New Directions in NET Management. Kjell Öberg MD, PhD Professor Endocrine Oncology ENETS Center of Excellence Uppsala University Hospital Sweden

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1 New Directions in NET Management Kjell Öberg MD, PhD Professor Endocrine Oncology ENETS Center of Excellence Uppsala University Hospital Sweden Sydney-May 2018

2 Overview of presentation DIAGNOSTICS THERAPEUTICS Biomarkers circulating markers tissue markers Immunotherapy Molecular Imaging new isotopes 2

3 Diagnostic Advances in NETs Oberg K, Modlin IM, De Herder W, et al. Consensus on biomarkers for neuroendocrine tumour disease. Lancet Oncol 2015; 16:e

4 New developments MicroRNAs Multiple Transcript Analysis in blood Circulating tumor cells Next Generation Sequencing Modlin et al, Plos One 2013; Khan M S et al. Clin Cancer Res 2011, Li et al, Mod Pathol

5 MicroRNA Testing in Neuroendocrine Tumors of the Gastrointestinal Tract family of small non-coding highly conserved singlestranded RNAs they are involved in the regulation of cell proliferation, differentiation,survival, and apoptosis dysregulation of micrornas is a hallmark of cancer Vincentini et al, Molecules 2014, 19, ; 5

6 mir-21 is significantly overexpressed in pancreatic NET and it is detectable in patients plasma samples By repressing pro-apoptotic genes (e.g PTEN or PDCD4) mir21 stimulates proliferation and tumor initiation Vincentini et al, Molecules 2014, 19,

7 Upregulated MiR-96,-182,-183,-196a & - 200a in serum samples a possible new biomarker to detect disease? Courtesy Valeria Giandomenico, Uppsala 7

8 Liquid Biopsy: The Promise Early detection Markers for recurrence Prognosis Screening New Targets Tumor heterogeneity Identification of targets Monitoring Response Resistance Targeted Therapy Molecular assays Molecular typing Personalized Therapeutics Drug Testing Xenografts?

9 Liquid Biopsy Plasma: proteins, hormones, circulating DNA/RNA (mrna/mirna) Cellular compartment: WBCs, platelets Circulating Tumor Cells Oncosomes Tumor Exosomes Circulating Nucleic Acids 0+ /10ml 100+ /10ml /10ml pg-ng/10ml Whole blood captures the tumor signature 9

10 Circulating Neuroendocrine Tumor Signature Neuroendocrine Tumor Transcriptomes Consensus NET-Specific Tumor Tissue Gene Network Adenocarcinoma Transcriptomes + - NET GENE NETWORK AC GENE NETWORK TUMOR TISSUE mrna Upregulated tumor tissue genes Blood-Based Evaluation RT-PCR (75 candidates) NETest 51 mrna liquid biopsy TUMOR BLOOD mrna Upregulated Blood NET genes?

11 Performance Metrics Intra-assay: % Sensitivity >90% Specificity >90% Inter-assay: 0.5-2% AUROC >90% Bloodbased PCR assays Accuracy >93% Positive Predictive Value >90% Negative Predictive Value >90% Accurate, robust, reliable?

12 From Gene Expression in Blood to Omics Algorithmic Analysis Normal Gene expression 51 markers (Heat map) Patients (n=200) TUMOR Proliferation Growth Factor signaling Gene Cluster Analysis OMIC Clusters Secretion SSTR Epigenetic Regulation? Pluripotency Apoptosis Metabolism

13 Methodology: Sample to Score MOLECULAR QUANTIFICATION Circulating Gene Expression Fingerprint Diagnostic Analysis Tumor Score Gene Cluster Analysis Omic Index NETest Score (0-100%) qpcr: Multi Gene cdna synthesis mrna isolation Whole Blood Sample 0 HRS MATHEMATICAL ANALYSIS 8 HRS?

14 Diagnostic NETest score: AUC= CgA levels: AUC= Z-statistic: , p< NETest: sensitivity (85-98%), specificity (93-97%)?

15 *p<0.001 vs CgA, Dopamine (DA) or Noraderenaline (NA) AUROC: 0.99 Stable versus Progressive Disease NETest can diagnose PPGL and elevated levels can differentiate stable from progressive disease?

