1. PHARMACOTHERAPY IN METASTATIC THYROID CANCER VOICHIŢA MOGOŞ

2. Differentiated thyroid cancers may be among the most effectively treated malignancies in patients present with disease localized in the thyroid bed. In this stage the disease may be cured or contained indefinitely with surgery combined with radioiodine thyroid remnants ablation where indicated. 10 % of thyroid cancer patients either present with advanced disease or later have recurrences after primary therapy.

3. Advanced thyroid cancer is defined as: Presence of distant metastases not including cervical lymph node metastases Recurrent locally invasive disease of the neck According to American Association Joint Committee on Cancer Classification advanced thyroid cancer includes: Stage IV disease Stage II disease (distant metastases) in patients older than 45 years. Any occurrence of anaplastic thyroid cancer is also considered a advanced disease because of its extremely poor prognosis even after aggressive surgical resection

4. Clinic of Endocrinology – Iasi: 326 cases of thyroid cancer – clinical data at presentation

5. Most important sites of metastases in DTC lung - 45 %** - 49 %* bone only- 24%*- 39 %** other single sites – 4 %**- 8%* multiple sites – 12 %**- 19 %* papillary cancer in 51 % of cases follicular cancers in 49 % of cases Clinical symptoms in patients with metastases from DTC (differentiated thyroid cancer) may be present especially from bone metastases: swelling, pain, fractures (80 %) Hag M., Harmer C.: Clin.Endocrinol.(Oxf), 2005, v.63, nr.1, pp: 87-93 Samson E., Brieley J., Le LW., Rotstein L., Thang RW.Cancer 2007, v.110, nr.7, pp: 1451-1456

6. Prognostic factors in patients with metastastatic DTC at the time of diagnosis age 100 % survival before 45 years and 62 % survival after 45 years site of metastases : bone metastases or brain metastases are of worse prognosis histology: papillary – 75 %, follicular – 62 % iodine avidity of metastases: iodine avid – 82 %, non iodine avid – 57 % 3 years actuarial survival - 69 % 5 year actuarial survival - 50 % 5 year disease - free patients - 16 % 71 % of death are produced by disease progression

8. Treatment of advanced DTC Patients with advanced thyroid cancer have a few therapeutic options for effective disease control 131 I therapy is the mainstay of initial therapy for advanced differentiated thyroid cancer. Unfortunetely the metastatic disease is frequntly unresponsive either primary or secondarily to radioiodine therapy. Other modalities: aggressive surgery external beam irradiation gamma knife radiosurgery may be useful in selected patients especcialy in respect to symptom palliation chemotherapy Morris JC., Bible KC., Smallridge RC.: Clinical trials for advanced thyroid cancer. Mayo Clinic Endocrinology Update 2007,www.mayoclinic.org/endocrinology Pacini F., Schlumberger M et al. Eur.J.Endocrinol. 2006, v.2, 154- 203 Samson E., Brieley J., Le LW., Rotstein L., Thang RW.Cancer 2007, v.110, nr.7, pp: 1451-1456

9. DTC – lung metastases In the case of radioiodine uptake treatment consists of 131 I administration after prolonged suppressive T4 therapy withdrawal or rhTSH 131 I activity ranges from 1.7 to 7.4 MBq or higher every 4-8 month in the first 2 years and thereafter at longer intervals. WBS will be performed 2-5 days after iodine administration in order to assess the response to treatment toghether with thyroglobulin monitoring, thus quiding further treatment. WBS is not required before treatment because it does not modifie indication for treatment and may induce stunning effect (reduction of uptake of the subsequrnt therapeutic iodine dose). There is no maximum cumulative dose of radioiodine that can be given in patints with persistent disease. However most remissions are obtained with an activity equal or lower that 22 GBq (600 mCi Litium may be useful as an adjuvant therapy of metastatic DTC increasing accumulation and retention of radioiodine in the lesion. Pacini F., Schlumberger M et al. Eur.J.Endocrinol. 2006, v.2, 154- 203

