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Spatial UBE2N Protein Expression Indicates Genomic Instability In Gemoll et al. BMC Cancer (2019) 19:710 https://doi.org/10.1186/s12885-019-5856-1 RESEARCH ARTICLE Open Access Spatial UBE2N protein expression indicates genomic instability in colorectal cancers Timo Gemoll1* , Elena Miroll1, Oliver Klein2, Annette Lischka1, Murat Eravci3, Christoph Thorns4 and Jens K. Habermann1,5 Abstract Background: One major hallmark of colorectal cancers (CRC) is genomic instability with its contribution to tumor heterogeneity and therapy resistance. To facilitate the investigation of intra-sample phenotypes and the de novo identification of tumor sub-populations, imaging mass spectrometry (IMS) providesapowerfultechniqueto elucidate the spatial distribution patterns of peptides and proteins in tissue sections. Methods: In the present study, we analyzed an in-house compiled tissue microarray (n = 60) comprising CRCs and control tissues by IMS. After obtaining protein profiles through direct analysis of tissue sections, two validation sets were used for immunohistochemical evaluation. Results: A total of 28 m/z values in the mass range 800–3500 Da distinguished euploid from aneuploid CRCs (p < 0.001, ROC AUC values < 0.385 or > 0.635). After liquid chromatograph-mass spectrometry identification, UBE2N could be successfully validated by immunohistochemistry in the initial sample cohort (p = 0.0274, ROC AUC = 0.7937) and in an independent sample set of 90 clinical specimens (p = 0.0070, ROC AUC = 0.6957). Conclusions: The results showed that FFPE protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value for improved molecular classification. Particularly, the protein expression of UBE2N was validated in an independent clinical cohort to distinguish euploid from aneuploid CRCs. Keywords: Imaging mass spectrometry, Genomic instability, Aneuploidy, UBE2N Background rearrangements and deviations from the euploid chromo- Colorectal cancer (CRC) is the third most frequent type of some number seem to contribute to intratumor hetero- cancer with a worldwide incidence of 1.4 million people geneity being increasingly recognized as a common [1]. Nearly every CRC arises from three pathways, namely, phenomenon across human carcinomas [7]. Intratumor chromosomal instability (CIN), microsatellite instability heterogeneity is histologically indistinguishable at the (MSI) and CpG island methylation (CIMP) [2–4]. While microscopic level [8] but is thought to result in unique the MSI pathway is defined by failures in the DNA mis- molecular subtypes that drive tumor progression and de- match repair system (MMR) and an increased rate of mu- termine the disease outcome of the patient [9, 10]. In this tations [5], the CIMP pathway is characterized by the context, aneuploidy remains underestimated when analyz- altered methylation of genes, which results in modified ing mechanisms of tumor progression and relapse and transcription, e.g., the silencing of tumor suppressor genes when used as a parameter for individualized medicine. [2]. In contrast, the CIN pathway displays a high rate of Several studies have revealed that CIN onset precedes chromosome segregation errors that lead to aneuploidy subclonal expansion and metastatic dispersion in several and affects more than 80% of all CRCs [6]. Chromosomal cancers and shares the same chromosomal aberrations be- tween intratumoral regions [11–13]. Hence, it is of ab- solute importance to elucidate aneuploidy-associated * Correspondence: [email protected] colorectal carcinogenesis respecting its role of intratu- 1Section for Translational Surgical Oncology and Biobanking, Department of Surgery, University of Lübeck and University Medical Center mor heterogeneity for individualized medicine. Schleswig-Holstein, Campus Lübeck, Lübeck, Germany Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gemoll et al. BMC Cancer (2019) 19:710 Page 2 of 8 Thus far, the emphasis of research into molecular tumor subdivided into carcinomas with lymph node positive heterogeneity has been on the genomic and transcriptomic and negative metastasis, euploid and aneuploid cancers, levels, including evolutionary divergence between primary UICC I/II and UICC III/IV cancers and patients with a tumors and metastatic outgrowths [14]. Traditionally, survival of less and more than 60 months. Patients with spatial heterogeneity in the tissue samples has been limited palliative treatment (R1 resection), survival < 30 days after to