3D connectomes of reactive and neoplastic CD30 positive lymphoid cells and surrounding cell types.

ABSTRACT

Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas in Western Europe. It is diagnosed on the basis of histological sections by pathologists using a light microscope. The tumor cells, the Hodgkin- and Reed Sternberg cells (HRS), are visualized by morphology and positive response for the CD30-antigen. The same antigen can also be detected by immunohistochemistry on a reactive counterpart, showing CD30+ cells in special immunoreactions, such as inflammations of lymph nodes (lymphadenitis). CD30+ cells in reactive and neoplastic conditions are surrounded by lymphocytes and histiocytes, forming a micromilieu that enables the survival of the tumor cells, as well as their reactive counterparts. This study deals with an investigation of CD30+-surrounding cells using a confocal laser technology, visualizing the contacts of reactive and neoplastic CD30+ cells with CD68+ macrophages and CD163+ macrophages as well as to PD1+ lymphocytes and B cells (CD20+). CD4 immunostains were not included, because CD4+ cells were too numerous for clear dissection of single cells. 3D images visualized the, so-called, connectomes. Clear differences in the number of contacts between CD30-reactive and neoplastic cells (HRS) with macrophages and B lymphocytes were visible. Lymphadenitis and Mixed Cellularity type of classical Hodgkin Lymphoma (cHL) differed in that Mixed Cellularity (MC) cHL had more connections to macrophages (CD163+) and lower number of connections to B cells (CD20+). The connectomes of both Hodgkin variants MCcHL and Nodular Sclerosis cHL (NScHL) mainly differed in the number of contacts to CD163+ macrophages, which was higher in MCcHL. Investigating the volumes of CD30+ -reactive and neoplastic cells, we found out that reactive cells showed lesser volumes, which correlated with the number of contacts. The comparison between 2D and 3D images, including 3D prints, demonstrated clear advantages of the 3D method. 3D images visualized significantly more and clearly defined intercellular contacts. Complicated cellular networks and their contacts became especially evident in volume and surface evaluations, as well as in 3D prints.