1. Maria Chiara Zatelli Sezione di Endocrinologia Dip. di Scienze Biomediche e Terapie Avanzate Università di Ferrara Università di Ferrara Direttore: Prof. Ettore degli Uberti Analisi genetica del nodulo tiroideo: implicazioni per l’esecuzione del linfonodo sentinella Ferrara, 20 gennaio 2012

2. …molecular marker…? …a molecular marker (genetic marker) is a DNA sequence associated to a part of the genome, used in molecular biology and biotechnology experiments to identify a very highly specific DNA sequence… What relevance in thyroid nodules? EFE 2012 Thyroid molecular markers & sentinel lymphnode

3. Fine-needle aspiration biopsy (FNAB) is currently the most sensitive and specific tool for the presurgical differential diagnosis of thyroid malignancy but but has also substantial limitations Eszlinger et al. Mol Cell Endocrinol 2010;322:29 thyroid surgery 75% benign lesions 5% malignant lesions 20% follicular proliferation FAFTCfvPTC EFE 2012 Thyroid molecular markers & sentinel lymphnode

4. MOLECULAR BIOLOGY study of biology at molecular level pathology Does it improve cytological fine needle aspiration diagnosis of thyroid nodules? EFE 2012 Thyroid molecular markers & sentinel lymphnode

5. RNA DNA cytology somatic mutation analysis rearrangement studies BRAFN-RAS H-RAS K-RAS RET/PTC1 RET/PTC3 PPAR  /PAX8 Highly feasible in clinical settings EFE 2012 Thyroid molecular markers & sentinel lymphnode

6. PAPILLARY CARCINOMA DNA BRAF V600E point mutation [K601E and V599Ins] 45-80% of PTC, mainly tall cell and classic hystology 45-80% of PTC, mainly tall cell and classic hystology  extrathyroidal invasion  extrathyroidal invasion higher stage higher stage  recurrence (with reduced I up-take)  recurrence (with reduced I up-take)  de-differentiation  de-differentiation resticted to PTC Nikiforova et al. Exp Rev Mol Diagn 2008, 8: 83 Lupi et al. J Clin Endocrinol Metab. 2007;92:4085 EFE 2012 Thyroid molecular markers & sentinel lymphnode

7. do they improve diagnosis? do they improve diagnosis? genetic tests Kim et al. J Clin Endocrinol Metab, 2011, 96:658 BRAFV600E molecular test CytologyBRAF Cytology + BRAF Sensitivity10077.389.664.089.686.7 Specificity36.498.895.510095.598.8 PPV92.992.199.410099.492.9 NPV10095.952.593.752.597.5 Accuracy93.395.490.295.490.296.9 K value 0.51 ± 0.11 0.81 ± 0.02 0.63 ± 0.07 0.76 ± 0.05 0.63 ± 0.07 0.88 ± 0.01 Zatelli et al. Eur J Endocrinol 2009, 161:467 EFE 2012 Thyroid molecular markers & sentinel lymphnode

8. Kim et al. J Clin Endocrinol Metab, 2011, 96:658 do they improve diagnosis? do they improve diagnosis? genetic tests BRAFV600E molecular test BRAF mutation analysis may help especially in small nodules EFE 2012 Thyroid molecular markers & sentinel lymphnode

9. Section of Endocrinology University of Ferrara (1436 ♀ ; 420 ♂ ) age 52 ± 0,32 (12-73) 2421 FNAs Biomolecular analysis Pathology US & clinical evaluation 1856 pz suspected: 829 NON suspected: 1529 BRAFV600E molecular test EFE 2012 Thyroid molecular markers & sentinel lymphnode

10. cytologyS%NS% tot82934,2159265,7 Malignant rate 11513,91036,5 PTC/malignant11297,49895,1 BRAF + 647,7503,1 BRAF +/ Malignant64/11555,750/10348,5 FNAB per se detects a high percentage of malignant lesions in unsuspected nodules Martina Rossi et al. submitted All nodules should undergo FNAB EFE 2012 Thyroid molecular markers & sentinel lymphnode

11. BRAFV600E molecular test CytologyBRAF Cytology + BRAF SNSSNSSNS Sensitivity76,869,456,65192,984,7 Specificity99,799,910010099,799,9 PPV97,798,610010098,198,8 NPV96,59893,696,998,999 Accuracy96,698,194,19798,899 BRAF testing significantly increases FNAB sensitivity also in nodules clinically non suspected BRAFV600E molecular test increases diagnostic sensitivity for PTC 15 PTC patients “rescued” by BRAF analysis EFE 2012 Thyroid molecular markers & sentinel lymphnode Martina Rossi et al. submitted

