Clairette RAHARISOLO VOLOLONANTENAINA1 , Genotypes identification of human papillomaviruses in paraffin-embedded cervical cancer samples Clairette RAHARISOLO VOLOLONANTENAINA1 , Richter RAZAFINDRATSIMANDRESY2, Maherisoa RATSITORAHINA3, Jean-Michel HERAUD2 1 Department Pathology, Institut Pasteur de Madagascar, Antananarivo, Madagascar 2 Virology Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar 3 Epidemiology Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar 2nd Edition UNESCO-Merck Africa Research Summit 28 and 29 november 2016 Addis Ababa

Introduction (1) Situation in the world Cervical cancer (CC): 2nd most frequent cancer in woman worldwide Over 80% of new cases diagnosed in developing countries WHO Globocan 2012: 528, 000 CC cases worldwide reported developing regions: 445,000 CC cases, 2nd position after breast cancer developed regions: 83,000 CC cases, 11th position 266,000 deaths Situation in Madagascar: Madagascar: no official data on cancers Oncology service of CHU/HJRA Antananarivo: recorded 470 new cases of CC/year, 2nd after breast cancer (unpublished report 2011), Department Pathology of Institut Pasteur de Madagascar: 400 new cases of CC recorded during the last 10 years, 2nd after breast cancer , the majority was diagnosed on advanced stage

Introduction (2) Human papilloma virus (HPV) is the most common sexually transmitted virus Among the 200 HPV genotypes described, only 40 are related to the anogenital mucosa [Munoz et al., 2003], with two categories of HPV types: Low-risk HPV (LR-HPV: 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81) are associated with genital warts. HPV6 and 11 are the most frequent types found in genital warts. High-risk HPV (HR-HPV: 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 73 and 82) are associated with low and high-grade cervical intraepithelial lesions HPV 16 & 18 are responsible of 80% CC,

Introduction (3) Low-grade squamous intraepithelial lesion (LSIL): 59% to 82% HPV positive High-grade squamous intraepithelial lesion: 80% HPV positive Persistent infection with High-Risk-HPV types has been recognized as a factor for CC development Prophylactic HPV vaccines have thus been developed to protect against HPV infection Madagascar: HPV vaccination is still not available to all the population; a demonstration vaccination program was provided by GAVI The Vaccine Alliance and implemented by the Ministry of Health in two pilot districts in 2013.

Human papillomavirus and cervical neoplasia among female sex workers in Madagascar. (Smith JS1 et al. Int J Gynecol Cancer 2010) Studied population: 90 female sex workers (FSWs), mean age: 32,6y,o (ranged 18 to 58 years) from Antananarivo, Madagascar in 2004 Samples: exfoliated cervical cell specimen in standard transport media HPV prevalence: 36.7% HPV-52 (11.1%), HPV-31 (5.6%), HPV 39 (5.6%), and HPV-16 (3.3%). and HPV 83 (3.3%). Prevalence and associated Factors to Human Papillomaviruses Types 16 and 18 among Malagasy Women in Fianarantsoa, Madagascar (Rakotozafy JCR et al, 2013 Studied population: 1712 women consenting, median age: 36,5 y,o (ranged 18–60 years); study was conducted in a cohort of women from three rural area of Fianarantsoa, Madagascar HPV-16 positive: (1.3%); HPV-18. positive: (0.9%)

Human papillomavirus prevalence and type-specific distribution of high- and low-risk genotypes among Malagasy women living in urban and rural areas. (Catarino R et al. Cancer Epidemiol. 2016) Studied population: 1075 women, mean age: 44 (ranged 30-65 years) Samples: self-colleting vaginal sample Prevalence of all 19 types HR-HPV infection: 39.3% The most common HR- HPV types were HPV-53 (6.2%) and HPV-68 (5.8%), followed by HPV-52 (5.2%), HPV-35 (4.5%), HPV-73 (3.4%), HPV-31 (3.4%), HPV-16 (3.1%), and HPV-18 (3.1%).

