Lucerne, November 2nd-3rd, 2012 RET:

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1 SAMO Interdisciplinary Workshop on Oncogenic Driver Lucerne, November 2nd3rd, 2012 RET: Thyroid Malignancies Rossella Elisei, MD Department of Endocrinology Univeristy of Pisa, Italy

2 THYROID GLAND: 2 CELLULAR TYPES Colloid Parafollicular cells Follicular cells Blood vessel FOLLICULAR CELLS: 99% C CELL or PARAFOLLICULARLAR CELLS: 1%

3 THYROID CANCER IS RARE TUMOR AND REPRESENTS ONLY 1% OF ALL HUMAN TUMORS A l l h u m a n c a n c e r T h y r o i d c a n c e r MOST FREQUENT CANCER AMONG ALL ENDOCRINE TUMORS!!!

4 THYROID CANCER HISTOTYPE (Department of Endocrinology, Pisa) TSH dependance % Iodine uptake Tg secretion DIFFERENTIATED THYROID CARCINOMA PAPILLARY FOLLICULAR MEDULLARY UNKNOWN ANAPLASTIC LYMPHOMA

5 Genetic alterations in thyroid carcinoma Genetic alteration PTC FTC ATC MTC RET rearrangement 1343% RET mutation NTRK1 rearrangement 3050% (MEN2: 100%) 513% BRAF mutation 2969% 1035% RAS mutation 021% 4053% 2060% PPAR rearrangement 2563% P53 mutation 6788% Modified from Kondo T, et al.nat Rev Cancer. 2006

6 RET PROTOONCOGENE : tyrosine kinase receptor

7 Nature 1987: A new oncogene in human thyroid papillary carcinomas and their lymphnodal metastases (A. Fusco et al.) RET D10S170 ( H 4 ) RET/D10S17 0 ( PTC ) H 4 protoret SP TM tyrosine kinase H 4 tyrosine kinase PTC1 The PTC in which the RET/PTC rearrangement was described belonged to an irradiated patient

8 Ret Oncogene Activation in Human Thyroid Neoplasms Is Restricted to the Papillary Cancer Subtype Massimo Santoro et al J Clin Invest 1992 RET Acitvation in Human Thyroid Tumors Tumor type Positive/analyzed USA France Italy Total Papillary 11/65 8/70 14/42 33/177 Follicular 0/11 0/13 0/13 0/37 Anaplastic 0/2 0/5 0/8 0/15 Medullary 0/0 0/3 0/15 0/18 Adenoma 0/0 0/18 0/16 0/34 *Other 0/0 0/1 0/4 0/5 Total * Squamous cell carcinoma, sarcomatoid carcinoma.

9 CHERNOBYL APRIL 26, 1986

10 12 Incidence per in Belarus 11,3 Adolescents 10 Children (0 14) Adolescents (15 18) Adults (19 34) 9,5 9.7 Cases per ,8 2,9 2,3 1,2 1,4 1,0 4 3,8 2,9 3,2 1,9 1,4 3,8 3 4,2 3,1 2,6 2,1 6,6 5,7 5,7 5,6 4,9 3,4 2,5 1,7 2,6 0,7 6,9 Young adults Children 0,3 0,3 0,2 0,1 0 0,4 0, Cardis E et al, J. Radiol Prot 26: , 2006

11 ALMOST SIMULTANEOUSLY Klugbauer et al. ( 1995) 8/12 (66.6%) 2/12 PTC2 (16.6%) 6/12 PTC3 (50.0%)

12 Ionizing Radiation Effects on DNA Double strand breaks Rearrangements (RET/PTC) Chromosomes exposed to gamma rays

13 SCHEMATIC RAPPRESENTATION OF RET/PTC REARRANGEMENTS SP EC TM TK ProtoRET Most frequently involved in thyroid tumorigenesis H4 R1a ELE1 Golga5 htif1 htif1 ELKS ELKS/RET KTN1 RFG9 PCM1 RFP HOOK3 RET/PTC1 RET/PTC2 RET/PTC3 RET/PTC5 RET/PTC6 RET/PTC7 RET/PTC8 RET/PTC9 PCM1/RET RFP/RET HOOK3/RET and others

14 Distinct pattern of RET oncogene rearrangements in morphological variants of radiation induced and sporadic thyroid papillary carcinomas in children Nikiforov et al Cancer Res 1997 Prevalence of RET rearrangements in radiation induced and sporadic childhood tumors RET/PTC3 significantly more frequent than RET/PTC1

