Immunotherapy for Glioblastoma: Updates and Future Directions

Transcription

1 Immunotherapy for Glioblastoma: Updates and Future Directions Stephen Bagley, MD, MSCE Instructor of Medicine Friday, June 8, 2018

2 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

3 Overview 1) Brief background: cancer immunotherapy 2) Recent key immunotherapy studies in glioblastoma 3) Specific challenges in glioblastoma immunotherapy 3

4 4

5 FDA approvals for immune checkpoint inhibitors (as of 3/23/18) 5 Ribas, et al Science 2018

6 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

7 Cancer evasion of immunosurveillance Glioblastoma 7 Vonderheide, Cancer Cell 2018

8 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

9 Local and systemic immunosuppression in glioblastoma 9 Lim et al, Nat Rev Clin Oncol 2018

10 Overview 1) Brief background: cancer immunotherapy 2) Recent key immunotherapy studies in glioblastoma 3) Specific challenges in glioblastoma immunotherapy 10

11 Classes of Cancer Immunotherapy 11 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 Interferon-beta treatment has been shown to yield a net immune stimulating effect against glioblastoma initiating cells (stem cells) In older clinical trials, only limited clinical activity of interferon-beta and interferon-alpha therapies in combination with alkylating chemotherapy for high grade glioma Phase III trial ongoing in China Interferon-gamma is currently being studied in combination with DNX-2401 (oncolytic adenovirus) for recurrent glioma IFN-gamma very poorly tolerated, did not seem to improve outcomes compared to DNX-2401 alone (Lang et al, SNO 2017) IL-12 and GM-CSF used as vaccine adjuvants Wolpert et al,, PLoS One 2015; Yamamuro et al, Int J Oncol 2015 Buckner et al, Cancer 2001; Groves, et al, Br J Cancer 2009 Motomura et al, Cancer 2011

12 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 12

13 Vaccine Therapies for Glioblastoma Previously considered one of the most promising approaches to improving the outcomes of patients with glioblastoma Negative results from several phase II and phase III trials have challenged the concept of vaccination as a single-modality immunotherapy Cold Hot 13 Johnson et al, Clin Cancer Res 2017

14 Rindopepimut (CDX-110, PEPvIII) Peptide vaccine that mimics EGFR variant III (EGFRvIII), a constitutively active mutant form of EGFR expressed exclusively on GBM cells in 25 30% of patients Advantage: neoantigen expressed exclusively on tumor cells, limiting risk of on-target, off-tumor toxicities Disadvantage: EGFRvIII is heterogeneously expressed (both spatially and temporally) on GBM cells in vivo ACT IV Trial: Randomized, Phase III trial in newly diagnosed EGFRvIII+ GBM N=745, GBM s/p maximal surgical resection and standard chemoradiation without progression 405 patients with MRD (<2cm 2 contrast enhancing tumor) 1:1 randomization to rindopepimut vs. control via monthly intradermal injection concurrent with TMZ Primary endpoint: OS in the MRD subgroup 14 Del Vecchio et al, Oncogene 2013 Weller et al, Lancet Oncol 2017

15 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? 15 *** 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 Reardon et al, ASCO 2015

16 ICT-107: a multipeptide dendritic cell vaccine 16 Patient-derived dendritic cells (post-surgery apheresis) incubated ex vivo with six peptides commonly overexpressed in GBM cells compared to nonmalignant tissues ( warehouse approach using tumor-associated antigens): Melanoma-associated antigen 1 (MAGEA1) HER2 interferon-inducible AIM2 I-dopachrome tautomerase (DCT) melanocyte protein (PMEL) IL-13 receptor subunit-alpha2 (IL- 13Ralpha2) Phuphanich, et al, Cancer Immunol. Immunother 2013 Randomized, Phase II Trial N=124, newly diagnosed GBM <1cm 3 residual tumor, completed first-line chemoradiation 77 patients (62%) HLA-A2 positive Phase III trial was opened for Randomized 2:1 to ICT-107 or control patient (autologous DCs accrual not exposed in to 2016 the GBM but antigens) was induction terminated QWx4 vaccinations, due followed to lack by maintenance vaccinations plus TMZ of funding June 21, 2017 Median PFS improved with ICT-107 (HR 0.57, p=0.01). Median OS favored ICT-107 by 1.6 months in ITT group (NOT statistically significant; p=0.64) HLA-A2+ subgroup: median OS 18.3 months with ICT-107 v months with control Wen et al, SNO Annual Meeting 2014

