Efficacy and Safety of Oral Metronomic Chemotherapy in Recurrent Refractory Advanced Gynaecological Cancer: An Experience From the Regional Cancer Centre of Eastern India.

INTRODUCTION: The outcome of recurrent/metastatic gynaecological malignancy has drastically improved with the introduction of poly(ADP-ribose) polymerase inhibitors and immunotherapy, but the use of these drugs in routine practice is complicated due to access barriers and their high cost in developing countries. The purpose of this study is to present the clinical response, outcome and safety of oral metronomic chemotherapy (OMCT) in resource-limited, financially constrained populations. METHODS: This is a retrospective study on patients with advanced gynaecological cancer treated at Chittaranjan National Cancer Institute, Kolkata, India, from 2021 to 2023. The patients were treated with one of these two regimens: a split-dose course of cyclophosphamide (50 mg orally once daily for 21 days) and capecitabine (500 mg twice daily continuous) or a fixed-dose combination (capecitabine 1800 mg and cyclophosphamide 80 mg orally for 14 days in every 21 days) until disease progression or unacceptable toxicities occurred. All data was captured from the hospital's medical records until June 2023. Toxicity data was reported per the Common Terminology Criteria for Adverse Events (CTCAE) v5.1, and progression-free survival (PFS) was estimated using Kaplan-Meier methods. RESULTS: Among 34 screened patients, 10 were excluded due to noncompliance. This study analysed 24 patients with a median age at diagnosis of 56 years (IQ range 44-75). Sixteen (67%) patients were at stage IV disease with an Eastern Cooperative Oncology Group (ECOG) performance status of 3. Ovarian and cervical cancers were 80% and 20%, respectively; among them, 16 (67%) patients were platinum-refractory. Forty-two per cent of patients received three lines of chemotherapy before OMCT. A split course versus fixed dose was given to 67% versus 33% of the population; the best responses per the Response Evaluation Criteria in Solid Tumours v1.1 were complete response in 12%, partial response in 67% and stable disease in 21%. The most common toxicities were grade I anaemia (54%), grade I chemotherapy-induced nausea and vomiting (46%), grade I fatigue (42%) and grade I neutropenia (21%). Twenty-five per cent of patients were offered next-line systemic therapy after progression. The entire cohort had a median PFS of nine months (95%, CI: 5.2-12.7). Cox regression analysis identified a median PFS of 12 months (95%, CI: 6.2-17.7) among platinum-refractory groups. CONCLUSION: OMCT was a well-tolerated, affordable regimen with durable clinical response and survival outcome (median PFS of nine months) in recurrent, refractory advanced gynaecological cancer and can be offered to patients at resource-limited centres.

Down-regulation of FA-BRCA Pathway in Cervical Carcinoma Gradually Reversed During the Development of Chemo-tolerance: Clinical Implications.

Cervical cancer is one of the leading causes of cancer death among females, worldwide. The contributory role of different cellular pathways in the process of carcinogenesis is still poorly understood. Our study was focused here to understand the functional evaluation of key regulatory genes of FA-BRCA pathway in the development of CACX and their role in chemo-tolerance of the disease by analyzing the molecular profile of the genes both in normal and tumour tissue of our sample pool, also validated in in silico datasets. Later on, prognostic importance of the genes was further evaluated in plasma DNA and cisplatin-treated in vitro system. We found that expression profile of FA-BRCA pathway genes was gradually reduced from undifferentiated basal-parabasal layers of normal tissue towards the progression of the disease. Further analysis revealed that frequent promoter methylation [32-55%] and deletion [34-52%] events were the plausible reasons for their reduced expression in CACX. Noticeably, invasion of promoter methylation of the genes [11-17%] in plasma CTCs of CACX patients was positively correlated [p < 0.001] with poor prognosis among patients. On the other hand, functional upregulation of these genes at higher concentrations [IC50-70] of cisplatin was a predictor for the development of drug tolerance, as evaluated in our in vitro study. This finding was supported further by low prevalence of γ-H2X foci formation and reduced expression of DNMT1 at higher concentrations of cisplatin. In totality, we discovered that the FA-BRCA pathway must be inactivated for cancer formation. In contrast, elevated gene expression played a substantial role in building of chemo-tolerance and might be associated with developing increased risk of disease recurrence among patients.

Immunogenicity and safety of a new quadrivalent HPV vaccine in girls and boys aged 9-14 years versus an established quadrivalent HPV vaccine in women aged 15-26 years in India: a randomised, active-controlled, multicentre, phase 2/3 trial.

