Synopsis Presentation 13/06/2016

1. Synopsis Presentation13/06/2016

2. Detection and Genotyping of Human papillomavirus and Its Role in Cervical Cancer in Sexually Active Women Attending Tertiary Care Hospital Varsha Saxena PhD Scholar, 156/Jan 2015 Dept. of Microbiology Yenepoya Medical College

3. Guide: Dr. Vidya Pai Professor and Head Dept. of Microbiology Yenepoya Medical College Mangalore. Co- guide: Dr. Rajagopal K Professor and Head Dept. of OBG Yenepoya Medical College Mangalore.

4. Contents: • Introduction • Review of Literature • Social Relevance • Aim and Objectives • Material and Methods • Work Plan • Statistical Analysis • Time Plan • Budget • References

5. Abbreviations: • HPV – Human papillomavirus • HR – High Risk • LR – Low Risk • DNA – Deoxyribonucleic acid • CIN – Cervical Intraepithelial Neoplasia • WHO – World Health Organization • Pap – Papanicolaou Staining

6. Introduction: • Human papillomavirus (HPV) is one of the most common sexually transmitted pathogen. (Woodman et al., 2007) • It is estimated that around 75% of sexually active women are infected by this virus. • Over 100 different types of HPVs are identified. • Classified in two groups depending on their tissue tropism (Woodman et al., 2007,Hausen HZ., 2002) i.e. • those which are causing genital infections and • those which are causing cutaneous infections.

7. Contd.. • Non-enveloped, icosahedral particles, 55 nm in diameter in size with double-stranded DNA genome.(Chung SH et al., 2010) • Along with women, this virus is even contracted by men and they also do have the ability to transmit it. • Unlike women in men there is no confirmed lab test available such as women do have Pap smear tests.(Dunne EF et al., 2006)

8. Contd.. • Viral persistence for a prolonged period of time ultimately leads to cervical cancer.(Ho Gloria YF et al., 2004) Sometimes it stays inside the body for years. • The different subtypes of HPV with distinguished variations in its genetic and oncogenic potential are known. • The subtypes which specifically affect the anogenital tract are: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 66, 69 etc.(Khan S et al., 2007)

9. Contd.. • These subtypes are further classified as high-risk (HR) and low-risk (LR) types and possess a remarkable ability to cause malignancy and play a pivotal role in development of various tumors.(Bernard HU et al., 2010) • HPV type 6 and 11 - low risk genital types while type 16 and 18 contribute towards malignancy and fall under the high risk genital types.(Porras C et al., 2010)

10. Contd.. • In the acquisition of HPV infection sexual behavior plays a important role.(Panatto D et al., 2012) • Other factors include : impaired immune response, persistence of virus, smoking, extensive use of oral contraceptives and administration of steroid hormones. (Stanely M., 2006) • HPV encodes eight genes, among which two early HPV genes E6 and E7 are known to play crucial role in tumor formation.(Hariri S et al., 2011)

11. Contd.. • E6 binds with p53 and prevents its translocation and thereby inhibit the ability of p53 to activate or repress target genes. • E7 binds to hypophosphorylated Rb and thereby induces cells to enter into premature S-phase by disrupting Rb-E2F complexes.(Shaikh F et al., 2012) • The continued expression of E6/ E7 genes is required for maintenance of the cancerous state.(Park S et al., 2016)

12. Contd.. • HPV screening is very important and the careHPV test has clear advantages in certain settings and is worthy of further study. • Its simplicity and cost-effectiveness make it worth promoting as a cervical cancer screening method for improved healthcare in areas with few public health resources. (Ying H et al., 2014)

13. Review of literature • Cervical cancer is one of the most prevalent cancer in women and one of the main cause of death, especially in young women.(Yasmeen A et al., 2010) • Persistent HR-HPV infections are associated with CIN that are graded into CIN1, 2 and 3, depending upon the severity of the lesion.(Schmeink CE etal., 2011)

14. Contd.. • Other than persistent HPV infection:(Bell MC et al., 2011) • younger age • lower socioeconomic status • earlier sexual debut • multiple sexual partners • contraception • other sexually transmitted infections • multiple childbirths • Smoking • Immunosuppression and • poor nutrition.

15. Contd.. • Non compliance with cervical cancer screening and diagnostic programs is one of the major hurdles faced by the clinicians.(Chase DM et al., 2012) • In 2004, cervical cancer was the 5th most common cause of cancer death among women in the world, with 489,000 new cases reported and 268,000 deaths.(Parkin DM et al., 2005)

16. Contd.. • Faridi et al.,(24) in their study have mentioned that, in the coming year cervical cancer is going to be one of the most common cause of death especially in young women. • In western countries this ratio is significantly high however it varies throughout the world.(Faridi et al., 2011) • According to WHO statistics, every year approx. 500,000 new cases are being registered out of which 250,000 are fatal.(Faridi et al., 2011)

