Immunotherapy for Glioblastoma: Updates and Future Directions

Stephen Bagley, MD, MSCE Instructor of Medicine

Friday, June 8, 2018 Disclosures

I have research funding support from Incyte, Novocure, Eli Lilly, and Tesaro. I have no financial conflicts of interest related to this presentation.

Please note that some of the studies reported in this presentation were published as an abstract and/or presented at a conference. These data and conclusions should be considered to be preliminary until published in a peer-reviewed journal.

2 Overview

1) Brief background: cancer immunotherapy

2) Recent key immunotherapy studies in glioblastoma

3) Specific challenges in glioblastoma immunotherapy

3 4 FDA approvals for immune checkpoint inhibitors (as of 3/23/18)

5 Ribas, et al Science 2018 Chimeric antigen receptor (CAR) T cells for hematologic malignancies

• CAR T cells FDA approved for: • Refractory pre-B cell acute lymphoblastic leukemia • Refractory diffuse large B cell lymphoma

6 Cancer evasion of immunosurveillance

Glioblastoma

7 Vonderheide, Cancer Cell 2018 Classes of Cancer Immunotherapy

• Systemic cytokine therapy • Interferons • Interleukins • Vaccines • Peptide • Dendritic cell • DNA • Monoclonal antibodies • Immune checkpoint inhibitors • Co-stimulatory receptor agonists • Small molecules • IDO inhibitors, many others • Oncolytic viral therapy and immunotoxins • Adoptive T cell Transfer

8 Vonderheide, Cancer Cell 2018 Local and systemic immunosuppression in glioblastoma

9 Lim et al, Nat Rev Clin Oncol 2018 Overview

1) Brief background: cancer immunotherapy

2) Recent key immunotherapy studies in glioblastoma

3) Specific challenges in glioblastoma immunotherapy

10 Classes of Cancer Immunotherapy

• Systemic cytokine therapy • Interferons • Interferon-beta treatment has been shown to • Interleukins yield a net immune stimulating effect against • Vaccines glioblastoma initiating cells (stem cells) • Peptide • In older clinical trials, only limited clinical activity • Dendritic cell of interferon-beta and interferon-alpha therapies in combination with alkylating • DNA chemotherapy for high grade glioma • Monoclonal antibodies • Phase III trial ongoing in China • Immune checkpoint inhibitors • Interferon-gamma is currently being studied in • Co-stimulatory receptor agonists combination with DNX-2401 (oncolytic • Small molecules adenovirus) for recurrent glioma • IFN-gamma very poorly tolerated, did not seem to • IDO inhibitors, many others improve outcomes compared to DNX-2401 alone • Oncolytic viral therapy and (Lang et al, SNO 2017) immunotoxins • IL-12 and GM-CSF used as vaccine adjuvants • Adoptive T cell Transfer

Wolpert et al,, PLoS One 2015; Yamamuro et al, Int J Oncol 2015 11 Buckner et al, Cancer 2001; Groves, et al, Br J Cancer 2009 Motomura et al, Cancer 2011 Classes of Cancer Immunotherapy

12 Vaccine Therapies for Glioblastoma

• Previously considered one of the most promising approaches to improving the outcomes of patients with glioblastoma “Cold” “Hot”

• Negative results from several phase II and phase III trials have challenged the concept of vaccination as a single-modality immunotherapy

13 Johnson et al, Clin Cancer Res 2017 Rindopepimut (CDX-110, PEPvIII)

• Peptide vaccine that mimics EGFR ACT IV Trial: Randomized, Phase III trial variant III (EGFRvIII), a in newly diagnosed EGFRvIII+ GBM constitutively active mutant form of EGFR expressed exclusively on • N=745, GBM s/p maximal surgical resection and GBM cells in 25–30% of patients standard chemoradiation • Advantage: neoantigen expressed without progression • 405 patients with MRD exclusively on tumor cells, limiting (<2cm2 contrast enhancing risk of “on-target, off-tumor” tumor) toxicities • 1:1 randomization to rindopepimut vs. control via • Disadvantage: EGFRvIII is monthly intradermal injection heterogeneously expressed (both concurrent with TMZ spatially and temporally) on GBM • Primary endpoint: OS in the MRD subgroup cells in vivo

14 Del Vecchio et al, Oncogene 2013 Weller et al, Lancet Oncol 2017 Rindopepimut: Issues and Future Directions

• Is a certain level of tumor tissue needed to drive immune responses? • RT and TMZ-induced lymphopenia • Spontaneous loss of antigen expression, even in the control arm, as observed in patients undergoing repeat surgery • Failure of a strong humoral immune response to translate into clinical benefit • Are vaccines the right approach in GBM?