BACKGROUND: To meet global cervical cancer elimination efforts, a wider range of affordable and accessible vaccines against human papillomavirus (HPV) are needed. We aimed to evaluate the immunogenicity and safety of a quadrivalent HPV vaccine (targeting HPV types 6, 11, 16, and 18), developed and manufactured by the Serum Institute of India (SIIPL). Here we report outcomes in the 9-14 years cohort. METHODS: This randomised, active-controlled, phase 2/3 trial was conducted at 12 tertiary care hospitals across India. Healthy participants aged 9-14 years or 15-26 years with no history of HPV vaccination were eligible for enrolment. Female participants were randomly assigned (1:1) with an interactive web response system, by use of a central computer-generated schedule and block randomisation (block sizes of 2, 4, 6, and 8), to receive the SIIPL quadrivalent HPV vaccine (Cervavac; SIIPL, Pune, India) or the comparator quadrivalent HPV vaccine (Gardasil; Merck Sharp & Dohme, Harleem, the Netherlands). Participants, investigators, laboratory technicians, and sponsors were masked to treatment allocation of female participants. Male participants were given the SIIPL quadrivalent HPV vaccine in an open-label manner. Study vaccines were administered intramuscularly with a two-dose schedule (at day 0 and 6 months) in the cohort aged 9-14 years, and with a three-dose schedule (at day 0, month 2, and month 6) in the cohort aged 15-26-years. Immunogenicity was assessed 30 days after the last dose by use of multiplexed ELISA. The primary outcome was the non-inferiority of immune response in terms of the geometric mean titre (GMT) of antibodies against HPV types 6, 11, 16, and 18 generated by the SIIPL quadrivalent HPV vaccine in girls and boys (aged 9-14 years) compared with the GMT generated by the comparator quadrivalent HPV vaccine in women aged 15-26 years at month 7 in the modified per-protocol population (ie, all participants who received all doses of study vaccines per assigned treatment group and had both day 0 and 1-month immunogenicity measurements after the last dose following protocol-defined window periods with no major protocol deviations). Non-inferiority was established if the lower bound of the 98·75% CI of the GMT ratio was 0·67 or higher. The co-primary outcome of occurrence of solicited adverse events (within 7 days of each dose) and unsolicited adverse events (up to 30 days after the last dose) was assessed in all participants who were enrolled and received at least one dose of study vaccine. The trial is registered with the Clinical Trials Registry - India (CTRI/2018/06/014601), and long-term follow-up is ongoing. FINDINGS: Between Sept 20, 2018, and Feb 9, 2021, 2341 individuals were screened, of whom 2307 eligible individuals were enrolled and vaccinated: 1107 (738 girls and 369 boys) in the cohort aged 9-14 years and 1200 (819 women and 381 men) in the cohort aged 15-26 years. No race or ethnicity data were collected. 350 girls and 349 boys in the SIIPL quadrivalent HPV vaccine group and 338 women in the comparator vaccine group were included in the modified per-protocol population for the primary endpoint analysis. The median follow-up for the analyses was 221 days (IQR 215-231) for girls and 222 days (217-230) for boys in the SIIPL quadrivalent HPV vaccine group, 223 days (216-232) for girls in the comparator vaccine group, and 222 days (216-230) for women in the comparator vaccine group. GMT ratios were non-inferior in girls and boys receiving the SIIPL quadrivalent HPV vaccine compared with women receiving the comparator vaccine: GMT ratios for girls were 1·97 (98·75% CI 1·67-2·32) for HPV type 6, 1·63 (1·38-1·91) for HPV type 11, 1·90 (1·60-2·25) for HPV type 16, and 2·16 (1·79-2·61) for HPV type 18. For boys the GMT ratios were 1·86 (1·57-2·21) for HPV type 6, 1·46 (1·23-1·73) for HPV type 11, 1·62 (1·36-1·94) for HPV type 16, and 1·80 (1·48-2·18) for HPV type 18. The safety population comprised all 1107 participants (369 girls and 369 boys in the SIIPL quadrivalent HPV vaccine group, and 369 girls in the comparator group). Solicited adverse events occurred in 176 (48%) of 369 girls and 124 (34%) of 369 boys in the SIIPL vaccine group and 179 (49%) of 369 girls in the comparator vaccine group. No grade 3-4 solicited adverse events occurred within 7 days of each dose. Unsolicited adverse events occurred in 143 (39%) girls and 147 (40%) boys in the SIIPL vaccine group, and 143 (39%) girls in the comparator vaccine group. The most common grade 3 unsolicited adverse event was dengue fever, in one (<1%) girl in the SIIPL vaccine group and three (1%) girls in the comparator group. There were no grade 4 or 5 adverse events. Serious adverse events occurred in three (1%) girls and three (1%) boys in the SIIPL vaccine group, and five (1%) girls in the comparator vaccine group. No vaccine-related serious adverse events were reported. There were no treatment-related deaths. INTERPRETATION: We observed a non-inferior immune response with the SIIPL quadrivalent HPV vaccine in girls and boys aged 9-14 years and an acceptable safety profile compared with the comparator vaccine. These findings support extrapolation of efficacy from the comparator vaccine to the SIIPL quadrivalent HPV vaccine in the younger population. The availability of the SIIPL quadrivalent HPV vaccine could help meet the global demand for HPV vaccines, and boost coverage for both girls and boys globally. FUNDING: Biotechnology Industry Research Assistance Council, Department of Biotechnology (DBT), Government of India, and Serum Institute of India.

