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 BLOTTING HYBRIDIZATION DDS. TUAN NGUYEN 1
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, 4/100. 000 male Ø Prevalent cancer, high mortality 3, 6/100. 000 female 7, 8/100. 000 3
Multiple reasons HPV infection 4
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 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 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, 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 - 800 C Brush 10 -20 times 9
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
RESULTS & DISCUSSION 12
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, 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 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 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 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 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-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 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 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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