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Working group Prof. Klopfleisch

Staff

Research focus:

 

Digital pathology

Pathologists have been using light microscopes to diagnose diseases in tissue sections since the development of modern pathology by Rudolf Virchow in 1858, but the rapid development of digital information technology over the last three decades has now reached classical pathology, like all other aspects of modern life.

The relatively new technology of slide scanners makes it possible to digitize histological sections on glass slides, visualize them on a computer screen, analyze them automatically and exchange them quickly over long distances via networks. Currently, fully digitized scans of glass slides, so-called Whole Slide Images (WSI), are increasingly used in the routine diagnostics of pathology institutions and it can be assumed that they will be an integral component of veterinary pathology in the future. Furthermore, automated image recognition algorithms are used in particular for the automated quantification of objects in tissue sections, e.g. mitotic cells, immunohistochemistry-positive cells, etc.

Our group is currently involved in various projects for the evaluation and validation of digital pathology methods in the fields of diagnostics, research and education and also aims to develop new software approaches for the automatic image analysis of histological and cytological preparations. One focus here is on tumor diagnostics and toxicological pathology and, in particular, the automatic recognition of mitotic figures in tumor tissues for tumor grade assessment, the development of automatic tumor recognition and differentiation and the automated preselection of pathologically altered tissue areas in tissue samples from toxicity tests on HE sections.

ResearchGate project: https://www.researchgate.net/project/Digital-Pathology-New-Approaches-to-the-Automated-Image-Analysis-of-Histologic-Slides

Publications:

  1. Bertram CA, Aubreville M, Gurtner C, Kershaw O, Meier A, …., Kiupel M, Maier A, Klopfleisch R. Computerized Calculation of Mitotic Count Distribution in Canine Cutaneous Mast Cell Tumor Sections: Mitotic Count is Area-Dependent. Vet Pathol, 2020, doi: 10.1177/0300985819890686, PMID: 31808382

  2.  Bertram CA, Aubreville M, Marzahl C, Maier A, Klopfleisch R. A large-scale dataset for mitotic figure assessment on whole slide images of canine cutaneous mast cell tumor. Sci Data. 2019 Nov 21;6(1):274. doi: 10.1038/s41597-019-0290-4. PMID: 31808382

  3. Bertram CA, Gurtner C, Dettwiler M, Kershaw O, Dietert K, Pieper L, Pischon H, Gruber AD, Klopfleisch R. Validation of Digital Microscopy Compared With Light Microscopy for the Diagnosis of Canine Cutaneous Tumors. Vet Pathol. 2018, 55(4):490-500. doi: 10.1177/0300985818755254. PMID: 29402206

  4. Bertram CA, Klopfleisch R. The Pathologist 2.0: An Update on Digital Pathology in Veterinary Medicine. Vet Pathol. 2017 Sep;54(5):756-766. doi: 10.1177/0300985817709888. PMID: 28578626

  5. Bertram CA, Firsching T, Klopfleisch R. Virtual Microscopy in Histopathology Training: Changing Student Attitudes in 3 Successive Academic Years. J Vet Med Educ. 2018; 45(2):241-249. doi: 10.3138/jvme.1216-194r1. PMID: 29099317

  6. Aeffner F, Adissu HA, Boyle MC, Klopfleisch R, Newbigging S, Schaudien D, Turner O, Wilson K. Digital Microscopy, Image Analysis, and Virtual Slide Repository. ILAR J. 2018; 59(1):66-79. doi: 10.1093/ilar/ily007. PMID: 30535284

  7. Aubreville M, Bertram CA, Jabari S, Marzahl C, Klopfleisch R, Andreas Maier. Learning New Tricks from Old Dogs -- Inter-Species, Inter-Tissue Domain Adaptation for Mitotic Figure Assessment. (https://arxiv.org/pdf/1911.10873.pdf)

  8. Aubreville M, Bertram CA, Marzahl C, Gurtner C, Dettwiler M, Schmidt A, Bartenschlager F, Merz S, Fragoso M, Kershaw O, Klopfleisch R, Maier A. Field of Interest Prediction for Computer-Aided Mitotic Count, 2019. (https://arxiv.org/pdf/1902.05414.pdf)

  9. Marzahl C, Aubreville M, Bertram CA,…., Klopfleisch R, Maier A. Deep Learning-Based Quantification of Pulmonary Hemosiderophages in Cytology Slides. (https://arxiv.org/pdf/1908.04767.pdf)

  10. Aubreville M,  Bertram CA, Klopfleisch R, Maier A. SlideRunner - A Tool for Massive Cell Annotations in Whole Slide Images. (https://arxiv.org/pdf/1802.02347v1.pdf)