A Study of the Impact and Restitutive Efforts of Cervical Cancer Screening during the COVID-19 Pandemic in a Regionsl Cancer Centre in Eastern India.

AIM: To evaluate the feasibility and efficacy of switching to a self-sampling based screening as compared to ongoing provider-collection based screening using HPV DNA test and assess the compliance of HPV positive women for further treatment during the COVID 19 pandemic. METHOD: The study participants were women aged 30-60 years from rural and semi-urban communities around Kolkata, who underwent screening followed by HPV testing by Hybrid Capture II test. In the pre pandemic era, the women who attended the health centres where trained health workers that collected cervical samples. Following lockdown, the health workers distributed the self-sampling device to the women during home visits and counselled them to collect their samples by themselves. Thereafter the self collected screened positive women were brought to the hospital for further treatment instead of community clinics. RESULTS: From April 2018 to March 2020, 12,718 women underwent screening using either HPV DNA test or visual inspection with acetic acid. HPV samples were either provider collected (62.7%) or self-collected (37.2%). The HPV positivity and CIN2+ detection rate were 5.4% and 2%. From April 2020 to February 2022, 10,792 women underwent screening using self-sampling only. The HPV positivity rate and CIN2+ detection rate were found to be 5.1% and 1.9 % . CONCLUSION: Cervical cancer screening by HPV self-sampling advocates participation of more women especially in rural areas, while accelerating progress towards elimination of cervical cancer.

Role of MLH1 and MSH2 deficiency in the development of tumorigenesis and chemo-tolerance of cervical Carcinoma: Clinical implications.

Cervical cancer (CACX) is one of the top causes of cancer death in women globally. The involvement of several cellular pathways in carcinogenesis is still poorly understood. Here, we focused to evaluate the contributory role of Mismatch Repair (MMR) pathway genes-MLH1 and MSH2 in CACX and their association with chemo-tolerance of the disease. For this purpose, molecular profiles (expression/promoter methylation/deletion) of the genes were analysed in both normal cervical epithelium and tumour tissue, also validated in in-silico dataset as well. Later on, prognostic importance of the genes was identified through analysis of their methylation/expression status in plasma DNA of circulating tumour cells (CTCs) and cisplatin-tolerant CACX cell lines respectively. It was found that the expression profile of MLH1 and MSH2 genes was considerably reduced from undifferentiated basal-parabasal layers of normal cervical epithelium towards progression of the disease. Further analysis showed that frequent deletion [34-48%] and promoter methylation events [28-46%] of the genes were the plausible reasons for their reduced expression during tumorigenesis. Incidentally, the prevalence of MLH1 [32%] and MSH2 [27%] promoter methylation found in CTCs of plasma of the clinically advanced CACX patients implicated their prognostic importance of the disease. In addition, the patients having high alterations of those genes resulted in poor patient outcomes even after the therapy. In in-depth analysis of this result in cisplatin-tolerant CACX cell lines, we discovered that increased promoter methylation frequency of those genes at higher concentrations of cisplatin and gradual accumulation of the cells in the G2/M phase of the cell cycle were the rational causes for their reduced expression and MMR deficiency in the system. Hence, it is possible to conclude that the gradual down-regulation of MLH1 and MSH2 proteins may be a key event for MMR pathway inactivation in CACX. This might also be associated with chemo-tolerance and overall poor survival among the patients.

Prevalence of novel gamma HPV types 223 and 225 in oral cavity and skin of Indian normal and neoplastic participants.

Gamma-papillomaviruses, though traditionally classified as cutaneotropic, actual tissue tropism is largely unexplored. This study aimed to evaluate the tissue-specific prevalence of two novel-HPV 223 and 225 in samples of oral mucosa and keratinized epithelium of varied skin parts from 226 female and male subjects, with or without neoplastic/dysplastic lesions in oral cavity or cervix. The gamma-human papillomavirus (gamma-HPV) 223 and 225 DNA presences were determined by polymerase chain reaction (PCR) ursing the HPV type-specific primers and confirmed by Sanger sequencing. Viral load in the HPV 223 and HPV 225 positive samples were determined by absolute real-time quantification method. Alpha-HPV DNA prevalence was also checked in oral mucosa to ascertain coinfection status. Novel HPV 223 was present in 4.4% (10/226) oral mucosal samples of the study population; interestingly all were females with no prevalence in their corresponding skin swab samples. Whereas, the prevalence of HPV 225 was found both in the skin and oral mucosa of 28.2% (N = 37/131) female and 17.9% (N = 17/95) male participants. Alongside, HPV 223 viral load was found to be significantly higher (p = 0.02 < 0.05) in the oral mucosa of diseased participants, whereas, HPV 225 viral load was higher in the oral mucosa of normal participants. Our results suggest that gamma-HPV 223 has its prevalence only in the oral mucosal epithelium, whereas, HPV 225 has its prevalence on both mucosal and keratinized skin epithelium, indicating its dual tropism nature.

