Guiding treatment and clinical management of patients with CNS lymphomas by minimal-invasive detection of circulating tumor DNA in cerebrospinal fluid

Guiding treatment and clinical management of patients with CNS lymphomas by minimal-invasive detection of circulating tumor DNA in cerebrospinal fluid | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Guiding treatment and clinical management of patients with CNS lymphomas by minimal-invasive detection of circulating tumor DNA in cerebrospinal fluid Florian Scherer, Samuel Weinschenk, Ulrike Philipp, Julia Kuehn, and 26 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5099294/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Various clinical scenarios preclude or delay invasive stereotactic biopsies and subsequent histopathological assessment for the diagnosis of central nervous system lymphoma (CNSL), necessitating innovative minimal-invasive strategies. We designed a digital droplet PCR (ddPCR) assay for minimal-invasive identification of CNSL in clinical practice by detecting MYD88 L265P mutations in circulating tumor DNA (ctDNA) of cerebrospinal fluid (CSF). After assay approval by the national accreditation authority, this laboratory-developed test (LDT) was first independently validated in a cohort of 128 patients with confirmed malignant or inflammatory/infectious brain diseases, revealing a sensitivity of 67% and specificity of 100% for correct CNSL diagnosis. Following implementation in a clinical laboratory environment, the LDT was applied to 205 CSF samples from 182 independent patients, reporting results to treating physicians with a median turnaround time of 5 days. The MYD88 L265P mutation was detected in 33% of CSF specimens, obviating the need for invasive surgical biopsies in 37% of patients and guiding lymphoma-specific treatment in 48% of evaluable cases. 94% of patients undergoing CNS-directed treatment based on CSF-ctDNA profiling objectively responded to therapies. Collectively, our results demonstrate that minimal-invasive identification of CNSL by ctDNA genotyping in CSF can effectively guide clinical management and has practice-changing impact for a substantial subset of patients with unknown CNS lesions. Health sciences/Oncology/Cancer/CNS cancer Health sciences/Biomarkers/Diagnostic markers Health sciences/Oncology/Cancer/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/B-cell lymphoma Figures Figure 1 Figure 2 Figure 3 Main text Central nervous system lymphomas (CNSL) are aggressive extranodal Non-Hodgkin lymphomas confined to the CNS compartment and classified as large B-cell lymphomas of immune-privileged sites in the current 2022 WHO classification 1 . In contrast to other primary CNS tumors or brain metastases that are mainly treated with surgery and radiotherapy, the primary curative treatment strategy for CNSL is based on intensive immunochemotherapies followed by high-dose consolidation regimens 2 – 5 . Due to this unique therapeutic approach, stereotactic biopsy of the tumor lesion followed by histopathological assessment is the gold standard for CNSL diagnosis, which reduces the risk associated with more extensive surgical procedures performed for other brain cancer types 3 . While neurosurgical biopsies are generally characterized by low complication rates, various clinical scenarios preclude or delay this invasive procedure and final CNSL diagnosis. For example, tumor localization in eloquent brain areas or surgical high-risk situations associated with patient frailty might result in unacceptable perioperative morbidity. Furthermore, concurrent corticosteroid or antiplatelet treatment often cause a significant delay of stereotactic procedures and might lead to indeterminate histopathological findings 6 – 8 . In these critical scenarios and due to a rapid decline of neurological recovery and therapy response rates associated with a delay of treatment initiation in this aggressive lymphoma type, minimal-invasive identification of CNSL from cerebrospinal fluid (CSF) could have transformative impact on the clinical management of patients with unknown CNS lesions 3 , 6 , 9 . This also extends to patients with suspected secondary involvement of systemic lymphomas or relapse of primary CNSL (PCNSL), who would benefit from surgery-free confirmation of diagnosis to tailor further treatment. Conventional CSF analyses such as cytopathology (CP) or flow cytometry (FC) and diagnostic magnetic resonance imaging (MRI) have shown low sensitivities and discriminative capacity to facilitate biopsy-free CNSL diagnosis, highlighting the need for more innovative technologies 6 , 10 – 16 . Several previous research studies have demonstrated reliable detection of the lymphoma-specific MYD88 L265P mutation in circulating tumor DNA (ctDNA) from CSF of CNSL patients and showed a potential treatment-guiding effect in anecdotal cases or in small case series 6 , 17 – 26 . However, the value of a liquid biopsy approach for surgical planning and treatment management in large patient cohorts and daily clinical practice has never been prospectively explored. Therefore, we here developed and implemented a sensitive and highly specific digital droplet PCR (ddPCR) assay in a clinical laboratory environment for minimal-invasive detection of MYD88 L265P from CSF and evaluated its impact on patient clinical management. Results MYD88 L265P ddPCR assay development and independent validation To develop a ddPCR assay as a laboratory-developed test (LDT) for the analysis of MYD88 L265P in ctDNA of body fluids, we underwent an approval 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. The assay settings, performance parameters, and technical results that led to approval of the LDT by the DAkKS for its use in clinical routine are described in detail and depicted in the Supplementary Methods section, Supplementary Fig. 1 , and Supplementary Tables 1–6 . This included the assessment of the limit of detection (LOD), limit of blank (LOB), analytical sensitivity and specificity, linearity, accuracy, and inter-/intra-assay reliability ( Supplementary Fig. 1, Supplementary Tables 1–6 ). Collectively, the development process revealed a LOB of 0.5 detected mutant copies per mL sample volume and a LOD of 0.05% mutant allele frequency (AF) as key parameters of our assay that were applied to all subsequent analyses to faithfully determine the presence of MYD88 L265P in ctDNA of body fluids (Supplementary Methods). Next, we tested and validated the performance of the MYD88 L265P ddPCR assay in an independent research study cohort of patients whose clinical situations reflected one of the intended applications of the LDT (Fig. 1 a). We applied the assay to 77 CSF specimens (median volume: 1.9 mL, range: 0.2–9.9 mL) and 91 plasma samples (median volume: 8.5 mL, range: 2.7–11.8 mL) collected from 128 patients with contrast-enhancing brain lesions and verified histopathological diagnosis to evaluate its diagnostic sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in both compartments (Fig. 1 a, Supplementary Fig. 2a , and Supplementary Table 7 ). Two-thirds of these patients ( n = 87, 68%) were diagnosed with either primary CNSL (PCNSL, 88%) or isolated secondary CNSL (iSCNSL, 12%) (defined as ‘CNSL patients’), while 41 patients showed a broad variety of other brain diseases, including primary brain tumors (glioblastoma, lower-grade gliomas and others, 46%), brain metastases (25%), or inflammatory/infectious diseases such as multiple sclerosis (MS, 29%) (defined as ‘Non-CNSL patients’) (Fig. 1 a,b and Supplementary Table 7 ). In this setting, which is agnostic to the tumor mutation status (‘tumor-agnostic approach’), we minimal-invasively detected MYD88 L265P with a sensitivity of 67% from CSF and 37% from plasma samples of patients with confirmed CNSL (Fig. 1 c). In comparison, conventional CSF analyses including CP and FC identified CNSL infiltration with a sensitivity of 22% ( n = 46, Supplementary Fig. 2b ). Importantly, the hotspot variant was never detected in Non-CNSL patients, revealing a specificity and PPV of 100% for both analytes (Fig. 1 c). The amount of ctDNA was 24-fold higher in MYD88 L265P-positive CSF samples than in plasma, with a median CSF-ctDNA AF of 5.5% compared to 0.23% in blood plasma ( p = 0.0001, Supplementary Fig. 2c ). In 49 CNSL cases, the MYD88 L265P mutation was detected in a matched tumor specimen by targeted-capture next-generation sequencing (NGS, Fig. 1 a). Limiting our analyses to CSF/plasma samples with corresponding tumors and known MYD88 L265P positivity (‘tumor-informed approach’), we