Milano 25.05.2007

2. WHO Classification 2000 Well differentiated endocrine tumors (benign or low grade malignancy) Well differentiated endocrine carcinomas Poorly differentiated endocrine carcinomas (small cell carcinomas) Mixed exocrine and endocrine carcinomas Tumor-like lesions Who classification distinguished five principal groups of neuroendocrine tumors , but really three of them have interest in clinical paractice Who classification distinguished five principal groups of neuroendocrine tumors , but really three of them have interest in clinical paractice

3. WHO Classification 2000 ::

4. Neuroendocrine tumorsFrequency

6. WHO Classification 2000 Well differentiated endocrine tumors (benign or low grade malignancy) Well differentiated endocrine carcinomas Poorly differentiated endocrine carcinomas (small cell carcinomas) Mixed exocrine and endocrine carcinomas Tumor-like lesions secondsecond

7. WHO Classification 2000 Well differentiated endocrine carcinomas

8. Medical treatment Biotherapy ? Chemotherapy ?

9. Medical treatment Biotherapy

10. Objectives of Medical Treatment Efficacy Symptom control Biochemical control Control of tumor burden

12. Clinical Presentation: non functioning tumours Symptoms related to the mass effect: Bowel obstruction GI bleeding (rare)

13. Clinical Presentation: functioning tumours Carcinoid syndrome < 20%: Cutaneous flushing: upper part of the body (80%) Watery diarrhea and abdominal cramp (80%) Bronchospasm Endocardial fibrosis( 30-40 %): arrhythmia. Right heart insufficiency.

14. Well Differentiated carcinomaand biotherapy Rationales: NETs carry receptor(s) for growth factor responsable in cellular proliferation, angiogenesis,hormone secretion and clinical symptoms: Insulin like growth factor-1 PDGF-alpha TGF-alpha TGF-beta VEGF expression 80-90% of NETs show high-affinity somatostatin receptors

15. Octreotide Octreotide LAR Pasireotide SOM 230 Lanreotide Lanreotide autogel (Lan ATG)

18. Somatostatin analogs

19. Somatostatin analogs

20. Carcinoid Syndrome and  somatostatin analogs

21. Carcinoid Syndrome and  somatostatin analogs

24. Interferon-a/somatostatin analoguesin combination: randomized trials

25. Interferon-a/somatostatin analoguesin combination: Non randomized trials

26. WHO Classification 2000 Well differentiated endocrine tumors (benign or low grade malignancy) Well differentiated endocrine carcinomas Poorly differentiated endocrine carcinomas (small cell carcinomas) Mixed exocrine and endocrine carcinomas Tumor-like lesions

27. WHO Classification 2000 Poorly differentiated endocrine carcinomas

28. Medical treatment Chemotherapy

29. Cytotoxic therapy for carcinoid tumors

30. Cytotoxic therapy for carcinoid tumors

31. Chemotherapy of endocrine pancreatic tumors

40. Targeted Therapy Monoclonal antibody anti-VEGF: Bevacizumab: suppression of tumor blood flow and prolungation of PFS. Yao et al ASCO 2005 Small multi-TK inhibitors: Sunitinib:RR 15% islet cell, 2% carcinoid, SD JCO 2006 Sorafenib, Vatalanib : Phase II ongoing Imatinib (Gleevec): no activity Gross et al .Endocrine related Cancer 2006 Endostatin: only SD. Kulke et al JCO 2006 EGFR inhibitors: Gefitinib: no Object.resp. ASCO 2005

42. Role of angiogenesis in NET Angiogenic growth factors contribute to tumor growth VEGF is found in 84% of carcinoid and 59% of islet cell tumors VEGFR is found in 71% of carcinoid and 67% of islet cell tumors Suggests autocrine stimulation in carcinoid and islet cell tumors The mTOR pathway regulates production of angiogenic growth factors and the proliferation of vascular endothelial cells The antiangiogenic activity of mTOR inhibitors is another reason to investigate them in NET. NET are highly vascularized. The production of angiogenic growth factors by NET drive angiogenesis and may also drive tumor growth by paracrine-autocrine growth stimulation. Reference Hobday T, Rubin J, Goldberg R, et al. Molecular markers in metastatic gastrointestinal neuroendocrine tumors. Proc Am Soc Clin Oncol. 2003;22:269. Abstract 1078.The antiangiogenic activity of mTOR inhibitors is another reason to investigate them in NET. NET are highly vascularized. The production of angiogenic growth factors by NET drive angiogenesis and may also drive tumor growth by paracrine-autocrine growth stimulation. Reference Hobday T, Rubin J, Goldberg R, et al. Molecular markers in metastatic gastrointestinal neuroendocrine tumors. Proc Am Soc Clin Oncol. 2003;22:269. Abstract 1078.