16 Surgery *p<0.02 versus Pre-treatment NETest correlates with tumor removal and elevated scores >30 days post-surgery predict? recurrent disease #p<0.05 vs. Group II

17 Surgical Resection & Residual disease London (GEP-NETs) Milan (P-NETs) Turin (BP-NETs) Poster: ENETS 2018 EJCTS 2018 Tumor resection correlates directly with decrease in NETest blood levels?

18 Response to SSA Therapy *p<0.001 versus Imagedetected disease progression Elevations in NETest accurately predict somatostatin analog therapy failure at least 3 months earlier than imaging? #p<0.002 vs. changes in CgA

19 Monitoring Response to PRRT FUP = Follow-up at 6 months R = responder NR = non-responder *p<0.05 versus cycle I #p<0.05 versus responders NETest can monitor effectiveness of PRRT?

20 Blood mrna Clinical Utility in the Oncological space High sensitivity and specificity as a diagnostic tool. Methodology is robust/rigorous and validated Confirm complete resection and identify minimum residual disease postsurgery Stratify patients with stable and progressive disease for treatment Monitoring treatment efficacy and identify the risk of treatment failure and disease progression Function as a complementary diagnostic/specific predictor signature for the efficacy of a specific drug or targeted isotope therapy?

21 Circulating Tumor Cells Loberg et al. Neoplasia Jul; 6(4):

22 Prognostic relevance of CTCs in NETs Circulating Tumor Cells As Prognostic Markers in Neuroendocrine Tumors Khan et al. J Clin Oncology 2012:31; patients: Pancreatic 42 Small intestine 101 Bronchial 17 Hindgut 3 CUP 12

23 The number of CTCs which can be found in blood is correlated to survival PFS and OS in patients with NET (n 175) Survival curves according to (A, B) presence of circulating tumor cells (CTCs), (C, D) grade, and (E, F) chromogranin A (CgA) demonstrating differences in (A, C, E) progression-free and (B, D, F) overall survival. Khan et al JCO 2012

24 Molecular Genetics 24

25 Molecular profiling of NEC NGS of 63 patients 11 P-NET 29 nonp-net 23 NEC 50 cancer-related genes were tested for mutations Vijayvergia N et al. Br J Cancer 2016;115:564-70

26 Molecular Profiling 26

27 Core pathways in PanNETs 27

28 Molecular Imaging- New Tracers 28

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30 CRS Page 30

31 Courtesy of Prof. Richard Baum, Zentralklinik, Bad Berka, Germany 31

32 Therapeutic Advances in NETs E V E N T S Pasireotide 1st Clinical Trial PROMID Everolimus pnet CLARINET SUTENT pnets Everolimus All NETs Oncolytic Virus AdVince Telostritat Ethyl PRRT Lanreotide Approved Europe Temozolomide STZ Oberg STZ Approved US Lanreotide Europe Octreotide Publ. Kvols/Oberg Octreotide Approved US Octreotide LAR Approved STZ Moertel Octreotide Oberg IFN Oberg

33 Science (editors) - Breakthrough of the year 2013 Immune checkpoint blockade antibodies (checkpoint inhibitors) block inhibitory signals in T cells, thereby enabling T cells to attack and eradicate tumor cells Oncolytic viruses, which induce immunogenic tumor cell death can make non-responding patients susceptible to checkpoint inhibitors Adoptive transfer of patient-derived T cells, engineered ex vivo with a chimeric antigen receptor (CAR) can eradicate leukemia and lymphoma

34 PD-1/PD-L1 checkpoint inhibitors extend the life-span of preexisting tumor-reactive T-cells Pembrolizumab, Nivolumab (a-pd1) - Malignant melanoma - Lung cancer (NSCLC) - Renal cell carcinoma - Head/neck cancer - Hodgkin s lymphoma - Gastric cancer - Hepatocellular carcinoma - Microsatellite instability-high (MSI-H) or mismatch repair deficient (dmmr) solid tumors (both adult and pediatric) Atezolizumab, Aveluzumab, Durvalumab (a-pdl1) - Bladder cancer - Melanoma - Lung cancer - Merkel cell carcinoma

