CLIA Laboratory Testing of Urinary BRAF V600E DNA mutations: Application in the Management of Patients with Histiocytic Diseases

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1 CLIA Laboratory Testing of Urinary BRAF V600E DNA mutations: Application in the Management of Patients with Histiocytic Diseases Adriana Muniz 1, Mariko Matsutani 1, Karena Kosco 1, John Spinosa 1, Cecile Rose T. Vibat 1, Vlada Melnikova 1, Filip Janku 2 and Mark G. Erlander 1 Mark Erlander, PhD October 8, 2015

2 Company Overview Significant technology around circulating tumor DNA (ctdna) Ultra-sensitive Mutant Allele Enrichment Quantification for dynamic monitoring Optimized for degraded DNA and compatible with multiple specimen types Non-invasive ctdna technology addresses high unmet clinical need Detection of mutation Monitoring for emergence of resistant mutation Monitoring for tumor load CLIA certified, CAP accredited lab to offer diagnostic services NASDAQ: TROV Founded in 1999 NASDAQ listing 2012 Focused on cancer management Headquartered in San Diego, CA Growing body of clinical evidence 2

3 Circulating Tumor DNA (ctdna) Tumor cells Main Advantages of ctdna Captures intratumor heterogeneity Systemic overview of cancer Frequent sampling options for monitoring applications Different analyte options depending on clinical context 3

4 Clinical Utility Opportunities for Circulating Tumor DNA Minimal Residual Disease (MRD) Detection Quantitate Drug Response Monitoring Current Cancer Standard of Care Surgery Adjuvant Therapy Tissue Biopsy Therapy Imaging (every 6-8 weeks) Trovagene PCM adds information that imaging does not currently provide at much earlier time points. TROVAGENE PCM Monitoring for Minimal Residual Disease Tumor Recurrence Molecular Detection of Clinically Actionable Mutations Week 1: Assess Tumor Cell Kill by Therapy Beyond Week 1: Monitor Tumor Mutation Burden Monitoring for Progression and Emergence of Resistance TROVAGENE CLINICAL UTILTY Earlier Detection of Metastatic Disease Alternative to Tissue Biopsy Right Patients Treated with Right Therapy Eliminates Patient Morbidity due to Tissue Biopsy Procedure Immediate Assessment of Drug Effect on Tumor Predict Best Response Weeks in Advance of Imaging Enables Early Switch from Ineffective Therapy Anticipates and Enables Next Therapy to Target Tumor Resistance Copyright 2015 Trovagene, Inc. Confidential 4

5 Circulating Tumor DNA and ECD/LCH CLINICAL YTILITY ECD/LCH ALL STAGES Molecular diagnosis of BRAF mutation patients are placed on targeted therapy Re-staging of cancer ECD/LCH Tissue Biopsy First Line Treatment Imaging (every 2-3 months) TROVAGENE Detection BRAF V600E Monitoring Therapeutic Response BRAF V600E Copyright 2015 Trovagene, Inc. Confidential 5

6 SM Trovagene Precision Cancer Monitoring (PCM ) Platform Extraction and Isolation of ctdna Mutant Allele Enrichment Platform Agnostic detection Detection and Quantification 59% ctdna 2.0 ng/ul gdna 1.4 ng/ul 92% ctdna 4.9 ng/ul gdna 0.04 ng/ul Proprietary error rate detection and normalization. ~0.7µg ctdna/ 100 ml of urine >100-fold enrichment Integration with Commercially Available Platforms Single Molecule Detection Ultra-Sensitive Detection and Quantitative Monitoring 6

7 Proprietary Mutant Allele Enrichment Method Mutants Wild Type Blocker TROVAGENE TECHNOLOGY Platform independent Works with ddpcr or sequencing Sample type agnostic Works on tissue, blood and urine Non-allele specific technology, specific to hotspot of interest Copyright 2014 Trovagene, Inc. Confidential 7

8 Urine BRAFV600E cfdna Assay STEP ONE: Pre-amplification with wild-type suppression to decrease amplification of WT patient DNA STEP TWO: Amplification with primers complimentary to A,B tags (droplet digital PCR) Two-Step Design for 31 bp BRAF V600E Assay tag A A M WT Blocker WT M M M M M M tag B TaqMan Probe B mL urine collected DNA extracted widely variable, 60ng loaded into assay Two-step PCR (1) suppress WT allele amplification (2) determination prevalence of enriched mutant allele Ratio V600E:WT fragments is reported Cutoffs for detection based on 20 BRAF+ and 50 healthy controls

9 Technology Pipeline Single Assays for Monitoring (Ultrasensitive, Quantitative) Multiplex Panel KRAS (Exons 2,3,4) NRAS (Exons 2,3,4) KRAS (5 assays) BRAF (V600E) NRAS (5 assays) PIK3CA (H1047R) EGFR (6 assays) EGFR (Exon 19 Del) BRAF (1 assay) EGFR (Exon 20 T790M) PIK3CA (2 assays) EGFR (Exon 21 L858R) ctrna Detection for Gene Rearrangements ALK RET ROS Multiplex Find-It Panel 100+ genes Copyright 2015 Trovagene, Inc. Confidential 9