43. mTOR in neuroendocrine tumors

44. mTOR and angiogenesis This is a depiction of the signaling and stimulatory functions of mTOR and the mechanisms for both direct and indirect effects of RAD001 on tumor, endothelial cell, and smooth muscle cell (pericyte) growth and proliferation. In a growing tumor, inhibition of mTOR directly reduces tumor cell growth and proliferation and directly inhibits the growth and proliferation of stromal cells involved in angiogenesis (eg, endothelial cell and smooth muscle cells)1 RAD001 also indirectly inhibits angiogenesis through suppression of the production of hypoxia-inducible factor 1a (HIF-1a), a component of a factor (HIF-1) that regulates transcription of hypoxia-related genes including those for vascular endothelial growth factor (VEGF), other angiogenic growth factors (eg, platelet-derived growth factor [PDGF]), and proteins used in glucose transport into the cell2 References Guba et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med. 2002;8:128-135. O’Reilly TM, Wood JM, Littlewood-Evans A, et al. Differential anti-vascular effects of mTOR or VEGF pathway inhibition: a rational basis for combining RAD001 and PTK787/ZK222584. Proc Am Assoc Cancer Res. 2005;46:715. Abstract 3038. This is a depiction of the signaling and stimulatory functions of mTOR and the mechanisms for both direct and indirect effects of RAD001 on tumor, endothelial cell, and smooth muscle cell (pericyte) growth and proliferation. In a growing tumor, inhibition of mTOR directly reduces tumor cell growth and proliferation and directly inhibits the growth and proliferation of stromal cells involved in angiogenesis (eg, endothelial cell and smooth muscle cells)1 RAD001 also indirectly inhibits angiogenesis through suppression of the production of hypoxia-inducible factor 1a (HIF-1a), a component of a factor (HIF-1) that regulates transcription of hypoxia-related genes including those for vascular endothelial growth factor (VEGF), other angiogenic growth factors (eg, platelet-derived growth factor [PDGF]), and proteins used in glucose transport into the cell2 References Guba et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med. 2002;8:128-135. O’Reilly TM, Wood JM, Littlewood-Evans A, et al. Differential anti-vascular effects of mTOR or VEGF pathway inhibition: a rational basis for combining RAD001 and PTK787/ZK222584. Proc Am Assoc Cancer Res. 2005;46:715. Abstract 3038.

