CaCx Cases Research Articles

Prevalence of human papilloma virus and Chlamydia trachomatis in endometrial and cervical carcinoma: a comparative study in North Indian women

Cervical cancer (Cacx) is the second and endometrial cancer (Ec) is the third most common gynecological cancer worldwide. The present study aims to understand the complex and unexplored conditions occurring in cervix and endometrium of the female genital tract caused due to the infection of the human papilloma viruses (HPVs) and Chlamydia trachomatis (CT). A total of 300 tissue biopsy samples of cervix and endometrium were included in the present study and tested for the presence of HPV and CT deoxyribonucleic acid (DNA) by using polymerase chain reaction (PCR) technique. The odds ratios and 95% confidence interval were considered for the calculation of the association of HPV and CT infection with the risk of cervical or Ec. Among endometrial patients, samples were 5% positive for HPV and 5% positive for CT infection. Among endometrial control group, no sample was found positive for either HPV or CT infection. Among cervical patients, 72% samples were positive for only HPV infection and 1% samples were positive for only CT infection. Among control group, 7% of samples were positive for only HPV infection and 3% were positive for only CT infection. The co-infection of CT with HPV in 9% of Cacx cases and in 2% of cervical control samples was also observed. This is the first study in Indian women to detect the prevalence of HPV and CT infections in endometrium cases and control. An updated estimate regarding the HPV and CT prevalence in cervix cases and control samples was also provided.

Brachytherapy Capacity for Management of Cervical Cancer in Low- and Middle-Income Countries: An Updated IAEA Database Analysis

<h3>Purpose/Objective(s)</h3> Cervical cancer (ca-cx) is a leading cause of cancer death in women worldwide. Low and Middle-Income countries (LMICs) account for 90% of case mortalities annually. Treatment include surgery, RT +/- chemotherapy. RT consist of EBRT +/- brachytherapy (BT) or exclusive BT for stage IA. LMIC access to RT is crucial for the global control of ca-cx. However, most studies on RT capacity in LMICs have focused on EBRT. This study assesses progress in the BT capacity in LMICs. It analyzes the current demand and deficits based on the updated survey of global RT facilities and epidemiological trends of ca-cx. <h3>Materials/Methods</h3> We projected the 2022 incidence of ca-cx for each LMIC from GLOBOCAN, a database of global cancer registries maintained by the IACR. Using an optimal BT utilization (BTU) of 71.4%, we estimate the demand for BT in each LMIC. The BTU is the proportion of cases with BT indication. Evidence-based estimates informed BTU of 71.4% for ca-cx in LMICs. We obtained the number of BT units in each LMIC from the IAEA DIRAC. Per IAEA, an HDR unit can deliver 1,920 RT frx a year. Prior studies show an average of 3 frx per RT course for ca-cx in LMICs. The required BT for each LMIC was computed as (I * BTU * 3) / (1,920). Deficit was analyzed from available BT as of Feb 2022 and compared to 2014. <h3>Results</h3> 2022 anticipates 591,610 new ca-cx cases in 133 LMICs, for which 422,410 would require BT. 80 LMICs had BT, and 53 had none. There are 1,199 BT in all LMICs, a 198% increase from 402 in 2014 and exceeds the demand of 661 needed to control ca-cx. However, there was an inequitable geographic distribution & regional deficit. Sub-Sahara Africa (SSA) and East Asia /Pacific (EA/P) had deficit of 114 and 110 BT respectively. These regions meet only 36% and 47% of the needed BT. They have also had the slowest progress from 2014. An analysis at the economic level showed that low-income economies (LIEs) were more likely to have BT deficit. Only 3 of 27 LIEs meet full coverage. BT coverage in LIEs is 6.5% which pales in comparison to 279% and 168% for upper and lower-middle-income economies, respectively. <h3>Conclusion</h3> There is progress in BT access for ca-cx in LMICs from last decade. However, a regional deficit persists. The shortage is most dire in the LIEs located in the EA/P and SSA subcontinents. The findings of this study should be taken with some caution. The IAEA-DIRAC captures RT facilities worldwide but is self-reported data. Also, this study, like prior IAEA studies, analyzed with HDR-BT capacity. HDRs are more efficient than LDR which the available resource in some LMICs. There is a limited report of the BT specifications on DIRAC. Finally, ca-cx is not sole indication for BT, even though it is the main indication in LMICs. While previous analyses on BT capacity in LMICs have only utilized epidemiological data of ca-cx, a growing BT utilization for prostate and other gynecologic cancers should be noted. We, therefore, anticipate a slight decrease in our estimated capacities when all other indications are factored, although likely inconsequential.