*** Non-comparative phase II trial (ReACT): • Bevacizumab-naïve recurrent GBM patients (n=72) with EGFRvIII+ tumors randomized to bevacizumab plus double-blinded injection of rindopepimut or control (KLH) • PFS-6 of 27% versus 11% (p=0.048) • Median OS 12.0 vs. 8.8 months (p=0.02) Weller et al, Lancet Oncol 2017 15 Reardon et al, ASCO 2015 ICT-107: a multipeptide dendritic cell vaccine

• Patient-derived dendritic cells Randomized, Phase II Trial (post-surgery apheresis) • N=124, newly diagnosed GBM incubated ex vivo with six • <1cm3 residual tumor, completed first-line peptides commonly over- chemoradiation •Phase77 patients III (62%) trial HLA was-A2 positive opened for expressed in GBM cells compared • Randomized 2:1 to ICT-107 or control to nonmalignant tissues patient(autologous DCsaccrual not exposed in to 2016 the GBM but (“warehouse” approach using antigens) • wasinduction terminated QWx4 vaccinations, due followed to lackby tumor-associated antigens): ofmaintenance funding vaccinations on June plus TMZ 21, 2017 • Melanoma-associated antigen 1 (MAGEA1) • Median PFS improved with ICT-107 (HR 0.57, p=0.01). • HER2 • interferon-inducible AIM2 Median OS favored ICT-107 by 1.6 months in ITT • I-dopachrome tautomerase (DCT) group (NOT statistically significant; p=0.64) • melanocyte protein (PMEL) • HLA-A2+ subgroup: median OS 18.3 months with • IL-13 receptor subunit-alpha2 (IL- ICT-107 v. 12.9 months with control 13Ralpha2)

16 Phuphanich, et al, Cancer Immunol. Immunother 2013 Wen et al, SNO Annual Meeting 2014 DCVax-L (Northwest Biotherapeutics): whole tumor lysate used to pulse autologous dendritic cells • Serves as a source for the • Two prior phase I/II studies at UCLA entire spectrum of tumor • N=20 newly diagnosed GBM antigens (“personalized” • Median PFS ~24 months • Median OS ~36 months (with reported “tail” of vaccine), but logistically survivalStill curve) no published data challenging (requires • As of July, 2011, 33% of patients had reached or (orexceeded anything 4 years’ median peer OS and reviewed 27% had reached collection of tumor sample or exceeded 6 years median OS from each individual • Most forrecently presentation the company reported two at patients a have patients for processing and exceeded 10 years’ survival • Phase III scientifictrial ongoing (started meeting) 12/2006): DC stimulation) most patients on control arm crossed over to DCVax • 25 patients treated in an Information Arm (“indeterminate progressors”) after vaccine was already produced: • median OS 21.5 months, with 24% of patients Liau et al, Clin Cancer Res 2005 exceeding 48 month OS 17 https://www.nwbio.com/dcvax-l/ Other Vaccines: ongoing trials and future directions

• IDH peptide vaccines • 2 phase I trials ongoing (NOA-16 and RESIST): IDH1-R132H-mutated peptide vaccines • HSPPC-96 (antigenic tumor peptides bound to HPS-96) • Phase I, multicenter phase II trials of HSPPC-96 reported in recurrent GBM; 90% of patients with OS > 6 months. • However, randomized PhII trial of HSPPC-96 with bev vs. bev alone was negative (Bloch et al, SNO Annual Meeting 2017) • DNA vaccines: • VXM01: VEGFR2 expression plasmid encoded in live, attenuated Salmonella bacteria (NCT02718443) • INO-5401: WT1, hTERT, and PSMA (given via IM electroporation) (NCT03491683) • Viral Antigens: • High percentage of CMV gene products expressed in malignant gliomas (but not surrounding brain tissue) • Pp65: dominant CMV epitope expressed in 50-70% of newly diagnosed GBM: • Phase I study: 6 patients treated with DCs pulsed with CMV pp65 RNA (preconditioned the vaccine site with tetanus/diphtheria (Td) toxoid): • 50% OS rate at 40 months • Use of adjuvants aimed at stimulating myeloid compartment • SL-701, e.g.: an emulsion of multivalent glioma-associated antigens (short synthetic peptides, including IL13R, survivin, others) administered with poly-ICLC adjuvant • Preliminary phase II data presented at SNO 2017 Annual Meeting • Partial and complete responses demonstrated, tail to survival curve

