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. 2025 Oct 6;15(10):2002-2018.
doi: 10.1158/2159-8290.CD-24-1788.

Detection of Human Brain Cancers using Genomic and Immune Cell Characterization of Cerebrospinal Fluid through CSF-BAM

Alexander H Pearlman #  1   2   3   4 , Yuxuan Wang #  1   2   3   4 , Anita Kalluri  5 , Megan Parker  5 , Joshua D Cohen  1   2   3   4 , Jonathan Dudley  1   2   3   4   6 , Jordina Rincon-Torroella  1   2   3   4   5 , Yuanxuan Xia  1   2   3   4   5 , Ryan Gensler  5 , Melanie Alfonzo Horowitz  5 , John N Theodore  5 , Lisa Dobbyn  1   2   3   4 , Maria Popoli  1   2   3   4 , Janine Ptak  1   2   3   4 , Natalie Silliman  1   2   3   4 , Kathy Judge  1   2   3   4 , Peter A Calabresi  7   8 , Mari Groves  5 , Christopher M Jackson  5 , Eric M Jackson  5 , George I Jallo  9 , Michael Lim  10 , Mark Luciano  5 , Debraj Mukherjee  5 , Jarushka Naidoo  11 , Sima Rozati  12 , Cole H Sterling  1   4 , Jon Weingart  5 , Carl Koschmann  13 , Alireza Mansouri  14 , Michael Glantz  14 , David Kamson  4   7 , Karisa C Schreck  4   7 , Carlos A Pardo  7 , Matthias Holdhoff  1   4 , Maximilian F Konig  2   15   16 , Suman Paul  1   2   4 , Kenneth W Kinzler  1   2   3   4 , Nickolas Papadopoulos  1   2   3   4 , Bert Vogelstein  1   2   3   4   17 , Christopher Douville  1   2   3   4 , Chetan Bettegowda  1   2   3   4   5
Affiliations

Detection of Human Brain Cancers using Genomic and Immune Cell Characterization of Cerebrospinal Fluid through CSF-BAM

Alexander H Pearlman et al. Cancer Discov. .

Abstract

Patients with 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 invasive and dangerous procedure. In this study, we present a new workflow termed "CSF-BAM" that simultaneously identifies B-cell or T-cell receptor sequences, aneuploidy, and mutations using amplification of both strands of the DNA from cerebrospinal fluid (CSF) samples. We applied CSF-BAM to a validation set of 209 samples from patients with brain cancers. Among the 129 samples from patients with the most common aggressive cancer types, the sensitivity of detection was 81%. None of 30 CSF-BAM assays were positive in CSF samples from patients without brain cancers (100% specificity). CSF-BAM provides an integrated approach to identify neoplasia in the central nervous system, provides information about the genetics and immune environment, and has the potential to inform patient management.

Significance: There is a paucity of technologies beyond surgical biopsy that can accurately diagnose central nervous system neoplasms. We developed a novel, sensitive, and highly specific assay that can detect brain cancers by comprehensively identifying somatic mutations, chromosomal copy-number changes, and adaptive immunoreceptor repertoires from samples of CSF. See related commentary by Weiss, p. 1976.

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Conflict of interest statement

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. P.A.C is a principal investigator on grants to Johns Hopkins University from Genentech. P.A.C. has received consulting honoraria for serving on scientific advisory boards for Biogen, Novartis, and Lilly. M.F.K. is a consultant to Allogene, Argenx, Atara Biotherapeutics, Bristol Myers Squibb, Revel Pharmaceuticals, Sana Biotechnology, and Sanofi. M.F.K. receives research support from Blackbird Labs. 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.

Figures

Figure 1.
Figure 1.
CSF-BAM overview. CSF is obtained and DNA is extracted from the entire sample. CSF-BAM examines three analytes simultaneously: B cell or T cell receptor rearrangements, Aneuploidy, and Mutations using PCR-mediated amplification of both strands of the DNA.
Figure 2.
Figure 2.
Schematic of CSF-BAM. A, Independent libraries are generated from each strand of the original DNA template molecules in a manner where unique molecular identifiers (UIDs) allow the strands from the two libraries to be mapped back to their original duplex. B, BCRs are evaluated with SafeBSeqS. Illustrated here is amplification of BCRs with a primer targeting the IGHJ1 segment, among the multiplex set of 4 total IGHJ primers. C, Aneuploidy is evaluated with WGS through amplification of total libraries using adapter-specific primers. D, Mutations are evaluated with SaferSeqS. Illustrated here is amplification of IDH1 using an IDH1-specific primer, among the multiplex set of 120 total gene-specific primers.
Figure 3.
Figure 3.
Performance of CSF-BAM and each analyte. The sensitivity of each analyte within CSF-BAM is demonstrated across the major classes of tumors tested. The composite sensitivity of CSF-BAM is demonstrated in black.
Figure 4.
Figure 4.
A, BCR clonality (clonality for non-evaluable samples with total UIDs ≤20 defined as 0), B, estimated tumor fraction by aneuploidy, and C, mutant allele frequency for each sample evaluated with CSF-BAM.
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