p16INK4a-expression in relation to E6/E7-mRNA-expression of human

papillomaviruses in tonsillar squamous cell carcinoma (TSCC)

Anna Sophie IhloffJournal Club

Kopf- und NeurozentrumKlinik für Hals-, Nasen-, OhrenheilkundeDirektor Prof. Dr. med. R. Knecht

HPV prevalence in head and neck (HN) SCC all anatomical sites 20 % - 30 % tonsillar carcinoma 50 % - 60 %

Hoffmann et al., Acta Otolarygol (Stckh) 1998Gillison et al., J Natl Cancer Inst. 2000 Klussmann et al., Cancer 2001Hoffmann et al., Cancer Lett 2005

Basics

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 1998Gillison et al., Semin Oncol 2004 Klussmann et al., Am J Pathol 2003Ritchie et al., Int J Cancer 2003 Hoffmann et al., Cancer Lett 2005

Basics

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 2001Hafkamp et al., Int J Cancer 2003Braakhuis et al., J Natl Cancer Inst. 2004Begum et al., Clin Cancer Res 2005

Basics

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

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

Results

MY09/11-PCR Gelspur DNA Result

1 SmartLadder2 SiHa (250 ng) HPV163 SiHa (2.5 ng)4 SiHa (25 pg)5 Nr. 110 HPV166 Nr. 194 HPV7 Nr. 201 HPV168 Nr. 274 HPV169 Nr. 275 negative10 Nr. 295 HPV?11 Nr. 339 HPV?12 Nr. 362 HPV1613 Nr. 401 HPV1614 Nr. 417 negative15 H2O16 SmartLadder

Results

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

Results

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

Weak expression

Strong expression

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

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

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