Rampling R, Clin Cancer Res 2016 Bloch et al, Neuro Oncol 2014 18 Mitchell et al, Nature 2015 Reardon, Neuro Oncol 2017 Classes of Cancer Immunotherapy

19 Immune checkpoint inhibition… Must be preceded by… T cell priming

Salama et al, Clin Cancer Res 2011

20 FDA approvals for immune checkpoint inhibitors (as of 3/23/18)

21 Ribas, et al Science 2018 Neoantigens increase likelihood of T cell priming against the tumor

22 Mutational burden and benefit from immune checkpoint inhibition

Melanoma NSCLC

Johnson et al, Cancer Immunol Res 2016 Rizvi et al, Science 2015

23 Tumor mutational burden is generally low in GBM

Alexandrov et al, Nature 2013

Garg et al. Oncoimmunology 2017

***Caution must applied in interpreting animal models of immune checkpoint inhibition for glioma! -strong preclinical data, but orthotopic implantation of glioma cell lines with high mutational load might not enable accurate prediction of responses in patients with spontaneous GBM Garg et al, Oncoimmunology 2017

24 Randomized Phase 3 Study: Nivolumab vs Bevacizumab in Patients With Recurrent Glioblastoma (CheckMate 143)

• N=369 patients with no prior VEGF therapy • Randomized 1:1: nivolumab 3 mg/kg every 2 weeks or bevacizumab 10 mg/kg every 2 weeks • At baseline in both arms, ~80% of patients had measurable disease and ~40% of patients required corticosteroids • Grade 3–4 treatment-related adverse events: • 18% (nivolumab) • 15% (bevacizumab) • Primary endpoint was overall survival (OS) – no difference in median OS (9.8 mo nivo vs. 10.0 mo bev) or OS rate at 12 months

Reardon DA et al, World Federation of Neuro-Oncology Societies Quadrennial Meeting 25 Zurich, Switzerland 2017 Randomized Phase 3 Study: Nivolumab vs Bevacizumab in Patients With Recurrent Glioblastoma (CheckMate 143)

• Among patients who received nivolumab: • Response rate 8% • Median duration of response 11.1 months • 2 complete responses (clinical details for these patients not available, but possibility of hypermutation is likely)

Johanns et al, Cancer Discovery 2016

Reardon DA et al, World Federation of Neuro-Oncology Societies Quadrennial Meeting 26 Zurich, Switzerland 2017 Exploratory phase I cohorts from CheckMate 143

• 3 combined nivolumab and ipilimumab treatment arms (N=40): • NIVO3: nivolumab 3 mg/kg Q2W • NIVO1+IPI3: nivolumab 1 mg/kg + ipilimumab 3 mg/kg Q3W for 4 doses, then nivolumab 3 mg/kg Q2W • NIVO3+IPI1 nivolumab 3 mg/kg + ipilimumab 1 mg/kg Q3W for 4 doses, then nivolumab 3 mg/kg Q2W • AEs leading to discontinuation occurred in 10% (NIVO3), 30% (NIVO1+IPI3), and 20% (NIVO3+IPI1) of patients. Three patients (8%) achieved a partial response (NIVO3, n = 1; NIVO3+IPI1, n = 2) and 8 (20%) had stable disease for ≥12 weeks • This combination strategy is not being pursued further in phase III of CheckMate 143

27 Omuro et al, Neuro Oncol 2018 Ongoing Phase 3 Studies of nivolumab in GBM

• CheckMate 498: newly diagnosed GBM with unmethylated MGMT promoter • Nivolumab replaces temozolomide during and after radiation