45. mTOR regulates HIF-1a and HIF-2a expression HIF-1 and HIF-2 are transcription factors for hypoxic stress-related genes HIF-1a/2a are normally degraded by VHL protein HIF-1 and HIF-2 condition the tumor to adapt to growth under hypoxic conditions and promote angiogenesis and metastasis In pancreatic NET, HIFs may contribute to tumor growth through mechanisms unrelated to VEGF mTOR inhibitionAnti-NET activity via HIF-1? suppression mTOR activation stimulates translation of HIF-1?, which ultimately increases production of proangiogenic factors such as vascular endothelial growth factor-A (VEGF-A) and PDGF, and other molecules such as those involved in glucose transport. In well-oxygenated cells, HIF-1? is degraded by the von Hippel-Lindau (VHL) protein, which binds and targets it for destruction by the proteasome; loss of the VHL protein is a driving force in the development of some cancers, such as clear cell renal cancer.1,2 In hypoxic cells, such as those found in tumors, HIF-1? translocates to the nucleus and combines with HIF-1?, ultimately initiating the transcription of hypoxia-regulated genes, such as those for VEGF-A and inducible nitric oxide synthetase (iNOS), which promote.1,2 Cell survival under anaerobic conditions Angiogenesis Metastasis Overexpression of HIF-1? has been associated with aggressive disease and poor prognosis in cancers of the breast, ovary, cervix, esophagus, brain, and head and neck; loss of HIF-1 activity decreases tumor growth, vascularization, and energy metabolism.1,2 mTOR inhibition can decrease HIF-1? levels and inhibit VEGF production.1 In contrast to carcinomas, pancreatic NET are highly vascularized, reflecting the normal condition of the tissue in which they arise. However, these tumors are poorly angiogenic, and both VEGF production and the density of microvessels in the tumor decrease with tumor progression. HIF-1a expression, on the other hand, is high in these tumors, with localization moving from the cytoplasm to the nucleus as the tumor progresses.3 Recent studies suggest different roles for HIF-1a, depending upon the tumor microenvironment.4 In pancreatic NET, it appears that HIF-1a may contribute to tumor progression by mechanisms other than through VEGF, suggesting the therapies that target HIF-1a, rather than or in addition to VEGF, may be of particular benefit. References Powis G, Kirkpatrick L. Hypoxia inducible factor-1alpha as a cancer drug target. Mol Cancer Ther. 2004;3:647-654. Vaupel. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Oncologist. 2004;9(suppl 5):10-17. Couvelard A, O’Toole D, Turley H, et al. Microvascular density and hypoxia-inducible factor pathway in pancreatic endocrine tumours: negative correlation of microvascular density and VEGF expression with tumour progression. Br J Cancer 2005;92:94-101. Blouw B, Song H, Tihan T, et al. The hypoxic response of tumors is dependent on their microenvironment. Cancer Cell. 2003;4:133-146.mTOR activation stimulates translation of HIF-1?, which ultimately increases production of proangiogenic factors such as vascular endothelial growth factor-A (VEGF-A) and PDGF, and other molecules such as those involved in glucose transport. In well-oxygenated cells, HIF-1? is degraded by the von Hippel-Lindau (VHL) protein, which binds and targets it for destruction by the proteasome; loss of the VHL protein is a driving force in the development of some cancers, such as clear cell renal cancer.1,2 In hypoxic cells, such as those found in tumors, HIF-1? translocates to the nucleus and combines with HIF-1?, ultimately initiating the transcription of hypoxia-regulated genes, such as those for VEGF-A and inducible nitric oxide synthetase (iNOS), which promote.1,2 Cell survival under anaerobic conditions Angiogenesis Metastasis Overexpression of HIF-1? has been associated with aggressive disease and poor prognosis in cancers of the breast, ovary, cervix, esophagus, brain, and head and neck; loss of HIF-1 activity decreases tumor growth, vascularization, and energy metabolism.1,2 mTOR inhibition can decrease HIF-1? levels and inhibit VEGF production.1 In contrast to carcinomas, pancreatic NET are highly vascularized, reflecting the normal condition of the tissue in which they arise. However, these tumors are poorly angiogenic, and both VEGF production and the density of microvessels in the tumor decrease with tumor progression. HIF-1a expression, on the other hand, is high in these tumors, with localization moving from the cytoplasm to the nucleus as the tumor progresses.3 Recent studies suggest different roles for HIF-1a, depending upon the tumor microenvironment.4 In pancreatic NET, it appears that HIF-1a may contribute to tumor progression by mechanisms other than through VEGF, suggesting the therapies that target HIF-1a, rather than or in addition to VEGF, may be of particular benefit. References Powis G, Kirkpatrick L. Hypoxia inducible factor-1alpha as a cancer drug target. Mol Cancer Ther. 2004;3:647-654. Vaupel. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Oncologist. 2004;9(suppl 5):10-17. Couvelard A, O’Toole D, Turley H, et al. Microvascular density and hypoxia-inducible factor pathway in pancreatic endocrine tumours: negative correlation of microvascular density and VEGF expression with tumour progression. Br J Cancer 2005;92:94-101. Blouw B, Song H, Tihan T, et al. The hypoxic response of tumors is dependent on their microenvironment. Cancer Cell. 2003;4:133-146.