10. DTC – bone metastases Bone metastases may be treated with a combination of surgery and 131 I. When 131 I is taken in the metastases - 100-150 mCi of 131 I every 4-6 month, the effectiveness of higher doses remaind to be demonstrated. The success of the therapy depends of the effective half-life of radioiodine in the tumor. Other therapies for bone metastases include: external beam iradiation, bisphosphonates, embolisation and cement injections. Palliative surgery for bone metastases when there are neurological or ortopedic complications is usefull to debulk large tumors anf may be curative in patients with a single of a few bone metastases Pacini F., Schlumberger M et al. Eur.J.Endocrinol. 2006, v.2, 154- 203

11. DTC brain matastases Brain matastases are rare. When possible they will be resected and if non iodine avid or non resecatable external beam irradiation may provide palliation. Usually they carry a worse prognosis, having a mean post treatment survival of around 12 months.

12. Thyrotropin suppressive therapy Rationale and recommendations for THS suppressive therapy: to inhibit THS –dependent growth of thryroid cancer cells by decreasing TSH levels to less than 0.1 mIU/L TSH suppressve therapy is manditory in patients with persistent disease including those with detectable levels of thyroglobulin and no signs or symptoms and in high risk patients for 3-5 years. Adverse events of THS suppressive therapy: thyrotixicosis and bone loss

13. Human Recombinant TSH for diagnostic and therapeutic purposes in DTC Protocol for hr TSH adminsitration prior diagnostic or therapeutic 131 Iodine administration 0.9 mg hrTSH day 1 and 2 131 Iodine id given day 3 TG measurements in days 1-3-5 WBS in day 5 Adverse effects of hrTSH: nausea fatique headache No toxicity and antibodies directed against hrTSH have been seen. hrTSH provides a stimulation of tumor cells as good as T4 withdrawal.

14. Duntas LH et all. Hormones 2003, 2(3), 169-174 131 I WBS

15. hrTSH preserves quality of live of patients avoiding prologend hypothyroidism decreases morbidity associated with prolonged thyroid hormones withdrawal it is indicated in patients unable to rise endogenous TSH and in those in whom prolonged withdrawal is contraindicated in patients with spine metastases and spinal cord compression a short course of high dose corticoisteroids will avoid worsening of neurological symptoms hrTSH is particularly suitable in advanced reccurent or metastatic DTC, and in patients who are intolerant to TSH stimulation by L4 withdrawal

16. Stategies to enhance 131 I uptake and retainment into DTC neoplastic cells Litium therapy: 300 mg 3 times per day or 10 mg/kg.bw Redifferentiation in iodide non –avid DTC –Retionoids are derivatives of vitamin A(retinol) that increase NIS mRNA expression and iodide uptake in thyroid cancer line cell. They bind to RXR retinoid nuclear receptor that regulates cell growth and differentiation. In clinical trial retionoic acids reestablish iodide transport in 20-50 % of cases. Recommended doses are of 1-1.5 mg/ kg / day of isotretinoin or 300 mg per day bexarothene prior to 131 I uptake –Histone dyacethylase inhibitors indice through a unclear mechanism cell cycle arrest and dedifferentiation. Thay reestablish NIS expression, Tg mRNA and iodide accumulation –Valproic acid inhibits histone dyacetylase –Gene therapy is directed to introduce NIS code into less differentiated thyroid carcinoma cells by using a viral vector carrying the gene

17. Chemotherapy Classic chemotherapy has a little or no role in the management of advanced differentiated thyroid cancer. I is restricted to patients with progressive disease, uncontrolled by surgery and 131 iodine therapy. Studies with chemotherapy in radioresistant DTC are limited. Single agent doxorubicin or a combination of doxoribicin and cysplatin provide a 10-20 % partial and transient response without impact on prolonged survival in patients with metastatic DTC.. Recommanded doses of doxorubicin are 60-70 mg/m 2 every 3 weeks. Cysplatin and epirubicyn is another possible chemotherapy regimen Combined chemotherapy using carboplatin and epirubicin under THS endogenous or exogenous stimulation demonstrated a rate of complete and partial response of 37 %. Anthracycline may be indicated in patients wit bone nonfunctioning metastases with progressive disease.

