Sankhadeep Dutta

Genetic and epigenetic changes of HPV16 in cervical cancer differentially regulate E6/E7 expression and associate with disease progression

OBJECTIVE The study was aimed at understanding the complex interactions of genetic and epigenetic events in expression of HPV16 E6/E7 and progression of cervical carcinoma. For this, expression of E6/E7 was done in 36 samples, along with the physical status, methylation and LCR sequence variations. Later, the genetic and epigenetic studies were extended to 239 samples to find out the association of these factors with progression of cervical cancer. METHODS E6/E7 expression was quantified by real-time PCR. Physical status of HPV16 was determined by mutiplex-PCR of whole E2 ORF using overlapping primers and E6 ORF and validated by real-time PCR. Methylation status of P97 promoter/enhancer was analyzed by methylation sensitive restriction analysis (MSRA). Viral lineage and variations in LCR was ascertained by sequencing LCR/E6/E7 ORFs. RESULTS Samples with episomal unmethylated virus showed comparatively high expression of E6/E7 than episomal methylated, integrated unmethylated and integrated methylated forms of HPV16. Variations in the LCR, particularly in the binding sites of negatively regulating transcription factors, also contribute to high expression of E6/E7. The integrated form significantly increases with decrease of episomal form during tumor progression. Methylation of the promoter/enhancer gradually decreased with tumor progression and is inversely correlated to integration. Two novel variants were observed in E6 gene in European- and North-American-1-lineages. Log-rank test revealed better prognosis of the patients with episomal methylated HPV16 compared to the other forms. CONCLUSION Our results show higher expression of E6/E7 in samples with episomal unmethylated virus having sequence variations in LCR.

Clearance of Cervical Human Papillomavirus Infection by Topical Application of Curcumin and Curcumin Containing Polyherbal Cream: A Phase II Randomized Controlled Study

Curcumin and curcumin containing polyherbal preparations have demonstrated anti-microbial and anti- viral properties in pre-clinical studies. Till date no therapeutic intervention has been proved to be effective and safe in clearing established cervical human papillomavirus (HPV) infection. The present study evaluated the efficacy of Basant polyherbal vaginal cream (containing extracts of curcumin, reetha, amla and aloe vera) and of curcumin vaginal capsules to eliminate HPV infection from cervix. Women were screened by Pap smear and HPV DNA test by PCR. HPV positive women without high grade cervical neoplasias (N=287) were randomized to four intervention arms to be treated with vaginal Basant cream, vaginal placebo cream, curcumin vaginal capsules and placebo vaginal capsules respectively. All subjects were instructed to use one application of the assigned formulation daily for 30 consecutive days except during menstruation and recalled within seven days of the last application for repeat HPV test, cytology and colposcopy. HPV clearance rate in Basant arm (87.7%) was significantly higher than the combined placebo arms (73.3%). Curcumin caused higher rate of clearance (81.3%) than placebo though the difference was not statistically significant. Vaginal irritation and itching, mostly mild to moderate, was significantly higher after Basant application. No serious adverse events were noted.

Prevalence of human papillomavirus in women without cervical cancer: a population-based study in Eastern India.

Despite the high incidence of cervical cancer, population-based data on prevalence of human papillomavirus (HPV) are limited in India. This study aimed to evaluate the prevalence of any HPV type and type-specific prevalence of HPV 16/18 in women without cervical cancer. HPV viral load was measured and correlated with cytologic abnormalities of the cervix. A total of 2501 women between 25 and 65 years of age and without cervical cancer were screened by pap smear cytology. HPV DNA was detected from cervical scrapes by nested polymerase chain reaction. Detection of HPV 16/18 was carried out by polymerase chain reaction using type-specific primers and was confirmed by Southern hybridization. Viral load was determined by absolute real-time polymerase chain reaction. Population prevalence of any HPV was found to be 9.9%. The risk of HPV infection was higher in women aged 25 to 34 years (odds ratio, 1.11), in married women below 20 years of age (odds ratio, 1.80), and in women with parity ≥4 (odds ratio, 1.04). Prevalence of HPV 18 (1.4%) was greater than that of HPV 16 (0.6%) in the overall screened population. High-grade squamous intraepithelial lesion cytology was more frequent in women infected with HPV 16 than in those infected with HPV 18 and other types. A gradual increase in HPV copy numbers was associated with progressive cytologic severity. In this study, HPV prevalence is comparable to HPV prevalence reported by other studies among Indian and Asian women. Although the prevalence of HPV 18 was more than that of HPV 16, type 16 infection was associated with higher oncogenicity.