Polymorphism of interleukin-6 -174 G/C (rs1800795) & the corresponding interleukin-6 level as a prognostic marker of cervical cancer.

Background & objectives:Aetiology of cervical cancer (CaCx) is multifactorial. Besides human papillomavirus (HPV) infection, many immunogenetic factors are involved in this complex process. The present study was carried out to investigate one such factor, interleukin-6 (IL-6), a central pro-inflammatory cytokine and a polymorphism at its promoter region -174 G/C (rs1800795) with CaCx.Methods:HPV-infected women with or without CaCx were enrolled in group I and II, respectively. Another group of uninfected healthy women was also included as group III for comparison. Polymorphism in IL-6-174 G/C and IL-6 levels were analyzed by sequence-specific primer PCR (PCR-SSP) and ELISA, respectively.Results:Groups I (n=111) and II (n=87) had significantly higher frequency of IL-6-174 GG genotype [odds ratios (OR)=3.9; P<0.001 and OR=3.2; P<0.001, respectively] as compared to group III (n=163). Furthermore, individuals with GG or GC genotypes had high IL-6 levels than those with CC genotypes. IL-6 levels were significantly (P<0.001) elevated in group I. This was also significantly high in untreated cases as compared to treated (P<0.05) ones. IL-6 levels of treated group were comparable with groups II and III.Interpretation & conclusions:Our results suggested a possible association of IL-6-174 GG with CaCx, which was also associated with high IL-6 levels. Decreased levels of IL-6 following treatment indicate its possible prognostic use in CaCx cases.

Exploring the p53 connection of cervical cancer pathogenesis involving north-east Indian patients.

As per WHO, Cervical cancer (CaCx) is a global issue, being the fourth common cancer in women with incidence rate of 13.1 per 1 lakh women globally and accounting for 311000 deaths in the year 2018 itself globally. The molecular pathogenesis in Human papillomavirus (HPV) infected cases is inconclusive. The detection of molecular factors leading to progression of CaCx can be important in the diagnosis and management of the disease. p53 a known tumor suppressor gene having a regulative role in cell cycle has been highlighted as key factor in the prevention of cancer but its significance in CaCx cases has been variably documented. The present study therefore targeted to evaluate the significance of p53 profile in CaCx cases in ethnically distinct northeast Indian population. Blood and Tissue samples (N = 85) of cervical cancer patients were collected and screening for HPV was performed using PCR. Thereafter the differential mRNA expression(qPCR), Immunohistochemistry, Mutation (PCR direct sequencing method) of p53 was studied. Further p53 epigenetic profiling was done by Methylation specific PCR (MS-PCR) and western blotting by using p53 acetylation specific antibodies. Our findings revealed that the downregulation of p53 was associated with the progression of disease and the variation in downregulation based on p53 polymorphism was observed. Further hypermethylation and deacetylation of p53 was also found to be associated with the pathogenesis of CaCx. The downregulated expression and hypermethylation of p53 in lower grade of CaCx, together established its association with the progression of CaCx from lower to severe grade. Therefore, in CaCx patients of northeast Indian population, malfunctioning of p53 is found to have significant role in cervical cancer progression.

Impact of modulation of telomerase and cancer stem-cell marker OCT4 axis in cervical cancer pathogenesis with underlying HPV16 infection.

Lacunae exist in the molecular event(s) specificity associated with cervical cancer (CaCx) pathogenesis. The present study aimed to evaluate the significance of telomerase-cervical cancer stem cells (CSCs) modulation in CaCx pathogenesis with underlying HPV16 infection. The study included HPV16 positive cases only (N = 65) of the total enrolled cases from Northeast India. The analysis of viral load and the differential messenger RNA expression of E6, E7, hTERT, hTR, and cancer stem-cell markers was studied by real-time polymerase chain reaction. Further the protein and colocalization study for E6, hTERT, and oct4 was performed by immunofluorescence. The real-time polymerase chain reaction based analysis showed an upregulation of HPV16 viral oncoprotein E6 and E7, and telomerase component hTERT and hTR expression and their correlation in CaCx susceptibility and severity. The hTERT expression correlated with viral load; while the E6 and telomerase protein expression colocalized in the nucleus. The CSCs marker octamer-binding transcription factor 4 (OCT4) was significantly upregulated in CaCx cases, was associated with CaCx susceptibility and severity, and colocalized with E6 expression in the nucleus as revealed from the immunofluorescence studies. To conclude, the telomerase-OCT4 axis modulation holds key in HPV16 CaCx pathogenesis mediated by HPV16 E6 viral oncoprotein expression, and underlines its potential for therapeutic targeting.