• CheckMate 548: newly diagnosed GBM with methylated MGMT promoter • Nivolumab given in addition to temozolomide during and after radiation

28 Other emerging data with immune checkpoint inhibitors in GBM

• Phase I single agent data in recurrent GBM has also been presented (but not published) for pembrolizumab and durvalumab • 3% response rate and 13% progression-free at 1 year with durvalumab in bevacizumab-naïve recurrent GBM • 4% response rate with pembrolizumab in this same population) • Phase II study of durvalumab in bevacizumab-refractory recurrent GBM presented at SNO 2017 Annual Meeting (Reardon, et al): • No efficacy • Preliminary Phase II data for pembrolizumab and bevacizumab combination therapy in recurrent GBM presented at ASCO 2017 Annual Meeting (Reardon, et al): : • Well tolerated without DLT or unexpected toxicity

Reardon DA et al, Society for Neuro-Oncology Annual Meeting 2016 29 Lim M et al, ASCO Annual Meeting 2016 Reardon DA et al, Society for Neuro-Oncology Annual Meeting 2017 Biomarkers of response to PD-1/PD-L1 immune checkpoint inhibition

• PD-L1 expression? • Degree of expression 2%-88% depending antibody used, whether measuring exclusively on tumor cells, etc • Prognostic impact in GBM also controversial • Cut-off of 1% did not influence outcomes in subgroup analysis of CheckMate143 • Loss of PTEN, NF1 mutation, and mesenchymal subtype associated with higher expression • Most expression in GBM is on tumor infiltrating myeloid cells (TIMs), consisting mostly of M2 tumor-associated macrophages • High expression on circulating myeloid cells associated with poor response to HSP-96 vaccination in GBM (this association was NOT seen in patients treated with SOC, and was NOT seen with tumor PD-L1 expression)

• PD-L2 playing an increasingly important role in GBM and other tumors • Associated with survival in GBM, more robustly expressed than PD-L1, and more highly associated with pathways of T Antonios et al, Neuro Oncol 2017 cell activation in GBM than PD-L1

Reardon et al, Neuro Oncol 2017 Yearley et al, Clin Cancer Res 2017 30 Nduom et al, Neuro Oncol 2016 Ricklefs et al, Neuro Oncol 2017 Berghoff et al, Neuro Oncol 2015 Bloch et al, Clin Cancer Res 2017 Small molecule inhibitors previously studied for immunomodulation in GBM

• IDO inhibitors: • PF-06840003: single agent phase I trial for recurrent malignant glioma • 13 patients treated as of SNO 2017 Annual Meeting • Adequate CNS penetration as measured by CSF kynurenine/tryptophan ratio (Reardon et al, Neuro Oncol 2017) • CSF1R inhibitors • Despite exciting preclinical data, PLX3397 did not demonstrate efficacy as single agent in recurrent human GBM (Butowski et al, Neuro Oncol 2016) • TGF-Beta receptor 1 (TGFBR1) inhibitors • Randomized Phase II study: galunisertib monotherapy vs. galunisertib plus lomustine vs. lomustine alone in patients with recurrent GBM: • No differences in OS

31 Small molecule inhibitors in development for immunomodulation in GBM

• IDO inhibitors • Epacadostat (phase II in combination with nivolumab for selected advanced cancers) (NCT02327078) • Indoximod (Phase I/II with temozolomide for temozolomide-refractory brain tumors) (NCT02052648) • CSF1R inhibitors • FPA008 (phase I in combination with nivolumab for selected advanced cancers) (NCT02526017) • STAT3 inhibitors • WP1066 (phase I in recurrent glioma and brain metastases) (NCT01904123)

32 Immune co-stimulatory receptor agonists in development for GBM (alone or as adjuncts to immune checkpoint inhibitors)

• Anti-OX40 Agonists • Anti-CD137 Agonists • Preclinical data demonstrating efficacy in GBM: • Strong preclinical data led to ongoing trial through Adult Brain Tumor Consortium (ABTC 1501): Anti- LAG3 or urelumab (anti-CD137) alone and in combination with nivolumab in recurrent GBM (NCT02658981) • Anti-CD40 Agonists • Critical role in T cell priming (activating dendritic Jahan et al, Neuro Oncol 2018 cells, • Anti-GITR Agonists converting cold tumors to hot) • Preclinical data demonstrating efficacy in GBM: • Preclinical data demonstrating efficacy in GBM:

