Serum antibodies against tumor-specific frameshift peptides in Lynch syndrome as a potential tumor-related marker Miriam Reuschenbach, Nicoline Hoogerbrugge, Jolanda deVries, Mirjam Tariverdian, Martina Kadmon, Elke Holinski-Feder, Monika Morak, Verena Steinke, Nils Rahner, Matthias Kloor, Magnus von Knebel Doeberitz and The German HNPCC Consortium Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Germany and Clinical Cooperation Unit Applied Tumor Biology, German Cancer Research Center (DKFZ) Heidelberg, Germany InSiGHT Meeting, San Antonio, TX, USA, 2011 MSI-H colorectal cancers are particularly immunogenic • MSI-H CRC show dense infiltration with activated, cytotoxic lymphocytes • The infiltration with lymphocytes is particulary pronounced in the hereditary form of MSI-H CRC (Lynch syndrome) CD8 • Crohn‘s like reaction is often observed in MSI-H CRC • MSI-H CRC are associated with good prognosis and low number of distant metastases The immunogenicity of MSI-H CRC is caused by MMR deficiency-induced Frameshift Peptides (FSPs) coding microsatellite TGT . AAA . AAA . AAA . ACG . TGC . TGG . CTA . GCT . GA......... C K K K T C W L A FSP neo-sequence . . . SELECTION OF TARGETS Identification of coding microsatellites and prediction of frameshift peptides in silico Determination of mutation frequency Evaluation of immunogenic potential in vitro Analysis of immune responses in patients ex vivo Vaccination ? Diagnostic markers ? FSP specific serum antibody • prevalence is highest in MSI-H CRC patients and Lynch syndrome mutation carriers • levels are highest in MSI-H CRC patients peptide MSI-H CRC patients healthy mutation carriers healthy controls AIM2(-1) 5.8% (4/69) 0.0% (0/31) 3.8% (2/52) CASP5(-1) 4.3% (3/69) 6.5% (2/31) 1.9% (1/52) ZNF294(-1) 4.3% (3/69) 0.0% (0/31) 0.0% (0/52) MARCKS(-1) 2.9% (2/69) 0.0% (0/31) 1.9% (1/52) TGFBR2(-1) 10.1% (7/69) 9.7% (3/31) 5.8% (3/52) TGFBR(-1)-N 11.6% (8/69) 0.0% (0/31) 5.8% (3/52) TGFBR(-1)-C 7.2% (5/69) 19.4% (6/31) 3.8% (2/52) TAF1B(-1) 11.6% (8/69) 6.5% (2/31) 1.9% (1/52) Any FSP 29.0% (20/69) 29.0% (9/31) 15.4% (8/52) p=0.241 p=0.036 Mann Whitney test • Lynch syndrome patients with history of MSI-H colorectal cancer (n=69) • Healthy Lynch syndrome mutation carriers without tumor history (n=31) • Healthy controls, age matched (n=52) Reuschenbach et al (2010) healthy controls MSI-H cancer patients 8/25 32.0% normalized OD > 0.1 normalized OD between 0.07 and 0.1 2/34 5.9% Antibody levels in MSI-H CRC patients are highest • shortly after tumor resection and in advanced stages Association with time since tumor resection 60.0% (3/5) < 3 months (p=0.020, Fisher’s exact test ) 28.6% (2/7) 3 to 12 months 27.7% (10/36) > 12 months Association with tumor stage 42.9% (6/14) UICC III (p=0.051, Fisher’s exact test ) 13.3% (4/30) UICC I or II FSP-specific antibodies in sera withdrawn preoperatively Lynch syndrome patients with MSI-H cancer, preoperatively (n=7) TGFBR2(-1) AIM2(-1) TAF1B(-1) ZNF294(-1) MARCKS(-1) CASP5(-1) Patient 1 any FSP normalized OD > 0.1 Patient 2 Patient 3 normalized OD between 0.07 and 0.1 Patient 4 Patient 5 Patient 6 Patient 7 28.7% 0.0% 28.7% 28.7% 0.0% 0.0% 71.4% (5/7) New Multiplex Assay to detect FSP Antibodies • Extension of antigen panel to FSPs derived from other cMS-containing genes • Prospective serum sampling and correlation of antibodies with the course of disease 60 FSPs derived from cMS containing genes Summary and Discussion • Serum antibodies against FSPs can be found in patients with MSI-H CRC • High antibody levels are restricted to patients with short time interval between tumor resection and serum sampling • Low level antibody reactivity can be found in healthy Lynch syndrome mutation carriers • A high throughput multiplex assay is established Acknowledgements Department of Applied Tumor Biology Matthias Kloor Kathrin Bauer Magnus von Knebel Doeberitz Department of Surgery, University Heidelberg, Germany Mirjam Tariverdian Martina Kadmon Moritz Koch Jürgen Weitz Markus W. Büchler Radboud University, Nijmegen, The Netherlands Jolanda de Vries Nicoline Hoogerbrugge Institute of Human Genetics, Innsbruck, Austria Katharina Wimmer The German HNPCC Consortium Medizinisch Genetisches Zentrum Munich, Germany Monika Morak Elke Holinski-Feder Funded by the Deutsche Krebshilfe (German Cancer Aid) Institute of Human Genetics, University Bonn, Germany Verena Steinke Nils Rahner