BACKGROUND: Diagnosing primary or secondary CNS lymphoma (CNSL) is a clinical challenge due to the limitations of standard biopsy and imaging procedures despite established guidelines. Therefore, accurate biomarkers and analytical methods that are convenient for practical routine use are needed to diagnose and manage these aggressive lymphomas effectively. We evaluated the utility of minimally invasive circulating tumor DNA (ctDNA) detection in a prospective real-world scenario, moving this approach closer to clinical practice. METHODS: A total of 164 plasma, cerebrospinal fluid (CSF), and tumor samples from 56 CNSL patients were collected to analyze tumor DNA by the diagnostic next-generation sequencing (NGS) panel LYNX, enabling simultaneous analysis of gene variants, chromosomal aberrations, and antigen receptor rearrangements in targeted regions. RESULTS: The well-known genetic heterogeneity of CNSL was refined with integrative molecular data, showing the most frequent MYD88, PIM1, and KMT2D mutations and a broad spectrum of chromosomal aberrations, reflecting high genomic complexity. The multi-target approach achieved a substantially higher detection rate of CNS infiltration (90%) than tracking a single variant in gene MYD88 (46%). CSF clearly surpasses plasma if applying a routine (non-ultrasensitive) NGS approach and allows for more reliable evidence of CNS involvement than conventional flow cytometry (91% vs. 21%, p < 0.001). Parallel analysis of tumor DNA in both cell-free and cellular DNA from CSF makes the probability of primary or secondary CNS malignancy detection even higher. CONCLUSIONS: Our prospective, tissue-agnostic approach highlights the feasibility of ctDNA sequencing by a commonplace and affordable method, offering higher sensitivity to detect CNS infiltration with lymphoma than standard cell-analyzing techniques. We accentuate the benefit of a multi-target NGS approach and adequate CSF sampling to obtain satisfactory diagnostic yield. Less invasive liquid biopsy testing by comprehensive NGS complements standard procedures in the diagnostics and management of CNSL patients, especially when encountering limitations.

Center of Molecular Medicine CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Internal Medicine Hematology and Oncology University Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

Department of Medical Genetics and Genomics Faculty of Medicine Masaryk University and University Hospital Brno Brno Czech Republic

Department of Pathology University Hospital Brno Brno Czech Republic

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Khwaja J, Nayak L, Cwynarski K. Evidence-based management of primary and secondary CNS lymphoma. Semin Hematol. 2023;60:313–21. PubMed

Bobillo S, Khwaja J, Ferreri AJM, Cwynarski K. Prevention and management of secondary central nervous system lymphoma. Haematologica. 2023;108:673–89. PubMed PMC

Miller KD, Ostrom QT, Kruchko C, Patil N, Tihan T, Cioffi G, et al. Brain and other central nervous system tumor statistics, 2021. CA Cancer J Clin. 2021;71:381–406. PubMed

Green K, Munakomi S, Hogg JP. Central nervous system lymphoma. StatPearls. Treasure Island (FL): StatPearls Publishing; 2025. PubMed

Grommes C, DeAngelis LM. Primary CNS lymphoma. J Clin Oncol. 2017;35:2410–8. PubMed PMC

Savage KJ. Secondary CNS relapse in diffuse large B-cell lymphoma: defining high-risk patients and optimization of prophylaxis strategies. Hematol Am Soc Hematol Educ Program. 2017;2017:578–86. PubMed PMC

Roschewski M, Hodson DJ. Diffuse large B-cell lymphoma involving the central nervous system: biologic rationale for targeted therapy. Haematologica. 2024;109:388–400. PubMed PMC

Barajas RF, Politi LS, Anzalone N, Schöder H, Fox CP, Boxerman JL, et al. Consensus recommendations for MRI and PET imaging of primary central nervous system lymphoma: guideline statement from the international primary CNS lymphoma collaborative group (IPCG). Neuro-Oncol. 2021;23:1056–71. PubMed PMC

Quijano S, López A, Manuel Sancho J, Panizo C, Debén G, Castilla C, et al. Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin’s lymphoma: improved sensitivity of flow cytometry. J Clin Oncol. 2009;27:1462–9. PubMed

Baraniskin A, Schroers R. Liquid biopsy and other noninvasive diagnostic measures in PCNSL. Cancers. 2021;13:2665. PubMed PMC

Velasco R, Mercadal S, Vidal N, Alañá M, Barceló MI, Ibáñez-Juliá MJ, et al. Diagnostic delay and outcome in immunocompetent patients with primary central nervous system lymphoma in Spain: a multicentric study. J Neurooncol. 2020;148:545–54. PubMed

Bobillo S, Crespo M, Escudero L, Mayor R, Raheja P, Carpio C, et al. Cell free Circulating tumor DNA in cerebrospinal fluid detects and monitors central nervous system involvement of B-cell lymphomas. Haematologica. 2021;106:513–21. PubMed PMC

Mutter JA, Alig SK, Esfahani MS, Lauer EM, Mitschke J, Kurtz DM, et al. Circulating tumor DNA profiling for detection, risk stratification, and classification of brain lymphomas. J Clin Oncol. 2023;41:1684–94. PubMed PMC