observed a clinical sensitivity for our assay of 82% in CSF and 37% in plasma specimens (Fig. 1 d). Finally, we further assessed the reproducibility of our measurements and representation of allelic fractions across analytical platforms, comparing our results with those obtained from targeted-capture NGS of 75 CSF and plasma samples ( Supplementary Table 7 , Supplementary Methods). We found a high concordance rate of 89.3% for the detection of MYD88 L265P and a significant correlation of ctDNA concentrations between the technologies ( p < 0.0001, r = 0.99, Supplementary Fig. 2d ). Collectively, these data demonstrate robust performance of the ddPCR technology in a representative clinical setting, revealing superior sensitivity for minimal-invasive MYD88 L265P identification in the CSF compared to blood plasma, mainly due to markedly higher ctDNA concentrations in this compartment. Feasibility of MYD88 L265P analysis from CSF-ctDNA in clinical practice After implementation in our clinical laboratory environment, we applied the MYD88 L265P ddPCR assay to 205 CSF samples collected from 182 hospitalized patients and submitted to our laboratory between January 2022 and June 2024 ( Supplementary Fig. 3a and Supplementary Table 8 ). CSF samples were obtained at the discretion of the treating physician and submitted from hospitals of the University Medical Center Freiburg (Departments of Neurosurgery, Neurology, and Hematology/Oncology, n = 85) and 20 external referral centers ( n = 120) ( Supplementary Table 8 ). The number of CSF submissions steadily increased from a median of 8 samples per quarter in 2022 to a median of 46.5 samples per quarter in 2024 (Fig. 2 a). Submitted CSF volumes ranged from 0.5 to 10 mL, with a median of 2.9 mL (Fig. 2 b). We reported the results of the MYD88 L265P ddPCR assay to treating physicians with a median turnaround time of 5 days (average: 5 days), with more than a third of analyses (36%) being completed within 3 days (Fig. 2 c). We detected the hotspot variant in 68 CSF samples (33%), while 137 samples (67%) showed a negative result (Fig. 2 d and Supplementary Table 8 ). The mutational AFs ranged from 0.055–25.2% in ctDNA-positive CSF samples, with a median of 0.55% (Fig. 2 e). Guiding clinical management by CSF-ctDNA profiling 127 patients, providing 143 CSF samples, participated in our observational study, facilitating the evaluation of clinical, pathological, and radiological information as well as the assessment of clinical implications following CSF-ctDNA analyses ( Supplementary Fig. 3a and Supplementary Table 9 ). Reasons for CSF submission in the observational study included the presence of an unclear brain lesion with CNSL as differential diagnosis ([1], n = 49, 34%) and either surgical high-risk situation due to patient frailty or localization in an eloquent brain region ([1a], n = 25, 17%), delay of surgery due to concomitant steroid/antiplatelet treatment ([1b], n = 8, 6%), or the lack of a definitive diagnosis after biopsy and histopathological assessment ([1c], n = 16, 11%), a suspected secondary brain manifestation of systemic lymphoma either as occult involvement ([2a], n = 35, 24%), synchronous involvement ([2b], n = 6, 4%), or metachronous SCNSL relapse ([2c], n = 15, 10%), or a suspected PCNSL relapse ([2d], n = 8, 6%), and the request to profile CSF-ctDNA as a monitoring biomarker in patients with known CNSL ([3], n = 30, 21%) (Fig. 3 a and Supplementary Table 9 ). MYD88 L265P was detected in 52 of 143 CSF samples (36%) from patients participating in the observational study ( Supplementary Fig. 3a and Supplementary Table 9 ). In 31 CSF-ctDNA positive cases (60%), the ddPCR results directly guided or helped guiding further treatment and surgical management (Fig. 3 b-d). 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%) (Fig. 3 b-d and Supplementary Table 9 ). Specifically, 33 patients did not undergo surgery or biopsies were delayed due to surgical high-risk situations ([1a,1b], Fig. 3 b). Positivity of CSF-ctDNA ( n = 9, 27%) eliminated the need for neurosurgical interventions in 78% of these patients and was treatment-guiding in 56% of cases (Fig. 3 b). Two patients received an accelerated diagnosis of CNSL by 6 and 11 days, respectively; however, this had no effect on surgical planning or treatment initiation (Fig. 3 b and Supplementary Table 9 ). In 16 patients, histopathological assessment following stereotactic biopsies revealed no definitive diagnosis ([1c], Fig. 3 c). MYD88 L265P was detected in 6 of these patients (38%), leading to CNSL-specific treatment in 83% of cases without further pathological confirmation and supported the diagnosis of a suspected but not confirmed cerebral amyloidoma from Waldenstrom macroglobulinemia (Fig. 3 c and Supplementary Table 9 ). Finally, we received 64 CSF samples from patients with suspected CNS involvement of systemic lymphomas or suspected PCNSL relapse and found the CNSL-specific hotspot variant in 18 cases (28%, [2], Fig. 3 d). These positive findings contributed to surgical planning and obviated stereotactic biopsies in 50% of patients, while 15 patients (83%) received CNSL-specific treatment following CSF-ctDNA profiling (Fig. 3 d and Supplementary Table 9 ). CNS-directed therapies were initiated in 25 patients with the support of our analyses (Fig. 3 e). While 6 patients (24%) received palliative treatment, 19 patients (76%) underwent curative-intent immunochemotherapies that were mostly based on high-dose methotrexate (Fig. 3 e and Supplementary Table 9 ). 94% of patients with an evaluable CNS lesion by MRI responded to therapies ( n = 17), while two patients with no radiological follow-up or evaluable brain lesion showed a clinical response (Fig. 3 e). Yet, one patient experienced a rapid progression following palliative rituximab and radiotherapy that was initiated based on CSF-ctDNA results (Fig. 3 e and Supplementary Table 9 ). Supplementary Fig. 3b-e highlights four representative cases in which CNSL-specific therapies were applied based on minimal-invasive genotyping from CSF, leading to clinical responses of their brain lesions. Comparison with conventional CSF analyses and influence of corticosteroid treatment 142 CSF samples of the observational study were simultaneously analyzed by conventional CP and/or FC, revealing a positivity rate of 4% for the detection of CNSL ( Supplementary Fig. 4a,b ). In case of CSF-ctDNA positivity, the concordance rate with conventional CSF analyses was low (6%), while the vast majority of MYD88 L265 negative cases were CSF CP/FC negative (97%) and only three CSF samples with monoclonal CD19-positive lymphoma cells were ctDNA-negative (3%, Supplementary Fig. 4a,b ). In 26 patients of the observational study, a histopathological diagnosis was achieved after stereotactic biopsy of the CNS lesion, performed prior to or concurrently with CSF sampling ( Supplementary Table 9 ). MYD88 L265P was identified in 12 patients with verified CNSL (60%, 12/20) and never detected in 6 patients with other brain malignancies ( Supplementary Fig. 4b ). Finally, we explored the influence of corticosteroid treatment on CSF-ctDNA detection rates and concentrations. We found no differences in MYD88 L265P detection rates between patients who received corticosteroids prior to the lumbar puncture and those who did not undergo corticosteroid therapy (Fig. 3 f). Furthermore, we observed that cumulative corticosteroid doses preceding lumbar punctures did not correlate with CSF-ctDNA concentrations in mutated samples (Fig. 3 g). Discussion We here demonstrate that minimal-invasive identification of CNSL by ctDNA profiling from CSF can effectively guide treatment and surgical management in daily clinical practice and has practice-changing impact for a substantial subset of patients with unknown CNS lesions in challenging clinical situations. Due to the aggressiveness and localization of brain lymphomas, rapid diagnostic workup and treatment initiation are crucial to prevent neurological impairment and maintain favorable clinical outcomes 27 . Yet, various factors in clinical reality, which can be either patient-specific, treatment-related, or associated with the disease, often impair a timely and accurate diagnosis of CNSL. In these situations, there is a critical need for minimal-invasive approaches that utilize CSF biomarkers for the identification of CNSL. Current CNSL clinical guidelines such as the 2024 ‘EHA-ESMO Clinical Practice Guideline’ consider the assessment of CSF biomarkers including MYD88 L265P as a promising tool for CNSL diagnosis when