Cervical cancer subtypes harbouring integrated and/or episomal HPV16 portray distinct molecular phenotypes based on transcriptome profiling of mRNAs and miRNAs

Heterogeneity in cervical cancers (CaCx) in terms of HPV16 physical status prompted us to investigate the mRNA and miRNA signatures among the different categories of CaCx samples. We performed microarray-based mRNA expression profiling and quantitative real-time PCR-based expression analysis of some prioritised miRNAs implicated in cancer-related pathways among various categories of cervical samples. Such samples included HPV16-positive CaCx cases that harboured either purely integrated HPV16 genomes (integrated) and those that harboured episomal viral genomes, either pure or concomitant with integrated viral genomes (episomal), which were compared with normal cervical samples that were either HPV negative or positive for HPV16. The mRNA expression profile differed characteristically between integrated and episomal CaCx cases for enriched biological pathways. miRNA expression profiles also differed among CaCx cases compared with controls (upregulation—miR-21, miR-16, miR-205, miR-323; downregulation—miR-143, miR-196b, miR-203, miR-34a; progressive upregulation—miR-21 and progressive downregulation—miR-143, miR-34a, miR-196b and miR-203) in the order of HPV-negative controls, HPV16-positive non-malignant samples and HPV16-positive CaCx cases. miR-200a was upregulated in HPV16-positive cervical tissues irrespective of histopathological status. Expression of majority of the predicted target genes was negatively correlated with their corresponding miRNAs, irrespective of the CaCx subtypes. E7 mRNA expression correlated positively with miR-323 expression among episomal cases and miR-203, among integrated cases. miR-181c expression was downregulated only among the episomal CaCx cases and negatively correlated with protein coding transcript of the proliferative target gene, CKS1B of the significantly enriched “G2/M DNA Damage Checkpoint Regulation” pathway among CaCx cases. Thus, the two CaCx subtypes are distinct entities at the molecular level, which could be differentially targeted for therapy. In fact, availability of a small molecule inhibitor of CKS1B, suggests that drugging CKS1B could be a potential avenue of treating the large majority of CaCx cases harbouring episomal HPV16.

A meta-analysis of association of Human Leukocyte Antigens A, B, C, DR and DQ with Human Papillomavirus 16 infection

Human Papillomavirus (HPV) induced cervical cancer (CaCx) is a major health problem in women from both developing and developed regions of the world. This virus accounts for >95% of the CaCx cases with a preponderance of HPV type −16 (65%). Paradoxically HPV-16 is prevalent even in the cervix of healthier women and anti HPV-16 T-cell response is considered critical for the viral clearance. Studies on HLA association with HPV-16 infection and cervical cancer have yielded varied HLA associations in different epidemiological settings. To validate these associations, we performed a meta-analysis of HLA-A, B, C, DR and DQ association with HPV-16 infection. Of the 1409 studies retrieved, 26 qualified for meta-analysis based on stringent inclusion and exclusion criteria.HLA-B*47, B*57, DRB1*10, DRB1*15 and DQB1*0303 were significantly associated with HPV-16 infection (OR = 3.4, 1.8, 1.5, 1.1 and 1.5 respectively). HLA-B*49, B*39, A28 (serotype), C*04 and DRB1*13 were negatively associated with HPV-16 (OR = 0.5, 0.6, 0.7, 0.7, and 0.7 respectively). Certain HLA alleles such as B*07, DRB1*15, DRB1*11 and DRB1*07 showed weakly positive associations. A comprehensive analysis coupling HPV-16 antigenic diversity and the HLA variation in various global populations shall provide further insights into the immunogenetic predisposition to HPV-16 and shall help identify host-parasite co-evolution.

Deregulated TNF-Alpha Levels Along with HPV Genotype 16 Infection Are Associated with Pathogenesis of Cervical Neoplasia in Northeast Indian Patients.

