1. p16INK4a-expression in relation to E6/E7-mRNA- expression of human papillomaviruses in tonsillar squamous cell carcinoma (TSCC) Anna Sophie Ihloff Journal Club Kopf- und Neurozentrum Klinik für Hals-, Nasen-, Ohrenheilkunde Direktor Prof. Dr. med. R. Knecht

2. HPV prevalence in head and neck (HN) SCC  all anatomical sites 20 % - 30 %  tonsillar carcinoma50 % - 60 % Hoffmann et al., Acta Otolarygol (Stckh) 1998 Gillison et al., J Natl Cancer Inst. 2000 Klussmann et al., Cancer 2001 Hoffmann et al., Cancer Lett 2005 Basics

3. Patients with HPV positive tumours present with  more aggressive biological behaviour  better survival rates in comparison to HPV negative cases Andl et al., Cancer Research 1998 Gillison et al., Semin Oncol 2004 Klussmann et al., Am J Pathol 2003 Ritchie et al., Int J Cancer 2003 Hoffmann et al., Cancer Lett 2005 Basics

4. HPV infection leads to  inactivation of p53 by E6 => no p53-mutations  inactivation of pRb by E7 => reduced pRb-level => overexpression of p16  HPV positive SCC are considered to be a specific tumor entity in head and neck cancer Klussmann et al., Cancer 2001 Hafkamp et al., Int J Cancer 2003 Braakhuis et al., J Natl Cancer Inst. 2004 Begum et al., Clin Cancer Res 2005 Basics

5.  39 patients with TSCC  8 female & 31 male / 44-85 (62,1) years  30 palatine tonsil / 9 lingual tonsil Material and Methods  HPV-DNA-detection PCR MY09/11 and GP5+/6+ with Luminex Southern Blot Hybridization (SBH)  HPV-E6/E7-mRNA-detection RT-PCR with SBH Northern Blot Hybridization  p16-Immunohistochemistry paraffinembedded antibody MTS-1, BIOLOGO Klaes et al., Int J Cancer 2001

6. HPV-DNA  21/39 HPV-positive TSCC (53,8 %)  20/21 infected with HPV16  01/21 infected with HPV35 Results

7. MY09/11-PCR GelspurDNAResult 1SmartLadder 2SiHa (250 ng)HPV16 3SiHa (2.5 ng) 4SiHa (25 pg) 5Nr. 110HPV16 6Nr. 194HPV 7Nr. 201HPV16 8Nr. 274HPV16 9Nr. 275negative 10Nr. 295HPV? 11Nr. 339HPV? 12Nr. 362HPV16 13Nr. 401HPV16 14Nr. 417negative 15H 2 O 16SmartLadder   Results

8. HPV-E6/E7-mRNA  11/21 with strong expression of E6/E7-mRNA (52%) Results

9. p16-immunohistochemistry  12/39 with p16-overexpression  12/21 HPV-positive TSCC with p16-overexpression  9/21 HPV-positive TSCC without p16-overexpression 6/9 without E6/E7-mRNA-expression 3/9 with E6/E7-mRNA-expression-signals only after hybridization Results

10. Weak expression

11. Strong expression

12. p16 expression in correlation to HPV DNA and E6/E7 mRNA expression  elevated p16 expression could be assigned to HPV DNA positive tumours  correlation of p16 overexpression and HPV- positivity proofed to be statistically highly significant (p=0.0018)  10 HPV DNA positive cases with p16 overexpression expressed E6/E7 mRNA in the tumour tissue Results

13.  The predominant majority of HPV DNA positive TSCC show an E6/E7-mRNA-expression.  The detection of p16-overexpression correlates with the pattern of E6/E7-mRNA-expression, yet not with the HPV DNA status. Summary

14.  HPV DNA presence is correlated with viral mRNA expression and subsequent enhancement of p16 levels reflecting biological activity of the viruses in the infected tissue  p16-overexpression predicts translational active HPV- infection in head and neck cancers, thus giving rise to the opportunity to identify active HPV infections in HNSCC on a practicable and routine way applying p16 immuno- histochemistry Conclusion