DETERMINATION OF HPV DNA IN ORAL SQUAMOUS CELL

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DETERMINATION OF HPV DNA IN ORAL SQUAMOUS CELL CARCINOMA WITH BRUSH CYTOLOGY AND REVERSE

DETERMINATION OF HPV DNA IN ORAL SQUAMOUS CELL CARCINOMA WITH BRUSH CYTOLOGY AND REVERSE BLOTTING HYBRIDIZATION DDS. TUAN NGUYEN 1

CONTENT 1 INTRODUCTION 2 MATERIALS AND METHODS 3 RESULTS & DISCUSSION 4 CONCLUSION 2

CONTENT 1 INTRODUCTION 2 MATERIALS AND METHODS 3 RESULTS & DISCUSSION 4 CONCLUSION 2

Oral - Oropharyngeal cancer HCM city, 2007 - 2011: SQUAMOUS CELL CARCINOMA (OSCC): 12,

Oral - Oropharyngeal cancer HCM city, 2007 - 2011: SQUAMOUS CELL CARCINOMA (OSCC): 12, 4/100. 000 male Ø Prevalent cancer, high mortality 3, 6/100. 000 female 7, 8/100. 000 3

Multiple reasons HPV infection 4

Multiple reasons HPV infection 4

Ø Biopsy specimens: HPV infection: 37% of OSCC, 0% of normal oral mucosa (NOM)

Ø Biopsy specimens: HPV infection: 37% of OSCC, 0% of normal oral mucosa (NOM) (Cuc Tran, 2012) Ø 1999: Cytology in dentistry. Ø WHO: Reverse blotting hybridization 5

OBJECTIVES 1. To compare the HPV prevalence and its types in OSCC and in

OBJECTIVES 1. To compare the HPV prevalence and its types in OSCC and in normal oral mucosa (NOM) 2. To investigate the association between HPV infection and clinico-pathological features of OSCC. 6

DNA virus, Papillomaviridae Mechanism of carcinogenesis E 6 p 53 Apoptosis Cancer p. Rb

DNA virus, Papillomaviridae Mechanism of carcinogenesis E 6 p 53 Apoptosis Cancer p. Rb Proliferation E 7 http: //wiki. ggc. edu/images/9/93/HPV_genome. jpg 7

MATERIALS & METHODS OSCC: p = 37% (Cuc Tran, 2012) NOM: p = 6,

MATERIALS & METHODS OSCC: p = 37% (Cuc Tran, 2012) NOM: p = 6, 1% (Seifi, Iran 2013) OSCC: 122 cases • Diagnosis of squamous cell carcinoma • No specific therapy treatment Normal oral mucosa (NOM): 63 cases • Volunteer • Maching age (10 years)- sex • Normal mucosa, no antecedence of cancer 8

1 - Informed consent - Clinical examination 2 Cytology Interdental toothbrush Storage at -

1 - Informed consent - Clinical examination 2 Cytology Interdental toothbrush Storage at - 800 C Brush 10 -20 times 9

3 ØDNA extraction ØPCR HPV ØMolecular Hybrid DNA extraction Qiagen kit DNA quality (Nano.

3 ØDNA extraction ØPCR HPV ØMolecular Hybrid DNA extraction Qiagen kit DNA quality (Nano. Drop 1000) PCR-HLA: HLA-DQ PCR-HPV: MGP 5+/6+ RBH Electrophoresis 10

Reverse Blotting Hybridization (RBH) www. nature. com/nprot/journal/v 1/n 6/fig_tab/nprot. 2006. 404_F 1. html 11

Reverse Blotting Hybridization (RBH) www. nature. com/nprot/journal/v 1/n 6/fig_tab/nprot. 2006. 404_F 1. html 11

RESULTS & DISCUSSION 12

RESULTS & DISCUSSION 12

Group Age Male Female Total Average ± SD Cases (%) OSCC 55, 7 ±

Group Age Male Female Total Average ± SD Cases (%) OSCC 55, 7 ± 12, 5 87 (71, 3) 35 (28, 7) 122 (65, 9) NOM 54, 2 ± 13, 6 40 (63, 5) 23 (36, 5) 63 (34, 1) Mann-Whitney U : p > 0, 05 χ2 : p > 0, 05 13

