. Sixty-two percent of patients with antepartum LSILs had disease regression postpartum, 32% had persistent LSILs postpartum, and 6% experienced progression of an LSIL to an HSIL. All cases of HSIL that were diagnosed antepartum persisted on postpartum cytologic examination. Three patients were found to have microinvasive squamous cell carcinoma after postpartum conization. Five years of follow-up data were available for 98 patients (60%), 78 of whom had antepartum LSILs and 20 of whom had antepartum HSILs. Sixty percent of patients with antepartum LSILs detected on Pap smear developed recurrent LSILs within 5 years, and all 20 patients with antepartum HSILs developed recurrent HSILs within 5 years. CONCLUSIONS: Most cases of LSIL regressed or remained stable during pregnancy. All cases of HSIL diagnosed antepartum persisted in the postpartum period, and 11% of patients with antepartum HSILs were found to have invasive carcinoma postpartum. High rates of recurrence for both LSIL and HSIL were noted 2-5 years after the diagnosis of SIL in the antepartum.     

Improved therapeutic responses in a xenograft model of human B lymphoma (Namalwa) for liposomal vincristine versus liposomal doxorubicin targeted via anti-CD19 IgG2a or Fab' fragments.

PURPOSE: Monoclonal antibody-mediated targeting of liposomal anticancer drugs to surface antigens expressed on malignant B cells can be an effective strategy for treating B-cell malignancies. In a murine model of human B-cell lymphoma, we have made in vitro and in vivo comparisons of long-circulating sterically stabilized (Stealth) immunoliposome (SIL) formulations of two anticancer drugs, vincristine (VCR) and doxorubicin (DXR), with different mechanisms of action and drug release rates. EXPERIMENTAL DESIGN: SIL formulations of VCR or DXR were conjugated to the monoclonal antibody anti-CD19 (SIL[alphaCD19]) or its Fab' fragments (SIL[Fab']). Specific binding of SILs to Namalwa cells was studied using radiolabeled liposomes, and cytotoxicities of DXR- or VCR-loaded SILs were quantitated by a tetrazolium assay. Pharmacokinetic and drug leakage experiments were performed in mice using dual-labeled liposomes, and the therapeutic responses of SILs were evaluated in a Namalwa (human B lymphoma) cell xenograft model. RESULTS: SIL[alphaCD19] or SIL[Fab'] had higher association with and cytotoxicity against Namalwa cells than nontargeted liposomes. SIL[Fab'] had longer circulation times than SIL[alphaCD19], and VCR had faster release rates from the liposomes than DXR. SIL formulations of either VCR or DXR had significantly better therapeutic outcomes than nontargeted liposomes or free drugs. SILs loaded with VCR were superior to those loaded with DXR. SIL[Fab'] had better therapeutic outcomes than SIL[alphaCD19] for the drug DXR but were equally efficacious for the drug VCR. CONCLUSIONS: Treatment of a B lymphoma model with single injections of anti-CD19-targeted liposomal formulations of VCR resulted in high levels of response and long-term survivors. Responses to anti-CD19-targeted liposomal DXR were more modest, although the longer circulation times of SIL[Fab'] versus SIL[alphaCD19] led to superior therapeutics for DXR-loaded immunoliposomes.     

原发性肝癌患者可溶性IL—2受体水平,IL—2和NK活性的变化及意义

采用双抗体夹心ELlSA法检测43例原发性肝细胞癌(PHC)病人血清可溶性白细胞介素2受体(sIL-2R)水平,及其外周血单个核细胞(PBMC_s)的白细胞介素2(IL—2)活性和自然杀伤(NK)细胞活性.另将51例慢性乙型活动性肝炎和肝硬化病人做为对照.结果,上述病人血清sIL—2R水平明显升高,IL—2和 NK活性低于正常人,其中PHC的IL—2和NK活性的降低又为显著(P<0.001< O.001).表明PHC病人的细胞免疫功能受损程度最重.检测sIL_2R水平和IL—2活性,可望做为对PHC患者病情分析及判断预后的指标.     

Glutathione S-transferase (placental) as a marker of transformation in the human cervix uteri: an immunohistochemical study

Using an indirect immunohistochemical technique on paraffin sections, employing a polyclonal antibody to the acidic (placental) form of glutathione-S-transferase (GST), we have evaluated cytoplasmic and nuclear staining in a series of 67 cervical biopsies including normal non neoplastic tissue, immature squamous metaplasia, all grades of cervical intraepithelial neoplasia (CIN) and invasive carcinomas of keratinising and non-keratinising types. No differences in cytoplasmic staining between the varied lesions studied were seen. However, there were marked differences in nuclear staining. While normal non-neoplastic stratified squamous epithelium showed weak staining of the lower one-third of the epithelium only, in immature squamous metaplasia and in all grades of CIN there was intense nuclear staining in all layers of the epithelium. Invasive carcinomas showed generally less intense nuclear staining than CIN lesions. Endocervical cell nuclei also showed intense nuclear staining. These findings indicate that GST is of limited use as a marker of transformation in the human cervix uteri.     