17. Contd.. • The most recent reports from USA showed that the women are more prone to this infection than the men. • The overall reported percentage of getting infectionin women irrespective of races was quite high being 17.9% while the chances of getting infection in men being comparably low as 8%.(Faridi et al., 2011)

18. Contd.. • About 80% of the women live in developing countries that do not have adequate cervical cancer screening or treatment programs. • In these countries, only a minority of women receive effective screening and treatment for cervical cancer. • The HC2 assay can detect 13 types of high-risk HPV associated with cervical cancer: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68.(Ying H et al., 2014)

19. Contd.. • However, the Hybrid Capture 2(HC2) assay is expensive and requires laboratory equipment, so it is not commonly available in developing countries. (Zhao FH et al., 2013) • The careHPV test is a powerful, rapid, cheaper and precise HPV-DNA detection method in which antibodies bind to magnetic beads, rapidly capturing specific target HPV nucleic acid sequences, which are then detected using a chemiluminescence signal.

20. Contd.. • This method can detect 14 types of high-risk HPV(16,18,31,33,35,39,45,51,52,56, 58, 59, 66, and 68) and more than 80 specimens can be processed in 2.5 hours. This allows screening and follow- up to be completed in one day.(Zhao FH et al., 2013)

21. Indian scenario: • Cancer of the cervix constitutes about 15-51% of all female cancers and rates of incidence ranges from 17.2 to 55 per 100,000 women in different regions of India. • Infection of HPV 16/18 is estimated to account for more than 80% of invasive cervical cancer including CIN3 and for 50% of CIN2 lesions.(Franceschi S et al.,2003)

22. Contd.. • In India, HPV type 16 alone in cervical cancer is 70-90 per cent while occurrence of HPV type 18 varies from 3 to 20 percent.(Bhatla N et al., 2006) • Other high risk HPV types such as HPV 45, 33, 35, 52, 58, 59, and 73 have also been reported are rare and constitute only a minor group.(Peedicayil et al., 2006)

23. Contd.. • Interestingly, the peak of HPV infection, particularly HPV 16, appears to reach at later stage in third decade of sexual life at 26-35 years in Indian women in contrast to 18-25 years reported in western countries.(Schiffman M et al.,2005, Das BC et al., 2008) • In a community based study (Sasidharanpillai Set al., 2015) rural women from Karnataka, India are totally unaware of HPV infection and its health effects.

24. Social Relevance: • As HPV is one of the most common causes of cervical cancer in sexually active women and because of the less awareness regarding this infection and malignancy, the mortality rate is increasing. • Subjects participating in this study will be directed for treatment if positive for infection at an early stage. • So with this study we will be able to create awareness in our society.

25. Aim: This study is aimed to evaluate the feasibility of a probe-based assay to detect HPV at an early stage from cervical specimens suspected to have HPV infection and may progress to malignancy.

26. Objectives: • To determine genital HPV occurrence in sexually active women. • To evaluate the type specific viral DNA (High Risk- 16, 18 & Low Risk- 6, 11). • To detect the E6 & E7 oncoproteins, responsible for the malignancy in 90% of the cases. • Detection of cervical intra epithelial neoplasia. • To compare the Pap smear findings, careHPV test kit finding and PCR findings. • To determine the risk factors associated with genital HPV and its frequency of occurrence in sexually active women.

27. Material & Method: • Research Design: Prospective study • Study Site: Cervical brush samples will be collected from Dept. of OBG, Yenepoya hospital, Mangalore. • Study duration: 36 months.

28. Sample size: 155 (Symptomatic Patients) • Ethical clearance: Will be obtained from the Yenepoya University, Mangalore. • Informed consent: Will be taken in study. • Statistical assessment: Will be done with the help of SPSS windows software.

29. Inclusion Criteria All married, non-pregnant women attending gynecology OPD for any reason, who are ready to consent will be included in the study. • Exclusion Criteria Unmarried and pregnant women will be excluded from the study.

30. Sample Collection: • Two exfoliative cervical specimens will be obtained during a single pelvic examination.(Parada et al., 2010) • The 1stcervical specimen will be used to make a conventional Pap smear and 2nd cervical specimen will be stored in viral transport medium. • All genital samples will be collected by a trained doctor. • The samples will be stored at -20°C in the laboratory prior to DNA extraction.

31. Pap smear study: Cervical brush samples will be subjected to Pap staining and the results will be recorded according to the standard protocol.

32. Molecular Diagnosis: • DNA extraction: (Franceschi S et al., 2003) DNA extraction will be done from the entire specimen using the Qiagen DNA extraction kit. Divide samples in two identical aliquots (1 ml) and centrifuged at 14,000 rpm for 10 min Suspend pellets in 1 ml phosphate buffered saline, centrifuge (14,000 rpm for 10 min) Store one pellet at -20°C to account for potential problems in genotyping

33. Overnight incubate specimen at 37°C in the presence of Proteinase K (1.25 mg/ml) Extract DNA and purify according to the recommendations of the supplier • Two blank samples per plate (water without cervical cells) will be processed as negative control to assess possible contamination.