Diagnostic accuracy of VIA and HPV detection as primary and sequential screening tests in a cervical cancer screening demonstration project in India

Visual inspection after acetic acid application (VIA) and human papillomavirus (HPV) detection tests have been recommended to screen women for cervical cancer in low and middle income countries. A demonstration project in rural India screened 39,740 women with both the tests to compare their accuracies in real population setting. The project also evaluated the model of screening women in the existing primary health care facilities, evaluating the screen positive women with colposcopy (and biopsy) in the same setup and recalling the women diagnosed to have disease for treatment at tertiary center. Accuracy of VIA and HPV test used sequentially was also studied. VIA was performed by trained health workers and Hybrid Capture II (HC II) assay was used for oncogenic HPV detection. Test positivity was 7.1% for VIA and 4.7% for HC II. Detection rate of CIN 3+ disease was significantly higher with HC II than VIA. Sensitivities of VIA and HC II to detect 162 histology proved CIN 3+ lesions were 67.9 and 91.2%, respectively after adjusting for verification bias. Specificity for the same disease outcome and verification bias correction was 93.2% for VIA and 96.9% for HC II. Triaging of VIA positive women with HPV test would have considerably improved the positive predictive value (4.0 to 37.5% to detect CIN 3+) without significant drop in sensitivity. All VIA positive women and 74.0% of HC II positive women had colposcopy. There was high compliance to treatment and significant stage‐shift of the screen‐detected cancers towards more early stage.

Inactivation of CHEK1 and EI24 is associated with the development of invasive cervical carcinoma: Clinical and prognostic implications

To understand the importance of frequent deletion of chromosomal 11q23.3‐24.3 region in cervical carcinogenesis, alterations (deletion/methylation/mutation/expression) of the candidate genes LOH11CR2A, EI24 and CHEK1 located in the region were analyzed in 29 cervical intraepithelial neoplasia (CIN), 112 cervical carcinoma (CACX) samples and two CACX cell lines. The deletion frequency of these genes was low in CIN than in CACX [CIN: CHEK1: 28%, EI24: 21%, LOH11CR2A: 15% and CACX: CHEK1: 51%, EI24: 41%, LOH11CR2A: 36%]. Similar trend was seen in promoter methylation of these genes [CIN: CHEK1: 10%, EI24: 3%, LOH11CR2A: 3% and CACX: CHEK1: 55%, EI24: 31%, LOH11CR2A: 14%]. Mutations of the genes are a rare event. Overall alterations (deletion and methylation) of CHEK1 and EI24 were associated with progression of CACX. Quantitative mRNA expression analysis showed reduced expression of the three genes in concordance to their molecular alterations. A shorter isoform of CHEK1 lacking exon 8, hence impaired in substrate binding capacity, was found in two samples. Immunohistochemical analysis showed nuclear expression of Chek1, p‐Chek1 and Ei24 in tumor tissues, whereas the cell lines exhibited both nuclear and cytoplasmic expression of Chek1 and Ei24, as is also evident from Western blot analysis suggesting differential localization of the proteins. Alterations of CHEK1 and EI24 coupled with tumor stage and early sexual debut (≤19 years) predicted worst prognosis. Thus, our data suggest that inactivation of EI24 and CHEK1 through two independent mechanisms contributes to the development of CACX.

Physical and methylation status of human papillomavirus 16 in asymptomatic cervical infections changes with malignant transformation

Aims To evaluate how the genetic and epigenetic profile of human papillomavirus 16 (HPV16) changes from asymptomatic cervical infections to cervical cancer (CaCx) development. Methods HPV16 physical status, methylation of its early–late promoters and its upstream enhancer sequences were analysed in samples from asymptomatic cervical infections (n=89), pre-neoplastic lesions (low and high grade squamous intraepithelial lesions LSIL/HSIL, n=28) and primary CaCx (n=98). Results In asymptomatic infection (65%, 58/89) and LSIL/HSIL (57%, 16/28) samples, the episomal form of HPV16 was predominant whereas integration of HPV16 was significantly (p=0.01) higher in CaCx (59%, 57/98). The integrated viral form was also present in asymptomatic (27%, 24/89) and LSIL/HSIL (25%, 7/28) samples. The methylation of the enhancer region was comparable (29–34%) among asymptomatic, LSIL/HSIL and CaCx samples. The episomal form exhibited relatively higher methylation of the early promoter (52%) than that of the late promoter (40%) in asymptomatic infection but the integrated form in asymptomatic carriers showed the opposite methylation pattern (early promoter (42%) vs late-promoter (54%)). A similar pattern was observed in LSIL/HSIL samples, with comparable frequencies (44%) of early and late promoter methylation of the episomal form. However, irrespective of HPV16 physical status, higher methylation of late promoter than that of early promoter was observed in CaCx samples. An inverse correlation was observed between HPV16 integration and overall methylation of the early promoter–enhancer region in CaCx (p=0.05), LSIL/HSIL (p=0.09) and asymptomatic samples (p=0.09). Conclusions Our study indicates that integration of HPV16 along with changes in methylation pattern of early and late promoters is essential for neoplastic transformation of asymptomatic cervical infections.