35 Although checkpoint inhibitors can induce long-term complete responses only a minority of cancer patients respond to checkpoint inhibitors Immunologically hot Immunologically cold Checkpoint inhibitors requires presence of anti-tumor CD8 + T-cells T cell (CD8 + ) infiltrated T cell (CD8 + ) excluded Immune desert Hegde et al, Clin Cancer Res, 2016 Induce tumor-reactive CD8 + T cells to make patients susceptible to checkpoint inhibitors

36 Immunogenic cell death (ICD) converts immunologically cold tumors to hot tumors Oncolytic virus Radiotherapy Certain drugs ICD leads to release of intracellular danger signals that matures dendritic cells (DCs) to induce T cell responses against tumor antigens from dying tumor cells Gotwals P et al, Nature Rev Cancer, 2017

37 Mode of action Oncolytic virus immunotherapy The virus replicates in tumor cells, kills them and release progeny virus. Normal cells are not attacked PBS 0/5 A7/74 4/8 Normal cells Direct lytic action Cancer cells Dendritic cells Dendritic cells take up mutated proteins from killed cancer cells and educate T cells to recognize and attack cancer Immunological attack T cells. Progeny virus infects more cancer cells T cells kill cancer cells

38 Oncolytic viruses in clinical cancer trials T-VEC is an Herpes Simplex Virus (HSV)-1 virus engineered to secrete GM-CSF. Intratumoral injections of T-VEC received FDA approval for refractory melanoma It is the first oncolytic virus approved for cancer treatment in the Western World. Ongoing phase II and III trials to evaluate combination treatment of T-VEC and anti-pd1 or anti-ctla4 antibodies indicate promising synergistic effects. and Maraba ONYX-015/H101 Imlygic (T-vec) JX594 Reolysin PVS-RIBO MV-NIS ONCOS-102 Cattaneo et al, Nature Reviews Microbiology, 2008 Coxsackievirus A21 Cavatak Seneca Valley picornavirus SVV-001

39 Clinical responses for metastatic melanoma patients treated with a combination of T-VEC (oncolytic virus) and Pembrolizumab (checkpoint inhibitor) Oncolytic virus plus anti-pd-1 therapy favorably changed the tumor microenvironment A high overall response rate of 62% to the combination in metastatic melanoma A high complete response rate of 33% to the combination in metastatic melanoma Ribas et al, Cell, 2017 Phase 1b clinical trial

40 Immunotherapy of neuroendocrine tumors/cancers Checkpoint inhibitors are being evaluated (anti-pd1, anti-pdl1, anti-ctla4 antibodies) Oncolytic viruses are being evaluated (also in combination with checkpoint inhibitors) From clinicaltrials.gov NCT (recruiting at multiple hospitals in Spain, Grupo Espanol de Tumores Neuroendocrinos); Durvalumab (a-pdl1) plus Tremelimumab (a-ctla4) for the treatment of patients with advanced Neuroendocrine Neoplasms of GEP or Lung Origin NCT (recruiting at Fox Chase Cancer Center, Philadelphia, Merck); Pembrolizumab (a-pd1) in patients with NETs NCT (recruiting at multiple locations, Novartis); PDR001 (a-pd1) in patients with advanced or metastatic, well-differentiated, non-functional NETs of Pancreatic, Gastrointestinal, or Thoracic origin or poorly-differentiated GEP-NEC NCT (recruiting at Duke University, Merck & Ipsen); Study of Pembrolizumab (a-pd1) with Lanreotide (somatostatin analog) depot for GEP-NETs NCT (recruiting at multiple locations, National Cancer Institute); Nivolumab (a-pd1) and Ipilimumab (a-ctla4) in treating patients with Rare Tumors (including NETs) NCT (recruiting in Australia, Olivia Newton-John Cancer Research Institute, Bristol-Mayers Squibb); Nivolumab (a-pd1) and Ipilimumab (a-ctla4) in treating patients with Rare Tumors (including NETs)