Multiple factors are associated with human papillomavirus (HPV) infection related cervical anomalies and its progression to cervical carcinoma (CaCx), but data vary with respect to the underlying HPV genotype and with population being studied. No data are available regarding the role of immunological imbalance in HPV infected CaCx pathogenesis from Northeast India, which has an ethnically distinct population, and was aimed to be addressed through this study. The study included 76 CaCx cases, 25 cervical intraepithelial neoplasia (CIN) cases, and 50 healthy female controls. HPV screening and genotyping were performed by PCR. Differential expression of tumor necrosis factor alpha (TNF-α) was studied at serum level by enzyme-linked immunosorbent assay and tissue level by immunohistochemistry and messenger RNA (mRNA) level by real-time PCR. The data were correlated with interferon gamma (IFN-γ) and NF-κβp65 levels at protein level, as well as HPV16 E6 and E7 expression at transcript level statistically. HPV infection and HPV16 genotype were predominant in the studied cohort. TNF-α was found to be downregulated at both mRNA and protein levels in CaCx cases compared to controls; and the gradient downregulation correlated with progression of the disease from normal→CIN→CaCx. TNF-α expression correlated with insufficient modulation of both IFN-γ and NF-κβp65. The HPV16 E6 and E7 transcripts were found to be sharply upregulated in CaCx cases strongly inversely correlated with the TNF-α expression. Significant role of TNF-α downregulation associated with insufficient IFN-γ and total NF-κβp65 modulation and the resulting significant upregulation of viral transcripts E6 and E7 are key to the HPV16 infection mediated CaCx pathogenesis in northeast Indian patients.

Polymorphisms in Cytotoxic T-lymphocyte Associated Antigen 4 Gene Does Not Affect sCytotoxic T-lymphocyte Associated Antigen 4 Levels in Human Papillomavirus-Infected Women with or without Cervical Cancer

Background: Cervical cancer (CaCx) is the second most common cancer in Indian women. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) + 49 AA polymorphism is known to be associated with CaCx. Current attempt is to use immunotherapy for the treatment of metastatic melanoma and metastatic castration-resistant prostate cancer, i.e., blocking of CTLA-4 using a fully human monoclonal CTLA-4 antibody to disrupt its inhibitory signal. This allows the CTLs to destroy the cancer cells. There is no information available on the soluble level of CTLA-4 on which the immunotherapy is targeted. This is specifically in Indian population including cases with CaCx. Objective: The aim of this study is to evaluate the levels of soluble CTLA-4 (sCTLA-4) in human papillomavirus (HPV)-infected women with or without CaCx and their association with the polymorphism at CTLA-4 + 49 A/G and CTLA-4 −318 C/T genotypes. Materials and Methods: This is an exploratory case–control study involving two groups of HPV-infected women, the cases were with invasive CaCx and the control group was women with the healthy cervix. sCTLA-4 levels were measured using ELISA in 92 CaCx cases and 57 HPV-positive women with the healthy cervix. Results: Both cases and controls have similar sCTLA-4 levels. Comparison of CTLA-4 + 49A/G and −318 C/T genotypes with sCTLA-4 levels among cases and control also did not show any statistically significant difference. Conclusion: The present study suggests sCTLA-4 levels are not affected by a polymorphism at + 49 A>G CTLA-4. Hence, levels of CTLA-4 are similar in both CaCx cases and control group.

Impact of viral and host DNA methylations on HPV16-related cervical cancer pathogenesis.

Epigenetic alterations within human papillomavirus (HPV) and host cellular genomes are known to occur during cervical carcinogenesis. Our objective was to analyse the influence of (1) methylation within two immunostimulatory CpG motifs within HPV16 E6 and E7 genes around the viral late promoter and their correlation, if any, with expression deregulation of host receptor (TLR9) and DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and (2) global DNA methylation levels within CpGs of the repetitive Alu sequences, on cervical cancer (CaCx) pathogenesis. Significantly higher proportions of CaCx samples portrayed methylation in immunostimulatory CpG motifs, compared to HPV16-positive non-malignant samples, with cases harbouring episomal HPV16 showing decreased methylation compared to those with viral integration. A significant linear trend of TLR9 upregulation was recorded in the order of HPV-negative controls < HPV16-positive non-malignant samples < HPV16-positive CaCx cases. TLR9 upregulation in cases with episomal HPV16 was again higher among those with non-methylated immunostimulatory CpG motifs. Comparison of cases with HPV-negative controls revealed that DNMT3A was significantly downregulated only among integrated cases, DNMT3B was significantly overexpressed among both categories of cases, although at variable levels, while DNMT1 failed to show any deregulated expression among the cases. Global host DNA hypomethylation, also showed a significant linear increasing trend through the progressive CaCx development stages mentioned above and was most prominently higher among cases with episomal HPV16 as opposed to viral integration. Thus, HPV16 and host methylations appear to influence CaCx pathogenesis, with differential molecular signatures among CaCx cases with episomal and integrated HPV16.