biopsies are not possible 3 . A plethora of research studies have explored protein and genetic markers including MYD88 L265P for biopsy-free identification of CNSL from CSF 14 , 15 , 17 – 22 , 24 , 25 , 28 – 31 . However, despite exhibiting some clinical utility in anecdotal cases and small case series, these technologies have not been regularly and broadly applied in clinical routine yet, largely due to the lack of standardization, prospective validation, and uncertainties associated with limited inter-laboratory reproducibility. We here underwent an extensive regulatory development and validation process that led to the implementation of a MYD88 L265P ddPCR assay in our clinical laboratory environment for minimal-invasive detection of CNSL from CSF. This procedure was optimized for high specificity to minimize false-positive results and misclassification, which could result in inappropriate treatment of patients with other brain diseases. Previous reports suggested that the MYD88 L265P mutation might be present in patients with neurologic autoimmune conditions such as MS or associated with lymphoid clonal hematopoiesis of indeterminate potential (CHIP) 32 , 33 . In our study and in work from other groups, MYD88 L265P was never observed in patients with inflammatory or autoimmune brain diseases nor in the plasma of elderly healthy individuals, revealing 100% specificity across distinct and independent Non-CNSL cohorts 18 , 23 . We applied our LDT to 205 CSF samples over a period of 2.5 years in clinical practice and assessed its implications in an observational study. Results were communicated to treating physicians within 5 days (including weekends) and directly guided or helped guiding clinical management in 60% of CSF-ctDNA positive cases. Neurosurgical interventions were obviated or CNSL diagnosis was accelerated in 19 patients and a treatment-guiding effect was reported for 25 patients who received CNS-directed agents based on or supported by minimal-invasive CSF-ctDNA detection. Importantly, all but one patient objectively responded to CNSL-specific therapies, illustrating the clinical benefit of our liquid biopsy approach for a significant subgroup of patients whose final diagnosis otherwise might have remained unconfirmed or who would have faced high-risk surgical interventions to obtain a diagnosis. Of note, while positive CSF-ctDNA results can directly guide treatment or surgical strategies due to their high PPV, assessing the clinical impact of CSF-ctDNA negative results represents a considerable challenge, because a negative MYD88 L265P status does not exclude CNSL or help defining another diagnosis. Thus, knowing that CSF samples are negative for MYD88 L265P might help narrowing down differential diagnoses in certain scenarios, but it typically has no direct influence on the clinical management of patients with unknown CNS lesions. The most evident limitation of our technology can be attributed to the fact that approximately 30% of CNSL patients do not harbor the hotspot MYD88 L265P mutation, inevitably reducing applicability and sensitivity/NPV of our tumor-agnostic approach 31 , 34 – 37 . Alternative strategies have revealed higher sensitivities for CNSL identification, including assays that combine the detection of MYD88 L265P with protein biomarkers or immunoglobulin rearrangements, or next-generation sequencing-based technologies that capture hundreds or thousands of CNSL-specific genomic regions. However, despite showing promising results in retrospective studies, these methods require further regulatory standardization and have yet to prove their utility in clinical practice and large patient cohorts 6 , 17 , 25 , 31 . Another notable finding of our study was that the exposure to corticosteroids prior to lumbar punctures did not seem to have any impact on CNSL detection rates or the representation of MYD88 L265P allelic fractions. Corticosteroids are often used to manage neurological symptoms in CNSL patients but cause severe diagnostic delays and other challenges associated with steroid-induced transient lymphoma regression 7 , 8 . Our results indicate that CSF-ctDNA can be robustly detected irrespective of corticosteroid premedication, encouraging the use of minimal-invasive liquid biopsy technologies even in situations with radiological tumor reduction following steroid therapy. Yet, the duration of corticosteroid treatment and dosage were highly heterogeneous in our patient cohort, introducing some uncertainties around interpretability of the results and requiring further investigation of the corticosteroid effect for CSF-ctDNA detection in a prospective and standardized fashion. Methods Assay design and approval process We designed a ddPCR assay as a LDT for the detection of MYD88 L265P in the ctDNA of body fluids for its use in clinical practice. A detailed description of the assay development and the LDT approval process according to the DIN ISO 15189:2104-11 criteria of the national accreditation body of the Federal Republic of Germany (DAkkS, D-ML-13134-07-00) is available in the Supplementary Methods section of the Supplementary Information. Assay validation The MYD88 L265P ddPCR assay was comprehensively tested and validated in an independent clinical cohort of patients with contrast-enhancing brain lesions and verified histopathological diagnosis to evaluate its diagnostic sensitivity and specificity in both CSF and plasma samples. Samples from these patients were collected from patients participating in research studies conducted at the University Medical Center Freiburg, Germany (DRKS15307), and University Hospital of the Ludwig-Maximilians-University (LMU) Munich, Germany (Nr. 22 − 0008) that were approved by the ethics committees in accordance with the Declaration of Helsinki. In total, we collected 77 CSF samples and 91 plasma samples from 87 CNSL patients and 41 Non-CNSL patients with a broad variety of malignant, inflammatory, and infectious brain diseases. From 75 of these CSF/plasma samples and from 49 matched tumor specimens, the MYD88 L265P mutation status was additionally assessed by a targeted-capture NGS technology that has been described in previous studies (CAPP-Seq) 31 , 38 , 39 . Assay application in routine practice We analyzed CSF specimens obtained from hospitalized patients at 21 distinct centers between January 2022 and June 2024 by the approved MYD88 L265P ddPCR assay in our clinical laboratory at the University Medical Center Freiburg, Germany. The sample processing and analysis workflows are described in detail in the Supplementary Methods section. Results of the analyses were reported directly to treating physicians, who made treatment decisions and conducted communication of findings with patients. A subset of patients participated in our observational study, approved by the ethics committee according to the Declaration of Helsinki, that facilitated the use of clinical, pathological, and radiological information, and enabled the assessment of clinical implications following CSF-ctDNA analyses (DRKS00034686). Declarations Acknowledgements This work was supported by the Advanced Clinician Scientist Program of the Deutsche Gesellschaft für Innere Medizin (DGIM, to F.S.), the Deutsche Forschungsgemeinschaft (DFG, to F.S.), the Mertelsmann Foundation (to F.S.), the German Cancer Research Center (Deutsches Konsortium für Translationale Krebsforschung, DKTK, to F.S.), the Else Kröner-Fresenius-Stiftung (to F.S. and P.C.R), and Fraunhofer Society Germany (to P.C.R.). S.W. is supported by the José Carreras-DGHO fellowship. J.C.K. and N.N. are supported by the Berta-Ottenstein Programm Förderlinie Clinician Scientist of the University Medical Center Freiburg. We thank the FREEZE biobank Freiburg for their support. Ethics declarations Competing interests F.S. receives research funding from Gilead Sciences, Roche Sequencing Solutions, and Takeda, and received honoraria from AstraZeneca and Servier. S.K.A reports consulting for Foresight Diagnostics. E.S. received honoraria from SERB Pharmaceuticals. N.N. serves on advisory board for Servier and is consultant for B. Braun. P.C.R. received honoraria from Arcana and serves as consultant for Boston Scientific, Inomed, and Brainlab. All other authors did not report any conflicts of interest related to this work. Supplementary Information Supplementary Information exists for this manuscript and can be found in separate files (Supplementary Information [including Supplementary Methods and Supplementary Figures] and Supplementary Tables). References Alaggio R, Amador C, Anagnostopoulos I, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms. Leukemia. Jul 2022;36(7):1720–1748. doi: 10.1038/s41375-022-01620-2 Schorb E, Finke J, Ferreri AJ, et al. High-dose chemotherapy and autologous stem cell transplant compared with conventional chemotherapy for consolidation in newly diagnosed primary CNS lymphoma–a randomized phase III trial (MATRix). BMC Cancer. 