15 PREVALENCE OF RET REARRANGEMENTS IN MORPHOLOGICAL VARIANTS OF PEDIATRIC PAPILLARY CARCINOMA POSTCHERNOBYL (Nikiforov et al. Cancer Res, 1997) ret/ptci ret/ptc2 ret/ptc3 novel ret/ptc negativ e CLASSICAL FOLLICULAR SOLID MIXED SCLEROSING

16 PREVALENCE OF RET/PTC REARRANGEMENTS IN SPORADIC AND IRRADIATED PTC References Post Chernobyl n (%) Spontaneous n (%) Guerra et al 2011** nd 36% Hieber et al /22 (72) nd Hamatani et al /50 (22)* nd Rhoden et al 2006 nd 25/34 (73) Zhu et al 2006 nd 26/75 (34) Unger et al, /13 (77) nd Di Cristofaro et al, /17 (65) 9/21 (43) Rhoden et al, 2004 nd 18/25 (72) Puxeddu et al, 2003 nd 13/48 (27) Elisei et al, /25 (76) 11/47 (23) Cinti et al, 2000 nd 13/69 (19) Sheils et al, 2000 nd 12/50 (24) Chua et al, 2000 nd 44/62 (71) Thomas et al, /67 (55) nd Smida et al, /51 (49) nd Mayr et al, 1998 nd 8/99 (8) Tallini et al, 1998 nd 81/201 (40) Lam et al, 1998 nd 17/40 (43) Sugg et al, 1998 nd 51/86 (59) Nikiforov et al, /38 (87) 12/17 (70) Klugbauer et al, /15 (60) nd Fugazzola et al, /6 (66) nd Zou et al, 1994 nd 1/40 (2.5) Ishizaka et al, 1991 nd 1/11 (9) Fenton et al, 2000 nd 15/33 (45) Metanalysis 175/279 (62.7) 357/958 (37.2)

17 FREQUENCY OF RET/PTC IN IRRADIATED AND NOT IRRADIATED PAPILLARY THYROID CARCINOMA (Elisei et al. J. Clin. Endocrinol. Metab, 2001) % p = C h i l d r e n A d u l t s 2 0 n=25 n=17 n=25 n=22 0 IRRADIATED NOT IRRADIATED

18 Before 1997 After 1997 RET/PTC rearrangements BRAF point mutations Smyth et al Int J Surg Pathol 2005

19 THE PREVALENCE OF RET/PTC REARRANGEMENTS HAS DECREASED OVER THE YEARS An Italian series (n=860) 34 p< RET/PTC rearrang gements % Group 1 ( ) Group 2 ( ) Group 3 ( ) Romei et al JCEM 2012

20 RET/PTC rearrangements and clinical/pathological features of PTC Romei et al, End Rel Cancer, 2008 Age years mean median range Diameter mean median Female (58) Male (20) RET/PTC1+ RET/PTC1 p RET/PTC3+ RET/PTC3 p ns mean ns ns ns 3 13 ns Multicentricity 4 21 ns 3 22 ns Node metastases 6 16 ns 6 15 ns Variant Classic (37) Aggressive (14) Foll (13) n (%) 7 (19) 0 3(23) n (%) 30 (81) 14 (100) 10 (77) ns n (%) 5 (13) 1 (7) 3 (23) n (%) 32 (87) 13 (93) 10 (77) ns ns

21 RET/PTC rearrangements and mrna expression of thyroid differentiation genes Romei et al, End Rel Cancer, 2008 DCt RET/PTC3+ RET/PTC3 RET/PTC1+ RET/PTC1 p NIS Mean Median Tg Mean Median TPO Mean Median TSHR Mean Median TTF1 Mean Median GLUT1 Mean Median GLUT2 Mean Median NS NS NS NS NS NS NS

22 RET/PTC3 rearrangemets and pathological features Romei et al, End Rel Cancer, 2008 RET/PTC3 + RET/PTC3 n (%) n (%) Tumor size T1 (33) T2 (13) T3 (19) T4 (2) 5 (15) 3 (23) 2 (10) 2 (100) 28 (85) 10 (77) p= (90) 0 Class I (46) 5 (11) 41 (89) II (19) 5 (26) 14 (74) p=0.003 III (2) 2 (100) 0

23 RET protein expression has no prognostic impact on the long term outcome of papillary thyroid carcinoma P=NS P=NS Basolo et al 2001

