A Backwards Approach to GD2 Immunofluorescence in Human Neuroblastoma Tissue Samples: From Staining to Slicing.

backgroundThe disialoganglioside GD2, located at the plasma membrane, is selectively overexpressed in various solid tumors, where it contributes to tumor growth and the development of an aggressive tumor phenotype. Thus, over the last two decades GD2 has been gaining importance both as a tumor marker and a therapy target. In neuroblastoma, anti-GD2 monoclonal antibodies and CAR T-cells have become an integral part of the multimodal treatment for relapsed or refractory high-risk cases, which continue to associate with poor prognosis. GD2 characterization in neuroblastoma is well established for bone marrow aspirates and biopsies, but remains challenging in tumoral tissue samples, mostly due to epitope loss upon fixation.

aimsThe aim of our work was to assess a new protocol by staining GD2 in tissue specimens prior to fixation.

methodsPositive controls were tissue specimens from patients with histologically confirmed neuroblastoma and GD2 expression in bone marrow aspirate (

resultsGD2 signal was detected in all positive controls, while absent in all negative controls. After fixation, paraffinization and slicing no relevant signal loss was observed. Nevertheless, sufficient staining of 3D specimens required long incubation times, which led to increased cytolysis of the unfixed tissue.

conclusionsWe were able to establish and validate a novel protocol to reliably perform immunostaining of the membrane antigen GD2 in unfixed, primary neuroblastoma tissue. Although including few limitations, this staining workflow enables relatively quick assessment of GD2 status and thus, might represent a relevant diagnostic tool within the framework of tumor staging and precision medicine.