Current study Retrospective study on ICC cases diagnosed and archived in our laboratory from biopsies fixed to formaldehyde and included in paraffin according to the epidemiological profile of Malagasy women Objectives: To identify of the different genotypes of High-risk - HPV (HR-HPV) responsible for cervical cancer To have a reliable database that will be serve as a basis for establishing a large-scale study To determine the vaccine impact on the Malagasy female population

Study design Samples collection 2010 – 2012 90 cases of Invasive Cervical Cancer (ICC) histological diagnosed and archived in our laboratory DNA Extraction DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer’s instructions, preceding a high-heat treatment INNO-LiPA assay genotyping : designed for the identification of 28 HPV genotypes by detection of specific sequences in the L1 region of the HPV genome: Amplification of HPV using INNO-LiPA HPV genotyping Extra Amp kit (Innogenetics, Belgium) Statistical analysis: EpiInfo 7. p value less than 0.05 (p-value< 0.05) was considered as statistically significant.

Single infection HPV: 39% (32/82 infection HR-HPV positive) Results 90 cases invasive Cervical Cancer Prevalence Infection HPV: 91.1% (82/90 cases) Median age for HR-HPV infected participants: 55 years old [range: 46 - 63] 12 types of High Risk-HPV, of which types 16 (48.78%), 52 (35.37%), 18 (28.05%) , 39 (24.39%), 33 (25.61%) are the most frequent, Furthermore, genotypes -16 and/or -18 were detected in 73.83% (63/82) of HPV positive specimens Single infection HPV: 39% (32/82 infection HR-HPV positive) Multiple HPV infections: 60.98%% (50/82)

Results: Distribution of HPV genotypes detected in different types of ICC using INNO-LiPA test Squamous Cells Carcinoma (SCC) : 72 positive HPV cases Multiple infection: 61% (44/72 positive cases) Single infection: 39% (28/72 positive cases) HPV 16 is responsible for a single infection in more than half of the cases of SCC (57.14%), followed by HPV 52. Adenocarcinoma: 8 positive HPV cases. Multiple infection: 6 cases Single infection: 2 cases (HPV 16) Botryoid Rhabomyosarcoma: 1 case (HPV 16) HPV 18 was still co-infected with HPV16 or other HR-HPV, never observed in single infections. Low Risk -HPV54 was identified in twenty five % (25%) of tested samples, whose 90% were associated with HPV52.

Discussion (1) In our study, 82 (91.1%) of ICC were positive for HPV by genotyping assay. HPV16 was predominant followed by HPV52, HPV18, HPV39 and HPV-33. Our data showed a similar results, regarding the prevalence of HPV associated to ICC (91.1%), as previously described and HPV16. Indeed, Ennaifer et al. in 2015 (Asian Pac J Cancer Prev. 2015; 16(15):6769-72.) described 100% of HR-HPV positive amongst 89 Tunisian women with ICC cases Vidal et al. in 2011 (Vidal et al, Infectious Agents and Cancer, 2011, 6:20): indentified 89% positive HPV among Tanzanian women with ICC cases

Discussion (2) Multiple infection accounts for more than half of the cases is mainly dominated by HPV16 infection, which has been detected in both multiple and single infections followed by HPV 52, HPV 18, and HPV39. Unlike the three studies carried out in Madagascar and cited above, the prevalence of HR-HPV is clearly very high in our study compared to their finding, HPV 16 is responsible for a single infection in more than half of the cases of squamous cells carcinoma followed by HPV 52. HPV 18 was still co-infected with HPV16 or other High Risk-HPV, it was never observed in single infections HR-HPV 52 was the most detected in our study before HPV 18.