Heger J-M, Mattlener J, Schneider J, Gödel P, Sieg N, Ullrich F, et al. Entirely noninvasive outcome prediction in central nervous system lymphomas using Circulating tumor DNA. Blood. 2024;143:522–34. PubMed

Alig S, Macaulay CW, Kurtz DM, Dührsen U, Hüttmann A, Schmitz C, et al. Short Diagnosis-to-Treatment interval is associated with higher Circulating tumor DNA levels in diffuse large B-Cell lymphoma. J Clin Oncol. 2021;39:2605–16. PubMed PMC

Kurtz DM, Scherer F, Jin MC, Soo J, Craig AFM, Esfahani MS, et al. Circulating tumor DNA measurements as early outcome predictors in diffuse large B-Cell lymphoma. J Clin Oncol. 2018;36:2845–53. PubMed PMC

Scherer F, Kurtz DM, Newman AM, Stehr H, Craig AFM, Esfahani MS, et al. Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by Circulating tumor DNA. Sci Transl Med. 2016;8:364ra155. PubMed PMC

Boire A, Brandsma D, Brastianos PK, Le Rhun E, Ahluwalia M, Junck L, et al. Liquid biopsy in central nervous system metastases: a RANO review and proposals for clinical applications. Neuro-Oncol. 2019;21:571–84. PubMed PMC

Martínez-Ricarte F, Mayor R, Martínez-Sáez E, Rubio-Pérez C, Pineda E, Cordero E, et al. Molecular diagnosis of diffuse gliomas through sequencing of Cell-Free Circulating tumor DNA from cerebrospinal fluid. Clin Cancer Res. 2018;24:2812–9. PubMed

Olszewski AJ, Chorzalska AD, Petersen M, Ollila TA, Zayac A, Kurt H, et al. Detection of clonotypic DNA in the cerebrospinal fluid as a marker of central nervous system invasion in lymphoma. Blood Adv. 2021;5:5525–35. PubMed PMC

Yoon SE, Kim YJ, Shim JH, Park D, Cho J, Ko YH, et al. Plasma Circulating tumor DNA in patients with primary central nervous system lymphoma. Cancer Res Treat. 2022;54:597–612. PubMed PMC

Hattori K, Sakata-Yanagimoto M, Suehara Y, Yokoyama Y, Kato T, Kurita N, et al. Clinical significance of disease-specific MYD88 mutations in Circulating DNA in primary central nervous system lymphoma. Cancer Sci. 2018;109:225–30. PubMed PMC

Hiemcke-Jiwa LS, Leguit RJ, Snijders TJ, Bromberg JEC, Nierkens S, Jiwa NM, et al. MYD88 p.(L265P) detection on cell-free DNA in liquid biopsies of patients with primary central nervous system lymphoma. Br J Haematol. 2019;185:974–7. PubMed

Wright GW, Huang DW, Phelan JD, Coulibaly ZA, Roulland S, Young RM, et al. A probabilistic classification tool for genetic subtypes of diffuse large B cell lymphoma with therapeutic implications. Cancer Cell. 2020;37:551–e56814. PubMed PMC

Watanabe J, Natsumeda M, Okada M, Kobayashi D, Kanemaru Y, Tsukamoto Y, et al. High detection rate of MYD88 mutations in cerebrospinal fluid from patients with CNS lymphomas. JCO Precis Oncol. 2019;3:1–13. PubMed

Rossi D, Spina V, Bruscaggin A, Gaidano G. Liquid biopsy in lymphoma. Haematologica. 2019;104:648–52. PubMed PMC

Hiemcke-Jiwa LS, Ten Dam-van Loon NH, Leguit RJ, Nierkens S, Ossewaarde-van Norel J, de Boer JH, et al. Potential diagnosis of vitreoretinal lymphoma by detection of MYD88 mutation in aqueous humor with ultrasensitive droplet digital polymerase chain reaction. JAMA Ophthalmol. 2018;136:1098–104. PubMed PMC

Bravetti C, Degaud M, Armand M, Sourdeau E, Mokhtari K, Maloum K, et al. Combining MYD88 L265P mutation detection and clonality determination on CSF cellular and cell-free DNA improves diagnosis of primary CNS lymphoma. Br J Haematol. 2023;201:1088–96. PubMed

Navrkalova V, Plevova K, Hynst J, Pal K, Mareckova A, Reigl T, et al. LYmphoid NeXt-Generation sequencing (LYNX) panel: A comprehensive Capture-Based sequencing tool for the analysis of prognostic and predictive markers in lymphoid malignancies. J Mol Diagn. 2021;23:959–74. PubMed

R Core Team. R: A Language and environment for statistical computing. R Found Stat Comput. 2013:http://www.R-project.org/

Ollila TA, Kurt H, Waroich J, Vatkevich J, Sturtevant A, Patel NR, et al. Genomic subtypes May predict the risk of central nervous system recurrence in diffuse large B-cell lymphoma. Blood. 2021;137:1120–4. PubMed PMC

