1. Clairette RAHARISOLO VOLOLONANTENAINA1 ,
Genotypes identification of human papillomaviruses in paraffin-embedded cervical cancer samples
Clairette RAHARISOLO VOLOLONANTENAINA1 ,
Richter RAZAFINDRATSIMANDRESY2, Maherisoa RATSITORAHINA3, Jean-Michel HERAUD 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

2. 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Oncology service of CHU/HJRA Antananarivo: recorded 470 new cases of CC/year, 2nd after breast cancer (unpublished report 2011),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

3. 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,

4. 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.

5. 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%)

6. 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 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%).

7. 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

8. 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.

9. 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: ]
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)

10. 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.

11. 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): ) 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

12. 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.

13. 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

14. Thank you Clairette Raharisolo Vololonantenaina
Head of Department Pathology
Institut Pasteur de Madagascar
Contact:

15. 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)

16. 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

17. 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

18. 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

19. 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)