24 RET/PTC REARRANGEMENTS IN PTC RET/PTC rearrangements are specific for PTC RET/PTC rearrangements, mainly RET/PTC3, are more frequently found in radiation exposed PTC However, they are also present in sporadic cases with a variable prevalence from 2% to 42% The prevalence of RET/PTC rearrangements in PTC has been decreasing in the last 15 year RET/PTC rearrangements do not play any prognostic role although RET/PTC3 results to be associated with an advanced stage at diagnosis

25 THYROID CANCER HISTOTYPE (Department of Endocrinology, Pisa) % 710% 40 Survival % 60 FTC 50 5 MTC ATC PTC years PAPILLARY FOLLICULAR MEDULLARY UNKNOWN ANAPLASTIC LYMPHOMA

26 PREVALENCE OF DIFFFERENT FORMS OF MEDULLARY THYROID CARCINOMA SPORADIC 75% MTC MEN 2A FAMILIAL 25% MEN 2B FMTC

27 RET REarranged Transformed gene CHROMOSOME 10 KOBAYASHI, 1989: ISOLATED FROM CELLS TRANSFORMED IN VITRO BY TUMORAL DNA FROM A HUMAN LYMPHOMA

28 CHROMOSOME LOCALIZATION 10q11.2 RET AND MEN II!

29 PHENOTYPE DISTRIBUTION IN 477 KINDREDS WITH HEREDITARY MEDULLARY THYROID CARCINOMA ( 1994, INTERNATIONAL RET CONSORTIUM ) OTHERS (33,8%)!?! 161 M E N IIB FM TC (7,0 %) M E N IIA (3 ) M E N IIA (2 ) 96 (16,5%) 94 M E N IIA (1 ) (42,7%)

30 RELATIONSHIP BETWEEN THE PRESENCE OF RET MUTATION AND PHENOTYPE IN 477 KINDREDS AFFECTED WITH HEREDITARY MTC ( INTERNATIONAL RET CONSORTIUM, 1994 ) RET RET M EN2B M E N 2 A (1 ) M E N 2 A (2 ) M E N 2 A (3 ) FM TC OT HERS Eng et al, JAMA, 1994

31 RET MUTATIONS AND PHENOTYPES (INTERNATIONAL RET CONSORTIUM, 1994) Involved codons MEN IIA MEN IIA (1) (MTC + PHEO + HYPERP) MEN IIA (2) (MTC + PHEO) MEN IIA (3) (MTC + HYPERP) 6% 2% 92% 3% 4% 13% 80% 8% 15% 8% 69% 97% MEN IIB FMTC 7% 3% 33% 17% 30% 3% MTC sporadic* * Somatic mutations are found in 40% of sporadic MTC 95%*

32 2006 GENOTYPEPHENOTYPE ACCORDING TO KNOWN MUTATIONS deletions/insertion 8 FMTC Del G592G607 Del F612C MTC MTC Del D898E MTC MTC MTC MTC MTC MTC MEN2A MTC Data from Santoro M and Carlomagno F, Nature Clin Pract Endocrinol & Metab, 2006 CYSTEIN RICH DOMAIN Dupl E529C531 Del D631L Del E632C634 Del C630D631 Del E632L633 Del E632L633 Del E632A640 + insvrp C634insH/E/L/CR635 MUTATED CODONS PHENOTYPE EXTRACELLULAR DOMAIN EXONS TYROSINE KINASE DOMAIN MUTATED CODONS point mutations EXONS PHENOTYPE FMTC FMTC FMTCMEN 2A FMTCMEN 2A FMTCMEN 2A FMTCMEN 2A /633/ / FMTC MEN 2A MEN 2A MEN 2A FMTC FMTC FMTC MEN 2B FMTC FMTC MEN 2B

33 2011: many new RET mutations in MTC Exons 5, 8, 10, 11, different germline RET mutations 74 different somatic RET mutations 59 missense mutations 7 Deletions 8 Complex mutations

34 CORRELATION BETWEEN RET MUTATION AND THE PHENOTYPIC PRESENTATION OF HEREDITARY MTC MEN 2B MEN 2A V804L FMTC V804M Kouvaraki et al, 2005

35 CODON SPECIFIC AGE RELATED PROGRESSION IN MEN 2 Earliest age of manifestation (yr) MTC (95%) PCC (50%) (1%) (3%) (7%) (7%) Cys 630 (1%) 1 Cys 634 (68%) Glu 768 (1%) Leu 790 (5%) Tyr 791 (2%) Val 804 (2%) Ser 891 (2%) Met 918 (3%) Cys Cys Cys Cys exon 10 exon 11 exon 13 exon 14 exon 15 exon phpt (1030%) adapted from Machens and Dralle 2006