Conclusion In conclusion, understanding the epidemiology of multiple concurrent HPV infections and their impact on the natural history of cervical disease is essential for planning effective cervical cancer screening and HPV vaccination strategies. Except the limited sample size, our study demonstrated an extraordinarily high diversity and prevalence of multiple concurrent HPV infections in the invasive carcinomas of cervix from Malagasy women, with the predominance of the vaccine genotypes component

Thank you Clairette Raharisolo Vololonantenaina Head of Department Pathology Institut Pasteur de Madagascar Contact: +261 20 22 412 72 E-mail: claire@pasteur.mg

Distribution of HPV genotypes detected in different types of ICC using INNO-LiPA test DIAGNOSTIC N % Squamous cells carcinoma (SCC) 72 87.80 Adenocarcinoma (ADK) 8 9.76 Microinvasive carcinoma in situ (Micro.CIS) 1 1.22 Botryoid rhabdomyosarcoma (Rhabdo.) Total 82 100.00 Infection HPV types according to the cervical cancer diagnosed using INNO LIPa test DIAGNOSTIC INFECTION SCC N = 72 Pos (%) ADK N = 8 MICRO.CIS N = 1 RHABDO. TOTAL N = 82 Multiple 44 (61.11) 6(75.00) 50 (60.98) Single 32 (39.02) 2 (25.00) 1 (100.00)

Distribution of HPV genotypes detected in different types of ICC using INNO-LiPa test SCC N = 72 Pos (%) ADK N = 8 Micro- CIS N = 1 Rhabdo. Total N = 82 HR-HPV16 35 (48.61) 3 (37.50) 1 (100.00) 40 (48.78) HR-HPV18 21 (29.17) 2 (25.00) 23 (28.05) HR-HPV26 3 (4.17) HR-HPV33 18 (25.00) 21 (25.61) HR-HPV35 4 (5.56) 4 (4,88) HR-HPV39 1 (12.50) 22 (26.83) HR-HPV45 7 (9.72) 7 (8,54) HR-HPV51 1 (1.39) 1 (1,22) HR-HPV52 24 (33.33) 5 (62.50) 29 (35.37) HR-HPV56 HR-HPV66 3 (3,66) HR-HPV68 LR-HPV54 17 (23.61) 20 (24.39) LR-HPV74 LR-HPV6

HPV Genotypes detected amongst multiple HPV infections using INNO-LiPa test (1) MULTIPLE INFECTION GENOTYPES ADK N= 6 (%) SCC N=44 Total N=50 HPV16, HPV18, HPV39 3 (6.82) 3 (6) HPV16, HPV18, HPV39, HPV33, HPV52, HPV54 2 (4.55) 2 (4.00) HPV16, HPV18, HPV39, HPV52 1 (2.27) 1 (2.00) HPV16, HPV18, HPV33, HPV52, HPV54 HPV16, HPV26, HPV33 1 (2,27) HPV16, HPV33, HPV52, HPV54 4 (9.09) 4 (8.00) HPV16, HPV35 HPV16, HPV39 1 (16.67) HPV16, HPV45 HPV16, HPV45, HPV33, HPV52, HPV54 HPV16, HPV51

HPV Genotypes detected amongst multiple HPV infections using INNO-LiPa test (2)  MULTIPLE INFECTION  GENOTYPES ADK SCC Total HPV18, HPV39 9 (20.45) 9 (18.00) HPV18, HPV39, HPV33, HPV52, HPV54 1 (2.27) 1 (2.00) HPV18, HPV33 HPV18, HPV52 2 (33.33) 3 (6.00) HPV18, HPV54 HPV18, HPV56, HPV74 HPV26, HPV35 HPV33, HPV52, HPV54 3 (50.00) 4 (9.09) 7 (14.00) HPV33, HPV52, HPV54, HPV45 HPV33, HPV52, HPV54, HPV68, HPV39 HPV33, HPV45 HPV39, HPV52 HPV45, HPV54 HPV6, HPV45 HPV68, HPV39 TOTAL 6 (100) 44 (100) 50 (100) Co-infection with presence of HPV52+HPV54: 20 cases P<0.0000

HPV Genotypes detected amongst single HPV infections using INNO-LiPA test SINGLE INFECTION GENOTYPES ADK N=2 (%) MICRO.CIS N=1 RHABDO SCC N=28 Total N=32 HPV16 2 (100) 1 (100) 16 (57.14) 20 (62.50) HPV26 1 (3.57) 1 (3.13) HPV35 2 (7,14) 2 6,25) HPV39 1 (3,57) 1 (3,13) HPV45 HPV52 6 (21,43) 6 (18,75) HPV66 TOTAL 2 1 28 (100) 32 (100)