18. Novel therapies and clinical trials for DTC Target therapy is a new generation of anticancer treatment which planned to interfere with specific molecular targets, tipically a protein that is believed to play a crucial role in tumor growth and progression. Tumor progression is associated with more aggressive behavior, metastatoc spread, loss of iodine uptake ability that makes tumor resistant to conventional therapies. 1.Oncogene inhibitors 2.Angiogenesis inhibitors 3. Modulators of growth or apoptosis Cooper DS, Doherty GM et all. Thyroid, 2006, 16, 2, pp 4-22

19. Coelho SM et all. Arq.Bras.Endocrinol.Metab.2007, 51, 4, 612-624

20. Coelho Sm et al. Arq.Bras.Endocrinol.Metab.2007, 51, 4, 612-624

21. Strategies to inhibit RET. The different steps involved in RET activation, regulation, and synthesis are schematically depicted. Each step is associated with a potential means of inhibition for therapy. 1, Ligand binding and formation of ligand-GFR complex (antagonists, antibodies); 2, receptor dimerization (inhibitors); 3, autophosphorylation (tyrosine kinase inhibitors) 4, recruitment of adaptor proteins (phosphatases, inhibitors of protein- protein interaction) 5, intracellular signaling (various inhibitors) 6, internalization and nuclear translocation (antibodies, inhibitors); and 7, biosynthesis (gene therapy, RNAi). Jan Willem B. de Groot, Thera P. Links, John T. M. Plukker, Cornelis J. M. Lips and Robert M. W. Hofstra: RET as a Diagnostic and Therapeutic Target in Sporadic and Hereditary Endocrine Tumors Endocrine Reviews 27 (5): 535-560

22. ONCOGENE INHIBITORS RET tyrosine kinase inhibitors in DTC YD6474 – Zactima acts as antiangiogenic, RET blocking agent and inhibits EGFR (tested in PTC cell lines) Pyrazolopyridines PP1 and PP2 indolocarbazole derivatives inhibit RET in MTC cell lines Sorafenib (BAY 43-9006) multikinase inhibitor inhibitsd RET/PTC autophosphorilation

23. BRAF inhibitors BRAF kinase belongs to the RAF family of serine/threonine kinases ( BRAF – MAPK – ERK pathway) 36-69 % of PTC carry a point BRAF mutation. Sorafenib was tested in advanceed PTC and anaplastic thyroid cancer Antisense nucleotides designed to inhibit C-RAF ISIS 5132 (phase I study)

24. RAS inhibitors RAS is a GTP-binding protein involved in cell proliferation and survival. Mutations of RAS have been reported in thyroid cancer ISIS as first generation antisense nucleotide Farnesyl transferase inhibitors inactivate RAS Tipifarnib, Lonafarnib, Manumicin and paclitaxel

25. Other oncogene inhibitors MEK inhibitors CI – 1040 PD 03225901 ARRY – 142886 NTRK1 incodes for a high afinity receptor for NGF and its interraction with NGF is blocked by CEP – 701 Akt/mTOR (Akt/ mammalian target of rapamicin) acts through IP3 pathway. - rapamicin ( Sirolimus) - LY 294002 inhibitor of IP3 pathway was used in MTC cell lines

26. Angiogenesis inhibitors Inhibitors of VEGFR VEGF-R is the most prominent factor involved in tumor angiogenesis and is overexpressed in poorly differentiated and metastatic DTC recombinant human monoclonal antibodies against VEGF reduce angiogenesis Semaxanib, Vatalanib and Axitinib (USA clinical trial) are VEGFR inhibitors and tyrosine kinase inhibitors ZD6474 inhitis VEGF, RET oncogene an MEN 2a and 2 b Combrestatin is a tubulin –binding protein inhibitor with vascular targeting properties

27. Inhibitors of EGFR EGF-R is overexpressed in PTC and associated with poor prognosis It. Acts through RAS-RAF MAPK cascade and PI3 kinase pathway recombinant human monoclonal antibodies agains EGFR - Mab 4253 was testes in PTC cell lines AG1478 is a tyrosine kinase inhibitor taht antogonises EGF – mediated angiogenesis and local invasion Geftinib is a EGFR tyrosine kinase inhibitor in phase II clinical trial for iodine refractary DTC and advanced DTC Irressa (ZD1830) is a tyrosine kinase inhibitor that blocks EGFR