Differential association of hedgehog pathway in development of cervical carcinoma and its chemo-tolerance.

Cervical carcinoma (CACX) is still a dreadful threat to women in developing countries. Available conventional chemo-radiation therapies are not sufficient to restrict the disease recurrence. To unravel the mechanism of the disease recurrence, alteration of hedgehog self-renewal pathway was evaluated during development of CACX and in chemo-tolerance of the tumor. We have analyzed the alterations (expression/methylation/deletion) of some key regulatory genes (HHIP/SUFU/SHH/ SMO/GLI1) of hedgehog self-renewal pathway in cervical lesions at different clinical stages and compared with different datasets, followed by their clinico-pathological correlations. The changes in expression/methylation of the genes were then evaluated in two CACX cell lines (SiHa/HeLa) after treatment with chemotherapeutic drug cisplatin at different concentrations. Down regulation (mRNA/protein) of the antagonists HHIP and SUFU due to promoter methylation and/or deletion along with upregulation (protein) of agonists SHH, SMO and GLI1 was seen in early invasive lesions and subsequent clinical stages. Reduced protein expression of HHIP and SUFU showed significant association with high/intermediate expression of agonists SHH, SMO, GLI1 in the tumors and also poor prognosis of the patients. It was evident that cisplatin could restrict the growth of HeLa and SiHa cells through significant upregulation of antagonists HHIP and SUFU due to their promoter hypomethylation and down regulation of SHH in a concentration dependent manner without any significant changes in expression of SMO and GLI1, leading to the tumor cells in a dormant state. Thus, interplay of the agonists and antagonists has important role in activation of hedgehog pathway during development of CACX, whereas inactivation of the pathway due to upregulation of the antagonists is an important phenomenon in chemo-tolerance of the tumor. This suggests importance of epigenetic modification in chemo-resistance of CACX.

High nuclear expression of DNMT1 in correlation with inactivation of TET1 portray worst prognosis among the cervical carcinoma patients: clinical implications.

In this study, we aimed to understand the interplay of the epigenetic modifier genes DNMT1 and TET1 along with HPV infection in the cervical epithelium and how it changes during tumorigenesis. For this purpose, initially the bioinformatical analysis (methylation and expression profile) of DNMT1 and TET1 was analyzed in the TCGA dataset. Next genetic (deletion) and epigenetic profiling (promoter methylation) of DNMT1 and TET1 were done in our sample pool and also validated in CACX cell lines as well. The results were further correlated with different clinicopathological parameters. Our data revealed that HPV infection in basal/parabasal layers of cervical epithelium actually disrupts the epigenetic homeostasis of DNMT1 and TET1 proteins which ultimately leads to the high expression of DNMT1 along with further reduction in TET1 protein during the development of carcinoma. Further, in-depth look into the results revealed that comparatively low methylation frequency of DNMT1 coupled with high promoter methylation and deletion frequency [22-46%] of TET1 were the plausible reasons of their antagonistic expression profile during the progression of the disease. Interestingly, the prevalence of DNMT1 [9.1%] and TET1 promoter methylation [22.7%] found in both the plasma DNA of the respective CACX patients implicated its diagnostic importance in this study. Lastly, molecular alteration of TET1 alone or in combination with DNMT1 showed the worst overall survival among the patients. Hence, it may be concluded that an inverse molecular profile of DNMT1 and TET1 genes seen in the proliferative basal-parabasal layers of the cervical epithelium was aggravated during the development of CACX along with genetic and epigenetic changes due to HPV infection.

Screening technologies for cervical cancer: Overview.

Ever since the introduction of the Papanicolaou (PAP) smear test was published in 1941 in American Journal of Obstetrics and Gynecology, PAP test linked with definitive treatment has prevented millions of women from cervical cancer in the developed countries. Due to limited availability of resources, a lack of infrastructure and difficulty in getting highly trained professionals, widespread implementation of PAP test dependent cervical cancer screening program has not been established in low and middle income countries such as India. Therefore, after availability of non-cytological tests such as visual inspection on acetic acid (VIA) and human papillomavirus (HPV) DNA test, there is a paradigm shift in cervical cancer screening methods. In past two decades, various research work has convincingly established the utility of VIA and HPV test in developing countries. The evidences were evaluated by the World Health Organization (WHO) and recommendations have been recently published for comprehensive cervical cancer control strategies for the low and middle income countries. For any successful screening program, achieving high coverage (>70%) of the target population rather than frequent screening is the most important determinant. It is also equally important to ensure appropriate investigations of the screen positive women to establish the disease and treatment of the screen detected cases of cervical intra epithelial neoplasia (CIN) and cancer. HPV testing is the WHO recommended test for cervical cancer screening especially in view of widespread HPV vaccination in young population leading to lower prevalence of CIN and other HPV related diseases.