  11. Aubreville M,  Bertram CA, Klopfleisch R, Maier A. Augmented Mitotic Cell Count using Field Of Interest Proposal. https://arxiv.org/pdf/1810.00850.pdf

  12. Aubreville M,  Bertram CA, Klopfleisch R, Maier A. Field Of Interest Proposal for Augmented Mitotic Cell Count: Comparison of two Convolutional Networks. (https://arxiv.org/pdf/1810.09197.pdf)

  13. Aubreville M, Krappmann M, Bertram CA, Klopfleisch R, Maier A. A Guided Spatial Transformer Network for Histology Cell Differentiation. Eurographics Workshop on Visual Computing for Biology and Medicine At: Bremen, Germany, 2017, DOI: 10.2312/vcbm.20171233

  14. Krappmann, M.; Aubreville, M.; Maier, A.; Bertram, C.; Klopfleisch, R. Classification of Mitotic Cells Potentials: beyond the Limits of Small Data Sets. In: Bildverarbeitung für die Medizin 2018,– Andreas Maier (Hrsg.) (1 Aufl.), Vieweg: Springer, S. 245–250, ISBN: 978-3-662-56536-0

 

Methods in toxicological preclinical and drug safety studies

In cooperation with international research-based pharmaceutical companies and contract research organizations, we are currently developing innovative, more efficient methods for preclinical studies in the field of drug safety and efficacy. One main focus here is the development of more sensitive and reproducible methods of histopathological analysis using digital pathology and surrogate markers in order to achieve a reduction in the number of test animals in accordance with the 3R principle.

Furthermore, new approaches for the efficient performance of the micronucleus test (Giemsa, Acridine-Orange) and bone marrow analysis (myeloid-erythroid ratio) in rats and mice are being developed. QuPath, Sliderunner, and in certain projects Visiopharm, are used to annotate digital histological preparations and are used both in these software solutions and with various IT cooperation partners to develop deep learning algorithms for automatic image analysis.

 

Veterinary oncology

  • Metastasis-associated global mRNA and protein expression patterns in canine mammary and mast cell tumors
  • Circulating tumor cells as prognostic factors in canine mammary tumors
  • c-Kit dependent gene expression patterns in canine mast cell tumors

The molecular mechanisms of carcinogenesis of tumors in non-human mammals are still largely unknown. In various projects, mainly canine and feline tumors are investigated to identify gene mutations and gene expression changes in tumor cells with relevance for the development, diagnosis, prognosis and therapy of these tumor types.

Publications:

  1. Klopfleisch R (ed.), A Short Textbook of Veterinary Oncology / Kurzlehrbuch der Veterinäronkologie, Springer, available from 08/2016

  2. Bertram CA, Aubreville M, Gurtner C, Kershaw O, Meier A, Kiupel M, Maier A, Klopfleisch R. Computerized Calculation of Mitotic Count Distribution in Canine Cutaneous Mast Cell Tumor Sections: Mitotic Count is Area-Dependent. Vet Pathol, 2020

  3.  Aupperle-Lellbach H, Törner K, Staudacher M, Müller E, Steiger K, Klopfleisch R. Characterization of 22 Canine Pancreatic Carcinomas and Review of Literature. J Comp Pathol. 2019 ;173:71-82.

  4. Merz SE, Kershaw O, Petrick A, Gruber AD, Klopfleisch R, Breithaupt A. Tumour, but not Age-associated, Increase of Senescence Markers γH2AX and p21 in the Canine Eye. J Comp Pathol. 2019;173:41-48.

  5. Grassinger JM, Aupperle-Lellbach H, Erhard H, Merz S, Klopfleisch R. Detection of BRAF mutation in canine prostatic diseases. Tierarztl Prax Ausg K Kleintiere Heimtiere. 2019; 47(5):313-320.

  6. Grassinger JM, Merz S, Aupperle-Lellbach H, Erhard H, Klopfleisch R. Correlation of BRAF Variant V595E, Breed, Histological Grade and Cyclooxygenase-2 Expression in Canine Transitional Cell Carcinomas. Vet Sci. 2019, 19;6(1).

  7. Klopfleisch R, Kohn B, Gruber AD. Mechanisms of tumour resistance against chemotherapeutic agents in veterinary oncology. Veterinary Journal, 207:63-72, 2016.

  8. Klopfleisch R. Personalized medicine in veterinary oncology: one to cure just one. Veterinary Journal, 205:128-35, 2015. 