10 CLIA Experience Copyright 2015 Trovagene, Inc. Confidential 10

11 Detection: Histiocytic Disorders Urinary ctdna Accurately Identifies BRAFV600E-Positive Patients 1 70% BRAF mutational status obtained 100% Tissue BRAFV600E genotype Indeterminate Genotype BRAF Wildtype n=9 n=6 n=15 BRAFV600E Mutant Urinary ctdna BRAFV600E genotype Trovagene Assay Results Urinary ctdna N=30 p=0.005 Trovagene Assay Results Plasma ctdna N=19 p=0.02 BRAF mutational status obtained BRAF Wildtype n=14 n=16 BRAFV600E Mutant Initial Tissue Results Initial Tissue Results On BRAF inhibitor Wildtype Mutant Unknown On BRAF inhibitor Wildtype Mutant Unknown Allele burden significant different between mutant and WT BRAF mutational status obtained for 100% of patients by urine and only 70% of patients by tissue biopsy 100% concordance between tissue, urine and plasma in treatment naive patients 1 Hyman, Diamond, Janku, Abdel-Wahab et al., Cancer Discovery 2015 Jan;5(1):

12 Clinical Laboratory Experience Between November 2013 and September 2015, 77 urine specimens from 72 individual patients with, or suspected to have histiocytic disease were analyzed for the presence of the BRAFV600E mutation in urine. Five (5) patients had a second BRAFV600E urine test done within 1.5 months to 5 months of the first assessment. CLIA laboratory was blinded to the availability of biopsy or any results of tumor biopsy molecular testing for BRAFV600E. CLIA testing by validated qualitative PCR-enrichment-droplet digital PCR BRAFV600E test.

13 Summary Statistics Diagnosis Total # of Patients # BRAFV600E positive (%) ECD or suspected ECD LCH or suspected LCH ECD+LCH+ Myeloma # BRAFV600E negative (%) (53%) 21 (47%) 18 9 (50%) 9 (50%) 2 2 (100%) 0 (0%) Histiocytosis 3 0 (0%) 3 (100%) Non-ECD 1 1 (100%) 0 (0%) Non LCH 3 0 (0%) 3 (100%) Total (50%) 36 (50%) Copyright 2015 Trovagene, Inc. Confidential 13

14 Participating Institutions Countries USA France Israel Singapore Institutions DFCI Groupe Hospitalier Pitié-Salpêtrière, Paris Johns Hopkins Hospital Mayo Clinic MD Anderson Cancer Center Moffitt Cancer Center NIH Clinical Center Orlando Regional Medical Center Phoenix Children s Hospital Sheba Medical Center, Tel Aviv St. Jude Children's Research Hospital UCSF University of Colorado Hospital University of Mississippi Washington University School of Medicine Copyright 2015 Trovagene, Inc. Confidential 14

15 Longitudinal Monitoring with Applied Quantitation Algorithm Prospective Blinded Study Copyright 2015 Trovagene, Inc. Confidential 15

16 Monitoring: Histiocytic Disorders Dynamic Monitoring Captures Early Progression and Response 1 Successful monitoring tumor dynamics independent of drug class used Patient s tumor progressed within 1 wk of Anakinra withdrawal Demonstration of need for higher frequency urine-based testing to monitor One week Patient responded to Vemurafenib, BRAF inhibitor Demonstrates that continually monitoring patient enables optimal therapy over time 1 Hyman, Diamond, Janku, Abdel-Wahab et al., Cancer Discovery 2015 Jan;5(1):64-71 Copyright 2014 Trovagene, Inc. Confidential 16

17 Quantitative BRAFV600E Detection in Trovagene CLIA Laboratory Launch in CLIA - November 2015 Performance Characteristics ctdna Source Input DNA Performance Urine (specimen collection kit available, room temperature stability = 2 weeks) Plasma (Streck Whole Blood tube, BD Vacutainer K2 EDTA or CPT tube) Accurate quantitation of mutant alleles with any DNA input. Standardized reporting of #copies of mutant DNA per 10 5 genome equivalents (geq) Analytical Sensitivity Assay detects 5 copies of mutant DNA in a background of ~20,000 copies of wild-type DNA (0.025%) Reportable Range Precision Cancer Types Verified range of copies; 95% confidence intervals reported 4 fold discrimination within verified reportable range Histiocytic Disorders and Solid Tumore Copyright 2015 Trovagene, Inc. Confidential 17

18 Conclusions: Urine-based cfdna accurately identifies BRAFV600E mutations in ECD and LCH as compared to tissue-based diagnosis CLIA Experience: Testing of urine obtained from 72 patients with histiocytic diseases demonstrates that the frequency of BRAFV600E mutation detection in urinary ctdna is similar to that observed in tumor biopsies. As a convenient and non-invasive test, assessment of urinary ctdna may help identify patients eligible for the BRAFi therapy, especially in cases when biopsy is unavailable or unsuccessful. Copyright 2015 Trovagene, Inc. Confidential 18

19 Acknowledgements Filip Janku, MD Veronica Holley Goran Cabrilo Funda Meric-Bernstam Omar Abdel-Wahab, MD David Hyman, PhD MD Eli Diamond, MD Raajit Rampal Julie Feldstein Maria Arcila Mario Lacouture Minal Patel Hatim Husain, MD Razelle Kurzrock, MD Julia Johansen, MD Inna Chen, MD Afsaneh Barzi, MD Heinz-Josef Lenz, MD Eric Collisson. MD Margaret Tempero. MD 19

20 Thank you! Copyright 2015 Trovagene, Inc. Confidential 20