Experience of Human Papillomavirus Vaccination Project in a Community Set Up-An Indian Study.

BACKGROUND: Initial introduction of HPV vaccination from 2006 to 2008 was largely confined to high-income countries (HIC), such as Australia, the United States, and Europe, where cervical cancer incidence is lowest. Much of the post-introduction literature has come from HICs, with a focus on coverage levels achieved, provider acceptability and early impact of vaccination on disease endpoints. However, there are a few literature evaluating the mechanics of delivery, feasibility of the health system and acceptability from low and middle income countries (LMICs). The primary objective was to evaluate the feasibility, acceptability and safety of two dose HPV vaccination in adolescent girls between 9-14 years. METHODS: After an orientation camp followed by filling up of prevaccine questionnaires by parents on HPV related diseases and its vaccines and informed consent, girls between9-14years were vaccinated. They were asked to report any side effects in the next 24 hours after each dose. Parents were contacted on Day 7 and Day 30 to enquire about any side effects . Total 3 visits were required i.e two for the vaccination and one visit at 7th month post completion of second dose. To estimate the acceptability, successful completion of two doses by at least 80% of the girls were measured. For measurement of acceptability, either of the parents were recalled along with their daughter at 7th month and were asked to fill up a pre-set questionnaire. RESULTS: After institutional ethical clearance, 555 girls were recruited in the study from rural parts of West Bengal, India between July, 2017 to November, 2017. Out of which, 544 girls (98%) received their 2nd dose between January, 2018 and May, 2018 without any serious adverse effects. No serious adverse effect was reported on follow up till December, 2019. CONCLUSION: The introduction of HPV vaccination is feasible in large scale and the vaccine is well accepted and safe.
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A Prospective Randomized Trial to Compare Safety, Acceptability and Efficacy of Thermal Ablation and Cryotherapy in a Screen and Treat Setting.

BACKGROUND: The prospective randomized study aimed to compare the safety, acceptability and efficacy of thermal ablation (TA) to that of cryotherapy in screen and treat setting. METHODS: The participants were recruited prospectively in a community-based screening clinic in India. Women positive on visual inspection with acetic acid (VIA) test and/or Human Papillomavirus (HPV) test were assessed for eligibility for ablative treatment. Total 286 eligible women were randomized to receive either cryotherapy (N=150) or TA (N=136) performed by health workers. Colposcopy and cervical biopsy were performed on all, prior to treatment. Post-treatment follow-up was after one year with colposcopy and biopsy. RESULTS: Both the treatment methods had high acceptability. Significantly higher proportion of women treated by cryotherapy reported pain compared to women treated by TA, though intensity was mild in vast majority of them. Approximately 30% of women in both arms had histologic abnormalities, mainly CIN 1, and among those who attended follow-up 74.1% and 81.0% didn't have any CIN after cryotherapy and TA respectively. CONCLUSION: TA is as acceptable and safe as cryotherapy in screen and treat setting. TA has the logistic advantages for the low-resourced settings as the machines are more portable, do not require costly refrigerant gas and battery-driven models are available. The cure rates for CIN 1+ lesions in our study were comparable between cryotherapy and TA.

Cancer screening and early diagnosis in low and middle income countries : Current situation and future perspectives.

Limited health system capacities and competing health priorities in low and middle income countries (LMICs) necessitate a pragmatic approach to population-based cancer screening. Thus, the challenges faced by LMICs to implement a 'western' model of screening for common cancers and the possible means to overcome these challenges are presented. Breast cancer is the number one cancer with a rising trend in the majority of LMICs. Implementation of mass-scale mammography-based screening is not feasible and sustainable in most of them. While some LMICs have introduced breast cancer screening based on clinical breast examination (CBE), the programs need to be of appropriate quality. All LMICs should improve the capacity for early diagnosis of breast cancer along with other common cancers through community education, training of frontline health workers, facilitating prompt referrals and improving the infrastructure for cancer diagnosis and treatment. Resources permitting, the LMICs with high burden of cervical cancer may consider human papillomavirus (HPV) detection-based screening; a simple low-cost alternative is visual inspection with acetic acid (VIA). Regardless of the choice, a strong linkage should be established between screening and treatment with implementation of robust quality assurance. The few LMICs with a rising trend of colorectal cancers and adequate resources may implement demonstration projects to screen with fecal immunochemical tests (FIT). Oral cancer screening of habitual tobacco and/or alcohol users using oral visual examination (OVE) may be implemented in countries with high burden of the cancer, but primary prevention (i.e., tobacco/alcohol cessation) should be prioritized. Screenings for other cancers are not recommended for LMICs.

