Minimally invasive liquid biopsy analysis of B-cell non-Hodgkin lymphomas

The gold standard for the diagnosis of tumors are tissue biopsies. However, sometimes it is not feasible to obtain a tissue biopsy due to a high risk of complications or difficult anatomical localizations. This is especially true for tumors that are located in the central nervous system (CNS), which also includes the ocular compartment. In this thesis, we primarily focused on lymphomas of the CNS and studied alternatives for diagnosis as well follow-up in liquid instead of tissue biopsies. To this end, we analyzed feasibility of targeted MYD88 p.(L265P) mutation detection by highly sensitive droplet digital PCR (ddPCR) in low volume, low DNA content liquid biopsies, as this hotspot mutation is present in the majority (≥80%) of the lymphomas in our thesis. First, we analyzed cerebrospinal fluid (CSF) of patients with two different types of lymphoma: lymphoplasmacytic lymphoma (LPL) and primary CNS lymphoma (PCNSL). In both studies, we showed that most cases were well detectable in CSF by ddPCR for MYD88 p.(L265P) mutation analysis. In addition, we found that analysis of cell-free DNA in CSF is of added value to analysis of cellular DNA as well as routine biomarkers in CSF, such as flow cytometry and cytomorphology. Finally, we were able to detect lymphoma in plasma of PCNSL patients, a highly promising liquid biopsy for follow-up. Furthermore, we analyzed ocular fluids of patients with vitreoretinal lymphoma (VRL) of the eye, which is closely related to PCNSL and which metastasizes to the CNS in up to 90% of cases. We found that MYD88 p.(L265P) could be detected in easily accessible aqueous humor, which is less invasive than the routinely used vitreous fluid. In combination with interleukin analysis, VRL patients might be diagnosed and monitored in aqueous humor in the near future. As presence of MYD88 p.(L265P) in lymphomas can also be used for targeted therapy with Bruton’s tyrosine kinase (BTK) inhibitors, we also investigated feasibility of BTK inhibitor therapy in LPL with CNS involvement, for which it is currently not registered. We found that BTK inhibitor therapy was an effective treatment in a LPL patient with transformation and CNS localization and levels of the BTK inhibitor were detectable in the CSF. To further elucidate the mechanism of BTK resistance in LPL patients with additional CXCR4 mutations we performed CXCR4 immunohistochemistry. We found that LPL bone marrow samples with a CXCR4 mutation show cytoplasmic localization of the CXCR4 protein compared to nuclear localization in LPL samples without a CXCR4 mutation. Interestingly, those cases with cytoplasmic CXCR4 staining showed a heterogeneous, weak staining pattern of NFκB, compared to diffuse, strong NFκB staining in cases with nuclear CXCR4 staining. As BTK inhibitors eventually target NFκB, these results suggest that CXCR4 mutations in LPL patients result in production of an altered CXCR4 protein, which does not activate the NFκB pathway as it normally does, possibly leading to diminished response to BTK inhibitors