  9. Klopfleisch R, von Euler H, Sarli G, Pinho SS, Gärtner F, Gruber AD. Molecular carcinogenesis of canine mammary tumors: news from an old disease. Veterinary Pathology,48:98-116, 2011.

  10. Delcour N.M., Klopfleisch R., Gruber A.D. and Weiss A.T.A. (2013). Canine Cutaneous Histiocytomas are Clonal Lesions as Defined by X-linked Clonality Testing. Journal of Comparative Pathology 149: 192-198.

 

Cutaneous lupus erythematosus and pemphigus diseases in dogs, cats and horses

Autoimmune diseases are relatively rare diseases in pets. Nevertheless, they represent a frequently asked and thus to be confirmed/excluded clinical differential diagnosis, especially in dermatology. While, for example, a serological examination for circulating anti-nuclear antibodies (ANA) is often helpful in the diagnosis of systemic lupus erythemathosus, sufficiently sensitive and specific serological tests for cutaneous lupus and pemphigus are not yet available for pets. For these diseases, histopathology of skin biopsies and the detection of bound antibodies in the histological preparations is therefore the method of choice. In various projects, we are currently trying to refine the methods for detecting autoantibodies and correlate them with the clinical picture and therapeutic success in order to be able to provide better prognoses and recommendations for clinical colleagues in the future.

 

Foreign body reaction and M1 / M2 macrophage polarization in vivo

The foreign body reaction during the implantation of biomaterials is a double-edged sword. On the one hand, a certain reaction is necessary to ensure anchoring or resorption of the implant, but too strong a reaction leads to destruction of the implant or severe scar tissue formation.

Recent findings on the biology of macrophages have shown that macrophages are not just pure antigen-phagocytizing and -presenting and thus pro-inflammatory cells. On the contrary, both macrophage subtypes, the “pro-inflammatory” M1 macrophages, the various “anti-inflammatory” M2 macrophage subtypes and even the foreign body giant cells are necessary both for the defense against noxae and in their entirety for the regeneration and repair of almost all tissues. Depending on the implantation target, differently orchestrated and composed macrophage responses are therefore desirable to achieve the desired implant effect.

Most of the currently available data and differentiating M1 / M2 markers have been produced or discovered in in vitro models. In our current projects, we are working on verifying these findings obtained in the more artificial in vitro situation in the in vivo and ex vivo situation in the mouse, dog and pig.

Publications:

  1. Klopfleisch R. Macrophage reaction against biomaterials in the mouse model – phenotypes, functions and markers. Acta Biomaterialia, 1;43:3-13, 2016. PMID: 27395828

  2. Klopfleisch R, Jung F. The pathology of the foreign body reaction against biomaterials. Journal of Biomedical Material Research A, 105(3):927-940, 2017. PMID: 27813288

  3. Frede A, Neuhaus B, Klopfleisch R, Walker C, Buer J, Müller W, Epple M, Westendorf AM. Colonic gene silencing using siRNA-loaded calcium phosphate/PLGA nanoparticles ameliorates intestinal inflammation in vivo. Journal of Controlled Release, 222:86-96, 2016. PMID: 26699423

  4. Haase T, Klopfleisch R, Krost A, Sauter T, Kratz K, Peter J, Jung F, Lendlein A, Zohlnhöfer D, Rüder C. In vivo biocompatibility study of degradable homo- versus multiblock copolymers and their (micro)structure compared to an established biomaterial. Clin Hemorheol Microcirc. 2020 Jan 4. doi: 10.3233/CH-190748

 

Ageing in pets

Ageing is a physiological process that is associated with an increase in susceptibility to a wide range of diseases, such as various diseases of civilization and, in particular, the development of tumours. For example, it is assumed that the ageing of the peritumoral connective tissue in and around tumors has an important influence on the development and proliferation of tumors. In particular, the accumulation of senescent cells, which exhibit permanent cell cycle arrest, has an important influence on the development of tumors in their environment. In various projects, we are therefore currently investigating markers for senescent cells in dogs, the occurrence and association of senescent cells with aging-associated diseases, such as tumors.

Publications:

  1. Merz SE, Kershaw O, Petrick A, Gruber AD, Klopfleisch R, Breithaupt A. Tumour, but not Age-associated, Increase of Senescence Markers γH2AX and p21 in the Canine Eye. J Comp Pathol. 201;173:41-48. doi: 10.1016/j.jcpa.2019.10.004

  2. Merz SE, Klopfleisch R, Breithaupt A, Gruber AD. Aging and Senescence in Canine Testes. Vet Pathol. 2019;56(5):715-724. doi: 10.1177/0300985819843683