Independent Validation of the Prognostic Gene Expression Ratio Test in Formalin Fixed, Paraffin Embedded (FFPE) Mesothelioma Tumor Tissue Specimens

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1 Independent Validation of the Prognostic Gene Expression Ratio Test in Formalin Fixed, Paraffin Embedded (FFPE) Mesothelioma Tumor Tissue Specimens Assunta De Rienzo, Ph.D. 1, Robert W. Cook, Ph.D. 2, Jeff Wilkinson, Ph.D. 3, Corinne Gustafson, Ph.D. 1, William G. Richards, Ph.D. 1, Waqas Amin, M.D. 4,6, Clare E. Johnson, R.N. 2, Kristen M. Oelschlager, R.N. 2, Derek J. Maetzold 2,JohnF.Stone,Ph.D. 3,MichaelD. Feldman, M.D., Ph.D. 4,5, Michael J. Becich, M.D., Ph.D. 4,6, and Raphael Bueno, M.D. 1 1 Division of Thoracic Surgery, Brigham and Women s Hospital, and Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA 2 Castle Biosciences, Inc., 2014 San Miguel Dr., Friendswood 3 DNA Diagnostics, St. Joseph s Hospital and Medical Center, Phoenix, AZ 4 National Mesothelioma Virtual Bank, Pittsburgh, PA 5 Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 6 The Department of Biomedical Informatics at University of Pittsburgh, Pittsburgh, PA Brigham and Women s Hospital Harvard Medical School

2 Conflicts of Interest Investigator initiated grant from Castle Biosciences to RB at BWH Gene Ratio patent licensed to Castle Biosciences by BWH

3 Introduction Malignant Pleural Mesothelioma (MPM) is a rare and aggressive neoplastic disease Approximately 3,200 new cases are diagnosed in United States annually, and the incidence worldwide is estimated to rise in the next two decades Prognosis is generally poor for most MPM patients with a median survival between 4 and 12 months An optimal pre treatment stratification of MPM patients has not been yet defined We have developed a molecular algorithm able to translate comprehensive RNA expression profiling data into simple clinical tests based on the expression levels of a relatively small number of genes

4 The Gene Ratio Technique Tissue X Tissue Y A A b B Unknown Sample A B >1 Predict Tissue X <1 Predict Tissue Y Combine multiple ratios using geometric mean Gordon G.J. et al Cancer Res. 2002

5 Prognostic Gene Expression Ratio Test A four gene three ratio geometrical combination test {(KIAA0977/GDIA1)x(L6/CTHBP)x(L6/GDIA1)}⅓ has been validated in prospectively collected frozen tissue samples to predict postsurgical outcome for MPM patients Test and specimen properties have been established 0.91<score<1.15 = low confidence score range The test is reproducible, repeatable, and can be done with 10% tumor EPP Patients Pleurectomy Patients 100% Median OS Good risk: 16.8m Poor risk: 9.5m 80% p < % Median OS Good risk: 28.5m Poor risk: 7.0m 80% p = % 60% 40% 40% 20% 20% 0% Overall Survival (months) 0% Overall Survival (months) Gordon G.J. et al J Natl Cancer Inst. 2009

6 Overall Survival: Multivariable Model The test demonstrated to provide additional prognostic information to current pathological staging methods in the multivariable model Gordon G.J. et al J Natl Cancer Inst. 2009

7 Survival Curve by Prognostic Score Low risk: 31.9 m Intermediate risk: 19.9 m High risk: 6.9 m Value 1 Value 0 Predictive test high risk ( 1) low risk ( 1) Histological subtype mixed/sarcomatoid epithelial Gordon G.J. et al J Natl Cancer Inst Lymph node status N1 N2 N0

8 Aims of the Study To adapt the test for use with formalin fixed, paraffinembedded (FFPE) tissue on a high throughput microfluidics Real Time PCR (RT PCR) platform To assess the accuracy of the test when performed under CAP accredited, CLIA approved guidelines To validated the high throughput protocol in an independent multi center cohort of MPM tumor cases provided by the National Mesothelioma Virtual Bank

9

10 Relationship Between Gene Expression Scores from fresh frozen and FFPE MPM tissue

11 Concordance study to compare mesothelioma prognosis determined by BWH method and Castle method BWH prediction Gene ratio alone Castle prediction prognosis good poor good 55 4 poor % (61/77) concordance kappa = 0.31 (95% CI ) BWH prediction Gene ratio plus clinical factors Castle prediction prognosis low intermediate high low intermediate high % (70/77) concordance kappa = 0.81 (95% CI )

12 Validation in FFPE from NMVB % surviving Median survival Class 1 = 52 Class 2 = 14 Class 3 = 1

13 Conclusions The test has been successfully adapted to FFPE samples extending its utility beyond previous studies The test remains accurate when performed using a high throughput protocol under CAP accredited, CLIAapproved guidelines This study has validated the prognostic test and stratification strategy using independent samples collected from diverse centers

14 Acknowledgments Thoracic Surgery Oncology Laboratory Raphael Bueno, MD Corinne Gustafson, PhD Michael Archer, MD Yifan Zheng, MD Gavin Gordon, PhD Lingsheng Dong, MD Melissa Coleman, MD Madhu Mani, MD Nhien Dao Peter Sugarbaker Daniele Sciaranghella IMP David J. Sugarbaker, MD National Mesothelioma Virtual Bank Nancy Whelan BWH Tissue and Blood Repository Bank William G. Richards, PhD Thoracic Clinical Research Julianne Barlow Sara Rogers Biostats/Bioinformatics: Beow Y. Yeap, ScD Roederick V. Jensen, PhD John Quackenbush, PhD Yaoyu E. Wang, PhD Brigham and Women s Hospital Harvard Medical School

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