Impact of genetic variations and transcriptional alterations of HLA class I genes on cervical cancer pathogenesis.

In a novel attempt to understand the variations in DNA sequences underlying HLA class I alleles associated with HPV16-related CaCx, we determined the alleles by reconstructing SNP-based haplotypes from resequencing of the most polymorphic exons 2 and 3 of HLA-A, HLA-B and HLA-C. We also determined the impact of SNPs and transcriptional alterations of the genes on CaCx. A high density of SNPs was identified from resequencing. HLA expression was determined by real-time PCR. We identified that even a single associated HLA allele had many underlying SNP-based haplotypes. Out of the most frequent (≥5%) HLA class I alleles, HLA-B*40:06 and HLA-B*15:02 respectively imparted significant risk towards and protection from CaCx as well as HPV16 infection. Employing median-joining networks to detect clusters of sequence-variations for specific HLA alleles, we found the protective SNP-based signature, GAATTTA, in all SNP-based haplotypes of HLA-B*15:02 allele. The signature was derived from seven SNPs within HLA-B which were newly associated with the disease. Contrarily, similarly derived risk-signature, TTGCGCC, mapped only to 52% of SNP-based haplotypes of HLA-B*40:06 allele. This indicated that all SNP-based haplotypes underlying a particular associated HLA allele might or might not have a single signature of risk/protection. HLA-A, HLA-B and HLA-C expressions were downregulated among CaCx cases compared to asymptomatic infections and HPV-negative controls. HLA-A and HLA-B were repressed in both cases harbouring episomal and integrated HPV16, whereas HLA-C in only the latter. Novel genetic variations and differential downregulation-patterns of HLA class I have a significant bearing on HPV16-related CaCx pathogenesis.

Identification of genetic variation in the lncRNA HOTAIR associated with HPV16-related cervical cancer pathogenesis.

Previously, over-expression of the long noncoding RNA (lncRNA) HOTAIR has been found to be associated with the invasive and metastatic capacities of several epithelial cancers, including cervical cancer (CaCx). Here, we aimed at identifying functionally relevant genetic variants that may be employed to differentiate between clinically distinct CaCx subtypes, i.e., those exhibiting high HOTAIR levels and molecular signatures of metastasis and those lacking such signatures in the presence of low HOTAIR expression levels. Genomic DNA isolated from various cervical tissue samples (characterized by histopathology and HPV status) was used for HOTAIR amplicon sequencing, followed by validation of the findings by Sanger sequencing. The impact of the genetic variants found on the secondary structure of HOTAIR and the concomitant alterations in miRNA binding sites were determined through in silico analysis, followed by miRNA expression analysis by quantitative real-time PCR and confirmation of miRNA binding using a luciferase reporter assay. We found that rs2366152C was over-represented [ORage-adjusted = 2.58 (1.23-5.57); p = 0.014] in low HOTAIR expressing HPV positive CaCx cases compared to HPV negative controls. This genetic variant showed the propensity of a secondary structure alteration and gain of a miR-22 binding site in HOTAIR, which was found to be concordant with miR-22 over-expression in low HOTAIR CaCx cases compared to controls. We found that miR-22 expression negatively correlated with HOTAIR and E7 expression in HPV16 positive cases and in an E7 transfected HPV negative CaCx-derived cell line (C33A), but was not altered in high HOTAIR cases compared to controls. Reduced luciferase activity of a HOTAIR rs2366152C expression plasmid in C33A cells through miR-22 co-transfection confirmed the ability of miR-22 to specifically target rs2366152C-harbouring HOTAIR lncRNA in CaCx cells, ultimately leading to its down-regulation. Our data indicate that rs2366152C not only has the potential to serve as a marker for singling out CaCx cases lacking metastatic molecular signatures, but also to explain the functional inactivation of HOTAIR in these cases, including the mechanism of its down-regulation.

