1. Toward a molecular intra-operative diagnosis of SLN invasion R Garrel 1, V Burcia 1, J Solassol 2, V Costes 3, E Barbotte 4, D DeVerbizier 5, C Cartier 1, M Makeieff 1, L Crampette 1, N Boulle 2, T Maudelonde 5 B Guerrier 1. Montpellier ’s Teaching Hospitals FRANCE 1 ENT, 2 Molecular Biology, 3 Pathology, 4 Biostatistics, 5 Nuclear Medicine

2. Sentinel Lymph Node technique : sampling method n Lymph node Mapping –Lymphoscintigraphy n Lymph node Analysis n Serial sections + Immunohistochemistry n Lymph node Biopsy

3. Goals of SLN technique 1.staging improvement: n Full histopathological analysis 2. select the right treatment for the right patient n to limit neck dissections to pN+ cases only : ≈ 30% patients

4. Limitation n The lack of accuracy of the Intraoperative diagnosis – “the Missing Link” n Sensitivity of routine Frozen section – 20 % sensitivity * –In best hands with serial sections : 90 %** n but –loss of materials –Not applicable in routine  second surgery *Burcia et al. Otolaryngol Head and Neck Surgery 2010 **Tschopp et al. Otolaryngol Head and Neck Surgery 2005

5. The Role of molecular diagnosis ? n Advantages of molecular diagnosis for SLN staging Q-PCR –Not operator dependent –Automated → quicken → intraoperative use Questions are : #1 “Is the molecular staging as accurate as the gold standard ? ”. #2 “Which is the best Q-PCR marker ?”

6. Act 1 : Comparison between Q- PCR and Histopathology.................................................... Garrel et al. Clinical Cancer Research 2006............................................

7. Matched analysis SN section Snap frozen sections: 5µm/250µm IHC Q-PCR Cyto imprint n 3 cytokeratins : CK 5, 14 et 17

8. Q-PCR values / Size of SLN metastasis Garrel et al. Clinical Cancer Research 2006

9. = cutoff Patient Staging (18 patients -71 SLN) SN-SN+ Garrel et al. Clinical Cancer Research 2006

10. Key points n Results: –Q-PCR staging was as accurate as IHC: n positive neck vs negative neck: 100% se / sp n Background noise –No detection of minute micromestasis < 450 µm n in two patients but this inaccuracy was negated by the presence of larger metastases in another SLN being Q-PCR positive in the same two patients n Discriminatory power of Q-PCR is depending on the marker

11. Act 2 : The quest for the best Q-PCR marker

12. Literature: Q-PCR markers –Screening of 40 potential markers using primary tumor and gross metastatic deposits compared with benign nodes –Ferris Cancer Res et al. 2005.

13. Second run of Q-PCR (23 patients-85 SLN) n Comparison of the three markers –Pemphigus Vulgaris Ag / SCCAg / CK 17 n Q-PCR with absolute quantification (plasmide)

14. Patients neck Q-PCR staging SLN – (n=17) SLN + (n=6) 0 1 10 100 1000 10000 100000 1000000 4 6 13 PVA Cutoff value 562 SLN – (n=17) SLN + (n=6) 0 1 10 100 1000 10000 100000 1000000 12 1 4 6 SCCA Cutoff value 48 SLN – (n=17) SLN + (n=6) 0 1 10 100 1000 10000 100000 1000000 1 13 3 6 CK 17 Cutoff value 562 Solassol and Garrel BJC 2010

15. ROC curves analysis CK17/ SCCA/ PVA → Pemphigus Vulgaris Ag +++ 100% area under curve 0,000.250.500.751.00 0.00 0.25 0.50 0.75 1.00 PVA (AUC=100) SSC (AUC=97.9)) CK T17 (AUC=93.8) Sensitivity 1-Specificity Solassol and Garrel BJC 2010

16. Conclusion: PVA as the best Q-PCR marker n Larger cohorts to validate PVA for Q-PCR diagnosis n Evaluate the inter-laboratory variations of accuracy “path the way toward the intraoperative molecular staging of sentinel lymph nodes in head and neck squamous cell carcinomas” MicrometastasisMacrometastasis

17. Future study assessment of automated technique –Q-PCR –one-step nucleic acid amplification (OSNA) OSNA CK19 → PVA SN in Head and Neck as the gold standard ?

18. …Thank you