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Distinction of lymphoid and myeloid clonal hematopoiesis. Nat Med. Nov 2021;27(11):1921–1927. doi: 10.1038/s41591-021-01521-4 Oetjen LK, Bhattacharyya S, Galetta K. MYD88 L265P mutation in neurologic autoimmunity without evidence of malignancy. Mult Scler Relat Disord. Sep 2023;77:104868. doi: 10.1016/j.msard.2023.104868 Hernández-Verdin I, Kirasic E, Wienand K, et al. Molecular and clinical diversity in primary central nervous system lymphoma. Ann Oncol. Nov 16 2022;doi: 10.1016/j.annonc.2022.11.002 Chapuy B, Roemer MG, Stewart C, et al. Targetable genetic features of primary testicular and primary central nervous system lymphomas. Blood. Feb 18 2016;127(7):869–81. doi: 10.1182/blood-2015-10-673236 Radke J, Ishaque N, Koll R, et al. The genomic and transcriptional landscape of primary central nervous system lymphoma. Nat Commun . 05 10 2022;13(1):2558. doi: 10.1038/s41467-022-30050-y Nayyar N, White MD, Gill CM, et al. L265P mutation and. Blood Adv. Feb 12 2019;3(3):375–383. doi: 10.1182/bloodadvances.2018027672 Scherer F, Kurtz DM, Newman AM, et al. Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA. Sci Transl Med. Nov 09 2016;8(364):364ra155. doi: 10.1126/scitranslmed.aai8545 Kurtz DM, Scherer F, Jin MC, et al. Circulating Tumor DNA Measurements As Early Outcome Predictors in Diffuse Large B-Cell Lymphoma. J Clin Oncol. 10 2018;36(28):2845–2853. doi: 10.1200/JCO.2018.78.5246 Additional Declarations Yes there is potential Competing Interest. F.S. receives research funding from Gilead Sciences, Roche Sequencing Solutions, and Takeda, and received honoraria from AstraZeneca and Servier. S.K.A reports consulting for Foresight Diagnostics. E.S. received honoraria from SERB Pharmaceuticals. N.N. serves on advisory board for Servier and is consultant for B. Braun. P.C.R. received honoraria from Arcana and serves as consultant for Boston Scientific, Inomed, and Brainlab. All other authors did not report any conflicts of interest related to this work. Supplementary Files nreditorialpolicychecklist.pdf Article File - Editorial Policy Checklist SupplementaryInformation.pdf SupplementaryTables.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5099294","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":364055043,"identity":"e0fa2dce-4b11-4650-bc80-fe00e43f3127","order_by":0,"name":"Florian Scherer","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9635-5487","institution":"University of Freiburg","correspondingAuthor":true,"prefix":"","firstName":"Florian","middleName":"","lastName":"Scherer","suffix":""},{"id":364055044,"identity":"485f7bde-7d81-41d8-ad75-695d324841a8","order_by":1,"name":"Samuel Weinschenk","email":"","orcid":"","institution":"Department of Medicine I, Medical Center - 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(a) \u003c/strong\u003eOverview and consort diagram showing patients and specimens available from the independent validation cohort. \u003cstrong\u003e(b)\u003c/strong\u003e Pie charts depicting the distribution of brain diseases in the independent validation cohort. \u003cstrong\u003e(c) \u003c/strong\u003ePerformance parameters of the \u003cem\u003eMYD88\u003c/em\u003eL265P assay applied to CSF (left) and plasma samples (right) from patients of the independent validation cohort in a tumor-agnostic setting. \u003cstrong\u003e(d)\u003c/strong\u003e Sankey plots showing the \u003cem\u003eMYD88\u003c/em\u003e L265P positivity rate in CSF (top) and plasma (bottom) from CNSL patients with known \u003cem\u003eMYD88\u003c/em\u003eL265P-positive tumors (‘tumor-informed approach’). CNS, central nervous system; CNSL, CNS lymphoma; PCNSL, primary CNSL; iSCNSL, isolated secondary CNSL; CSF, cerebrospinal fluid; PPV, positive predictive value; NPV, negative predictive value.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5099294/v1/3fe36ffb4a207749f4e7fd52.png"},{"id":66550753,"identity":"0fc42a49-4b8a-4930-ab04-025053c09124","added_by":"auto","created_at":"2024-10-14 09:04:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":347439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMYD88 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eL265P ddPCR assay application in clinical practice. (a) \u003c/strong\u003eNumber of CSF specimens submitted from internal (orange) and external (blue) sites for routine \u003cem\u003eMYD88\u003c/em\u003eL265P testing (grey: total submissions), shown by quarter from quarter 1 in 2022 until quarter 2 in 2024. \u003cstrong\u003e(b) \u003c/strong\u003eScatter plot depicting submitted CSF volumes from all 205 samples in milliliters (mL). Lines highlight the median and range. \u003cstrong\u003e(c) \u003c/strong\u003ePie chart showing the distribution of turnaround times in days for \u003cem\u003eMYD88\u003c/em\u003eL265P analyses. \u003cstrong\u003e(d) \u003c/strong\u003eBar graph depicting the fraction of samples with positive (blue) and negative (gray) CSF-ctDNA results. \u003cstrong\u003e(e) \u003c/strong\u003eScatter plot highlighting CSF-ctDNA allele frequencies for all CSF analyses. Lines show the median and range. CSF, cerebrospinal fluid; Q, quarter; mL, milliliters; ctDNA, circulating tumor DNA.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5099294/v1/fc76688025f0278b4f388cba.png"},{"id":66553465,"identity":"e0fc4ca9-056e-4d6f-aba3-3a07cd2a6711","added_by":"auto","created_at":"2024-10-14 09:12:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":265877,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImplications of CSF-ctDNA analyses for clinical management of patients. (a)\u003c/strong\u003e Overview and graphical depiction of the reasons for CSF submissions for routine \u003cem\u003eMYD88\u003c/em\u003e L265P testing in CSF. Left [1]: Patients with unclear brain lesions and CNSL as differential diagnosis and either high-risk situation due to patient frailty or tumor lesions in eloquent brain regions [1a], delay of surgical intervention due to concomitant corticosteroid or antiplatelet treatment [1b], or unclear diagnosis after surgical intervention [1c]. Middle [2]: Patients with suspected secondary CNS involvement of systemic lymphomas either as occult [2a], synchronous [2b], or metachronous [2c] manifestation or suspected PCNSL relapse [2d]. Right [3]: CSF submitted from patients with known CNSL for CSF-ctDNA monitoring. \u003cstrong\u003e(b,c) \u003c/strong\u003eBar plots and pie charts showing the fraction of patients with CSF-ctDNA positivity (red) and negativity (grey) among patients in categories [1a,b] \u003cstrong\u003e(b)\u003c/strong\u003eand [1c] \u003cstrong\u003e(c)\u003c/strong\u003e and the effects of CSF-ctDNA detection for surgical planning and treatment initiation.\u003cstrong\u003e (d) \u003c/strong\u003eBar plot and pie chart showing the fraction of patients with CSF-ctDNA positivity (tourquoise) and negativity (grey) among patients in categories [2] and the effects of CSF-ctDNA detection for surgical planning and treatment initiation.\u003cstrong\u003e (e) \u003c/strong\u003eThis panel\u003cstrong\u003e \u003c/strong\u003ehighlights patients who received CNSL-specific treatment based on CSF-ctDNA positivity. The left bar plot shows the fraction of patients obtaining either curative-intent (dark blue) or palliative (light blue) CNS-directed therapies. Right: These two bar plots highlight the response to these therapies. Dark green: radiological CR, light green: radiological PR, lightest green: clinical response (radiologically LFU or no evaluable brain lesion), grey: no evaluable brain lesion by MRI due to occult involvement, red: radiological PD.\u003cstrong\u003e (f) \u003c/strong\u003eBar charts revealing the proportion of CSF samples being ctDNA positive (blue) or negative (grey) in patients with no corticosteroids (left) or with corticosteroid treatment (right) prior to and during lumbar puncture.\u003cstrong\u003e (g) \u003c/strong\u003eScatter plot comparing the cumulative corticosteroid dose on the x-axis with \u003cem\u003eMYD88\u003c/em\u003e L265P allele frequencies in CSF-ctDNA positive cases (y-axis). CNSL, central nervous system lymphoma; SCNSL, secondary CNSL; PCNSL, primary CNSL; CSF, cerebrospinal fluid; ctDNA, circulating tumor DNA; CR, complete response; PR, partial response; PD, progressive disease; mg, milligram.