Guiding Treatment and Clinical Management of Patients with CNS Lymphomas By Minimal-Invasive Detection of Circulating Tumor DNA in Cerebrospinal Fluid

Introduction: Central nervous system lymphomas (CNSL) are aggressive extranodal Non-Hodgkin lymphomas confined to the CNS compartment, mostly classified as diffuse large B-cell lymphomas. The gold standard for CNSL diagnosis is histopathological assessment of tumor tissue obtained by stereotactic brain biopsies. Yet, in high-risk situations or when patients obtain concurrent corticosteroids or antiplatelet therapy, biopsies are often delayed or a final diagnosis remains unconfirmed. In these scenarios, minimal-invasive CNSL identification from cerebrospinal fluid (CSF) could have transformative impact on the clinical management of these patients. Here, we developed a sensitive and highly specific digital droplet PCR (ddPCR) assay for the detection of MYD88 L265P in circulating tumor DNA (ctDNA) from CSF for its use in clinical routine. Methods: We underwent a certification process by the ‘National accreditation body of the Federal Republic of Germany’ (Deutsche Akkreditierungsstelle, DAkkS) in our accredited clinical laboratory at the University Medical Center Freiburg (Germany) to develop a ddPCR-based laboratory developed test (LDT) for the detection of MYD88 L265P in body fluids. The assay performance was independently validated in CSF and plasma samples collected from patients with contrast-enhancing CNS lesions and confirmed histopathological diagnosis. Following clinical implementation, our technology was applied to CSF specimens submitted from hospitalized patients between January 2022 and June 2024 for routine testing of MYD88 L265P. A subset of patients participated in our observational study (DRKS00034686), facilitating the evaluation of clinical, pathological, and radiological information as well as the assessment of clinical implications following CSF-ctDNA analyses. Results: The MYD88 L265P ddPCR assay development process revealed a limit of detection (LOD) of 0.05% mutant allele frequency (AF) and a limit of blank (LOB) of 0.5 detected copies per mL sample volume, leading to certification of the LDT by the DAkkS for its routine use in our clinical laboratory environment. Following this process, we comprehensively validated the technology's performance in 77 CSF specimens and 91 plasma samples from an independent cohort of 128 patients with contrast-enhancing brain lesions and verified CNSL (n=87) or other primary brain tumors and infectious/inflammatory brain diseases (‘Non-CNSL’, n=41). We identified the MYD88 L265P mutation with a sensitivity of 67% from CSF and 37% from plasma samples of patients with CNSL, while the hotspot variant was never detected in Non-CNSL patients, revealing a specificity and positive predictive value of 100% for both analytes. Levels of ctDNA were 24-fold higher in MYD88 L265P-positive CSF samples than in blood plasma (5.5% vs. 0.23%, p=0.0001). After certification and independent validation, we applied the ddPCR assay to 205 CSF samples collected from 182 hospitalized patients and submitted for routine testing of MYD88 L265P from 21 distinct centers. CSF volumes ranged from 0.5 to 10 mL, with a median of 2.9 mL. We detected the hotspot variant in 33% of CSF samples and reported the results to treating physicians with a median turnaround time of 5 days. 127 patients, providing 143 CSF samples, participated in our observational study. Reasons for CSF submission in the observational study included the presence of an unclear brain lesion with CNSL as differential diagnosis and surgical high-risk situation (n=49), suspected secondary CNSL involvement/relapse or PCNSL relapse (n=64), or ctDNA monitoring in patients with known CNSL (n=30). In 60% of CSF-ctDNA positive cases (31/52), our results guided or helped guiding further treatment and surgical management of patients. Specifically, minimal-invasive identification of MYD88 L265P obviated the need for neurosurgical biopsies or accelerated the diagnosis of CNSL in 19 patients (37%) and led to the initiation of CNSL-specific treatment in 25 cases (48%). 95% of patients receiving CNS-directed therapies based on CSF-ctDNA results showed radiographic and/or clinical response of their brain lesions. Conclusion: Our results demonstrate that minimal-invasive identification of CNSL by ctDNA profiling can effectively guide treatment and surgical management in clinical routine and has practice-changing impact for a subset of patients with unknown CNS lesions.