Abstract 1056: Impact of host methylome on cervical cancer pathogenesis

Abstract Identification of episomal HPV16 in cervical cancers (CaCx) besides the integrated forms, prompted us to investigate the global CpG methylation signatures among the different categories of CaCx samples, since alterations of CpG methylation in host genomes, common in many cancers, are likely to influence host pathogen interactions and the disease pathogenesis. We determined global DNA methylation patterns of the host genomes, in HPV16 (+) CaCx cases (11 episomal and 12 integrated) and controls (12 HPV16 (-) and 12 HPV16 (+), employing the Illumina Infinium 450K Bead Chip assay. Using Genome Studio software, we extracted the raw signal intensities of each probe. With statistical methods developed in-house, we performed color adjustment and normalization of the data using control probes. Differentially methylated CpG loci were identified by comparing methylation estimates (M-values) using a two-sample t-test followed by adjustment of p-values based on False Discovery Rate threshold of 0.01 among (i) CaCx cases, irrespective of HPV16 physical status, (ii) CaCx cases with episomal HPV16 and (iii) CaCx cases with integrated HPV16, compared to controls irrespective of HPV, since no difference was recorded among these two groups. Differentially methylated CpGs were identified and mapped using Illumina annotation to various genic regions (TSS1500, TSS200, 1st Exon, Gene Body, 5′ UTR and 3′ UTR). Irrespective of viral physical status, hypermethylation was more prominent across all genic regions than hypomethylation, albeit hypermethylation being lower in Gene Body and 3′-UTR regions only. Subsequently employing a Δβ threshold of 0.3 i.e. difference of β between two groups as 30%, differentially methylated CpGs mapped to 2536, 3222 and 3003 genes among total CaCx set, episomal and integrated CaCx cases, respectively. Ingenuity Pathway Analysis revealed a number of pathways that were significantly enriched among total CaCx cases, episomal or integrated, while some enriched pathways were recorded exclusively among episomal (Gap Junction Signaling, FGF Signaling, Huntington's Disease Signaling) and integrated (Chemokine Signaling etc.) CaCx cases. Expression of FGF2 gene (FGF Signaling pathway), PIK3R1 gene (Gap Junction Signaling pathway) and CXCL12 gene (Chemokine Signaling pathway) was found to be down regulated, based on Illumina -HT12_V4 array analysis on same set of samples. Significant negative correlation between hypermethylation in the promoter region, involving 5 CpGs of FGF and 1 CpG of CXCL12, were recorded (Pearson Correlation p-value&amp;lt;0.001). In case of PIK3R1, downregulated expression was negatively correlated with hypermethylation of 2 CpGs in the Gene Body. The study highlights the relevance of host methylation in cervical cancer pathogenesis involving Gene Body methylation in addition to promoters, and provides novel insights into HPV16 related CaCx pathogenesis involving episomal and integrated viral genomes. Citation Format: Shrinka Sen, Samsiddhi Bhattacharjee, Indranil Mukhopadhyay, Paramita Mandal, Sweta Sharma, Rahul Roy Chowdhury, Sharmila Sengupta. Impact of host methylome on cervical cancer pathogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1056. doi:10.1158/1538-7445.AM2015-1056

Bridging Links between Long Noncoding RNA HOTAIR and HPV Oncoprotein E7 in Cervical Cancer Pathogenesis.

Human Papillomavirus (HPV) type 16 oncoprotein E7 plays a major role in cervical carcinogenesis by interacting with and functionally inactivating various host regulatory molecules. Long noncoding RNA (lncRNA) HOTAIR is one such regulator that recruits chromatin remodelling complex PRC2, creating gene silencing H3K27 me3 marks. Hence, we hypothesized that HOTAIR could be a potential target of E7, in HPV16 related cervical cancers (CaCx). We identified significant linear trend of progressive HOTAIR down-regulation through HPV negative controls, HPV16 positive non-malignants and CaCx samples. Majority of CaCx cases portrayed HOTAIR down-regulation in comparison to HPV negative controls, with corresponding up-regulation of HOTAIR target, HOXD10, and enrichment of cancer related pathways. However, a small subset had significantly higher HOTAIR expression, concomitant with high E7 expression and enrichment of metastatic pathways. Expression of HOTAIR and PRC2-complex members (EZH2 and SUZ12), showed significant positive correlation with E7 expression in CaCx cases and E7 transfected C33A cell line, suggestive of interplay between E7 and HOTAIR. Functional inactivation of HOTAIR by direct interaction with E7 could also be predicted by in silico analysis and confirmed by RNA-Immunoprecipitation. Our study depicts one of the causal mechanisms of cervical carcinogenesis by HPV16 E7, through modulation of HOTAIR expression and function.