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5099294/v1/6df208f1e42ba886d91b0f13.png"},{"id":69107541,"identity":"d485916f-30ee-4508-aa07-1c561e2ecced","added_by":"auto","created_at":"2024-11-15 17:38:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1841682,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5099294/v1/f63f1bd6-935f-486b-afad-0226a9ab6a86.pdf"},{"id":66550751,"identity":"728eba17-f0e9-4171-af99-05dd5f202b8e","added_by":"auto","created_at":"2024-10-14 09:04:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1682412,"visible":true,"origin":"","legend":"\u003cp\u003eArticle File - Editorial Policy Checklist\u003c/p\u003e","description":"","filename":"nreditorialpolicychecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5099294/v1/291f5800761fb4eb24e3f092.pdf"},{"id":66550749,"identity":"b1766a6f-286f-4598-8cb3-2e40a66c8906","added_by":"auto","created_at":"2024-10-14 09:04:29","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":678229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5099294/v1/229693e5ecb8d8f406d49b13.pdf"},{"id":66550750,"identity":"690610c5-52dd-4714-a72f-44f4c5c5da96","added_by":"auto","created_at":"2024-10-14 09:04:29","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":77584,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5099294/v1/06b25ce5d05a01bd0a3c2dbe.xlsx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nF.S. receives research funding from Gilead Sciences, Roche Sequencing Solutions, and Takeda, and received honoraria from AstraZeneca and Servier. S.K.A reports consulting for Foresight Diagnostics. E.S. received honoraria from SERB Pharmaceuticals. N.N. serves on advisory board for Servier and is consultant for B. Braun. P.C.R. received honoraria from Arcana and serves as consultant for Boston Scientific, Inomed, and Brainlab. All other authors did not report any conflicts of interest related to this work.","formattedTitle":"Guiding treatment and clinical management of patients with CNS lymphomas by minimal-invasive detection of circulating tumor DNA in cerebrospinal fluid","fulltext":[{"header":"Main text","content":"\u003cp\u003eCentral nervous system lymphomas (CNSL) are aggressive extranodal Non-Hodgkin lymphomas confined to the CNS compartment and classified as large B-cell lymphomas of immune-privileged sites in the current 2022 WHO classification\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In contrast to other primary CNS tumors or brain metastases that are mainly treated with surgery and radiotherapy, the primary curative treatment strategy for CNSL is based on intensive immunochemotherapies followed by high-dose consolidation regimens\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Due to this unique therapeutic approach, stereotactic biopsy of the tumor lesion followed by histopathological assessment is the gold standard for CNSL diagnosis, which reduces the risk associated with more extensive surgical procedures performed for other brain cancer types\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. While neurosurgical biopsies are generally characterized by low complication rates, various clinical scenarios preclude or delay this invasive procedure and final CNSL diagnosis. For example, tumor localization in eloquent brain areas or surgical high-risk situations associated with patient frailty might result in unacceptable perioperative morbidity. Furthermore, concurrent corticosteroid or antiplatelet treatment often cause a significant delay of stereotactic procedures and might lead to indeterminate histopathological findings\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn these critical scenarios and due to a rapid decline of neurological recovery and therapy response rates associated with a delay of treatment initiation in this aggressive lymphoma type, minimal-invasive identification of CNSL from cerebrospinal fluid (CSF) could have transformative impact on the clinical management of patients with unknown CNS lesions\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This also extends to patients with suspected secondary involvement of systemic lymphomas or relapse of primary CNSL (PCNSL), who would benefit from surgery-free confirmation of diagnosis to tailor further treatment. Conventional CSF analyses such as cytopathology (CP) or flow cytometry (FC) and diagnostic magnetic resonance imaging (MRI) have shown low sensitivities and discriminative capacity to facilitate biopsy-free CNSL diagnosis, highlighting the need for more innovative technologies\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Several previous research studies have demonstrated reliable detection of the lymphoma-specific \u003cem\u003eMYD88\u003c/em\u003e L265P mutation in circulating tumor DNA (ctDNA) from CSF of CNSL patients and showed a potential treatment-guiding effect in anecdotal cases or in small case series\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. However, the value of a liquid biopsy approach for surgical planning and treatment management in large patient cohorts and daily clinical practice has never been prospectively explored. Therefore, we here developed and implemented a sensitive and highly specific digital droplet PCR (ddPCR) assay in a clinical laboratory environment for minimal-invasive detection of \u003cem\u003eMYD88\u003c/em\u003e L265P from CSF and evaluated its impact on patient clinical management.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMYD88\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eL265P ddPCR assay development and independent validation\u003c/span\u003e\u003c/p\u003e \u003cp\u003eTo develop a ddPCR assay as a laboratory-developed test (LDT) for the analysis of \u003cem\u003eMYD88\u003c/em\u003e L265P in ctDNA of body fluids, we underwent an approval 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. The assay settings, performance parameters, and technical results that led to approval of the LDT by the DAkKS for its use in clinical routine are described in detail and depicted in the Supplementary Methods section, \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e, and \u003cb\u003eSupplementary Tables\u0026nbsp;1\u0026ndash;6\u003c/b\u003e. This included the assessment of the limit of detection (LOD), limit of blank (LOB), analytical sensitivity and specificity, linearity, accuracy, and inter-/intra-assay reliability (\u003cb\u003eSupplementary Fig.\u0026nbsp;1, Supplementary Tables\u0026nbsp;1\u0026ndash;6\u003c/b\u003e). Collectively, the development process revealed a LOB of 0.5 detected mutant copies per mL sample volume and a LOD of 0.05% mutant allele frequency (AF) as key parameters of our assay that were applied to all subsequent analyses to faithfully determine the presence of \u003cem\u003eMYD88\u003c/em\u003e L265P in ctDNA of body fluids (Supplementary Methods).\u003c/p\u003e \u003cp\u003eNext, we tested and validated the performance of the \u003cem\u003eMYD88\u003c/em\u003e L265P ddPCR assay in an independent research study cohort of patients whose clinical situations reflected one of the intended applications of the LDT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We applied the assay to 77 CSF specimens (median volume: 1.9 mL, range: 0.2\u0026ndash;9.9 mL) and 91 plasma samples (median volume: 8.5 mL, range: 2.7\u0026ndash;11.8 mL) collected from 128 patients with contrast-enhancing brain lesions and verified histopathological diagnosis to evaluate its diagnostic sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in both compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cb\u003eSupplementary Fig.\u0026nbsp;2a\u003c/b\u003e, and \u003cb\u003eSupplementary Table\u0026nbsp;7\u003c/b\u003e). Two-thirds of these patients (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;87, 68%) were diagnosed with either primary CNSL (PCNSL, 88%) or isolated secondary CNSL (iSCNSL, 12%) (defined as \u0026lsquo;CNSL patients\u0026rsquo;), while 41 patients showed a broad variety of other brain diseases, including primary brain tumors (glioblastoma, lower-grade gliomas and others, 46%), brain metastases (25%), or inflammatory/infectious diseases such as multiple sclerosis (MS, 29%) (defined as \u0026lsquo;Non-CNSL patients\u0026rsquo;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b and \u003cb\u003eSupplementary Table\u0026nbsp;7\u003c/b\u003e). In this setting, which is agnostic to the tumor mutation status (\u0026lsquo;tumor-agnostic approach\u0026rsquo;), we minimal-invasively detected \u003cem\u003eMYD88\u003c/em\u003e L265P with a sensitivity of 67% from CSF and 37% from plasma samples of patients with confirmed CNSL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In comparison, conventional CSF analyses including CP and FC identified CNSL infiltration with a sensitivity of 22% (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;46, \u003cb\u003eSupplementary Fig.