Klanova M, Sehn LH, Bence-Bruckler I, Cavallo F, Jin J, Martelli M, et al. Integration of cell of origin into the clinical CNS international prognostic index improves CNS relapse prediction in DLBCL. Blood. 2019;133:919–26. PubMed PMC

Montesinos-Rongen M, Brunn A, Tuchscherer A, Borchmann P, Schorb E, Kasenda B, et al. Analysis of driver mutational hot spots in Blood-Derived Cell-Free DNA of patients with primary central nervous system lymphoma obtained before intracerebral biopsy. J Mol Diagn. 2020;22:1300–7. PubMed

Zviran A, Schulman RC, Shah M, Hill STK, Deochand S, Khamnei CC, et al. Genome-wide cell-free DNA mutational integration enables ultra-sensitive cancer monitoring. Nat Med. 2020;26:1114–24. PubMed PMC

Iriyama C, Murate K, Iba S, Okamoto A, Goto N, Yamamoto H, et al. Utility of cerebrospinal fluid liquid biopsy in distinguishing CNS lymphoma from cerebrospinal infectious/demyelinating diseases. Cancer Med. 2023;12:16972–84. PubMed PMC

Braggio E, Van Wier S, Ojha J, McPhail E, Asmann YW, Egan J, et al. Genome-Wide analysis uncovers novel recurrent alterations in primary central nervous system lymphomas. Clin Cancer Res. 2015;21:3986–94. PubMed PMC

Magnes T, Wagner S, Thorner AR, Neureiter D, Klieser E, Rinnerthaler G, et al. Clonal evolution in diffuse large B-cell lymphoma with central nervous system recurrence. ESMO Open. 2021;6:100012. PubMed PMC

Radke J, Ishaque N, Koll R, Gu Z, Schumann E, Sieverling L, et al. The genomic and transcriptional landscape of primary central nervous system lymphoma. Nat Commun. 2022;13:2558. PubMed PMC

Severson EA, Haberberger J, Hemmerich A, Huang RSP, Edgerly C, Schiavone K, et al. Genomic profiling reveals differences in primary central nervous system lymphoma and large B-Cell lymphoma, with subtyping suggesting sensitivity to BTK Inhibition. Oncologist. 2023;28:e26–35. PubMed PMC

Zhou Y, Liu W, Xu Z, Zhu H, Xiao D, Su W, et al. Analysis of genomic alteration in primary central nervous system lymphoma and the expression of some related genes. Neoplasia. 2018;20:1059–69. PubMed PMC

Belhouachi N, Xochelli A, Boudjoghra M, Lesty C, Cassoux N, Fardeau C, et al. Primary vitreoretinal lymphomas display a remarkably restricted Immunoglobulin gene repertoire. Blood Adv. 2020;4:1357–66. PubMed PMC

Riemersma SA, Oudejans JJ, Vonk MJ, Dreef EJ, Prins FA, Jansen PM, et al. High numbers of tumour-infiltrating activated cytotoxic T lymphocytes, and frequent loss of HLA class I and II expression, are features of aggressive B cell lymphomas of the brain and testis. J Pathol. 2005;206:328–36. PubMed

Bourbon E, Alcazer V, Cheli E, Huet S, Sujobert P. How to obtain a high quality ctdna in lymphoma patients: preanalytical tips and tricks. Pharm Basel Switz. 2021;14:617. PubMed PMC

Tumani H, Petereit HF, Gerritzen A, Gross CC, Huss A, Isenmann S, et al. S1 guidelines lumbar puncture and cerebrospinal fluid analysis (abridged and translated version). Neurol Res Pract. 2020;2:8. PubMed PMC

Hoang-Xuan K, Bessell E, Bromberg J, Hottinger AF, Preusser M, Rudà R, et al. Diagnosis and treatment of primary CNS lymphoma in immunocompetent patients: guidelines from the European association for Neuro-Oncology. Lancet Oncol. 2015;16:e322–332. PubMed

Schmitz N, Zeynalova S, Nickelsen M, Kansara R, Villa D, Sehn LH, et al. CNS international prognostic index: A risk model for CNS relapse in patients with diffuse large B-Cell lymphoma treated with R-CHOP. J Clin Oncol. 2016;34:3150–6. PubMed

Cisneros-Villanueva M, Hidalgo-Pérez L, Rios-Romero M, Cedro-Tanda A, Ruiz-Villavicencio CA, Page K, et al. Cell-free DNA analysis in current cancer clinical trials: a review. Br J Cancer. 2022;126:391–400. PubMed PMC

de Leval L, Alizadeh AA, Bergsagel PL, Campo E, Davies A, Dogan A, et al. Genomic profiling for clinical decision making in lymphoid neoplasms. Blood. 2022;140:2193–227. PubMed PMC

Mikolajewicz N, Yee PP, Bhanja D, Trifoi M, Miller AM, Metellus P, et al. Systematic review of cerebrospinal fluid biomarker discovery in Neuro-Oncology: A roadmap to standardization and clinical application. J Clin Oncol. 2024;42:1961–74. PubMed