28. Inhibitors of EGFR Thalidomide is an antiangiogenic factor was used in a phase II clinical trial including follicular, insular carcinoma refractory to radioiodine and MTC with distant metastases. 800 mg tolerated dose induced partial response in 18 % and stable disease in 32 % of cases with a median survavil of 23.8 months for responders and 11 months for non-responders Lenalidomide – immunomodulatory, antiangionegesis, clinical trial in USA

29. Modulators of growth or apoptosis HSP 90 is a multichaperone protein complex that mediates maturation and stability of different proteins involved in tumorigenesis: BRAF, Akt, CRAF, Her-2, EGFR, p53. Inhibition of HSP90 lead to degradation of these proteins and interruption of signal transduction that is essential for tumor progression. HSP 90 inhibitors: Geldamicin and ist derivatives 17 AAG and 17 DMAG ( phase I/II clinical trials) Macrolide radicicol and its derivative Cysplatin Novobiocin Tumor necrosis factor-related apoptosis inducing ligand TRIAL activates apoptosis by acting on its receptors TRIAL R1 and TRIAL R2 ans caspase pathway. TRIAL-induced apoptosis is inhanced by cycloheximide and paclitaxel Proteazome inactivators Bortezomib (USA clinical trial)

30. Modulators of growth or apoptosis Tiazolidindione and derivatives PPAR  is considered a tumor suppressor factor. PAX8/ PPAR  fusion oncogene was seen in follicular carcinoma and supresses wild type PPAR  activity. Troglitazone and rosiglitazone (USA clinical trial) inhibit tumor cell growth and upragulate NIS mRNA. High afinity PPAR  agonist RS5444 produces growth inhibition in anaplastic thyroid cancer cell lines. PPAR  agonists induce reversion of epidermal/mezenchymal transition which is critical for anaplastic transformation of differentiated thyroid cancer. They also increase cyclin-dependent apoptosis, increase cyclin-dependent kinase inhibitors P21 and P27, decrese BcL-x expression and decrease caspase 3 and caspase 7 activity. Cyclooxigenase 2 inhibitors Activation of COX 2 is overexpressed in different malignancies including thyroid carcinoma. A phase II clinical trial with celocoxib failed to prove any rezults in advanced thyroid cancer

31. Other targeting therapies Demetilating agents. Reducing aberant metilation of gene promoters that inhibit gene expression and is associated with loss of NIS, failure of 131 I treatmetn and aggressive behavior of DTC may be achieved by: 5 azacytidine 5 azo 21 deoxycytidine, decitabine (USA clinical trial) sodium butirate, phenylbutirate and phenyacetate that act through unknown mecanisms to induce NIS expression and iodine uptake

32. Coelho SM., Pines Carvalho D., Waisman M.: Arq. Bras. Endocrinol. Metab. 2007, v.51,nr.4, 612-624

33. Gene therapy Viral vectors were used to introduce NIS gene Wt (wild type) p53 gene that is critical regulator of cell cycle progression by activation the cascade of events that lead to apoptosis. Prodrug suicide gene therapy Thymidine kinase gene is introduced within the cells and placed under the control of thyroglobulin gene promoter that is expressed only in thyroid cells. Once activated thymidine kinase is able to react with Ganclyclovir resulting in DNA strand breaks and subsequent cell death.