Group OSCC NOM HPV + Cases (%) HPV Total Cases (%) p 29 (23,

Group OSCC NOM HPV + Cases (%) HPV Total Cases (%) p 29 (23, 8) 93 (76, 2) 122 (100) 0, 019 a 6 (9, 5) 57 (90, 5) 63 (100) OR (KTC 95%) 2, 609 b (1, 003 -6, 789) a: χ2 Test b: OR independently of exposure to potential habits 14

HPV rate compared with some studies Author Pintos (Canada 2008) Anaya-Saavedra, (Mexico 2008) This

HPV rate compared with some studies Author Pintos (Canada 2008) Anaya-Saavedra, (Mexico 2008) This study OSCC % NOM % 19, 4 4, 7 43, 5 17, 3 23, 8 9, 5 OR 3, 04 1, 0 -9, 3 6, 21 2, 98 -12, 97 2, 6 1, 00 -6, 79 15

HPV rate in OSCC Author Method case % Ritchie 2003 (USA) PCR-MY 09/11 10/94

HPV rate in OSCC Author Method case % Ritchie 2003 (USA) PCR-MY 09/11 10/94 10, 6 Rivero 2006 (Brazil) PCR-GP 5+/GP 6+ 0/40 0, 0 Pintos 2008 (Canada) PCR-PGMY 09/11 14/72 19, 4 Termine 2012(Italia) PCR-PGMY 09/11 21/83 25, 3 Gonzalez 2013(Mexico) PCR-MY 09/11 4/80 5, 0 Trần T. K. Cúc 2012(Việt Nam) Real-time PCR 40/109 36, 7 This study PCR-MGP 5+/6+ 29/122 23, 8 16

HPV rate in NOM Author Method Case % 16/169 9, 5 2/23 8, 7

HPV rate in NOM Author Method Case % 16/169 9, 5 2/23 8, 7 Lambropoulos 1997(Egypt) PCR-MY 09/MY 11 Furrer 2006(Argentina) PCR-GP 5+/6+ Anaya-Saavedra 2008(Mexico) PCR-MY 09/MY 11 43/248 17, 3 Goot-Heah 2012(Malaysia) PCR-PGMY 09/11 0/30 0 Gichki 2012 (Pakistan) Real-time PCR 47/192 24, 5 Seifi 2013 (Iran) PCR-MY 09/11 7/114 6, 1 Trần T. K. Cúc 2012 Real-time PCR 0/30 0 This study PCR-MGP 5+/6+ 6/63 9, 5 17

HPV type 18

HPV type 18

HPV type OSCC % NOM % -16 79, 3 50, 0 -35 0 16,

HPV type OSCC % NOM % -16 79, 3 50, 0 -35 0 16, 7 -51 0 16, 7 -53 10, 3 0 -58 6, 9 0 -43 3, 4 16, 7 -84 3, 4 0 HPV Type High risk Low risk Χ 2 Test : p > 0, 05 HPV NOM OSCC High risk -16, -35, -51, -53, -58 83, 3% 93, 1% Low risk -43, -84 16, 7% 6, 9% 19

HPV type NOM OSCC Zhang 2004(China) HPV-16: 68% Zhao, 2009(China) HPV-16: 64% Pattanshetty, 2014(India)

HPV type NOM OSCC Zhang 2004(China) HPV-16: 68% Zhao, 2009(China) HPV-16: 64% Pattanshetty, 2014(India) HPV-16: 72% St Guily, 2011 (France) HPV-16: 95% Cuc Tran, 2012 HPV 18: 48% HPV 16: 40% 20

CORRELATION BETWEEN HPV AND FEATURES OF OSCC HPV This study Age Sex Marriage habit

CORRELATION BETWEEN HPV AND FEATURES OF OSCC HPV This study Age Sex Marriage habit Occupation Site Grad lymph node Tumor size Clinical stage χ2 : p > 0, 05 Cruz , 1996 (Holland): ≤ 60 ys old Gonzalez, 2013 (Mexico): < 45 ys old p < 0, 05 Ali, 2009 (Pakistan): male > female St Guily, 2011 (France): male < female Soares, 2007 (Brazil) Reuschenbach, 2013 (Germany) No correlation 21

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 • HPV rate was 2 fold higher in case group, compared with control

• HPV rate was 2 fold higher in case group, compared with control group (23. 9% versus 9. 5%) • HPV infection were 2. 6 times more susceptible to OSCC • 7 HPV types including 5 high-risk HPV types: -16, -35, 51, -53, -58; and 2 low-risk types: - 43, -84 • Type 16 was the most common • Cytology: less invasive • RBH: fast, sensitive, accurate 23

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