36 DIFFERENT GEOGRAPHIC DISTRIBUTION OF DIFFERENT RET MUTATIONS

37 Modified from Machens and Dralle, Molecular Endocrinology, 2006 In vitro Transforming Activity N of foci ? 18?? 141 RET gene RET mutation Pisa N= Cys 630 Cys ex 13 Glu 768 Leu 790 Tyr ex 14 Val 804 Met Ser 891 Ala 883 Thr Ser 904 Thr Met ex 11 ex 13 ex 15 ex Halle N= ex 10 Cys Cys Cys Cys % of RET families EUROMEN N= no mut

38 Romei C et al (ItaMEN group), % of germline mutations Val804Met Val804Met Cys634Arg Cys634Tyr Ser891Ala Met918Thr Glu768Asp Leu790Phe Cys634Phe Cys618Ser No mut Cys634Gly Cys634Ser Cys618Arg Cys620Arg Cys634Trp Cys620Ser Val804Leu Cys630Tyr Cys609Tyr Cys618Gly Cys618Tyr Met918Val Tyr791Phe Val648Iso Ala883Thr Arg694Gln Cys515Ser Cys609Arg Cys609Gly Cys609Phe Cys609Ser Cys611Gly Cys620Gly Cys620Phe Cys630Arg Cys634Val Lys666Met Met848Thr Ser904Phe Thr338Ile Different types of germline RET mutation in Italian kindred

39 A NOVEL GERMLINE POINT MUTATION IN RET EXON 8 (GLY(533)CYS) IN A LARGE KINDRED WITH FAMILIAL MEDULLARY THYROID CARCINOMA Da Silva AM, Maciel RM, Da Silva MR, Toledo SR, De Carvalho MB, Cerutti JM J Clin Endocrinol Metab A NEWLY DETECTED MUTATION OF THE RET PROTOONCOGENE IN EXON 8 AS A CAUSE OF MULTIPLE ENDOCRINE NEOPLASIA TYPE 2A. Bethanis S, Koutsodontis G, Palouka T, Avgoustis C, Yannoukakos D, Bei T, Papadopoulos S, Linos D, Tsagarakis S. Hormones (Athens) 2007

40 RET MUTATIONS IN 100 TUMORAL TISSUES OF SPORADIC MEDULLARY THYROID CARCINOMA (Dept. Of Endocrinology, Pisa) n=34 40 Tumoral tissues N= n=1 n=7 n=1 0 Ex 16 Met 918 Ex 15 Glu 883 Ex 11 Cys 634 Ex 10 48bp deletion % n=57? RET+ somatic 43/100 (43%)

41 RET mutations and clinicalpathological features of sporadic MTC Age at diagnosis Sex (F/M) Tumor size (100) T1 (25) T2 (42) T3 (14) T4 (19) Node metastases (100) N1 (45) N0 (55) Distant metastases (100) M1 (20) M0 (80) Stage (100) I (19) II (32) III (27) IV (22) Outcome (100) disease free (39) persistent disease (50) dead (11) RET RET RET+ P 47.1 (20(2071) 53.1 (21(2183) NS 34/23 23/20 NS < Elisei et al, JCE&M, 2008

42 SURVIVAL CURVE IN MTC PATIENTS WITH AND WITHOUT RET SOMATIC MUTATIONS n= 100, mean followup 10 years Log rank p=0.006 Survival Functions Mutante 1,0 No RET Cum Survival 0,8 Sì Nocensored Sìcensored RET+ 0,6 0,4 0,2 0,0 0,00 5,00 10,00 15,00 20,00 25,00 30,00 Elisei et al, JCE&M, 2008

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44 Correlation of RET somatic mutations with clinicopathological features in sporadic medullary thyroid carcinomas Moura et al, British Journal Cancer 2009

45 COMBINED RET AND KI67 ASSESSMENT IN SPORADIC MEDULLARY THYROID CARCINOMA: A USEFUL TOOL FOR PATIENT RISK STRATIFICATION Caterina Mian et al EJE 2011 months months

46 MEN 2A typical mutations n= n=1 n=7 n=1 0 Ex 16 Met 918 Ex 15 Glu 883 Ex 11 Cys 634 Ex 10 48bp deletion %? MEN 2B typical mutations