34. Treatment of advanced MTC Classical chemotherpy Doxorubicin, bleomicin, caboplatin, cyclophosphamide, DTIC, epirubicin, etoposide, 5 fluorouracil, methotrexate, streptozitocin, vincrisine, vinsine. Combinations of drugs: DTIC + 5FU + epirubicin DTIC + cyclohosphamide+ vincristine Complete response: 3 % Partial response : 24 %, no benefit for survival. Embolisation of liver metastases is associated with regression of metastases and stabilization of the disease in 60 % of patients Somatostatin analoques have not proven efficacy

35. Immunotherapy Principles of immunotherapy: Stimulation of physiological immune response to tumor antigens To stimulate physiologic immune response by transfering dendritic cells able to combating, calcitonin, CEA. Dendritic cells are known to present antigens and induce immune response. To transfer immunostimulatory substances Vaccination with mature dendritic cells. It may be used in MTC patients not curecd by surgery. 2 studies performed with this treatment showed radiological response ( tumor reduction by 50 % assesse by Xray) in 1/7 and 4/10 patients, and biological response (reduction of CEA and calcitonin) in 3/7 and 7/10 patients. Hormonal and radiological stabilization of the disease was noticed in 4/7 and 3/10 patients. Treatment has a good tolerability. Transfer of immunostimulatory substances Studies on murine models were performed by introducing IL2 or IL12 into MTC cells by adenoviral vectors (murine model) Vezzosi D., Bennet A., Caron P.: Ann.Endocrinol.(Paris), 2007, v.68, pp:147-153

36. Radioimmunotherapy Intense expression by MTC cells of CEA allowed the use of anti CEA antibodies labeled with 131 I to target these cells. 9-30 treated patients were followed 12-121 months. Modereate regression (less than 50 %) was noticed in 7-29 %, and stable tumor in 35-73 %. Combined treatment with myelodepressant labeled anti-CEA antibodies coupled with autologous graft of hematopietic cells rezulted in reduction of tumor size in 10 of 12 treated patients. Vezzosi D., Bennet A., Caron P.: Ann.Endocrinol.(Paris), 2007, v.68, pp:147-153

37. MTC cell CEA anti CEA Ab 131 I or 90 Y CEA Ab 131 I labeled haptene CEA Ab Bivalnt antibodies In labeled haptehe

38. Treatments targeting RET gene or protein Germ line RET mutation are found in 95 % of hereditary MTC and 40-70 of sporadic MTC Ocogenic activity of RET may be inhibited by: dominant negative RET mutants that will create a RET protein without oncogenic activity or thyrosine kinase inhibitors, inhibitors of RET proteinkinase or inhibition of tumorigenic patways upstream of tyrosine kinase Dominant negative RET mutants Adenoviral vectors expressing dominant negative RET mutants have being used in some studies. Their use results in reduced quantity of RET protein at cell surface and reduced oncogenic capacity of RET Thyrosine kinase inhibitors ST 15 71, Imatinib (Glivec) reduces tumor size in animal model but the concentration needed to induce clinical efects in humans seems to be too high. Recently good rezults have been reported with Imatinib 600 mg per day in 9 patients A phase II study combining tyrosine kinase inhibitor Imatinib with chemothrapy (dacarbazine and capecitabine are now in trials) Other potential target agents: erlotinib, sorafenib, vandatinib, irinotecan (clinical trials), bevacizumab. Other combined therapies associate a RET tyrosine kinase inhibitor (Glivec) with FGFR tyrosine kinase inhibitor Other alternatives to inhibit RET oncogenic activity Use of riboyzmes (catalitic molecules) directed against mutated RET mRNA

39. Other treatments Use of suicide genes The suicide gene system involves a combination of herpex simplex virus type thymidine kinase and ganciclovir. Phisiologically, intracellular kinases will convert ganciclovir monophosphate in ganciclovir triphosphate wich in toxic for cells. In animal models the suicide gene therapy was effective in 75 % of cells. COX 1 and COX 2 inhibitors – indometacin could induce inhibition of tumor growth and calcitonin secretion in mTC cell lines. Radioiodine therapy following iodine symporter (NIS) gene expression induced by a viral virus. Vezzosi D., Bennet A., Caron P.: Ann.Endocrinol.(Paris), 2007, v.68, pp:147-153