The expression of cytokeratin No. 17 in squamous cell cancer of the cervix uteri: an immunohistochemical study of 19 cases]

Monoclonal antibodies E3 against cytokeratin No. 17 were used for immunohistochemical examination of samples from 19 squamous-cell carcinomas of the uterine cervix and 10 cases of squamous-cell metaplasia of the endocervix. The above antibodies selectively labelled reserve cells of the cervical canal mucosa, basal layer of mature metaplastic epithelium and parabasal and basal cells of immature metaplastic squamous epithelium. In large-cell keratinizing, non-keratinizing and small-cell carcinomas, the expression of cytokeratin No. 17 revealed tendency to basal orientation and was in many respects similar to that in metaplastic squamous epithelium. It was suggested that all the squamous-cell carcinomas studied originated from reserve (metaplastic) cells.     

p53 codon 72 ARG/PRO polymorphism is not related to HPV type or lesion grade in low- and high-grade squamous intra-epithelial lesions and invasive squamous carcinoma of the cervix.

A polymorphism at codon 72 of the p53 gene, resulting in either an arginine or a proline residue in the protein, has been reported to affect the susceptibility of p53 to human papillomavirus (HPV) E6-mediated degradation in cultured cells. However, the relevance of this polymorphism to naturally occurring HPV infection is unclear. Therefore, we analysed its relationship to infecting HPV type and lesion grade in a series of low- and high-grade squamous intra-epithelial lesions (SILs) and invasive carcinoma of the cervix. DNA extracted from morphologically characterised, paraffin-embedded tissues (30 normal cervices, 118 low-grade SILs, 118 high-grade SILs and 43 invasive carcinomas) was examined for the presence and type of HPV DNA, and the p53 genotype was identified by both allele-specific PCR and PCR-restriction fragment length polymorphism. There was no significant relationship between the frequency of p53 genotypes and either HPV type or lesion grade. Our data do not support the hypothesis that this p53 polymorphism is involved in the development of high-grade squamous cervical disease in this population.     

Expression of PTEN in the progression of cervical neoplasia and its relation to tumor behavior and angiogenesis in invasive squamous cell carcinoma

BACKGROUND AND OBJECTIVES: Loss of PTEN expression has been associated with tumor progression and adverse patient outcome. The purpose of this study was to evaluate PTEN expression in the successive steps of progression in cervical neoplasia and to determine its correlation with tumor angiogenesis and clinicopathologic features in squamous cell carcinoma of the uterine cervix. METHODS: Immunohistochemical staining with anti-PTEN antibody was performed in a total of 160 patients with 12 normal cervical epithelium, 63 cervical intraepithelial neoplasia (33 CIN I, 30 CIN III), and 85 cervical squamous cell carcinomas. Microvessels were immunohistochemically labeled with an antibody for CD34. Computerized image analysis was used to evaluate microvessel density (MVD). RESULTS: Reduced PTEN expression progressively increased along the continuum from normal epithelium to squamous cell carcinoma (P < 0.01). There was no significant correlation between PTEN expression and MVD. On univariate analysis, stage and reduced PTEN expression were significant prognostic factors for both disease-free and overall survival. However, stage was the only independent prognostic factor by multivariate analysis. CONCLUSIONS: Our results suggest that tumor progression in the cervical epithelium is accompanied by loss of PTEN protein expression. Reduced PTEN expression is not associated with tumor angiogenesis of squamous cell carcinoma of the uterine cervix.     

Tristetraprolin regulates interleukin-6, which is correlated with tumor progression in patients with head and neck squamous cell carcinoma

BACKGROUND:

Tumor‐derived cytokines play a significant role in the progression of head and neck squamous cell carcinoma (HNSCC). Targeting proteins, such as tristetraprolin (TTP), that regulate multiple inflammatory cytokines may inhibit the progression of HNSCC. However, TTP's role in cancer is poorly understood. The goal of the current study was to determine whether TTP regulates inflammatory cytokines in patients with HNSCC.

METHODS:

TTP messenger RNA (mRNA) and protein expression were determined by quantitative real‐time–polymerase chain reaction (Q‐RT‐PCR) and Western blot analysis, respectively. mRNA stability and cytokine secretion were evaluated by quantitative RT‐PCR and enzyme‐linked immunoadsorbent assay, respectively, after overexpression or knockdown of TTP in HNSCC. HNSCC tissue microarrays were immunostained for interleukin‐6 (IL‐6) and TTP.

RESULTS:

TTP expression in HNSCC cell lines was found to be inversely correlated with the secretion of IL‐6, vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE₂)_. Knockdown of TTP increased mRNA stability and the secretion of cytokines. Conversely, overexpression of TTP in HNSCC cells led to decreased secretion of IL‐6, VEGF, and PGE₂. Immunohistochemical staining of tissue microarrays for IL‐6 demonstrated that staining intensity is prognostic for poor disease‐specific survival (P = .023), tumor recurrence and development of second primary tumors (P = .014), and poor overall survival (P = .019).

CONCLUSIONS:

The results of the current study demonstrated that down‐regulation of TTP in HNSCC enhances mRNA stability and promotes secretion of IL‐6, VEGF, and PGE₂. Furthermore, high IL‐6 secretion in HNSCC tissue is a biomarker for poor prognosis. In as much as enhanced cytokine secretion is associated with poor prognosis, TTP may be a therapeutic target to reduce multiple cytokines concurrently in patients with HNSCC. Cancer 2011. © 2011 American Cancer Society.