47 TYROSINE KINASE INHIBITORS (TKI) The majority of TKI inhibit several receptors with different degree of inhibition DRUG IC50(nm) VEGFR1 VEGFR2 VEGFR3 RET MET KIT BRAF OTHER IMATINIB > ABL (38) AXITINIB VANDETANIB EGFR (500) MOTESANIB PDGFR (84) SUNITINIB FLT3 (21) GEFITINIB EGFR (14) SORAFENIB CRAF (6) E FGFR1 (25) CABOZANTINIB (XL184)

48 Vandetanib in patients with locally advanced or metastatic medullary thyroid cancer: a randomized, doubleblind phase III trial (ZETA) Samuel A Wells,1 Bruce G Robinson,2 Robert F Gagel,3 Henning Dralle,4 James A Fagin,5 Massimo Santoro,6 Eric Baudin,7 Rossella Elisei,8 Barbara Jarzab,9 James Vasselli,10 Jessica Read,11 Peter Langmuir10 Anderson J Ryan12 and Martin Schlumberger7, for the ZETA investigators* No. of events/no. of patients (%) 1.0 Vandetanib 300 mg Placebo All patients with sporadic disease V=66/203 (32.5%) P=49/95 (51.6%) RET mutation positive V=40/110 (36.4%) P=25/45 (55.6%) RET mutation negative V=1/2 (50.0%) P=5/6 (83.3%) RET mutation unknown V=25/91 (27.5%) P=19/44 (43.2%) 0.2 M918T mutation positive V=35/101(34.7%) P=25/41 (61.0%) 0.1 M918T mutation negative V=21/54 (38.9%) P=18/37 (48.6%) M918T mutation unknown V=10/48 (20.8%) P=6/17 (35.3%) Progressionfree free survival Time (months) Number of patients Vandetanib 300 mg Placebo Hazard ratio (95% confidence interval) A hazard ratio <1 favors vandetanib The analyses were performed using a log rank test with treatment as the only factor Significant increase of progression free survival RET negative MTC are responsive to Vandetanib BUT Ret positive cases are more responsive J Clin Oncol, in press

49 AZD6474ZACTIMAVANDETANIB CAPRELSA 6 APRIL 2011: APPROVED BY FDA 18 FEBRUARY 2012: APPROVED BY EMA ADVANCED METASTATIC MTC ALREADY AVAILABLE IN USA AND IN SEVERAL, BUT NOT ALL, EUROPEAN COUNTRIES

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51 CLINICAL CASE PHYSICAL EXAM 55 YEAR OLD WOMAN PAINLESS RIGHT ANTERIOR CERVIAL ADENOPATHY 3 RIGHT ANTERIOR CERVICAL LYMPH NODES AND 1 PALPABLE NODULE IN THE UPPERPOLE OF THE RIGHT THYROID LOBE

52 NECK ULTRASOUND VERY SUSPICIUOS!!!!!!! THYROID NODULE BILATERAL NECK LYMPH NODES

53 FINE NEEDLE ASPIRATION Tyr 3: indeterminate or follicular neoplasm (Bethesda cytological classification) Serum Ct: 328 pg/ml MTC

54 SPECIFIC LABORATORY TESTS IN MTC PATIENTS BLOOD URINE 24 hours urine for catecholamines and total methaneprhines WNL Calcitonin 450 pg/ml (VN < 10 pg/ml) CEA 10 ng/ml (VN nonsmoker <2.5 ng/ml) Calcium, PTH and VitD: WNL Fractionated plasma metanephrine: WNL

55 FAMILIAL MEDICAL HISTORY FAMILY HISTORY NEGATIVE FOR: THYROID CANCER PHEOCROMOCYTOMA HYPERPARATHYROIDISM APPARENTLY SPORADIC MTC

56 Germline RET mutation screening V804M

57 SURGICAL TREATMENT: pathology report TOTAL THYROIDECTOMY + BILATERAL CERVICAL LYMPHADENECTOMY MTC 2.2 x1.9 cm in the right lobe No extracapsular invasion 4/13 right and 1/9 left cervical lymph nodes with MTC metastases T2N1Mx

58 METASTATIC EVALUATION: STAGING NECK: BILATERAL CERVICAL ADENOPATHY CHEST AND ABDOMEN: NEGATIVE

59 14 months after TT+LNC Serum Ct: 800 pg/ml (it was 125 pg/ml 6.5 months before) TB CT scan: 1) mediastinal adenopathy in the upper retrosternal area, with no evidence of superior vena caval obstruction but persistent retrosternal pain 2) multiple pulmonary nodules (biggest: 1.5 cm) 3) no liver metastases

60 2. VANDETANIB AND MEDIASTINAL LYMPH NODE METASTASES 8/2/ /11/2004 IT WORKS!!!!

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