Differential Expression of HPV16 L2 Gene in Cervical Cancers Harboring Episomal HPV16 Genomes: Influence of Synonymous and Non-Coding Region Variations

We tested the hypothesis that (i) synonymous variations within the coding regions, and (ii) variations within the non-coding regions of HPV, influence cervical cancer (CaCx) pathogenesis under the impact of intact HPV16 genomes. Whole genome sequence analysis of HPV16 isolates within 70 CaCx cases and 25 non-malignant samples revealed that synonymous variations were significantly higher within the E6 (p = 0.014), E5 (p = 0.001) and L2 (p = 0.0002) genes of HPV16 isolates within cases, compared to isolates within non-malignant samples. All of the 25 (100%) humanized codons identified within L2 ORF of the samples analyzed, were harbored by CaCx cases, while 8 out of 25 (32%) were harbored by HPV16 positive non-malignant samples (p = 3.87105E-07). L2 (mRNA and protein) expression was evident only among cases with episomal viral genomes and L2 mRNA expression correlated significantly with E2 gene copy numbers suggesting expression from all episomal genomes. Among such cases, Asian American (AA) isolates portrayed all of the humanized codons (100%; 4–6/sample) recorded within L2, which was significantly higher (p = 2.02E-7) compared to the European (E) isolates (22.8%; none or 1–2/sample). Additionally, majority of E variant isolates within cases (54/57; 94.7%) portrayed a variation (T4228C) within the short non-coding region (NCR2) between E5 and L2 genes, which portrays a weak promoter activity specific for L2 mRNA expression. This resulted in loss of 9 out of 14 miRNA binding sites (hsa-miR-548 family), despite the significant overexpression of miR548a-5p and miR548d-5p among such cases (28.64 and 36.25 folds, respectively), in comparison to HPV negative control samples. The findings exemplify the biological relevance of sequence variations in HPV16 genomes and highlight that episomal HPV16 in CaCx cases employ multiple mechanisms to sustain L2 expression, thereby justifying the potential role of L2 in such cancers, as opposed to those harboring viral integration.

Some Novel Insights on HPV16 Related Cervical Cancer Pathogenesis Based on Analyses of LCR Methylation, Viral Load, E7 and E2/E4 Expressions

This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1∧E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1∧E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 CT/E4 CT. Relative quantification based on comparative CT (theshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 CT (p = 0.007) and E2 CT (p<0.0001), respectively, each normalized with ACTB CT, among episomal cases only. The k-means clustering analysis considering E7 CT from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis.

Lipid peroxidation and antioxidants in different stages of cervical cancer: Prognostic significance

Free radical Injury is associated with cancer, but how the extent of oxidative stress correlates with the FIGO (International Federation of Gynecology and Obstetrics) stage in Carcinoma Cervix (Ca Cx), and its significance as a prognostic marker, is not clear and needs an in-depth study. To correlate the blood levels of Lipid Peroxidation (LPO), Reduced Glutathione (GSH), Superoxide Dismutase (SOD), and Vitamin A and E levels with the clinical stage in Ca Cx. This is a Prospective Case Control Study. LPO, SOD, reduced GSH were estimated by Bio Chemical Assays and Vitamins by High Performance Liquid Chromatography (HPLC). The cases and controls were compared using One Way ANOVA and different stages over different time periods were individually compared by Repeated Measure Analysis of Variance. The results indicated a statistically significant increase of LPO vis-a-vis the FIGO stage of Ca Cx and control, while the antioxidant status as depicted by GSH and SOD decreased. Vitamin A and E levels were significantly lower in cancer cases as compared to the control. Increased LPO and reduced antioxidant levels may be taken as associated predictive markers, thus suggesting that Ca Cx cases should get nutritive supplements to contain the blood LPO level and maintain a positive balance of antioxidants for a better outcome in terms of delayed recurrence and better Quality of Life (QOL).

Characterization of sequence variations within HPV16 isolates among Indian women: Prediction of causal role of rare non-synonymous variations within intact isolates in cervical cancer pathogenesis