Patel et al, J Immunother Cancer 2016 Shoji et al, Neuro Oncol 2016

33 Classes of Cancer Immunotherapy

34 Oncolytic Viral Therapy

• Previously considered separate from immunotherapy; MOA predicated on selective viral replication and subsequent destruction of cancer cells. • However, anti-tumor immune responses induced as a result of oncolytic viral infections have blurred this distinction. 1. Immune activation through pathogen- associated molecular patterns and pattern recognition receptors 2. Macrophage activation through toll-like receptors (TLRs), resulted in improved T cel infiltration 3. Direct release of tumor antigen upon cell death

35 Lim et al, Nat Rev Clin Oncol 2018 Poliovirus (PVSRIPO)

• Breakthrough Therapy designation from FDA in 2016 on basis of Phase I Trial (ongoing; NCT01491893)

• Genetically engineered poliovirus • Internal ribosome entry site is replaced with that of human rhinovirus type 2 to eliminate neurovirulence • Infects and replicates in cells that express poliovirus receptor (an oncofetal cell adhesion molecule often expressed in GBM) • Administered intratumorally via convection enhanced delivery (CED)

• Preliminary data (ASCO 2016 and ASCO 2017): • N=52, adults with recurrent GBM, solitary tumor <5.5cm, KPS>=70 • One grade 4 intracranial hemorrhage, 17 other grade 3/4 AEs (33%), including cerebral edema, dystonia, seizure, thromboembolism • ~20% of patients alive at 3 years (compared to 4% in institutional historical controls) • Two patients alive at ~5 years post-poliovirus injection without having received any additional interventions

36 Desjardins et al, J Clin Oncol 2017 Toca 511 (vocimagene amiretrorepvec)

• Non-lytic, replicating retrovirus derived from the Moloney murine leukemia virus engineered to encode a modified yeast cytosine deaminase (CD) • Although the virus infects both normal and tumor cells, the tumor cells lack typical viral defense mechanisms that prevent viral DNA integration into their genome

37 https://tocagen.com/product-candidates/ Toca 511: Phase I Trial

• N=45 subjects undergoing surgery for recurrent or progressive high-grade glioma • Virus injected into tissues lining the resection cavity • 6 weeks later patients received Toca FC (extended release formula of prodrug 5- flurocytosine • Median OSToca5 13.6 months trial is (superior ongoing: to external Randomized lomustine control Phase group) II/III trial in • Not only directrecurrent tumoricidal GBM:effect, butpatients also virally undergo induced generalized resection, antitumor then immune response? • 2017 AACR-NCIrandomized-EORTC International to Toca Conference511 onand Molecular Toca TargetsFC versus and Cancer Therapeutics: • Subset of 23 patients physician’sin the phase I trial who choice received higher SOC doses treatment of Toca 511 • Median OS =14.4 mo • Five complete responses: median duration of response 35.7 months • Two patients recorded as having achieved partial responses were, over time, reclassified as complete responders • Preclinical data: high local concentrations of 5-FU generated through Toca 511 eliminate immunosuppressive myeloid cells

38 Cloughesy et al, Sci Transl Med 2016 Adenovirus (1)

• 5-Delta 24RGD (DNX-2401; tasadenoturev) • Live adenovirus (can only replicate in Rb-pathway deficient cells) • Achieves tumor cell targeting through a 24-base deletion of the transforming protein E1A and insertion of an Arg-Gly-Asp motif onto a viral capsid protein • Improves targeting towards alpha integrins • Phase I trial in combination with TMZ • Virus successfully killed GBM cells • 2% complete response rate (some up to 3.5 years and ongoing) • Pseudoprogression, increased immune cell infiltration noted • Phase I trial in combination with pembrolizumab (anti-PD1) (NCT02798406) ongoing • Phase I trial in combination with Interferon-gamma therapy (NCT02197169) ongoing • Preliminary results ASCO 2017: SD or better in 85% of 27 patients, with several robust partial responses, OS rate at 12 months = 33% • No improvement with addition of IFN-gamma

