The Role of Macrophages in Multiple Myeloma and 3D visualization in multiple myeloma bone marrow compartment analysis

Principal Investigator: Associate Professor Sanja Štifter

Department: Department of Pathology, Faculty of Medicine

Institution: University of Rijeka, Croatia


Key words: macrophages, multiple myeloma, 3D visualization, bone marrow

The aim of the study was to shed light more closely on the interaction between malignant plasma cells in multiple myeloma (MM) and macrophages in the bone marrow. In order to achieve this goal immunohistochemical (IHC) identification and quantification of macrophages was performed and we also start 3D histology specimen reconstruction for precise spatial element recognition. Method: The immunohistochemical analysis was performed on trephine bone marrow samples collected from archival material as well as on bone biopsy samples from newly diagnosed patients with MM at Department of Hematology, Faculty of Medicine, Clinical Hospital Centre Rijeka and were subsequently analyzed at Department of Pathology, Faculty of Medicine, University of Rijeka, Croatia. Selection of bone biopsies, after the initial evaluation and the completion of the diagnostic algorithm, depended on percentage of tumor infiltrate (30-50%) and immunohistochemistry staining with CD68/CD163? CD68/CD138, Mannose, IP10, VEGF-A and CD31 antibodies. The biopsies were analyzed for the absolute number of macrophages, the percentage of plasma cells and the ratio of their representation in the sample. Sections were evaluated using visual standard microscopic analysis and a computer assisted IHC quantification on 44 cases. All stained slides were scanned (Olympus VS110) and analyzed using Alphelys Spot Browser 2 integrated system, consisting of software controlled (Spot Browser 2.4.4, Alphelys, France) and motorized stage microscope (Eclipse 50i, Nikon, Japan) with mounted digital camera (1360x1024 resolution, 24bit, CFW1310C, Microvision, France). Overview images of the slides were taken at 20x magnification, analyzing images of the complete section area at 100x or 200x magnification, under constant light intensity, microscope and camera settings. In the further course of our study, identification and quantification of the macrophage polarization status is intended. The previously obtained image analysis results were correlated with angiogenesis as total vascular area (TVA) and clinical parameters, including Durie Salmon (DS) classification. The intention is to explore whether NoM correlates with DS and whether macrophage activation status of polarization (M1/M2) can be immunohistochemically determined more efficiently or does this ultimately influences the clinical outcome in MM.
Regarding three-dimensional (3D) viewing in histopathology, it presents to us tissue and cellular components what yields information regarding tissue organization, cellular function and disease processes, upon which histopathological diagnosis is largely based. It is vital to remember, however. That tissue constitutes a 3D structure and that disease is a 3D process. A tissue section provides us with an (almost) two-dimensional (2D) translucent slice and reduces the dimensionality of features which exist in 3D. Within macrophage and MM project we aimed to make 3D reconstruction of serial sections of bone marrow since they can be a useful method to observe the appearance of tissue structures in the third dimension giving us an opportunity to closely analyze cell to cell relationships. In order to determine whether macrophage activation status of polarization (M1/M2) ultimately influences the spatial distribution of multiple myeloma (MM) cells we performed 3D reconstruction of histology bone marrow specimens. This way we have obtained model which gives us real 3D construct of tissue and possibility to visualize cellular spatial interactions. However, though more basic indirect stereological methods exist that allow us to obtain quantitative information in nature such as volume, surface area and feature number from the 2D information seen in tissue sections. Some technical problems of visualizing data still persist but since we managed to scan slides and document such large pool of data the technology has become ready to follow such huge demand of the end users while clinicians seem to be ready to adopt new tools in clinical as well basic research.