34. 2. Multiplex PCR: (Siddiqa A et al., 2014) • The reaction will be performed under different condition: 0.2mM of each primer will be used. • The following time temperature profile will be chosen: • One initial cycle of denaturation for 5 min at 94◦C • 45 cycles of 30 sec at 94◦C, 30 sec at 60 ◦C, • 30 sec of extension at 72◦C.

35. Contd.. • After the thermal cycles, the amplicons will be detected with the help of PCR machine. • The following oligonucleotide sequences will be used as primers for the detection of HPV (16, 18, 6 & 11) and oncoproteins E6 & E7.

37. 3. careHPV test: (Ying H et al., 2014) Supplied lysate will be added to the specimens to dissolve the cells. Exposed ds- HPV DNA will be denatured to become ss- DNA by heating the lysate ss- DNA then hybridizes with the full-length complementary RNA to form HPV DNA/RNA hybrids. Add Magnetic beads coated with monoclonal antibody for HPV DNA/ RNA hybrid mixtures. Add alkaline phosphatase to combine with the monoclonal antibody and a chromogenic substrate will be acted upon by the alkaline phosphatase.

38. Contd.. Light intensity generated by the reaction of the chromogenic substrate will reflects the amount of HPV DNA contained in the specimen. The ratio of relative light unit (RLU) to the mean RLU of the minimum positive control (RLU/CO) will be used for diagnosis. NOTE: The standard definition for a sample to be positive for HPV DNA is a reading > 0.5 pg/ml in one specimen. RLU/CO = 1.0 is used as a cut-off point.

39. Work Plan: Patient Selection Clinical history of visible genital warts or any abnormality in genitalia(155 patients)  Assessment of the possible risk factors associated with the infection  Sample collection – Two cervical brush samples Pap smear testing Storage at -70°C Screening by careHPV test kit

40. Positive Negative DNA extraction Multiplex PCR for – E6/E7 Oncoproteins & HPV genotyping (16, 18, 6 & 11) Compare the results of Pap smear testing, careHPV test kit and PCR for the presence or absence of infection.

44. Statistical Analysis • Data will be analyzed with the help of SPSS windows software. • Descriptive statistics like frequency (%) distribution will be used. • Chi square test will be used to find the association between two categorical variables. • Logistic regression will be used to determine the risk factors associated with cervical cancer caused by HPV. • P value ˂ 0.05 will be considered significant.

45. Timeline:

46. Budget:

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48. Khan S, Jaffer NN, Khan MN, Rai MA, Shafiq M, et al. Human papillomavirus subtype 16 is common in Pakistani women with cervical carcinoma. Int J Infect Dis. 2007; 11: 313-317. • Bernard HU, Burk RD, Chen Z, Doorslaer KV, Hausen HZ and de Villiers EM. Classification of Papillomaviruses (PVs) Based on 189 PV Types and Proposal of Taxonomic Amendments. Virology. 2010; 401 (1): 70–79. • Porras C, Bennett C , Safaeian M , Coseo S , Rodríguez AC et al., Determinants of Seropositivity among HPV16/18DNA positive young women. BMC Infect Dis. 2010; 10: 238. • Panatto D, Amicizia D , Trucchi C , Casabona F , Luigi La P et al., Sexual behaviour and risk factors for the acquisition of human papillomavirus infections in young people in Italy: suggestions for future vaccination policies. BMC Public Health. 2012; 12: 623-631. • Stanely M. Immune responses to human papillomavirus: Vaccine 2006; 24 (l1): 10-16. • Hariri S, Dunne E, Saraiya M, Unger E and Markowitz L. Human Papillomavirus: Chapter-5. VPD Surveillance Manual. 5th Edition; 2011.

49. Shaikh F, Sanehi P and Rawal R. Molecular screening of compounds to the predicted Protein-Protein Interaction site of Rb1-E7 with p53- E6 in HPV. Bio-information. 2012; 8(13): 607-612. • Park S, Park JW, Pitot HC and Lambert PF. Loss of Dependence on Continued Expression of the Human Papillomavirus 16 E7 Oncogene in Cervical Cancers and Precancerous Lesions Arising in Fanconi Anemia Pathway-Deficient Mice. mBio. 2016; 7 (3). • Ying H, Jing F, Fanghui Z, Youlin Q and Yali H. High-risk HPV nucleic acid detection kit– the careHPV test –a new detection method for screening. Scientific Reports. 2014; 4: 4704, DOI: 10.1038/srep04704. • Yasmeen A, Alachkar A, Dekhil H, Gambacorti-Passerini C and Al Moustafa AE. Locking Src/AblTyrosineKinase activities regulate cell Differentiation and Invasion of Human cervical cancer cells expressing E6/E7 oncoproteins of High-risk HPV. Journal of Oncology. 2010. • Gomez DT, Santos JL. Human Papillomavirus infection and cervical cancer: Pathogenesis and Epidemiology. Communicating Current Research and Educational Topics and Trends in Applied Microbiology A. Méndez-Vilas (Ed.)

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