40. Treatment of anaplastic thyroid cancer 47 % of cases present with syncronous metasteses at diagnosis Classic chemotherapy Japanese Society of Thyroid Surgery Cysplatin: 40 mg/m 2 + etopiside: 60 mg/m 2 + etopiside: 100mg/m 2 + peplomicine 5 mg for 3 days preoperatively with GCSF (Ganulocyte Colony Stimulating Factor to prevent a risky granulocyte decrease). Survival: 3-11 month ofter treatment Ausperger 9 (1990): Vinblastine, cysplatine, doxorubicine and mitoxantrone. Survival: 9 % after 1 year Schlumberger (1991): chemotherapy as sensitizer for external beam therapy: Before 45 years: doxorubicine 60 mg/m 2 and cysplatine 90 mg/m 2 After 45 years> mitoxantrone 14 mg. 5 complete response with 3 patients survived more than 20 month Tezelman (cit.Giufrida 2000): Bleomicine and flurorouracyl 2 hours before each dose of radiotion therapy Kebebew E. 2005: hiperfractionated radiotherapy with 100cGy 4 times a day to the dose of 3600 cGy associeted with doxirubicine Doxorubicin and paclitaxel

41. Treatment of anaplastic thyroid cancer Proteayome inhibitors: bertozonib alone ore in association with TRIAL Inhibitors of VEGFR: Semaxanib and Vatalanib (USA clinical trial) are VEGFR inhibitors and tyrosine kinase inhibitors Sorafenib (RAF kinase) USA clinical trial Combrestatin (angiogenesis inhibitor) USA clinical trial Gene therapy adenoviral mediated: p 53 tumor suppressor gene: inhibits cell proliferation and restors Tg gene, thyroperoxidase and TSH receptor genes cr6/Gadd45  tumor suppressor gene

42. Conclusions Conventional chemotherapeutic agents have shown not substantive activity in differentiated thyroid cancer and MTC Researces directed at defining genetic and signaling patways deffects in different cancer types including thyroid cancer improved understanding of the pathogenesis of thyroid cancer and made possible to design and develop of agents that specifically target these deffects and pathways.

43. Drug/ agentDiseasetargetPrimary site Tanespimycin (17 AAG) DTCHSP90Mayo Clinic Rochster and Mayo Clinic Jacksonville DecitabineDTCDNA methylationMD Anderson Cancer Centre BortezomibDTCUbiquitin- proteasome pathway Memorial Anderson Cancer Centre RomidepsinDTCHystone deacetylaseMemorial Sloan Kettering Cancer Centre RosiglitazoneDTC PPAR  University of California San Francisco IrinotecanMTCTopoisomeraseJonh Hopkins University SunitinibDTC/MTCMultikinaseUniversity of Chicago CombrestatinATCAngiogenesisCase Western Reserve University SorafenibATCMultikinase (RAF-kinase) Case Western Reserve University SorafenibMTCMultikinase (RAF – kinase) Ohio StateUniversity LenalidomideDTCImmunomodulatory Angiogenesis University of Kentuky ImatinibATCMultikinaseUniversity of Michigan AxitinibDTCVGFR inhibitorMulticenter trial VandatinibMTCRET protooncogeneMayo Clinic Rochester and Mayo Clinic Jacksonville www.cancer.gov/search/clinical_trials

44. Suggestion reading Coelho SM., Pines Carvalho D., Waisman M.: New perspective in the treatment of differentiated thyroid cancer. Arq. Bras. Endocrinol. Metab. 2007, 51 (4), 612-624 Cooper DS, Doherty GM., haugen BR., Kloos RT., Lee SL., Mandel SJ., Mazzaferri EL., Melver B:; Sherman SI and the American Thyroid Association Guidelines Taskforce: Management Guidelines for patients with thyroid nodules and differentiated thyroid cancer Thyroid. 2006. v. 16 (2), 4-25 Pacini F., Schlumberger M., Dralle H., Elisei R., Smit WA., Wiersinga W. And the European Thyroid cancer Task Force: European Consensus for the management of patients with differentiated thyroid cancer of the follicular epitelium Eur. J. Endocrinol. 2006, v.2, pp: 154-203 Schlumberger M. : Papillary anf follicular thyroid carcinoma, Ann. Endocrinol. (Paris). 2007, 68, 120-128 Vezzosi D., Bennet A., Caron P.: Recent advances in treatment of medullary carcinoma Ann.Endocrinol.(Paris), 2007, 68, 147-153