46. Preliminary Results of a Phase 2 study with RAD001 and Octreotide LAR in patients with advanced NET (SMSUS52) Single-arm phase 2 Metastatic or unresectable well-differentiated NET No prior chemotherapy 5 mg: safe and active dose for phase 2 studies Patients had low-grade neuroendocrine carcinoma and pancreatic islet cell tumors performance status 0–2 (Zubrod); 27 patients: 16 carcinoid, 11 pancreatic islet cell tumors Prior Sandostatin LAR: 17 (13 of 16 carcinoid, 4 of 11 Islet cell tumors) Prior cancer therapy for pancreatic islet cell tumor: 8 of 11 PD at study entry: 21 of 27 Reference Yao JC, Phan AT, Chang DZ, et al. Phase II study of RAD001 and depot octreotide in patients with advanced low grade neuroendocrine carcinoma. ASCO 2006 Gastrointestinal Cancers Symposium. Abstract 178. Available at: http://www.asco.org. Patients had low-grade neuroendocrine carcinoma and pancreatic islet cell tumors performance status 0–2 (Zubrod); 27 patients: 16 carcinoid, 11 pancreatic islet cell tumors Prior Sandostatin LAR: 17 (13 of 16 carcinoid, 4 of 11 Islet cell tumors) Prior cancer therapy for pancreatic islet cell tumor: 8 of 11 PD at study entry: 21 of 27 Reference Yao JC, Phan AT, Chang DZ, et al. Phase II study of RAD001 and depot octreotide in patients with advanced low grade neuroendocrine carcinoma. ASCO 2006 Gastrointestinal Cancers Symposium. Abstract 178. Available at: http://www.asco.org.

47. Inhibiting NET growth pathways with combination therapy* Growth-stimulating signals originating from within and outside the cell are integrated through mTOR into processes that maintain cell viability and stimulate cell growth, cell division, and angiogenesis. mTOR is a sensor that acts as a biochemical switch, ensuring that supplies of energy and nutrients in the cell are sufficient to support these processes1,2 In cancer cells, one or more of the proteins shown upstream of mTOR may be deregulated, and this loss of regulation contributes to, and in some cases drives, the malignancy. These include overproduction of hormones, cytokines, and growth factors and aberrant expression of growth factor receptors and signaling molecules, such as PI3-K, PTEN, Akt, and TSC1/2 (and LKB1, which is not shown). Aberrant signaling in parallel signaling pathways also affects signaling through mTOR because these pathways are connected to the mTOR pathway. These connections are referred to as “cross-talk” and involve the Ras/Raf/MAPK pathway and Abl signaling2-5 References Bjornsti and Houghton. Nat Rev Cancer. 2004;4:335-348. Crespo and Hall. Microbiol Mol Biol Rev. 2002;66:579-591. Huang et al. Cancer Biol Ther. 2003;2:222-232. Mita et al. Clin Breast Cancer. 2003;4:126-137. Wullschleger et al. Cell. 2006;124:471-484. Growth-stimulating signals originating from within and outside the cell are integrated through mTOR into processes that maintain cell viability and stimulate cell growth, cell division, and angiogenesis. mTOR is a sensor that acts as a biochemical switch, ensuring that supplies of energy and nutrients in the cell are sufficient to support these processes1,2 In cancer cells, one or more of the proteins shown upstream of mTOR may be deregulated, and this loss of regulation contributes to, and in some cases drives, the malignancy. These include overproduction of hormones, cytokines, and growth factors and aberrant expression of growth factor receptors and signaling molecules, such as PI3-K, PTEN, Akt, and TSC1/2 (and LKB1, which is not shown). Aberrant signaling in parallel signaling pathways also affects signaling through mTOR because these pathways are connected to the mTOR pathway. These connections are referred to as “cross-talk” and involve the Ras/Raf/MAPK pathway and Abl signaling2-5 References Bjornsti and Houghton. Nat Rev Cancer. 2004;4:335-348. Crespo and Hall. Microbiol Mol Biol Rev. 2002;66:579-591. Huang et al. Cancer Biol Ther. 2003;2:222-232. Mita et al. Clin Breast Cancer. 2003;4:126-137. Wullschleger et al. Cell. 2006;124:471-484.