17 DCVax-L (Northwest Biotherapeutics): whole tumor lysate used to pulse autologous dendritic cells 17 Serves as a source for the entire spectrum of tumor antigens ( personalized vaccine), but logistically challenging (requires collection of tumor sample from each individual patients for processing and DC stimulation) Liau et al, Clin Cancer Res 2005 Two prior phase I/II studies at UCLA N=20 newly diagnosed GBM Median PFS ~24 months Median OS ~36 months (with reported tail of survival curve) Still no published data As of July, 2011, 33% of patients had reached or (or exceeded anything 4 years median peer OS and reviewed 27% had reached or exceeded 6 years median OS Most for recently presentation the company reported two at patients a have exceeded 10 years survival scientific meeting) Phase III trial ongoing (started 12/2006): 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 exceeding 48 month OS

18 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 (NCT ) INO-5401: WT1, htert, and PSMA (given via IM electroporation) (NCT ) 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 18 Rampling R, Clin Cancer Res 2016 Bloch et al, Neuro Oncol 2014 Mitchell et al, Nature 2015 Reardon, Neuro Oncol 2017

19 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 19

20 Immune checkpoint inhibition Must be preceded by T cell priming Salama et al, Clin Cancer Res

21 FDA approvals for immune checkpoint inhibitors (as of 3/23/18) 21 Ribas, et al Science 2018

22 22 Neoantigens increase likelihood of T cell priming against the tumor

23 Mutational burden and benefit from immune checkpoint inhibition Melanoma NSCLC Johnson et al, Cancer Immunol Res 2016 Rizvi et al, Science

24 Tumor mutational burden is generally low in GBM Alexandrov et al, Nature 2013 Garg et al. Oncoimmunology 2017 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 24

25 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 mo bev) or OS rate at 12 months 25 Reardon DA et al, World Federation of Neuro-Oncology Societies Quadrennial Meeting Zurich, Switzerland 2017

26 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 Reardon DA et al, World Federation of Neuro-Oncology Societies Quadrennial Meeting Zurich, Switzerland 2017

27 Exploratory phase I cohorts from CheckMate 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 Omuro et al, Neuro Oncol 2018

28 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

29 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 29 Reardon DA et al, Society for Neuro-Oncology Annual Meeting 2016 Lim M et al, ASCO Annual Meeting 2016 Reardon DA et al, Society for Neuro-Oncology Annual Meeting 2017

30 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 cell activation in GBM than PD-L1 Antonios et al, Neuro Oncol Reardon et al, Neuro Oncol 2017 Nduom et al, Neuro Oncol 2016 Berghoff et al, Neuro Oncol 2015 Yearley et al, Clin Cancer Res 2017 Ricklefs et al, Neuro Oncol 2017 Bloch et al, Clin Cancer Res 2017

31 Small molecule inhibitors previously studied for immunomodulation in GBM IDO inhibitors: PF : 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

32 Small molecule inhibitors in development for immunomodulation in GBM IDO inhibitors Epacadostat (phase II in combination with nivolumab for selected advanced cancers) (NCT ) Indoximod (Phase I/II with temozolomide for temozolomide-refractory brain tumors) (NCT ) CSF1R inhibitors FPA008 (phase I in combination with nivolumab for selected advanced cancers) (NCT ) STAT3 inhibitors WP1066 (phase I in recurrent glioma and brain metastases) (NCT ) 32