\u0026nbsp;2b\u003c/b\u003e). Importantly, the hotspot variant was never detected in Non-CNSL patients, revealing a specificity and PPV of 100% for both analytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The amount of ctDNA was 24-fold higher in \u003cem\u003eMYD88\u003c/em\u003e L265P-positive CSF samples than in plasma, with a median CSF-ctDNA AF of 5.5% compared to 0.23% in blood plasma (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001, \u003cb\u003eSupplementary Fig.\u0026nbsp;2c\u003c/b\u003e). In 49 CNSL cases, the \u003cem\u003eMYD88\u003c/em\u003e L265P mutation was detected in a matched tumor specimen by targeted-capture next-generation sequencing (NGS, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Limiting our analyses to CSF/plasma samples with corresponding tumors and known \u003cem\u003eMYD88\u003c/em\u003e L265P positivity (\u0026lsquo;tumor-informed approach\u0026rsquo;), we observed a clinical sensitivity for our assay of 82% in CSF and 37% in plasma specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Finally, we further assessed the reproducibility of our measurements and representation of allelic fractions across analytical platforms, comparing our results with those obtained from targeted-capture NGS of 75 CSF and plasma samples (\u003cb\u003eSupplementary Table\u0026nbsp;7\u003c/b\u003e, Supplementary Methods). We found a high concordance rate of 89.3% for the detection of \u003cem\u003eMYD88\u003c/em\u003e L265P and a significant correlation of ctDNA concentrations between the technologies (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, r\u0026thinsp;=\u0026thinsp;0.99, \u003cb\u003eSupplementary Fig.\u0026nbsp;2d\u003c/b\u003e). Collectively, these data demonstrate robust performance of the ddPCR technology in a representative clinical setting, revealing superior sensitivity for minimal-invasive \u003cem\u003eMYD88\u003c/em\u003e L265P identification in the CSF compared to blood plasma, mainly due to markedly higher ctDNA concentrations in this compartment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFeasibility of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eMYD88\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eL265P analysis from CSF-ctDNA in clinical practice\u003c/span\u003e\u003c/p\u003e \u003cp\u003eAfter implementation in our clinical laboratory environment, we applied the \u003cem\u003eMYD88\u003c/em\u003e L265P ddPCR assay to 205 CSF samples collected from 182 hospitalized patients and submitted to our laboratory between January 2022 and June 2024 (\u003cb\u003eSupplementary Fig.\u0026nbsp;3a\u003c/b\u003e and \u003cb\u003eSupplementary Table\u0026nbsp;8\u003c/b\u003e). CSF samples were obtained at the discretion of the treating physician and submitted from hospitals of the University Medical Center Freiburg (Departments of Neurosurgery, Neurology, and Hematology/Oncology, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;85) and 20 external referral centers (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;120) (\u003cb\u003eSupplementary Table\u0026nbsp;8\u003c/b\u003e). The number of CSF submissions steadily increased from a median of 8 samples per quarter in 2022 to a median of 46.5 samples per quarter in 2024 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Submitted CSF volumes ranged from 0.5 to 10 mL, with a median of 2.9 mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). We reported the results of the \u003cem\u003eMYD88\u003c/em\u003e L265P ddPCR assay to treating physicians with a median turnaround time of 5 days (average: 5 days), with more than a third of analyses (36%) being completed within 3 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). We detected the hotspot variant in 68 CSF samples (33%), while 137 samples (67%) showed a negative result (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and \u003cb\u003eSupplementary Table\u0026nbsp;8\u003c/b\u003e). The mutational AFs ranged from 0.055\u0026ndash;25.2% in ctDNA-positive CSF samples, with a median of 0.55% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGuiding clinical management by CSF-ctDNA profiling\u003c/h2\u003e \u003cp\u003e127 patients, providing 143 CSF samples, participated in our observational study, facilitating the evaluation of clinical, pathological, and radiological information as well as the assessment of clinical implications following CSF-ctDNA analyses (\u003cb\u003eSupplementary Fig.\u0026nbsp;3a\u003c/b\u003e and \u003cb\u003eSupplementary Table\u0026nbsp;9\u003c/b\u003e). Reasons for CSF submission in the observational study included the presence of an unclear brain lesion with CNSL as differential diagnosis ([1], \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;49, 34%) and either surgical high-risk situation due to patient frailty or localization in an eloquent brain region ([1a], \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;25, 17%), delay of surgery due to concomitant steroid/antiplatelet treatment ([1b], \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8, 6%), or the lack of a definitive diagnosis after biopsy and histopathological assessment ([1c], \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16, 11%), a suspected secondary brain manifestation of systemic lymphoma either as occult involvement ([2a], \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;35, 24%), synchronous involvement ([2b], \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6, 4%), or metachronous SCNSL relapse ([2c], \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15, 10%), or a suspected PCNSL relapse ([2d], \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8, 6%), and the request to profile CSF-ctDNA as a monitoring biomarker in patients with known CNSL ([3], \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;30, 21%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cb\u003eSupplementary Table\u0026nbsp;9\u003c/b\u003e). \u003cem\u003eMYD88\u003c/em\u003e L265P was detected in 52 of 143 CSF samples (36%) from patients participating in the observational study (\u003cb\u003eSupplementary Fig.\u0026nbsp;3a\u003c/b\u003e and \u003cb\u003eSupplementary Table\u0026nbsp;9\u003c/b\u003e). In 31 CSF-ctDNA positive cases (60%), the ddPCR results directly guided or helped guiding further treatment and surgical management (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-d). Minimal-invasive identification of \u003cem\u003eMYD88\u003c/em\u003e 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%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-d and \u003cb\u003eSupplementary Table\u0026nbsp;9\u003c/b\u003e). Specifically, 33 patients did not undergo surgery or biopsies were delayed due to surgical high-risk situations ([1a,1b], Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Positivity of CSF-ctDNA (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9, 27%) eliminated the need for neurosurgical interventions in 78% of these patients and was treatment-guiding in 56% of cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Two patients received an accelerated diagnosis of CNSL by 6 and 11 days, respectively; however, this had no effect on surgical planning or treatment initiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cb\u003eSupplementary Table\u0026nbsp;9\u003c/b\u003e). In 16 patients, histopathological assessment following stereotactic biopsies revealed no definitive diagnosis ([1c], Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). \u003cem\u003eMYD88\u003c/em\u003e L265P was detected in 6 of these patients (38%), leading to CNSL-specific treatment in 83% of cases without further pathological confirmation and supported the diagnosis of a suspected but not confirmed cerebral amyloidoma from Waldenstrom macroglobulinemia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cb\u003eSupplementary Table\u0026nbsp;9\u003c/b\u003e). Finally, we received 64 CSF samples from patients with suspected CNS involvement of systemic lymphomas or suspected PCNSL relapse and found the CNSL-specific hotspot variant in 18 cases (28%, [2], Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). These positive findings contributed to surgical planning and obviated stereotactic biopsies in 50% of patients, while 15 patients (83%) received CNSL-specific treatment following CSF-ctDNA profiling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cb\u003eSupplementary Table\u0026nbsp;9\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCNS-directed therapies were initiated in 25 patients with the support of our analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). While 6 patients (24%) received palliative treatment, 19 patients (76%) underwent curative-intent immunochemotherapies that were mostly based on high-dose methotrexate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and \u003cb\u003eSupplementary Table\u0026nbsp;9\u003c/b\u003e). 