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Serum autoantibodies in ovarian cancer Isabel K 1Oncimmune 1* Macdonald ,Celine 1 Parsy-Kowalska , Jane 1 McElveen , 1 Allen , Jared Caroline 2 Chapman & Andrea 1 Murray Ltd, Nottingham, United Kingdom; 2 Division of Medical Sciences and Faculty of Medicine & Health Sciences, University of Nottingham Graduate Entry Medicine, Nottingham, UK *Corresponding author; [email protected] 1. Ovarian Cancer 4. Methodology Patient Population: 225,000 new cases & 140,000 deaths per year worldwide Recombinant Proteins: 1-2% life time risk in developed world High risk groups?: Early Detection: Current Clinical path: Tumour associated antigens Identified through literature review and scoring >50 years old Human cDNA sequences Family History Gibson cloned into vector (assay and His6 purification tags) Genetic risk groups e.g. BRCA1&2 >70% present at late stage Recombinant proteins overexpressed in E.coli FPLC purification & characterisation >90% 5 year survival for Stage I&II ELISA: 10-30% 5 year survival for Stage III&IV Symptomatic presentation, no screening programme 5 point titration of antigen; captures sera autoantibodies Pilot Study I: NY-ESO-1, CAGE, p53, SOX2, GBU4-5 & Annexin I 63 malignant Ovarian Cancer samples10 High risk offered CA125 &/or HE4 on request Imaging for patients with abnormal Hx, exam or CA125/HE4: Diagnosis by surgery/removal of ovaries (6% mortality) 68% early stage (I&II) Pilot Study II: 22 antigens including NY-ESO-1, CAGE, p53 & SOX2 78 malignant ovarian cancer sera samples10 Disease monitoring following treatment (CA125/HE4) 78 age matched healthy control sera samples 68% early stage (I&II) 2. Autoantibodies Antibodies raised against ‘self’ proteins by host immune response: 5. Results Autoimmune disorders: abnormal immune response to normal protein Pilot Study I: Tumours: abnormal proteins detected Panel Sensitivity for ovarian Cancer of 33% Panel Specificity set at 90% N y e s o - 1 B IR A : 5 0 ( c ir c le ) & 1 6 0 n M ( s o lid c ir c le ) Normal cell 2 .0 Normal host protein Tumour Cell Abnormal ‘tumour associated’ antigen Tumour genesis O D @ 650nm 1 .5 Autoantibodies specific for TAA Produced early in tumour genesis prior to clinical symptoms 1 .0 0 .5 Biological amplifiers : Increase the detectable signal for the corresponding tumour antigen 0 .0 Absent or low concentrations in healthy & benign cohorts Stable unlike antigens which can degrade rapidly Half-lives ≤ 30 days N l r r n n a e e Ovarian Benign Healthy g g i i c c n n rm n n e e a a o Cancer B .B .cohort .C . Ncontrol . C O O O O O S S cohortS S S cohort E E E E E Y Y Y Y N N Y N N Y E S O .N o rm a l N Dot plots of ELISA signal for NY-ESO-1: separated by cohort Detection is minimally invasive and cost effective A n t ig e n ( S a m p le G r o u p ) Pilot Study II: Confirmed pilot study I findings Identified additional markers with high sensitivities of 8-16% per marker 3. EarlyCDT Diagnostic aid for lung cancer already in clinical use (EarlyCDT-Lung1-5) Autoantibody levels for a benign cohort (n=66) were comparable to healthy control populations ELISA to measure a panel of 7 tumour associated serum autoantibodies Substrate binding; colorimetric output Enzyme (HRP) 6. Conclusions Anti-human antibody Patient sera autoantibody Tumour associated antigen ELISA plate Highly reproducible Both pilot studies strongly support previous findings suggesting autoantibodies are suitable biomarkers for ovarian cancer Early stage disease detected Patients with benign tumours, like healthy patients, have low levels of autoantibodies compared to those with malignant ovarian tumours A preliminary EarlyCDT-Ovary panel of 6 autoantibody assays was determined resulting in a combined sensitivity of 41% and specificity of 93.6% for malignant ovarian cancer Detection up to 4 years before CT (current diagnosis pathway) Technically & clinically validated on numerous high risk case control cohorts6&7 92% accuracy ~40% sensitivity & 93% specificity for all stage disease Launched 2012 & clinical performance as expected: >100,000 tests performed in US Diagnostic aid for high risk patients Patient stratification of nodules identified by CT8 NHS prospective randomised controlled clinical screening trial ongoing (UK)9 US health insurance companies reimburse use 7. Future perspectives Autoantibody panels can detect early stage ovarian cancer, distinguishing malignant from benign disease with a very high specificity Research is ongoing at Oncimmune to identify additional autoantibody markers to improve EarlyCDT-Ovary test performance Identifying suitable cohorts is a major obstacle to this work: several large independent cohorts containing a high proportion of early stage disease are needed. Aggressive and treatment resistant sub types must also be represented. Autoantibody panels have the potential to meet requirements for nationwide screening programmes for ovarian cancer References 1. Lam, S., et al. EarlyCDT-Lung: an immunobiomarker test as an aid to early detection of lung cancer. Cancer Prev Res (Phila) 4, 1126-1134 (2011); 2. Healey, G.F., et al. Signal stratification of autoantibody levels in serum samples and its application to the early detection of lung cancer. J Thorac Dis 5, 618-625 (2013); 3. Chapman, C.J., et al. EarlyCDT(R)-Lung test: improved clinical utility through additional autoantibody assays. Tumour Biol 33, 1319-1326 (2012); 4. Macdonald, I.K., et al. Application of a high throughput method of biomarker discovery to improvement of the EarlyCDT((R))Lung Test. PLoS One 7, e51002 (2012); 5. Boyle O, W.D., Khuu A, Jett J, Detterbeck F, Kennedy T, Miller D, Fritsche H, Wood W, Hamilton-Fairley G, Robertson J, Edelsberg J. An autoantibody test to aid in early detection of lung cancer in high-risk patients is likely to be cost-effective. in CHEST, Vol. 138 4 (2010). Tammemagi, C.M., et al. Lung cancer risk prediction: Prostate, Lung, Colorectal And Ovarian Cancer Screening Trial models and validation. Journal of the National Cancer Institute 103, 1058-1068 (2011); 6. Boyle, P., et al. Clinical validation of an autoantibody test for lung cancer. Ann Oncol 22, 383-389 (2011); 7. Murray, A., et al. Technical validation of an autoantibody test for lung cancer. Ann Oncol 21, 1687-1693 (2010); 8. Massion, P. Biormarker-driven programs for lung cancer screening. in World Conference of Lung Cancer, Vol. 8 S6 (Journal of Thoracic Oncology, Sydney, Australia, 2013); 9. Hume, C. Detection in Blood of Autoantibodies to Tumour Antigens as a Case-finding Method in Lung Cancer using the EarlyCDT-Lung test (ECLS). in ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US), Vol. 2014 (University of Dundee, 2014); 10. Clinical Research Centre Cape Cod. For more information: www.oncimmune.com or +44(0)1158231869