12. BRAFV600E molecular test BRAFV600E molecular testS%NS% ACUS263,1784,9 PTC675,01037,0 BRAF + 519,200,0 FN354,2621,2 PTC525,0923,7 25,769,7 BRAF testing identifies as malignant 10% of FN Indication to total thyroidectomy EFE 2012 Thyroid molecular markers & sentinel lymphnode Martina Rossi et al. submitted

13. EFE 2012 Thyroid molecular markers & sentinel lymphnode Thyroid molecular markers increase diagnostic sensitivity of cytology for PTC and influence surgical approach CONCLUSION -1

14. any prognostic value? any prognostic value? genetic tests EFE 2012 Thyroid molecular markers & sentinel lymphnode

15. Xing et al. J Clin Oncol. 2009;27:2977-82 BRAFV600E molecular test EFE 2012 Thyroid molecular markers & sentinel lymphnode

16. BRAFV600E molecular test PTC persistence/recurrence prediction All PTC Conventional PTC sensitivity68%79% specificity66%60% PPV36%34% NPV88%92% Xing et al. J Clin Oncol 2009; 27:2977-82 BRAF mutation–positive patients are significantly more likely to have PTC persistence/recurrence EFE 2012 Thyroid molecular markers & sentinel lymphnode

17. significantly reduced disease-free probability in BRAF+ patients Xing et al. J Clin Oncol. 2009;27:2977-82 BRAF V600E genetic test EFE 2012 Thyroid molecular markers & sentinel lymphnode

18. significantly increased mortality in BRAF+ patients Elisei et al. J Clin Endocrinol Metab. 2008;93:3943 BRAF V600E genetic test EFE 2012 Thyroid molecular markers & sentinel lymphnode

19. EFE 2012 Thyroid molecular markers & sentinel lymphnode Thyroid molecular markers may predict patients outcome CONCLUSION -2

20. any surgical relevance ? any surgical relevance ? genetic tests EFE 2012 Thyroid molecular markers & sentinel lymphnode

21. EFE 2012 Thyroid molecular markers & sentinel lymphnode BRAF mutation 0 10 20 30 40 50 60 BRAF (+) 34 BRAF (-) 69 n. of PTC LN met (+)LN met (-) 20/26 BRAF+ LN  is a significant risk factor for locoregional lymph node metastasis Kim et al. Ann Surg 2006;244: 799  is not necessary for locoregional lymph node metastasis lymph node metastases may show de novo mutations Vasko et al. J Clin Endocrinol Metab 2005; 90: 5265 103 PTC !! also in microPTC !! !! also in microPTC !! Lin et al. Ann Surg Oncol 2010;17:3294

22. EFE 2012 Thyroid molecular markers & sentinel lymphnode extrathyroidal invasion lymph node metastasis local neck recurrence PTC recurrence complications  surgical strategy lobectomy total thyroidectomy lymph node dissection no dissection BRAF mutation High prognostic impact Xing Endocrine Reviews 2007; 28: 742

23. EFE 2012 Thyroid molecular markers & sentinel lymphnode BRAF mutation Indication for sentinel lymphnode Yip et al Surgery 2009; 146: 1215 PREDICTOR OF LYMPHNODE METASTASES

24. decision making BRAFV600E molecular test more aggressive surgery neck lymph node dissection less aggressive surgery + - BRAF mutation-based risk stratification PTC management Xing et al. J Clin Oncol. 2009;27:2977-82 EFE 2012 Thyroid molecular markers & sentinel lymphnode

25. EFE 2012 Thyroid molecular markers & sentinel lymphnode Positivity of thyroid molecular markers as indication for sentinel lymphnode procedure CONCLUSION -3

26. May address patients with persistent/recurrent disease to therapy with BRAF-specific inhibitors Cantwell-Dorris et al. Mol Cancer Ther 2011, 10: 385 BRAF V600E genetic test EFE 2012 Thyroid molecular markers & sentinel lymphnode

27. EFE 2012 Thyroid molecular markers & sentinel lymphnode CONCLUSIONS increase cytology diagnostic sensitivity for PTC increase cytology diagnostic sensitivity for PTC may predict patients outcome may predict patients outcome influence surgical approach influence surgical approach their positivity is an indication for sentinel lymphnode procedure their positivity is an indication for sentinel lymphnode procedure Thyroid molecular markers

28. THANKS Ettore degli Uberti Section of Endocrinology Dept. of Biomedical Sciences and Advanced Therapies University of Ferrara Laboratorio di Fisiopatologia Endocrina Ambulatorio Ecografia/ Agoaspirati PersonaleMedicoTecnicoInfermieristico Assegnisti di ricerca DottorandiStudenti Degenza Day Hospital Ambulatori