Colposcopy telemedicine: live versus static swede score and accuracy in detecting CIN2+, a cross-sectional pilot study.

BACKGROUND: This cross-sectional pilot study evaluates diagnostic accuracy of live colposcopy versus static image Swede-score evaluation for detecting significant precancerous cervical lesions greater than, or equal to grade 2 severity (CIN2+). METHODS: VIA or HrHPV positive women were examined using a mobile colposcope, in a rural clinic in Kolkata, India. Live versus static Swede-score colposcopy assessments were made independently. All assessments were by gynecologists, junior or expert. Static image assessors were blinded to live scoring, patient information and final histopathology result. Primary outcome was the ability to detect CIN2+ lesions verified by directed biopsies. Diagnostic accuracy was calculated for live versus static Swede-score in detecting CIN2+ lesions, as well as for interclass correlation. RESULTS: 495 images from 94 VIA positive women were evaluated in this study. Thirteen women (13.9%) had CIN2+ on biopsy. No significant difference was found in the detection of CIN2+ lesions between live and static assessors (area under curve = 0.69 versus 0.71, p = 0.63). A Swede-score of 4+, had a sensitivity of 76.9% (95% CI 46.2-95.0%) and 84.6% (95% CI 54.6-98.1%), for live- and static-image assessment respectively. The corresponding positive predictive values were found to be 90.9% (95% CI 75.7-98.1%) and 92.6% (95% CI 75.7-99.1%). The interclass correlation was good (kappa statistic = 0.60) for the senior static assessors. CONCLUSIONS: Swede-score evaluation of static colposcopy images was found to reliably detect CIN2+ lesions in this study. Larger studies are needed to further develop the colposcopy telemedicine concept which may offer reliable guidance in management where direct specialist input is not available. TRIAL REGISTRATION: Ethical approval of the study was obtained by the Chittaranjan National Cancer Institute (CNCI) Human Research Ethics Committee (4.311/27/2014). The trial was retrospectively registered in the Clinical Trails Registry of India CTRI/2018/03/012470 .

Deregulation of LIMD1-VHL-HIF-1α-VEGF pathway is associated with different stages of cervical cancer.

To understand the mechanism of cellular stress in basal-parabasal layers of normal cervical epithelium and during different stages of cervical carcinoma, we analyzed the alterations (expression/methylation/copy number variation/mutation) of HIF-1α and its associated genes LIMD1, VHL and VEGF in disease-free normal cervix (n = 9), adjacent normal cervix of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 32), cancer of uterine cervix (CACX; n = 174) samples and two CACX cell lines. In basal-parabasal layers of normal cervical epithelium, LIMD1 showed high protein expression, while low protein expression of VHL was concordant with high expression of HIF-1α and VEGF irrespective of HPV-16 (human papillomavirus 16) infection. This was in concordance with the low promoter methylation of LIMD1 and high in VHL in the basal-parabasal layers of normal cervix. LIMD1 expression was significantly reduced while VHL expression was unchanged during different stages of cervical carcinoma. This was in concordance with their frequent methylation during different stages of this tumor. In different stages of cervical carcinoma, the expression pattern of HIF-1α and VEGF was high as seen in basal-parabasal layers and inversely correlated with the expression of LIMD1 and VHL. This was validated by demethylation experiments using 5-aza-2'-deoxycytidine in CACX cell lines. Additional deletion of LIMD1 and VHL in CIN/CACX provided an additional growth advantage during cervical carcinogenesis through reduced expression of genes and associated with poor prognosis of patients. Our data showed that overexpression of HIF-1α and its target gene VEGF in the basal-parabasal layers of normal cervix was due to frequent inactivation of VHL by its promoter methylation. This profile was maintained during different stages of cervical carcinoma with additional methylation/deletion of VHL and LIMD1.

Activation of Wnt-ß-catenin pathway in basal-parabasal layers of normal cervical epithelium comparable during development of uterine cervical carcinoma.