48. Efficacy (RECIST): Phase 2 study in advanced NET (SMSUS52) Responses assessed at 12 weeks by Response Evaluation Criteria in Solid Tumors (RECIST) The slide shows responses to RAD001 in both carcinoid and islet cell tumor patients. Responses in NET are difficult to achieve with any existing treatment modalities. However, since these tumors are indolent, it is not clear that disease stabilization was related to RAD001 therapy. Independent review downgraded a fourth PR in a pancreatic islet cell tumor from 31% unidimensional decrease to 20% decrease. Among the 10 patients not receiving prior Sandostatin LAR, there were 2 responses (3 if downgraded tumor included)—1 pancreatic, 1 carcinoid. Among 17 patients previously treated with Sandostatin, there was 1 PR in a carcinoid patient. There was 1 PR among 8 pancreatic islet cell tumor patients previously treated with chemotherapy. 71% PFS at 24 weeks was also encouraging. Reference Yao JC, Phan AT, Chang DZ, et al. Phase II study of RAD001 and depot octreotide in patients with advanced low grade neuroendocrine carcinoma. ASCO 2006 Gastrointestinal Cancers Symposium. Abstract 178. Available at: http://www.asco.org.Responses assessed at 12 weeks by Response Evaluation Criteria in Solid Tumors (RECIST) The slide shows responses to RAD001 in both carcinoid and islet cell tumor patients. Responses in NET are difficult to achieve with any existing treatment modalities. However, since these tumors are indolent, it is not clear that disease stabilization was related to RAD001 therapy. Independent review downgraded a fourth PR in a pancreatic islet cell tumor from 31% unidimensional decrease to 20% decrease. Among the 10 patients not receiving prior Sandostatin LAR, there were 2 responses (3 if downgraded tumor included)—1 pancreatic, 1 carcinoid. Among 17 patients previously treated with Sandostatin, there was 1 PR in a carcinoid patient. There was 1 PR among 8 pancreatic islet cell tumor patients previously treated with chemotherapy. 71% PFS at 24 weeks was also encouraging. Reference Yao JC, Phan AT, Chang DZ, et al. Phase II study of RAD001 and depot octreotide in patients with advanced low grade neuroendocrine carcinoma. ASCO 2006 Gastrointestinal Cancers Symposium. Abstract 178. Available at: http://www.asco.org.

49. RAD001 in advanced NET: Waterfall plot

50. RAD001 Safety:Phase 2 study in advanced NET (SMSUS52) 34 patients evaluable for toxicity (CTC v3.0) Most frequent AE: mild aphthous ulceration Grade 3/4 Fatigue (n = 3) Aphthous ulcers, diarrhea, rash (each n = 2) Anemia, thrombocytopenia, neutropenia, leukocytosis, hyperglycemia, hypoglycemia, hypokalemia, hypophosphatemia, nausea, pruritus (each n = 1) Reference Yao JC, Phan AT, Chang DZ, et al. Phase II study of RAD001 and depot octreotide in patients with advanced low grade neuroendocrine carcinoma. ASCO 2006 Gastrointestinal Cancers Symposium. Abstract 178. Available at: http://www.asco.org. Reference Yao JC, Phan AT, Chang DZ, et al. Phase II study of RAD001 and depot octreotide in patients with advanced low grade neuroendocrine carcinoma. ASCO 2006 Gastrointestinal Cancers Symposium. Abstract 178. Available at: http://www.asco.org.

51. Conclusions (I) Management with either biotherapy and chemotherapy can be guided by WHO classification in patients with malignant carcinoid. Ki-67 proliferation index might be considered as an additional parameter for choosing between chemotherapy or biotherapy Combination chemotherapy with Cisplatin, lederfolin, fluororuracil represents a valid therapeutic option in malignant carcinoid, having a good therapeutic index and favourable toxicity profile

52. Conclusions (II) Among targeted therapies, RAD001 may arrest NET growth by blocking downstream signaling through IGF-1R, TSC1/2, and mTOR and, in combination with Octreotide LAR, may act synergistically to arrest NET growth and alleviate symptoms

53. Open Questions A standard chemotherapy is still not in existence because a small number of patient cases and consequently a small number of randomized trials What is the best treatment for Endocrine Pancreatic Tumors? How can we select the best method of treatment for patients in the grey area (Ki-67 2-15%) ?