33 Immune co-stimulatory receptor agonists in development for GBM (alone or as adjuncts to immune checkpoint inhibitors) Anti-OX40 Agonists Preclinical data demonstrating efficacy in GBM: Jahan et al, Neuro Oncol 2018 Anti-GITR Agonists Preclinical data demonstrating efficacy in GBM: Anti-CD137 Agonists 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 (NCT ) Anti-CD40 Agonists Critical role in T cell priming (activating dendritic cells, converting cold tumors to hot) Preclinical data demonstrating efficacy in GBM: Patel et al, J Immunother Cancer 2016 Shoji et al, Neuro Oncol

34 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 34

35 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 pathogenassociated 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

36 Poliovirus (PVSRIPO) Breakthrough Therapy designation from FDA in 2016 on basis of Phase I Trial (ongoing; NCT ) 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

37 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

38 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 OS Toca months trial is (superior ongoing: to external Randomized lomustine control Phase group) II/III trial in Not only direct recurrent tumoricidalgbm: effect, but patients also virally undergo induced generalized resection, antitumor then immune response? randomized to Toca 511 and Toca FC versus physician s choice SOC treatment 2017 AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics: Subset of 23 patients in the phase I trial who received higher doses 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

39 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) (NCT ) ongoing Phase I trial in combination with Interferon-gamma therapy (NCT ) 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

40 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

41 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 (NCT ) 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

42 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 Primary modality: CAR T cells (covered in detail by Dr. Don O Rourke earlier today) Three targets with published clinical trial results to date: EGFRvIII (O Rourke et al, Sci Transl Med 2017) IL-13 R alpha 2 (Brown et al, N Engl J Med 2016; Brown et al, Clin Cancer Res 2015) HER-2, virus-specific (Zhang et al, JNCI 2016) Many other targets in various stages of clinical development 42

43 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 (NCT ) DNAmethyltransferase inhibitors 43

44 Overview 1) Brief background: cancer immunotherapy 2) Recent key immunotherapy studies in glioblastoma 3) Specific challenges in glioblastoma immunotherapy 44

45 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

46 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

47 Pseudoprogression with GBM immunotherapy: an already pressing problem made worse 67 year old male with recurrent GBM treated with nivolumab monotherapy on phase I trial: 60 year old male with recurrent GBM treated with nivolumab monotherapy on phase I trial: 47 Omuro et al, Neuro Oncol 2017 Roth et al, Neuro Oncol 2016

48 Circulating tumor material ( liquid biopsy ) in GBM 48 Bettegowda et al, J Molec Diag 2017

49 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) ideal for studying dynamic changes in tumor homeostasis Wan et al, Nature Reviews Cancer Bettegowda et al, J Molec Diag 2017

50 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

51 Circulating DNA as a proxy for disease response/progression (1) Melanoma (allele-specific ctdna) (n=36): 51 Schreuer et al, J Trans Med 2016

52 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

53 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

54 cfdna concentration in IDH mutant gliomas is related to tumor grade and enhancing tumor volume (n=80) 54 Boisselier et al, Neurology 2012

55 cfdna concentration 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 55 Time

56 Schema Collaboration with Carpenter lab cfdna blood draw #1 taken prior to surgery cfdna blood draw #2 cfdna blood draw #3 cfdna blood draw #4 cfdna blood draw #5 Screening Biopsy proven or radiographically suspected high grade glioma Day of Surgery Going to OR for resection at Penn Post-op MRI Standard radiation and temozolomide Start radiation/temozolomide End radiation/temozolomide Phase II Four week treatment break First post-radiation MRI Temozolomide maintenance cfdna blood draws every 8 weeks through time of disease progression Blood drawn approximately at same time as surveillance MRI scans

57 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

58 Thank you Questions? 58

59 Supplemental Slides 59

60 ctdna in primary brain tumors using Next Gen Sequencing 60 Piccioni et al, ASCO 2015 Annual Meeting Schwaederle et al, Oncotarget 2016