In this study, importance of Wnt-ß-catenin pathway in the development of uterine cervical carcinoma was evaluated. For this purpose, the profiles (expression/methylation/deletion) of ß-catenin, p-ß-catenin (Y654), Wnt3a, and APC were studied in disease free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of primary tumors (n = 70), CIN (n = 28), CACX (n = 102) samples, and two CACX cell lines (HeLa and SiHa). Immunohistochemical analysis revealed high/medium (74-95%) expression of ß-catenin/p-ß-catenin (Y654) and Wnt3a and low expression (23-26%) of APC in proliferating basal-parabasal layers contrary to differentiated spinous layer in normal cervix irrespective of HPV16 infection. The expression profile of the genes in the basal-parabasal layers did not change significantly during development of CACX. High (66%) promoter methylation of APC was seen in basal-parabasal layers and the cervical lesions (42-69%), unlike in spinous layers (25%). The promoter methylation status of APC was validated by in vitro demethylation experiments using 5-aza-dC in CACX cell lines. However, additional deletion of APC was significantly increased from CIN (12%) to stage I/II (40%) and became comparable in stage III/IV (48%) of the tumor. Patients with alterations (deletion/methylation) of APC and high/medium expression of Wnt3a/ß-catenin/p-ß-catenin (Y654) showed significantly poor survival. Thus our data indicate that cumulative effect of Wnt3a overexpression and APC inactivation are needed for overexpression of ß-catenin during the development of CACX.

Identification of Changes in the Human Papilloma Virus 16 (HPV16) Genome During Early Dissemination of Cervical Cancer Cells May Complement Histological Diagnosis of Lymph Node Metastasis.

Cancer of the uterine cervix (CACX) is one of the most common carcinoma affecting women worldwide. During treatment, histologically lymph node (LN) metastasis and presence of HPV DNA in blood plasma act as a major prognostic factor. Despite the lack of apparent LN involvement, some early-invasive CACX patients have shown recurrences and poor survival. This is suggestive of undetected early dissemination of cancer cells characterized by presence of HPV DNA in histologically non-metastatic LNs which finally progresses into histologically visible metastasis. This present study investigated the status and origin of HPV genome during early dissemination by molecular analysis in primary tumor (PT), histologically non-metastatic pelvic lymph nodes (LNs) and blood plasma (BP) of same patient. First, CACX patients showing signs of early dissemination was identified by detection of HPV in PT (n = 22) and their corresponding histologically non-metastatic pelvic LNs (n = 45) and BP (n = 18) followed by typing of HPV16/18. This was followed by comparative analysis of the physical, copy number and methylation (enhancer/early/late) status of HPV16 genome present in LNs and BP with that of PT. Our study revealed for the first time that the HPV16 genome were frequently present in the integrated form though the copy number was low in both non-metastatic LNs and BP. However, the methylation pattern of PT was discordant with that of corresponding LNs and BP in majority of the cases. Critical assessment of HPV16 profiles established that the presence of hrHPV may be due to the early dissemination of PT cells having significant pathological implications.

Risk of high-grade precancerous lesions and invasive cancers in high-risk HPV-positive women with normal cervix or CIN 1 at baseline-A population-based cohort study.

Infection with high-risk human papillomavirus (HR-HPV) is transient and clears on its own in majority of the women. Only a few women who have persistent infection may finally develop cervical intraepithelial neoplasia (CIN) or cervical cancer in later years. The risk of progression in the HR-HPV-positive women with normal cervix or low-grade lesion on colposcopy and histopathology at baseline is less studied. We performed a longitudinal study on 650 HR-HPV-positive women with colposcopy and/or histopathology-proved normal or CIN1 diagnosis at baseline to assess the cumulative risk of development of high-grade CIN. After a mean follow-up of 2.1 person years of observation (PYO) (range 0.1-5.1), the cumulative incidence of CIN2+ (6.4%; 3.0/100 PYO) was significantly higher in women who had persistent HR-HPV infection compared to those who cleared the infection (adjusted HR 6.28; 95% CI 2.87-13.73). The risk of viral persistence in women aged 50-60 years was two times higher compared to women aged 40-49 years and three times higher compared to women aged 30-39 years. The probability of having persistent infection increased progressively with higher viral load at baseline (adjusted HR 3.29, 95% CI 2.21-4.90 for RLU ≥100; adjusted HR 2.69, 95% CI 1.71-4.22 for RLU 10-100). Women with increasing viral load at follow-up had four times higher risk of developing CIN2 or worse lesions as compared to those with decreasing load (20.9% vs 4.8%; p < 0.001). In the context of developing countries where cytology or genotyping triaging is not feasible, colposcopy referral of HR-HPV-positive women with advancing age, viral persistence, and increasing viral load may be considered.

Association between high risk human papillomavirus infection and co-infection with Candida spp. and Trichomonas vaginalis in women with cervical premalignant and malignant lesions.

