Ranajit Mandal

Accuracy of human papillomavirus testing in primary screening of cervical neoplasia: Results from a multicenter study in India

The knowledge that cervical neoplasia are caused by human papillomavirus (HPV) infection has led to the evaluation of its role in screening. We evaluated the accuracy of HPV testing by Hybrid capture II (HC II) method in detecting cervical intraepithelial neoplasia grade 2 and 3 (CIN 2 and 3) lesions in 4 cross‐sectional studies with common protocol and questionnaire in 3 different locations (Kolkata, Mumbai and Trivandrum) in India. These studies involved 18,085 women aged 25–65 years. The reference standard for final diagnosis was a combination of colposcopy/biopsy. All women were investigated with colposcopy and 3,116 received directed biopsy. The sensitivity of HPV testing for detecting CIN 2–3 lesions varied from 45.7% to 80.9% across the study sites; the specificity varied from 91.7% to 94.6% and the positive predictive value from 6.7% to 13.7%. Retesting of 298 randomly chosen denatured samples in France revealed an agreement rate of 85.9% and a κ‐value of 0.72. Although HPV testing seems to be a promising approach for cervical cancer prevention, a large range in sensitivity was observed in our study, possibly due to variations in the quality of specimen collection and reference standards. A higher sensitivity was associated with the center performing the test well. Further developments in terms of more reproducible, less expensive and less sophisticated testing are essential to make the test feasible and effective in low‐resource settings.

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.

Evaluation of downstaging in the detection of cervical neoplasia in Kolkata, India

Unaided visual inspection or “downstaging” has been suggested as a potential alternative method for cervical cancer screening in developing countries. Our study was designed to evaluate the accuracy of downstaging to detect cervical neoplasia in a low‐resource setting. A total of 6,399 women aged 30–64 years were screened with downstaging by trained nonmedical health workers. Two thresholds were used to define positive downstaging: “low threshold” when any visible abnormality on the cervix was considered positive and “high threshold” when selected abnormalities such as bleeding on touch, bleeding erosion, hypertrophied oedematous cervix, congested stippled cervix and growth or ulcer constituted the positive test. All women underwent a colposcopy examination. Biopsies were directed when colposcopy revealed abnormal lesions. True disease status was defined as histologically proven moderate dysplasia and worse lesions. Since all the participants received a diagnostic (reference) investigation (biopsy and/or colposcopy), sensitivity, specificity and predictive values were estimated directly. Low‐ and high‐threshold downstaging were positive in 1,585 (24.8%) and 460 (7.2%) women, respectively. The sensitivities of low‐ and high‐threshold downstaging to detect high‐grade precursors and invasive cancers were 48.9% and 31.9%, respectively. The specificities were 75.8% and 93.3%, respectively. These results indicate that downstaging is not suitable as an independent primary screening modality for cervical neoplasia.

Prevalence of Human Papillomavirus and Co-Existent Sexually Transmitted Infections among Female Sex Workers, Men having Sex with Men and Injectable Drug abusers from Eastern India

BACKGROUND Human papillomavirus (HPV) is a very common sexually transmitted disease affecting both men and women and is responsible for different ano-genital cancers in either sex. Co-existing sexually transmitted infections (STI) including HIV have been considered as important co-factors for carcinogenesis induced by HPV. The purpose of this study was to determine the prevalence of any HPV, HPV 16 and HPV 18 and also concomitant STIs among female sex workers (FSW), men having sex with men (MSM) and injectable drug users (IDU). MATERIAL AND METHOD This cross-sectional study was conducted among 45 FSWs, 26 MSMs and 58 IDUs who attended the STI or de-addiction clinics. Genital scrape samples collected from glans penis and coronal sulcus in males and cervical squamo-columnar junction in females were tested for HPV DNA by PCR using HPV L1 consensus primer. Type specific PCR to detect HPV 16 and 18 was done on the samples positive on consensus PCR. All participants were tested for associated STIs including HIV and hepatitis B and cervical cytology was done on all females. RESULTS Among the FSWs, HPV was detected in 73.3% and HPV 16 and 18 was detected in 25.7%. Though the HPV prevalence was similarly high among MSMs (69.2%) and IDUs (72.4%), the prevalence of HPV 16 and 18 was much lower in these groups compared to the FSWs. Prevalence of cervico-vaginal infection with Trichomonas vaginalis and syphilis was significantly higher in the HPV positive women compared to the HPV negative women. There was no statistically significant difference in the prevalence of other STIs among HPV positive and negative women and men. CONCLUSION HPV infection is highly prevalent among FSW, MSM and IDUs. Trichomonas vaginalis infection is more frequent in HPV positive women.

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.

Phase 2 Randomized Controlled Trial of Radiation Therapy Plus Concurrent Interferon-Alpha and Retinoic Acid Versus Cisplatin for Stage III Cervical Carcinoma

PURPOSE Because a combination of retinoic acid, interferon-alpha, and radiation therapy demonstrated synergistic action and effectiveness to treat advanced cervical cancers in earlier studies, we designed this randomized phase 2 open-label trial to assess efficacy and safety of interferon alpha-2b (IFN) and 13-cis-retinoic acid (RA) administered concomitantly with radiation therapy (IFN-RA-radiation) to treat stage III cervical cancer. METHODS AND MATERIALS Stage III cervical cancer patients were randomized to study and control groups in a 1:1 ratio. All patients were treated with radiation therapy; study arm patients received IFN (3 × 10(6) IU subcutaneously) 3 times a week for 4 weeks and daily RA (40 mg orally) for 30 days starting on day 1 of radiation, whereas control arm patients received weekly cisplatinum (40 mg/m(2)) for 5 weeks during radiation. Patients were followed for 3 years. The primary endpoint was overall survival at 3 years. RESULTS Patients in the study (n=104) and control (n=105) groups were comparable for clinicopathological characteristics, radiation therapy-related variables and treatment response. Proportions of disease-free patients in the study and control groups were 38.5% and 44.8%, respectively, after median follow-up of 29.2 months. Hazard ratios were 0.67 (95% confidence interval [CI]: 0.44-1.01) and 0.69 (95% CI: 0.44-1.06) for overall and disease-fee survival, respectively, comparing the study group to control, and demonstrated an inferior outcome with RA-IFN-radiation, although differences were statistically nonsignificant. Kaplan-Meier curves of disease-free and overall survival probabilities also showed inferior survival in the study group compared to those in the control. Acute toxicities of chemoradiation were significantly higher with 2 acute toxicity-related deaths. CONCLUSIONS Treatment with RA-IFN-radiation did not demonstrate survival advantage over chemoradiation despite being less toxic. The trends predicted an inferior outcome with the RA-IFN combination.

Is interferon-α and retinoic acid combination along with radiation superior to chemo-radiation in the treatment of advanced carcinoma of cervix?

Locally advanced cervical cancers comprise a large majority of the gynecologic cancers in India and other developing countries. Concurrent chemo-radiation has improved the survival of high risk stage I and stage II cervical cancers. There is no evidence that the same survival benefit has been achieved with chemo-radiation in stage III and stage IV disease. Interferon-alpha and Retinoic acid have synergistic anti-proliferative activity. In combination with radiation, they substantially enhance the sensitivity of the squamous carcinoma cells to radiation. Based on these observations from the in vitro studies, a few clinical trials have evaluated the combination of interferon-alpha and Retinoic acid, concomitant with radiation, to treat cervical cancers. The results from these early trials were encouraging and the combination had minimal toxicities. However, till date, no phase III randomized controlled trial has been done to evaluate this therapeutic modality.

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.

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 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.