39 Adenovirus (2)

• Aglatimagene besadenovec (AdV-tk) – “gene-mediated cytotoxic immunotherapy” (GMCI) • Replication-incompetent adenovirus transfected to express the HSV thymidine kinase (HSV-TK) gene • converts the prodrug ganciclovir/valacyclovir into a toxic nucleotide analogue that kills replicating tumor cells • Phase I trial (BrTK01): • N=12, newly diagnosed GBM or AA, intratumoral injection at time of surgery followed by valacyclovir • Well tolerated; OS rate at 3 years = 25% • Two Phase II trials • BrTK02 : intratumoral AdV-tk administration and valacyclovir • HGG-01: intra-arterial AdV-tk and ganciclovir • Both are demonstrating favorable PFS and OS outcomes

40 Chiocca et al, J Clin Oncol 2011 Recombinant Immunotoxins (RITs): Antibody variable fragment (or specific ligand) connected to an engineered toxin fragment that kills target cells upon internalization

• D2C7-IT (D2C7 immunotoxin): • Delivered intratumorally by CED • scFv monoclonal ab with high affinity for EGFRwt and EGFRvIII+ GBM tumor cells, bound to PE38KDEL (Pseudomonas Exotoxin A) • In Phase I trial (NCT02303678) • MDNA55 • Fusion protein of a modified IL-4 protein linked to modified version of Pseudomonas Exotin A (PE) • Binds to IL4R, overexpressed by cancer cells and non-malignant cells of TME, and delivers the PE cell-killin agent • Delivered intratumoraly by CED • In multicenter Phase 2b study for recurrent GBM

41 Classes of Cancer Immunotherapy

• Systemic cytokine therapy • Interferons • Interleukins • Vaccines • Peptide • Dendritic cell • DNA • Primary modality: CAR T cells (covered in • Monoclonal antibodies detail by Dr. Don O’Rourke earlier today) • Immune checkpoint inhibitors • Three targets with published clinical trial results to date: • Co-stimulatory receptor agonists • EGFRvIII (O’Rourke et al, Sci Transl Med 2017) • Small molecules • IL-13 R alpha 2 (Brown et al, N Engl J Med 2016; Brown • IDO inhibitors, many others et al, Clin Cancer Res 2015) • Oncolytic viral therapy and immunotoxins • HER-2, virus-specific (Zhang et al, JNCI 2016) • Many other targets in various stages of clinical • Adoptive T cell Transfer development

42 Other systemic approaches to be considered in GBM immunotherapy…

• Modulating other cytokines and chemokines • Anti-IL-6 • Anti IL-10 • CXCL12, CCL2, CXCR4, CCL2, CCR2, and other chemokine/chemokine receptor inhibitors (migration/infiltration of immune cells) • Targeting angiogenesis • Anti-VEGF • Targeting immune cell metabolism (glucose metabolism, amino acid catabolism) • Dichloroacetate (DCA) – induces shift from glycolysis to oxidative phosphorylation • Arginase (ARG1) inhibitors (targets arginase depletion in TME) • IDO2/TDO – blocks conversion of tryptophan to kynurenine • MCT1/2 inhibitors - targets lactate transporters to avoid acidic milieu • MTOR inhibitors – suppresses glycolytic metabolism • A2AR inhibitors - decreases effect of excess adenosine, an immune inhibitory molecule in TME • Targeting the epigenome • results in changes in expression of tumor-associated antigens, components of the antigen processing and presentation machinery, immune checkpoints, and chemokines • HDAC inhibitors: currently ongoing trial of vorinostat with pembrolizumab in newly diagnosed GBM (NCT0342689) • DNAmethyltransferase inhibitors

43 Overview

1) Brief background: cancer immunotherapy

2) Recent key immunotherapy studies in glioblastoma

3) Specific challenges in glioblastoma immunotherapy

44 Major challenges for glioblastoma immunotherapy

• Integration with current standard of care • Radiation • TMZ • Tumor-treating fields • Steroids • bevacizumab • Blood brain barrier • Cold tumor with low mutational burden and paucity of intratumoral T cells • Defects in antigen-presentation machinery • Abundance of immunosuppressive cell populations • GBM tumor heterogeneity • Neurotoxicity • Pseudoprogression

