Patients who have radiographically detectable lesions in their brain or other symptoms compatible with brain tumors pose challenges for diagnosis. The only definitive way to diagnose such patients is through brain biopsy, an obviously invasive and dangerous procedure. Here we present a new workflow termed "CSF-BAM" that simultaneously identifies B cell or T cell receptor rearrangements, Aneuploidy, and Mutations using PCR-mediated amplification of both strands of the DNA from CSF samples. We first describe the details of the molecular genetic assessments and then establish thresholds for positivity using training sets of libraries from patients with or without cancer. We then applied CSF-BAM to an independent set of 206 DNA samples from patients with common, aggressive cancer types as well as other forms of brain cancers. Among the 126 samples from patients with the most common aggressive cancer types (high grade gliomas, medulloblastomas, or metastatic cancers to the brain), the sensitivity of detection was >81%. None of 33 CSF-BAM assays (100% specificity, 90% to 100% credible interval) were positive in CSF samples from patients without brain cancers. The sensitivity of CSF-BAM was considerably higher than that achieved with cytology. CSF-BAM provides an integrated multi-analyte approach to identify neoplasia in the central nervous system, provides information about the immune environment in patients with or without cancer, and has the potential to inform the subsequent management of such patients.

C.M.J. is a cofounder of Egret therapeutics with equity interests in the company and has received research support from Grifols, Biohaven, and InCephalo. E.M.J. is a consultant to Integra Lifesciences. K.C.S. reports consulting for Springworks, Nurix Therapeutics, and Novartis and research funding to Johns Hopkins from Fore Biotherapeutics, Springworks Therapeutics, and Lantern Pharma. C.H.S. and S.R. are consultants to Kiowa Kirin. C.H.S. has served on advisory boards for Acrotech Biopharma. C.H.S. has received honoraria from Medical Logix and Haymarket Medical Education. J.N. reports disclosures for AstraZeneca: research funding, consulting/advisory board, data safety monitoring board, honoraria; Bristol Myers Squibb: research funding, consulting/advisory board, data safety monitoring board, honoraria; Roche/Genentech: research funding, consulting/advisory board, honoraria; Amgen: research funding, consulting/advisory board; Arcus Biosciences: research funding, consulting/advisory board/steering committee; Mirati: research funding, honoraria; Novartis: research funding; Takeda research funding, consulting/advisory board; Pfizer: research funding, consulting/advisory board; Daiichi Sankyo: consulting/advisory board, data safety monitoring board, honoraria; NGM Pharmaceuticals: consulting/advisory board; Bayer: consulting/advisory board; Regeneron: consulting/advisory board; Elevation Oncology: consulting/advisory board; Abbvie: consulting/advisory board; Kaleido Biosciences: consulting/advisory board. S.P. is a consultant to Merck, owns equity in Gilead and received payments from IQVIA and Curio Science. B.V., K.W.K., and N.P. are founders of Thrive Earlier Detection, an Exact Sciences Company. K.W.K., N.P., and C.D. are consultants to Thrive Earlier Detection. B.V., K.W.K., N.P., and C.D. hold equity in Exact Sciences. N.P. and C.D. are consultants to Thrive Earlier Detection. B.V., K.W.K., J.D.C., and N.P. are founders of and own equity in Haystack Oncology. B.V., K.W.K., and N.P. are founders of and own equity in ManaT Bio. K.W.K. and N.P. are consultants to Neophore. K.W.K., B.V., and N.P. hold equity in and are consultants to CAGE Pharma. B.V. is a consultant to and holds equity in Catalio Capital Management. C.B. is a consultant to Depuy-Synthes, Bionaut Labs, Haystack Oncology, and Privo Technologies. C.B. is a cofounder of OrisDx. C.B. and C.D. are cofounders of Belay Diagnostics. The companies named above, as well as other companies, have licensed previously described technologies related to the work described in this paper from Johns Hopkins University. B.V., K.W.K., N.P., C. B., C.D., J.D.C., Y.W., and A.H.P. are inventors on some of these technologies. Licenses to these technologies are or will be associated with equity or royalty payments to the inventors and to Johns Hopkins University. Patent applications on the work described in this paper may be filed by Johns Hopkins University. The terms of all these arrangements are being managed by Johns Hopkins University in accordance with its conflict of interest policies.

Alexs Lemonade Stand Foundation (C. B.) American Society of Hematology Scholar award (S.P.) Benjamin Baker Endowment 80049589 (C.D. Y.W.) Burroughs Wellcome Career Award for Medical Scientists (C. B.) Commonwealth Fund (C.B. N.P.) Conrad R. Hilton Foundation (K.W.K. N.P. B.V.) DOD award W81XWH-21-1-0251 (K.C.S.) Doris Duke Career Development Award (K.C.S.) Jerome Greene Foundation (C.H.S.) Leukemia Lymphoma Society Translation Research Program award (S.P.) Lustgarten Foundation for Pancreatic Cancer Research (B.V.) NIH grant CA06973 (K.W.K. N.P. B.V.) NIH grant DRP80057309 (C.D.) NIH grant K08CA270403 (S.P.) NIH grant RA37CA230400 (C. B.) NIH grant R01CA276221 (C. B.) NIH grant R21TR004059 (C. B.) NIH grant T32GM136577 (J.D.C. A.H.P.) NIH grant U01CA230691 (C. B. N.P.) Sol Goldman Sequencing Facility at Johns Hopkins (B.V.) Swim Across America Translational Cancer Research Award (C.D. S.P.) Thomas M. Hohman Memorial Cancer Research Fund (C. B.) Virginia and D.K. Ludwig Fund for Cancer Research (C. B. C.D. K.W.K. N.P. B.V.)

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