New Directions in NET Management

Kjell Öberg MD, PhDProfessor Endocrine Oncology

ENETS Center of ExcellenceUppsala University Hospital

SwedenSydney-May 2018

Overview of presentation

DIAGNOSTICS THERAPEUTICS

Biomarkers Immunotherapy

circulating markers

tissue markers

Molecular Imaging

new isotopes

2

Diagnostic Advances in NETs

3

Oberg K, Modlin IM, De Herder W, et al. Consensus on biomarkers for neuroendocrine tumour disease. Lancet Oncol 2015; 16:e435-446

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 2013

4

MicroRNA Testing in Neuroendocrine

Tumors of the Gastrointestinal Tract

family of small non-coding

highly conserved single-

stranded 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, 2458-2468; 5

miR-21 is significantly overexpressed in

pancreatic NET and it is detectable in

patients’ plasma samples

Vincentini et al, Molecules 2014, 19, 2458-2468

By repressing pro-apoptotic genes (e.g PTEN or PDCD4) miR21 stimulates

proliferation and tumor initiation

6

Upregulated MiR-96,-182,-183,-196a & -

200a in serum samples – a possible

new biomarker to detect disease?

Courtesy Valeria Giandomenico, Uppsala7

?

Liquid Biopsy: The Promise

Screening

Monitoring

Personalized

Therapeutics

Targeted

Therapy

New

Targets

Early detection Markers for recurrence Prognosis

Response Resistance

Drug Testing Xenografts

Molecular assays Molecular typing

Tumor heterogeneity Identification of targets

Liquid Biopsy

Plasma: proteins, hormones, circulating DNA/RNA (mRNA/miRNA)

Cellular compartment: WBCs, platelets

Circulating Tumor Cells

0+ /10ml

“Oncosomes”

100+ /10ml

Tumor Exosomes

1010-1012/10ml

Circulating

Nucleic Acids

pg-ng/10ml

Whole blood captures the tumor signature9

?

Neuroendocrine TumorTranscriptomes

NET GENENETWORK

AdenocarcinomaTranscriptomes

AC GENENETWORK

-+

Consensus NET-SpecificTumor Tissue Gene Network

Upregulatedtumor tissue genes

TUMORTISSUEmRNA

UpregulatedBlood NET genes

TUMORBLOODmRNA

Blood-Based EvaluationRT-PCR (75 candidates)

NETest51 mRNA liquid biopsy

Circulating Neuroendocrine Tumor Signature

?

Performance Metrics

Accurate, robust, reliable?

Blood-based PCR

assays

Sensitivity>90%

Specificity>90%

Negative Predictive Value

>90%Accuracy>93%

AUROC>90%

Inter-assay:0.5-2%

Intra-assay:0.4-1.5%

Positive Predictive Value

>90%

?

?

Normal

TUMOR

OMICClusters

Proliferation

Epigen

eticR

egulatio

n

MetabolismApoptosis

Pluripotency

SSTRGrowth Factor

signaling

Secretion

From Gene Expression in Blood to Omics

Gene expression

51 markers(Heat map)

Patients (n=200)

Algorithmic Analysis

Gene ClusterAnalysis

Whole Blood Sample

mRNA isolation

cDNA synthesis

qPCR: Multi Gene

Circulating Gene Expression

Fingerprint

Diagnostic Analysis

“Tumor Score”

Gene Cluster Analysis

“Omic Index”

Methodology: Sample to Score

0 HRS

?

MOLECULAR QUANTIFICATION

MATHEMATICAL ANALYSIS

8 HRS

NETestScore

(0-100%)

?

?

Diagnostic

NETest: sensitivity (85-98%), specificity (93-97%)

NETest score: AUC=0.95-0.98

CgA levels: AUC=0.62-0.64

Z-statistic:

6.97-11.42,

p<0.0001

?

??

*p<0.001 vs CgA,Dopamine (DA) orNoraderenaline (NA)

AUROC: 0.99Stable versus Progressive

Disease

NETest can diagnose PPGL and elevated levels can differentiate

stable from progressive disease

??

*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

Surgery

?

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

??

*p<0.001 versus Image-detected 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

Response to SSA Therapy

??

*p<0.05 versus cycle I

#p<0.05 versus responders

FUP = Follow-up at 6 monthsR = responder

NR = non-responder

NETest can monitor effectiveness of PRRT

Monitoring Response to PRRT

?

• High sensitivity and specificity as a diagnostic tool.

• Methodology is robust/rigorous and validated

• Confirm complete resection and identify minimum residual disease post-

surgery

• 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

Blood mRNA –

Clinical Utility in the Oncological space

?

Circulating Tumor Cells

Loberg et al. Neoplasia. 2004 Jul; 6(4): 302–309

Prognostic relevance of CTCs in

NETs

Circulating Tumor Cells As Prognostic Markers

in Neuroendocrine Tumors

Khan et al. J Clin Oncology 2012:31;365-72

175 patients: Pancreatic 42

Small intestine 101

Bronchial 17

Hindgut 3

CUP 12

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

Molecular Genetics

24

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

Molecular Profiling

26

Core pathways in PanNETs

27

Molecular Imaging-New Tracers

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21.05.2018CRS Page 30

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

Therapeutic Advances

in NETs

32

STZ

Moertel

STZ

Oberg

STZ

Approved

US

Lanreotide

Europe

Lanreotide Approved

Europe

Pasireotide 1st Clinical Trial

PROMID

Everolimus

pNET

CLARINET

Octreotide

Oberg

IFN

Oberg

Octreotide Publ.