microscopy-based assessment, e.g., immunofluorescence surgery, and neoadjuvant radiotherapy for rectal tumors and fluorescence in situ hybridization. These targeted tech- were excluded. Patients matching the Amsterdam II- niques are limited (i) to very low throughput, (ii) to few criteria for the hereditary-non-polyposis-colorectal-cancer molecules, (iii) in quantifying expression levels and (iv) in (HNPCC) syndrome were also excluded. The study was comparing heterogeneity between samples. On the prote- conducted in adherence to the approval by the local Eth- ome, lipidome and metabolome level, matrix-assisted laser ical Committee of the University of Lübeck (#07–124). desorption/ionization (MALDI) imaging mass spectrometry (IMS) has emerged and combines de novo identifications Genomic instability assessment with unsupervised and unlabeled spatial resolution. By cor- Genomic instability was assessed by nuclear DNA ploidy relating molecular information with histology at spatial measurements by means of image cytometry using Feul- resolution, MALDI-IMS enables the analysis of both the gen stained imprints as described previously [15, 17]. relative abundance and distribution of proteins. In this con- Briefly, images of at least 500 nuclei were selected manu- text, we could already show that ploidy measurements and ally to create DNA ploidy histograms using the ACAS im- MALDI-IMS using fresh frozen material can identify prog- aging system (Ahrens ACAS, Hamburg, Germany). nostic markers in CRC, e.g. thymosin beta-4 [15]. However, Histograms were classified in euploid and aneuploid cell routine diagnostics are based on FFPE material [16]. Thus, populations according to Auer [17]. Hereby, DNA histo- we now aimed to identify prognosis-associated proteins in grams of types I (diploid), II (tetraploid), and III (diploid- FFPE material by MALDI-IMS. Depicted targets could pro- proliferating) were characterized as euploid cell popula- vide new insights into genomic instability, tumor hetero- tions. All DNA histograms were evaluated by three inde- geneity and treatment options for individualized medicine. pendent investigators who were unaware of the clinical and histopathological data of the patients. Methods Patient cohorts MALDI-imaging mass spectrometry experiments For the protein profiling by IMS and the first validation Prior to trypsin and matrix application, optical images of by IHC, one tissue microarray (TMA) was compiled the training TMA were obtained using a standard flatbed containing 40 human colorectal carcinomas (CRC) and scanner (ScanMaker 9800X2, Microtek, Germany). A total 20 normal human colorectal mucosa samples (training of 200 μl of the trypsin solution (20 μg, 20 mM ammo- TMA, Table 1) representing 20 patients in total. nium bicarbonate/acetonitrile 9:1) was applied directly A second TMA of 60 human CRCs and 30 normal onto the section using an automated spraying device human colorectal mucosa samples was used as a subse- (ImagePrep, Bruker Daltonik, Germany) according to the quent independent validation set for immunohisto- manufacturer’s protocol, with 39% power ± 0% modula- chemistry only (validation TMA, Table 2). For both tion. Tissue incubation with the trypsin solution was per- TMAs, all of the colorectal cancer samples were equally formed for 3 h at 37 °C in a moist chamber. Following Table 1 The clinical characteristics of colon cancer samples used for tissue microarray (TMA) analysis (training set) by means of MALDI-imaging and immunohistochemistry Clinical parameter Samples of adjacent non-neoplastic mucosa Samples of CRCsa, b Sex [male/female] 14/6 20/20 Age [years] 64.5 66.2 Ploidy [euploid/aneuploid] 20/20 T-status [1/2/3/4] 0/4/28/8 N-status [0/1/2] 20/10/10 M-status [0/1] 36/4 Grading [1/2/3] 0/28/12 Survival status [alive/dead] 18/22 Survival [months] 77.4 a Two samples could not be included in the SPTBN1 staining b Three samples could not be included in the UBE2N staining Gemoll et al. BMC Cancer (2019) 19:710 Page 3 of 8 Table 2 Clinical characteristics of colon cancer samples used for tissue microarray (TMA) analysis (validation set) by means of immunohistochemistry Clinical parameter Samples of adjacent non-neoplastic mucosa Samples of CRCsa Sex [male/female] 16/14 30/30 Age [years] 56.7 68.7 Ploidy [euploid/aneuploid] 30/30 T-status [1/2/3/4] 2/11/39/8 N-status [0/1/2] 30/23/7 M-status [0/1] 60/0 Grading [1/2/3] 3/41/16 Survival status [alive/dead] 33/27 Survival [months] 68.1 a
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