Differentiating Between Epstein-Barr Virus-positive Lymphoid Neoplasm Relapse and Post-transplant Lymphoproliferative Disorder After Sex-mismatched Hematopoietic Stem Cell Transplantation

After allogeneic hematopoietic stem cell transplantation (HSCT), accurate differentiation between donor-derived post-transplant lymphoproliferative disorder (PTLD) and relapse of recipient-derived lymphoproliferative disorder (LPD) is crucial for determining treatment. Conventional diagnostic approaches for PTLD include histopathological examination, flow cytometry, and chimerism analysis of bulk tumor tissue. However, these methods are inconclusive in cases in which the primary disease is an Epstein-Barr virus (EBV)-positive LPD and is of the same lineage as that of the post-HSCT LPD tumor cells. Particularly, in cases where the number of tumor cells in the tissue is low, it is difficult to determine the origin of tumor cells. In this study, we developed a new method to simultaneously detect signals using sex chromosome fluorescence in situ hybridization, immunofluorescence staining, and EBV-encoded small RNA in situ hybridization on a single section of formalin-fixed paraffin-embedded histopathological specimen. The utility of the method was validated using specimens from 6 cases of EBV-positive LPD after sex-mismatched HSCT that were previously difficult to diagnose, including Hodgkin lymphoma-like PTLD that developed after HSCT for Hodgkin lymphoma and recurrence of chronic active EBV infection. This method successfully preserved the histologic structure after staining and allowed accurate determination of tumor cell origin and lineage at the single-cell level, providing a definitive diagnosis in all cases. This method provides a powerful tool for the diagnosis of LPDs after sex-mismatched HSCT.