94% of patients with an evaluable CNS lesion by MRI responded to therapies (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17), while two patients with no radiological follow-up or evaluable brain lesion showed a clinical response (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Yet, one patient experienced a rapid progression following palliative rituximab and radiotherapy that was initiated based on CSF-ctDNA results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and \u003cb\u003eSupplementary Table\u0026nbsp;9\u003c/b\u003e). \u003cb\u003eSupplementary Fig.\u0026nbsp;3b-e\u003c/b\u003e highlights four representative cases in which CNSL-specific therapies were applied based on minimal-invasive genotyping from CSF, leading to clinical responses of their brain lesions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparison with conventional CSF analyses and influence of corticosteroid treatment\u003c/h3\u003e\n\u003cp\u003e142 CSF samples of the observational study were simultaneously analyzed by conventional CP and/or FC, revealing a positivity rate of 4% for the detection of CNSL (\u003cb\u003eSupplementary Fig.\u0026nbsp;4a,b\u003c/b\u003e). In case of CSF-ctDNA positivity, the concordance rate with conventional CSF analyses was low (6%), while the vast majority of \u003cem\u003eMYD88\u003c/em\u003e L265 negative cases were CSF CP/FC negative (97%) and only three CSF samples with monoclonal CD19-positive lymphoma cells were ctDNA-negative (3%, \u003cb\u003eSupplementary Fig.\u0026nbsp;4a,b\u003c/b\u003e). In 26 patients of the observational study, a histopathological diagnosis was achieved after stereotactic biopsy of the CNS lesion, performed prior to or concurrently with CSF sampling (\u003cb\u003eSupplementary Table\u0026nbsp;9\u003c/b\u003e). \u003cem\u003eMYD88\u003c/em\u003e L265P was identified in 12 patients with verified CNSL (60%, 12/20) and never detected in 6 patients with other brain malignancies (\u003cb\u003eSupplementary Fig.\u0026nbsp;4b\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eFinally, we explored the influence of corticosteroid treatment on CSF-ctDNA detection rates and concentrations. We found no differences in \u003cem\u003eMYD88\u003c/em\u003e L265P detection rates between patients who received corticosteroids prior to the lumbar puncture and those who did not undergo corticosteroid therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Furthermore, we observed that cumulative corticosteroid doses preceding lumbar punctures did not correlate with CSF-ctDNA concentrations in mutated samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe here demonstrate that minimal-invasive identification of CNSL by ctDNA profiling from CSF can effectively guide treatment and surgical management in daily clinical practice and has practice-changing impact for a substantial subset of patients with unknown CNS lesions in challenging clinical situations. Due to the aggressiveness and localization of brain lymphomas, rapid diagnostic workup and treatment initiation are crucial to prevent neurological impairment and maintain favorable clinical outcomes\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Yet, various factors in clinical reality, which can be either patient-specific, treatment-related, or associated with the disease, often impair a timely and accurate diagnosis of CNSL. In these situations, there is a critical need for minimal-invasive approaches that utilize CSF biomarkers for the identification of CNSL. Current CNSL clinical guidelines such as the 2024 \u0026lsquo;EHA-ESMO Clinical Practice Guideline\u0026rsquo; consider the assessment of CSF biomarkers including \u003cem\u003eMYD88\u003c/em\u003e L265P as a promising tool for CNSL diagnosis when biopsies are not possible\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. A plethora of research studies have explored protein and genetic markers including \u003cem\u003eMYD88\u003c/em\u003e L265P for biopsy-free identification of CNSL from CSF\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, despite exhibiting some clinical utility in anecdotal cases and small case series, these technologies have not been regularly and broadly applied in clinical routine yet, largely due to the lack of standardization, prospective validation, and uncertainties associated with limited inter-laboratory reproducibility.\u003c/p\u003e \u003cp\u003eWe here underwent an extensive regulatory development and validation process that led to the implementation of a \u003cem\u003eMYD88\u003c/em\u003e L265P ddPCR assay in our clinical laboratory environment for minimal-invasive detection of CNSL from CSF. This procedure was optimized for high specificity to minimize false-positive results and misclassification, which could result in inappropriate treatment of patients with other brain diseases. Previous reports suggested that the \u003cem\u003eMYD88\u003c/em\u003e L265P mutation might be present in patients with neurologic autoimmune conditions such as MS or associated with lymphoid clonal hematopoiesis of indeterminate potential (CHIP)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In our study and in work from other groups, \u003cem\u003eMYD88\u003c/em\u003e L265P was never observed in patients with inflammatory or autoimmune brain diseases nor in the plasma of elderly healthy individuals, revealing 100% specificity across distinct and independent Non-CNSL cohorts\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe applied our LDT to 205 CSF samples over a period of 2.5 years in clinical practice and assessed its implications in an observational study. Results were communicated to treating physicians within 5 days (including weekends) and directly guided or helped guiding clinical management in 60% of CSF-ctDNA positive cases. Neurosurgical interventions were obviated or CNSL diagnosis was accelerated in 19 patients and a treatment-guiding effect was reported for 25 patients who received CNS-directed agents based on or supported by minimal-invasive CSF-ctDNA detection. Importantly, all but one patient objectively responded to CNSL-specific therapies, illustrating the clinical benefit of our liquid biopsy approach for a significant subgroup of patients whose final diagnosis otherwise might have remained unconfirmed or who would have faced high-risk surgical interventions to obtain a diagnosis. Of note, while positive CSF-ctDNA results can directly guide treatment or surgical strategies due to their high PPV, assessing the clinical impact of CSF-ctDNA negative results represents a considerable challenge, because a negative \u003cem\u003eMYD88\u003c/em\u003e L265P status does not exclude CNSL or help defining another diagnosis. Thus, knowing that CSF samples are negative for \u003cem\u003eMYD88\u003c/em\u003e L265P might help narrowing down differential diagnoses in certain scenarios, but it typically has no direct influence on the clinical management of patients with unknown CNS lesions.