Sensitivity of APTIMA HPV E6/E7 mRNA test in comparison with hybrid capture 2 HPV DNA test for detection of high risk oncogenic human papillomavirus in 396 biopsy confirmed cervical cancers

The sensitivity of E6/E7 mRNA‐based Aptima HPV test (AHPV; Hologic, Inc.) for detection of cervical cancer has been reported based on only a small number of cases. We determined the sensitivity of AHPV in comparison with the DNA‐based Hybrid Capture 2 HPV test (HC2; Qiagen) for the detection of oncogenic HPV in a large number of cervical cancers at the time of diagnosis using cervical samples obtained in ThinPrep (Hologic). Samples yielding discordant results were genotyped using Linear Array assay (LA; Roche). Of 396 cases tested, AHPV detected 377 (sensitivity, 95.2%; 95%CI: 93.1–97.3), and HC2 376 (sensitivity, 94.9%; 95%CI: 92.7–97.1) with an agreement of 97.2% (kappa 0.7; 95%CI: 0.54–0.87). Among six AHPV+/HC2‐ cases, LA identified oncogenic HPV types in four including a type 73 and was negative in two. Among five AHPV‐/HC2+ cases, LA detected oncogenic HPV types in two including a type 73 and was negative in three. Of 14 AHPV‐/HC2‐ cases, 13 were genotyped. LA detected oncogenic HPV types in six, non‐oncogenic types in three, and was negative in four. This is the largest study to demonstrate the sensitivity of AHPV for the detection of invasive cervical cancer and this assay showed equal sensitivity to HC2. J. Med. Virol. 88:1271–1278, 2016.

Alteration of Human Papillomavirus Type 16 Genetic and Epigenetic Profiles in Cervical Cancer Patients Is Indicative of Poor Disease Prognosis: A Cohort Analysis.

Objective Aim of this study was to assess the changes in genetic and epigenetic profiles of human papillomavirus type 16 (HPV16), if any, in primary cervical cancer (CaCx) and corresponding plasma before and after therapy for possible prognostic evaluation. Methods The genetic (integration status) and epigenetic (methylation of enhancer, early promoter, and late promoter sequences) profiles of HPV16 were analyzed in pretherapy CaCx (n = 46), corresponding plasma, posttherapy cervical swabs (n = 39), and corresponding plasma from a single patient cohort. Quantitative viral load was also measured in these HPV16-positive primary CaCx and posttherapy cervical swabs. Results Presence of HPV16 in the patients’ plasma before/after therapy was significantly (P = 0.03) associated with higher viral load in the primary tumor site. Human papillomavirus type 16 integration and hypomethylation of the early (14 of 29, Z = 4.47, P < 0.01) and late promoters (20 of 29, Z = 3.74, P < 0.01) were more prevalent in the plasma than the corresponding pretherapy CaCx samples. However, the dissimilarity in integration status (5 of 24) was less evident between posttherapy cervical swabs and corresponding plasma, although hypomethylation of the early promoter and hypermethylation of the late promoter (8 of 24, Z = 2.6, P < 0.01) was seen in posttherapy plasma samples. Whereas in the posttherapy swabs, integrated (22 of 29) or mixed (7 of 29) form of HPV16 prevailed with hypomethylation of the enhancer (6 of 29, Z = 2.0, P < 0.05) and late promoter (18 of 29, Z = 4.4, P < 0.01) compared with the corresponding primary tumors. The patients having high HPV16 copy number in pretherapy and posttherapy cervical lesions and hypomethylation of early promoter/late promoter in the corresponding plasma showed increased disease recurrence with distant metastases. Conclusions The genetic-epigenetic profile of HPV16 in pretherapy/posttherapy CaCx samples showed significant association with disease prognosis.