BACKGROUND: Human papillomavirus (HPV) is the necessary cause of cervical cancer. Cervico-vaginal infection with pathogens like Chlamydia is a likely cofactor. The interactions between HPV, Trichomonas vaginalis (TV) and Candida spp. are less understood, though inflammation induced by these pathogens has been demonstrated to facilitate oncogenesis. OBJECTIVE: Our study aimed to evaluate the association between Candida spp. and TV co-infection with HPV in cervical oncogenesis. STUDY DESIGN: Women with normal cervix who were high-risk HPV-negative (N=104) and HPV-positive (N=105); women with CIN 1 (N=106) and CIN 2/CIN 3 (N=62) were recruited from a community based cervical cancer screening program. Cervical cancer patients (N=106) were recruited from a tertiary care oncology clinic. High-risk HPV was detected by Hybrid Capture II technique; Candida spp. and TV were detected by culturing the high vaginal swabs followed by microscopic examination in all. The disease status was established by histopathology in all the women. RESULT: HPV-positive women had significantly higher risk of having precursor lesions (of any grade) and cancer compared to HPV-negative women. Candida spp. or TV infection did not alter the risk of low grade or high grade lesions among HPV- positive women. HPV positive women co-infected with TV had higher risk of cervical cancer but not those co-infected with Candida spp. CONCLUSION: The higher risk of cancer observed in the women co-infected with HPV and TV without any enhanced risk of CIN 3 suggests secondary infection of the malignant growth by TV rather than any causal role. Co-infection with Candida spp. and/or TV infection did not increase the carcinogenic effect of HPV on cervix.

Evaluation of a compact, rechargeable, magnifying device to triage VIA and HPV positive women in a cervical cancer screening program in rural India.

PURPOSE: Many limited-resourced countries have either introduced cervical cancer screening programs or are contemplating to do so using visual inspection after acetic acid application (VIA) or human papillomavirus (HPV) detection tests. Both tests have high false-positivity and a suitable triaging strategy is required. Colposcopy triaging is not practicable in most resource-limited settings due to several reasons. We evaluated a portable, battery-operated, magnifying device (GynocularTM) to triage screen positive women in community setting in India. METHODS: Women positive on VIA or oncogenic HPV test were examined with Gynocular by clinicians in primary health clinics. Findings were documented using the International Federation for Cervical Pathology and Colposcopy (IFCPC) terminology. Swede score was also calculated. Biopsy was performed irrespective of Gynocular findings. The accuracy of Gynocular to detect high-grade lesions or cancer (HSIL+) was estimated. The suitability of Gynocular to correctly triage screen positive cases for immediate ablative treatment was also evaluated by creating simulated scenarios. RESULTS: Sensitivity and specificity of Gynocular were 96.4 and 47.1 %, respectively, to detect HSIL + at the threshold of IFCPC grade 1 findings. Increasing threshold to grade 2 changed sensitivity and specificity to 92.9 and 94.1 %, respectively. Optimum combination of sensitivity and specificity as determined by the receiver operating curve analysis was at the cut-off Swede score of 5. Triaging of VIA/HPV positive women to treatment using grade 2 criteria would have resulted in modest overtreatment and missing of very few high-grade lesions. CONCLUSION: Gynocular can be used as an effective triaging device for VIA/HPV positive women.

Association of P16-RBSP3 inactivation with phosphorylated RB1 overexpression in basal-parabasal layers of normal cervix unchanged during CACX development.

To understand the molecular mechanism of RB1 phosphorylation in basal-parabasal layers of normal cervix and during cervical cancer (CACX) development, we analyzed the alterations (expression/methylation/deletion/mutation) of RB1/phosphorylated RB1 (p-RB1) (ser807/811 and ser567) and two RB1 phosphorylation inhibitors, P16 and RBSP3, in disease-free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 28), CACX (n = 102) samples and two CACX cell lines. Immunohistochemical analysis revealed high/medium expression of RB1/p-RB1 (ser807/811 and ser567) and low expression of P16 and RBSP3 in proliferating basal-parabasal layers of majority of normal cervical epitheliums, irrespective of HPV16 infection. Interestingly, 35-52% samples showed high/medium expression of P16 in basal-parabasal layers of normal and had significant association with deleterious non-synonimous SNPs of P16. Methylation of P16 and RBSP3 in basal-parabasal layers of normal cervix (32 and 62%, respectively) showed concordance with their respective expressions in basal-parabasal layers. The methylation frequency of P16 and RBSP3 in basal-parabasal layers of normal did not change significantly in CIN and CACX. The deletion frequency of P16 and RB1 increased significantly with CACX progression. While, deletion of RBSP3 was high in CIN and comparable during CACX progression. P16 showed scattered and infrequent mutation in CACX. The alteration of P16 and RBSP3 was synergistic and showed association with overexpression of p-RB1 in tumors and associated with poor prognosis of patients. Thus, our data suggest that overexpression of p-RB1 in basal-parabasal layers of normal cervical epithelium was due to methylation/low functional-linked non-synonimous SNPs of P16 and RBSP3. This pattern was maintained during cervical carcinogenesis by additional deletion/mutation.