45 “Pseudoprogression” in GBM

Ellingson BM et al (J Neurooncol 2017):

“…Increase in contrast enhancement on post-contrast T1-weighted images mimicking tumor progression, which improves or stabilizes without further intervention or on histopathology as gliosis and reactive changes without any evidence of viable tumor”

-Wide range of reported incidence, depending on definitions used: -as low as 11%, as high at 64%

46 “Pseudoprogression” with GBM immunotherapy: an already pressing problem made worse

67 year old male with recurrent 60 year old male with recurrent GBM GBM treated with nivolumab treated with nivolumab monotherapy on monotherapy on phase I trial: phase I trial:

47 Omuro et al, Neuro Oncol 2017 Roth et al, Neuro Oncol 2016 Circulating tumor material (“liquid biopsy”) in GBM

48 Bettegowda et al, J Molec Diag 2017 Plasma cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA)

• In healthy patients, most DNA fragments are cleared by phagocytes, but a background level of cell-free DNA (cfDNA) exists in the circulation

• In cancer patients, production of circulating tumor DNA (ctDNA) fragments outpaces clearance mechanisms and results in accumulation of cfDNA

• Short half-life (between 16 min and <2.5 hours) Wan et al, Nature Reviews Cancer 2017 • ideal for studying dynamic changes in tumor homeostasis

49 Bettegowda et al, J Molec Diag 2017 How is cfDNA/ctDNA being used in other solid tumors?

• Molecular profiling (ddPCR, targeted next generation sequencing) of paired tumor/ctDNA samples has revealed high concordance rates in breast, colorectal, and lung cancers

• In breast, colon, and ovarian cancers and melanoma, ctDNA levels are correlated with clinical outcomes

• Measurement of cfDNA levels appears to be a viable proxy for tumor burden and a noninvasive means of disease monitoring

50 Siravegna et al, Nature Reviews Clinical Oncology 2017 Spindler et al, Clinical Cancer Res 2012 Circulating DNA as a proxy for disease response/progression (1)

Melanoma (allele-specific ctDNA) (n=36):

51 Schreuer et al, J Trans Med 2016 Circulating DNA as a proxy for tumor burden

Ovarian Cancer (TP53 mutant allele fraction): Melanoma (cfDNA concentration) (n=15):

52 Parkinson et al, PLoS Med 2016 Gangadhar et al, Pigment Cell Melanoma Res 2017 Detecting ctDNA in glioma

A landmark study assessing digital PCR-based technologies for detecting and quantifying ctDNA across human cancers:

***FINE PRINT: Of the 27 gliomas, only 9 were glioblastoma (grade IV). Most were IDH mutant low grade gliomas.

53 Bettegowda et al, Sci Transl Med 2014 cfDNA concentration in IDH mutant gliomas is related to tumor grade and enhancing tumor volume (n=80)

54 Boisselier et al, Neurology 2012 cfDNA quantification (ng/ml) as a surrogate of GBM tumor burden

• Primary Aim: To determine the correlation between cfDNA concentration and radiographic tumor burden using regression modeling

• Multiple imaging parameters • RANO criteria; contrast-enhancing disease, non-enhancing disease

• AV shunting protocol cfDNA

concentration Time

55 Schema

Collaboration with Carpenter lab cfDNA blood draw cfDNA blood cfDNA blood cfDNA blood cfDNA blood #1 taken prior to draw #2 draw #3 draw #4 draw #5 surgery

Day of First Screening Post-op MRI post-radiation Surgery MRI Standard radiation Temozolomide and temozolomide Four week maintenance • Biopsy proven or treatment radiographically break • cfDNA blood draws every suspected high Start End 8 weeks through time of radiation/temozolomide grade glioma radiation/temozolomide disease progression • Blood drawn • Going to OR for Phase II approximately at same resection at Penn time as surveillance MRI scans Preliminary results: plasma cfDNA concentration is closely correlated with tumor burden at time of initial diagnosis of GBM

57 Bagley, et al, ASCO 2018 Annual Meeting Thank you

Questions?

E-mail: [email protected]

58 Supplemental Slides

59 ctDNA in primary brain tumors using Next Gen Sequencing

60 Piccioni et al, ASCO 2015 Annual Meeting Schwaederle et al, Oncotarget 2016