We re-sequenced HPV16 genome (~ 6 kb) implicated in cervical carcinogenesis (LCR, E2, E5, E6, E7, L1, L2) to prioritize sequence variants for functional validation as biomarkers, using CaCx cases ( n = 74) and asymptomatic controls ( n = 24). Of the nucleotide variations recorded ( n = 271), non-synonymous changes in L2 region were significantly higher ( p = 0.005) among cases (2.67%) compared to controls (1.27%). Using SIFT database, 29 non-synonymous changes (frequency = 0.01–0.03) predicted as deleterious to protein functions were identified. Haplotype analysis considering 110 polymorphic variations (frequency ≥ 0.05) within intact viral isolates (53 CaCx cases and 21 controls) using NETWORK software, confirmed Asian–American (AA, 14.86%) and European (E, 85.14%) variants, differing at 78 positions. The E-variants portrayed thirty-six haplotypes, of which, E-12 was most prevalent within cases (38.1%; 16 / 42) and controls (28.57%; 6 / 21) harboring polymorphic variations at 10 positions, in contrast to HPV16R. Cases of the E-12 haplotype harbored 7 deleterious mutations distributed within L1 ( n = 1), E2 ( n = 1), E5 ( n = 1), and L2 ( n = 4), while none within similar controls. Thus rare deleterious variations within genes implicated in productive infection over the E-12 haplotype background of intact HPV16 isolates might be of causal relevance for CaCx development.

Predisposition to HPV16/18‐related cervical cancer because of proline homozygosity at codon 72 of p53 among Indian women is influenced by HLA‐B*07 and homozygosity of HLA‐DQB1*03

This study was undertaken to examine whether predisposition to human papillomavirus (HPV)16/18-related cervical cancer (CaCx) because of p53 proline homozygosity (Pro72Pro) among Indian women was mediated singly or jointly with immunogenetic risk factors such as HLA-B*07 or homozygosity of HLA-DQB1*03. Molecular detection of all three genetic factors was performed by polymerase chain reaction-restriction fragment length polymorphism using DNA from (i) 114 CaCx samples (78 HPV16/18 positive) and (ii) 195 cytologically normal cervical scrapes (116 HPV-negative and 79 HPV16/18-positive samples). Multiple logistic regression analyses were performed to examine independent effects of the three factors and to determine age-adjusted odds ratio (OR) [95% confidence interval (CI)] and P-values. HLA-B*07 was observed to be significantly associated with HPV16/18 infection in asymptomatic controls (OR(age-adjusted) = 4.73; 95% CI: 1.55-14.45; P = 0.006) and CaCx (OR(age-adjusted) = 6.14; 95% CI: 2.15-17.53; P < 0.001) in this enhanced sample set of CaCx cases. There was a lack of association between HLA-B*07 and HLA-DQB1*03 in our study samples. The association of p53Pro72Pro with CaCx was non-significant in the absence of HLA-B*07 in HPV16/18-positive women. In this group, prevalence of p53Pro72Pro and HLA-B*07 together was significantly higher (7.0%) among CaCx cases (OR(age-adjusted) = 14.05; 95% CI: 1.11-177.30; P = 0.04), compared with controls (1.3%) lacking both factors. HLA-DQB1*03 homozygosity (OR(age-adjusted) = 4.75; 95% CI: 1.17-19.30; P = 0.03) or p53Pro72Pro (OR(age-adjusted) = 5.84; 95% CI: 1.18-28.99; P = 0.03) was found to be significantly associated with CaCx, each in the absence of the other in this group but not when present jointly in contrast to those lacking both factors (P = 0.214). Thus, modulation of p53Pro72Pro-mediated susceptibility to CaCx by immunogenetic factors could possibly be mediated through cross talk between HPV16/18-induced immune evasion and cell transformation.

CpG methylation of HPV 16 LCR at E2 binding site proximal to P97 is associated with cervical cancer in presence of intact E2

Human papillomavirus type 16 (HPV-16) E2 protein negatively regulates transcription of the E6 and E7 genes. This study was done to test the hypothesis that methylation of the HPV 16 long control region (LCR) is overrepresented among cervical cancer (CaCx) cases compared to cytologically normal controls harboring intact E2 gene. Methylation of the E2 binding site (E2BS-I), proximal to the P97 promoter, was assessed by HpaII/ MspI restriction digestion while McrBC digestion was used to assess LCR-E6 (7289–540) for 57 CaCx samples and 15 normal controls. E2BS-I methylation was found to be significantly higher (56.14%) in cases compared to (20%) controls [OR age-adjusted (95% CI): 4.53 (1.05–19.43) p = 0.042]. The difference between cases (54.39%) and controls (40%) with respect to LCR-E6 methylation status [OR age-adjusted (95% CI): 1.77(0.5–6.3); p = 0.38] was not significant. Sequencing of a randomly selected set of 13 methylated malignant samples revealed absence or rare presence, of methylation at CpGs 7579, 7535, 7683 and 7862 respectively. Methylation was found to be more at CpGs within E2 binding sites proximal to the P97 promoter. These results indicate the involvement of E2 binding site methylation in presence of intact E2, leading to loss of E2 repressor activity in CaCx.