Kvols/Oberg

Octreotide

Approved US

Octreotide LAR

Approved

Temozolomide

SUTENT

pNETs

Oncolytic

Virus

AdVince

PRRT

Everolimus

All NETs

Telostritat

Ethyl

1970 1980 1982 1983 1985 1987 1988 1994 1998 2000 2007 2010 2011 2015 2016 2017

E

V

E

N

T

S

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

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

Although checkpoint inhibitors can induce long-term complete responses

only a minority of cancer patients respond to checkpoint inhibitors

Hegde et al, Clin Cancer Res, 2016

T cell (CD8+) infiltrated T cell (CD8+) excluded Immune desert

Checkpoint inhibitors requires presence of anti-tumor CD8+ T-cells

Induce tumor-reactive CD8+ T cells to make

patients susceptible to checkpoint inhibitors

Immunologically “cold”Immunologically “hot”

Immunogenic cell death (ICD) converts

immunologically “cold” tumors to “hot” tumors

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

Oncolytic virus

Radiotherapy

Certain drugs

PBS 0/5

A7/74 4/8

Dendritic cells take up mutated

proteins from killed cancer cells

and educate T cells to recognize

and attack cancer

Direct lytic action

Immunological attack

Progeny virus

infects more

cancer cells

Normal cells

Cancer cells

Dendritic cells

T cells .

T cells kill cancer cells

Mode of action – Oncolytic virus immunotherapy

The virus replicates in tumor cells,

kills them and release progeny virus.

Normal cells are not attacked

Oncolytic viruses in clinical cancer trials

Cattaneo et al, Nature Reviews Microbiology, 2008

and Maraba

Coxsackievirus A21

Cavatak

ReolysinJX594ONYX-015/H101

ONCOS-102

Imlygic (T-vec)

Seneca Valley picornavirus

SVV-001

MV-NISPVS-RIBO

• 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 2015.

• 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.

Ribas et al, Cell, 2017

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

Phase 1b clinical trial

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)

• NCT03095274 (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

• NCT02939651 (recruiting at Fox Chase Cancer Center, Philadelphia, Merck);

Pembrolizumab (a-PD1) in patients with NETs

• NCT02955069 (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

• NCT03043664 (recruiting at Duke University, Merck & Ipsen);

Study of Pembrolizumab (a-PD1) with Lanreotide (somatostatin analog) depot for GEP-NETs

• NCT02834013 (recruiting at multiple locations, National Cancer Institute);

Nivolumab (a-PD1) and Ipilimumab (a-CTLA4) in treating patients with Rare Tumors (including NETs)

• NCT02923934 (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)

From clinicaltrials.gov

Immunotherapy of Merkel cell carcinoma (MCC)

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 1.1 8 CR (9%), 20 PR (23%), SD 9 (10%), PD 32 (36%)

• 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

Kaufman et al, Lancet Oncology, 2016

Approved March 2017 by the FDA !!!

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

Immunotherapy of Merkel cell carcinoma

• 2 patients treated with TVEC (Oncolytic HSV-1 encoding GM-CSF)

TVEC intratumorally into all detectable metastases

Initial dose 2x106 PFU (plaque-forming unit) of TVEC

Then 2x108 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 2x106 PFU

Then 2x108 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

Cattaneo et al, Nature Reviews Microbiology, 2008

Oncolytic viruses in clinical cancer trials

Genetic modification of adenovirus for selective killing of tumor cells

Hexon

Fiber

C

C KT

FW

linker-YGRKR

K

Rlinker-RRRQ

Binding motifs in virus coat proteins

Retargeted Virus InfectionRetargeted Virus Replication

Control E1A expression

Promoter E1AmiRNA

target

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Ad[CgA-E1A] can reduce neuroendocrine tumor growth Treatment of luciferase-

expressing carcinoids (BON)

Leja et al., Clin Cancer Res 13: 2455-2462,

2007

46

Ad[CgA-E1A] provides prolonged survival of nude mice with

subcutaneous neuroendocrine tumors (BON)

Leja et al., Clin Cancer Res 13: 2455-2462,

2007 47

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 thirdline therapy in most patients

The virus will be infused via catheterinto the liver artery

48

2016- 03-05 2016-05-16

64mm 55mm

50

Defining Effective Combinations

of Immune Checkpoint Blockade

and Oncolytic Virotherapy.

Rojas J, Sampath P, Hou W, Thorne SH. Clin

Cancer Res. 2015.

PD-1 + Oncolytic virus

51

Rojas J, Sampath P, Hou W, Thorne SH. Defining Effective

Combinations of Immune Checkpoint Blockade and Oncolytic

Virotherapy. Clin Cancer Res. 2015

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

THANK YOU!

Centre of Excellence Endocrine Tumors

Uppsala University Hospital

http://www.endocrinetumors.org/

endocrinetumors.org

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