\u003c/p\u003e \u003cp\u003eThe most evident limitation of our technology can be attributed to the fact that approximately 30% of CNSL patients do not harbor the hotspot \u003cem\u003eMYD88\u003c/em\u003e L265P mutation, inevitably reducing applicability and sensitivity/NPV of our tumor-agnostic approach\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Alternative strategies have revealed higher sensitivities for CNSL identification, including assays that combine the detection of \u003cem\u003eMYD88\u003c/em\u003e L265P with protein biomarkers or immunoglobulin rearrangements, or next-generation sequencing-based technologies that capture hundreds or thousands of CNSL-specific genomic regions. However, despite showing promising results in retrospective studies, these methods require further regulatory standardization and have yet to prove their utility in clinical practice and large patient cohorts\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnother notable finding of our study was that the exposure to corticosteroids prior to lumbar punctures did not seem to have any impact on CNSL detection rates or the representation of \u003cem\u003eMYD88\u003c/em\u003e L265P allelic fractions. Corticosteroids are often used to manage neurological symptoms in CNSL patients but cause severe diagnostic delays and other challenges associated with steroid-induced transient lymphoma regression\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Our results indicate that CSF-ctDNA can be robustly detected irrespective of corticosteroid premedication, encouraging the use of minimal-invasive liquid biopsy technologies even in situations with radiological tumor reduction following steroid therapy. Yet, the duration of corticosteroid treatment and dosage were highly heterogeneous in our patient cohort, introducing some uncertainties around interpretability of the results and requiring further investigation of the corticosteroid effect for CSF-ctDNA detection in a prospective and standardized fashion.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAssay design and approval process\u003c/h2\u003e \u003cp\u003eWe designed a ddPCR assay as a LDT for the detection of \u003cem\u003eMYD88\u003c/em\u003e L265P in the ctDNA of body fluids for its use in clinical practice. A detailed description of the assay development and the LDT approval process according to the DIN ISO 15189:2104-11 criteria of the national accreditation body of the Federal Republic of Germany (DAkkS, D-ML-13134-07-00) is available in the Supplementary Methods section of the Supplementary Information.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssay validation\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eMYD88\u003c/em\u003e L265P ddPCR assay was comprehensively tested and validated in an independent clinical cohort of patients with contrast-enhancing brain lesions and verified histopathological diagnosis to evaluate its diagnostic sensitivity and specificity in both CSF and plasma samples. Samples from these patients were collected from patients participating in research studies conducted at the University Medical Center Freiburg, Germany (DRKS15307), and University Hospital of the Ludwig-Maximilians-University (LMU) Munich, Germany (Nr. 22\u0026thinsp;\u0026minus;\u0026thinsp;0008) that were approved by the ethics committees in accordance with the Declaration of Helsinki. In total, we collected 77 CSF samples and 91 plasma samples from 87 CNSL patients and 41 Non-CNSL patients with a broad variety of malignant, inflammatory, and infectious brain diseases. From 75 of these CSF/plasma samples and from 49 matched tumor specimens, the \u003cem\u003eMYD88\u003c/em\u003e L265P mutation status was additionally assessed by a targeted-capture NGS technology that has been described in previous studies (CAPP-Seq)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAssay application in routine practice\u003c/h3\u003e\n\u003cp\u003eWe analyzed CSF specimens obtained from hospitalized patients at 21 distinct centers between January 2022 and June 2024 by the approved \u003cem\u003eMYD88\u003c/em\u003e L265P ddPCR assay in our clinical laboratory at the University Medical Center Freiburg, Germany. The sample processing and analysis workflows are described in detail in the Supplementary Methods section. Results of the analyses were reported directly to treating physicians, who made treatment decisions and conducted communication of findings with patients. A subset of patients participated in our observational study, approved by the ethics committee according to the Declaration of Helsinki, that facilitated the use of clinical, pathological, and radiological information, and enabled the assessment of clinical implications following CSF-ctDNA analyses (DRKS00034686).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Advanced Clinician Scientist Program of the Deutsche Gesellschaft f\u0026uuml;r Innere Medizin (DGIM, to F.S.), the Deutsche Forschungsgemeinschaft (DFG, to F.S.), the Mertelsmann Foundation (to F.S.), the German Cancer Research Center (Deutsches Konsortium f\u0026uuml;r Translationale Krebsforschung, DKTK, to F.S.), the Else Kr\u0026ouml;ner-Fresenius-Stiftung (to F.S. and P.C.R), and Fraunhofer Society Germany (to P.C.R.). S.W. is supported by the Jos\u0026eacute; Carreras-DGHO fellowship. J.C.K. and N.N. are supported by the Berta-Ottenstein Programm F\u0026ouml;rderlinie Clinician Scientist of the University Medical Center Freiburg. We thank the FREEZE biobank Freiburg for their support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.S. receives research funding from Gilead Sciences, Roche Sequencing Solutions, and Takeda, and received honoraria from AstraZeneca and Servier. S.K.A reports consulting for Foresight Diagnostics. E.S. received honoraria from SERB Pharmaceuticals. N.N. serves on advisory board for Servier and is consultant for B. Braun. P.C.R. received honoraria from Arcana and serves as consultant for Boston Scientific, Inomed, and Brainlab. All other authors did not report any conflicts of interest related to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Information exists for this manuscript and can be found in separate files (Supplementary Information [including Supplementary Methods and Supplementary Figures] and Supplementary Tables).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlaggio R, Amador C, Anagnostopoulos I, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms. Leukemia. 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J Clin Oncol. 10 2018;36(28):2845\u0026ndash;2853. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1200/JCO.2018.78.5246\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2018.78.5246\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5099294/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5099294/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVarious clinical scenarios preclude or delay invasive stereotactic biopsies and subsequent histopathological assessment for the diagnosis of central nervous system lymphoma (CNSL), necessitating innovative minimal-invasive strategies. We designed a digital droplet PCR (ddPCR) assay for minimal-invasive identification of CNSL in clinical practice by detecting \u003cem\u003eMYD88\u003c/em\u003e L265P mutations in circulating tumor DNA (ctDNA) of cerebrospinal fluid (CSF). After assay approval by the national accreditation authority, this laboratory-developed test (LDT) was first independently validated in a cohort of 128 patients with confirmed malignant or inflammatory/infectious brain diseases, revealing a sensitivity of 67% and specificity of 100% for correct CNSL diagnosis. Following implementation in a clinical laboratory environment, the LDT was applied to 205 CSF samples from 182 independent patients, reporting results to treating physicians with a median turnaround time of 5 days. The \u003cem\u003eMYD88\u003c/em\u003e L265P mutation was detected in 33% of CSF specimens, obviating the need for invasive surgical biopsies in 37% of patients and guiding lymphoma-specific treatment in 48% of evaluable cases. 94% of patients undergoing CNS-directed treatment based on CSF-ctDNA profiling objectively responded to therapies. Collectively, our results demonstrate that minimal-invasive identification of CNSL by ctDNA genotyping in CSF can effectively guide clinical management and has practice-changing impact for a substantial subset of patients with unknown CNS lesions.\u003c/p\u003e","manuscriptTitle":"Guiding treatment and clinical management of patients with CNS lymphomas by minimal-invasive detection of circulating tumor DNA in cerebrospinal fluid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-14 09:04:24","doi":"10.21203/rs.3.rs-5099294/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cbf49ea7-f0d8-44fc-a79b-655c5843dcd1","owner":[],"postedDate":"October 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38728844,"name":"Health sciences/Oncology/Cancer/CNS cancer"},{"id":38728845,"name":"Health sciences/Biomarkers/Diagnostic markers"},{"id":38728846,"name":"Health sciences/Oncology/Cancer/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/B-cell lymphoma"}],"tags":[],"updatedAt":"2024-11-15T17:30:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-14 09:04:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5099294","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5099294","identity":"rs-5099294","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}