41 Immunotherapy of Merkel cell carcinoma (MCC) Merkel cell carcinoma is caused by the Merkel Cell Polyoma Virus (MCPyV) The virus is immunogenic and MCPyV-specific T-cells can often be found in MCC patients MCC cells often express PDL1 and the MCPyV-specific T-cells express PD1 Multicenter phase II, open label; 88 patients - patients with stage IV chemotherapy-refractory, histologically confirmed Merkel cell carcinoma Avelumab (anti-pdl1 antibody), 10 mg/kg body weight, every two week Evaluation by RESIST CR (9%), 20 PR (23%), SD 9 (10%), PD 32 (36%) Approved March 2017 by the FDA!!! Kaufman et al, Lancet Oncology, 2016

42 Immunotherapy of Merkel cell carcinoma Multicenter phase II, non-controlled study; 25 patients - First line treatment Pembroluzimab (anti-pd1 antibody), 2 mg/kg body weight every three week 14 patients (56%) experienced objective response (RESIST 1.1); 4 CR (16%), 10 PR (40%) 12 of the14 confirmed responses were ongoing at last follow-up Nghiem et al, NEJM, 2016

43 Immunotherapy of Merkel cell carcinoma 2 patients treated with TVEC (Oncolytic HSV-1 encoding GM-CSF) TVEC intratumorally into all detectable metastases Initial dose 2x10 6 PFU (plaque-forming unit) of TVEC Then 2x10 8 PFU/mL at 2-week intervals on 3 occasions Toxicity was limited to mild fatigue CR 2 weeks after the last dose PET/CT complete response 5 months later TVEC intratumorally into all palpable metastases Initial dose 2x10 6 PFU Then 2x10 8 PFU at 2-week intervals on 7 occasions Toxicity - mild fatigue, nausea, injection site tenderness PR by RECIST 1.1 with 62% reduction in index lesions Symptomatic 7 months after the last TVEC dose Blackmon et al, JAAD Case Report, 2017

44 Oncolytic viruses in clinical cancer trials Cattaneo et al, Nature Reviews Microbiology, 2008

45 Genetic modification of adenovirus for selective killing of tumor cells Retargeted Virus Replication Control E1A expression Retargeted Virus Infection Binding motifs in virus coat proteins Fiber C C F W K T Hexon linker-ygrk linker-rrrq K R R Promoter E1A mirna target 45

46 PBS Ad[CgA-E1A] PBS Ad[CgA-E1A] PBS Ad[CgA-E1A] PBS Ad[CgA-E1A] PBS Ad[CgA-E1A] Ad[CgA-E1A] can reduce neuroendocrine tumor growth Treatment of luciferaseexpressing carcinoids (BON) Day 11 Day 15 Leja et al., Clin Cancer Res 13: , Day 20 Day 26 Day 32 46

47 Ad[CgA-E1A] provides prolonged survival of nude mice with subcutaneous neuroendocrine tumors (BON) Leja et al., Clin Cancer Res 13: ,

48 Study Layout in NET-patients AdVince-study Phase I/IIa Patients with different types of NETs The patient will present liver dominant disease The treatment will be given as third line therapy in most patients The virus will be infused via catheter into the liver artery 48

49 mm 55mm

50 Defining Effective Combinations of Immune Checkpoint Blockade and Oncolytic Virotherapy. Rojas J, Sampath P, Hou W, Thorne SH. Clin Cancer Res

51 PD-1 + Oncolytic virus Rojas J, Sampath P, Hou W, Thorne SH. Defining Effective Combinations of Immune Checkpoint Blockade and Oncolytic Virotherapy. Clin Cancer Res

52 Conclusion Immunotherapy might be a new promising treatment for NETs Trials with PD-1 antibodies are ongoing in NETs Oncolytic viruses are tempting agents with dual actions, direct cell killing and stimulation of the immune system Immunotherapy can be combined with other therapies, chemotherapy, PRRT, targeted agents 52

53 THANK YOU! Centre of Excellence Endocrine Tumors Uppsala University Hospital 53 endocrinetumo