MALDI mass spectrometric imaging based identification of clinically relevant signals in prostate cancer using large-scale tissue microarrays Stefan Steurer 1† , Carina Borkowski 1† , Sinje Odinga 1 , Malte Buchholz 1 , Christina Koop 1 , Hartwig Huland 2 , Michael Becker 3 , Matthias Witt 3 , Dennis Trede 4 , Maryam Omidi 5 , Olga Kraus 5 , Ahmad S. Bahar 1 , A. Shoaib Seddiqi 1 , Julius M. Singer 1 , Marcel Kwiatkowski 5 , Maria Trusch 6 , Ronald Simon 1 , Marcus Wurlitzer 5 , Sarah Minner 1 , Thorsten Schlomm 2 , Guido Sauter 1 and Hartmut Schl€ uter 5 1 Institute of Pathology, University Medical Center Hamburg-Eppendorf, Germany 2 Martini-Clinic at University Medical Center Hamburg-Eppendorf, Germany 3 Bruker Daltonik GmbH, Bremen, Germany 4 Steinbeis Innovation Center SCiLS (Scientific Computing in Life Sciences), Bremen, Germany 5 Institute of Clinical Chemistry, Mass Spectrometric Proteomics, University Medical Center Hamburg-Eppendorf, Germany 6 Institute of Organic Chemistry, Mass Spectrometry, University of Hamburg, Germany To identify molecular features associated with clinico-pathological parameters and TMPRSS2-ERG fusion status in prostate cancer, we employed MALDI mass spectrometric imaging (MSI) to a prostate cancer tissue microarray (TMA) containing forma- lin-fixed, paraffin-embedded tissues samples from 1,044 patients for which clinical follow-up data were available. MSI analysis revealed 15 distinct mass per charge (m/z)-signals associated to epithelial structures. A comparison of these signals with clinico-pathological features revealed statistical association with favorable tumor phenotype such as low Gleason grade, early pT stage or low Ki67 labeling Index (LI) for four signals (m/z 700, m/z 1,502, m/z 1,199 and m/z 3,577), a link between high Ki67LI for one signal (m/z 1,013) and a relationship with prolonged time to PSA recurrence for one signal (m/z 1,502; p 5 0.0145). Multiple signals were associated with the ERG-fusion status of our cancers. Two of 15 epithelium-associated signals including m/z 1,013 and m/z 1,502 were associated with detectable ERG expression and five signals (m/z 644, 678, 1,044, 3,086 and 3,577) were associated with ERG negativity. These observations are in line with substantial molecular differences between fusion-type and non-fusion type prostate cancer. The signals observed in this study may characterize molecules that play a role in the development of TMPRSS2-ERG fusions, or alternatively reflect pathways that are activated as a consequence of ERG-activation. The combination of MSI and large-scale TMAs reflects a powerful approach enabling immediate prioritization of MSI signals based on associations with clinico-pathological and molecular data. Prostate cancer is a leading cause of cancer-related mortality in males. Worldwide, more than 600,000 men are diagnosed with prostate cancer each year. 1 Although the majority of prostate cancers are detected at early stages as a result of prostate specific antigen (PSA) screening, many patients present with advanced and metastatic cancer at the time of diagnosis. 2 It is hoped that a better understanding of the molecular biology of prostate cancer will help to improve prostate cancer diagnosis and therapy. Screening methods such as DNA, RNA, and protein arrays have led to the identification of multiple candidate gene altera- tions that occur in usually small fractions of prostate cancers. 3–5 Most of these alterations have not been further analyzed for clini- cal significance, partly because the necessary tissue samples with clinical annotation were lacking in the laboratories where the screening methods were utilized. Matrix-assisted laser desorp- tion/ionization (MALDI) mass spectrometric imaging (MSI) on a tissue microarray (TMA) is an interesting concept for screening for clinically relevant biomarkers. 6–8 This approach combines a systematic search for novel biomarkers by mass spectrometry with a simultaneous analysis of the newly found parameters on hundreds of tumors with clinical follow-up data. In this study, we applied MSI to a TMA containing 1,044 prostate cancers and identified multiple signals associated with clinical outcome in this disease. Key words: MALDI imaging, prostate cancer, tissue microarray, ERG, formalin-fixed tissue, Ki67, prognosis, antigen retrieval Additional Supporting Information may be found in the online version of this article. † S.S. and C.B. contributed equally to this work Grant sponsor: Bremen Economic Development (WFB, project “3D MALDI-Imaging basic experiment); Grant number: FUE0485B; Grant sponsor: European Union Seventh Framework Programme (project “UNLocX”); Grant number: 255931 DOI: 10.1002/ijc.28080 History: Received 13 Dec 2012; Accepted 21 Jan 2013; Online 5 Feb 2013 Correspondence to: Hartmut Schl€ uter, PhD, Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Hamburg Germany, Tel.: 149-40741058795, Fax: 149-405741040097, E-mail: hschluet@uke.de Early Detection and Diagnosis Int. J. Cancer: 133, 920–928 (2013) V C 2013 UICC International Journal of Cancer IJC