Preanalytical variables and analytes in liquid biopsy approach for brain tumors: A comprehensive review and recommendations from the RANO Group and the Brain Liquid Biopsy Consortium

Chetan Bettegowda¹, Houtan Noushmehr², Alessandra Affinito³, Manmeet S Ahluwalia⁴, Olaf Ansorge⁵, Katayoun Ayasoufi⁶, Stephen Bagley⁷, Jill Barnholtz-Sloan⁸, Myron Best⁹, Dieta Brandsma¹⁰, Chaya Brodie¹¹, Anke Brüning-Richardson¹², Ana Valeria Castro^{2

13}, Susan M Chang¹⁴, Gerolama Condorelli³, Ahmad Daher¹⁵, Vineet Datta¹⁶, John de Groot¹⁴, Pim French¹⁷, Evanthia Galanis¹⁸, Anna Golebiewska¹⁹, Petra Hamerlik²⁰, C Oliver Hanemann²¹, Matthias Holdhoff²², Jason Huse²³, Mustafa Khasraw⁶, Suzanne LeBlang²⁴, Beatrice Melin²⁵, Florent Mouliere²⁶, Claire O'Leary²⁷, Janusz Rak²⁸, Amitava Ray²⁹, Stephen Robinson³⁰, Ola Rominiyi³¹, Federico Roncaroli³², Roberta Rudà³³, Joan Seoane³⁴, Nik Sol¹⁰, Martin J van den Bent³⁵, Michael A Vogelbaum³⁶, Tobias Walbert^{37

38}, Colin Watts³⁹, Tobias Weiss³⁷, Michael Weller³⁷, Patrick Y Wen⁴⁰, Victoria Wykes⁴¹, Stephen Yip⁴², Susan C Short⁴³, Riccardo Soffietti⁴⁴

Affiliations

¹ Department of Neurosurgery, Johns Hopkins Khatib Brain Tumor Center, Johns Hopkins University, Baltimore, USA.
² Department of Neurosurgery, Henry Ford Health System, Detroit, Michigan, USA.
³ Department of Molecular Medicine and Medical Biotechnology, Federico II University, Napoli, Italy.
⁴ Miami Cancer Institute, and Florida International University, Miami, Florida, USA.
⁵ Neuropathology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
⁶ Department of Neurosurgery, Duke University, Durham, North Carolina, USA.
⁷ University of Pennsylvania, Philadelphia, Pennsylvania, USA.
⁸ Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Maryland, USA.
⁹ Department of Neurosurgery, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
¹⁰ Department of Neuro-Oncology, Netherlands Cancer Institute, Amsterdam, The Netherlands.
¹¹ Faculty of Life Sciences and the Institute of Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat-Gan, Israel.
¹² School of Applied Sciences, University of Huddersfield, Huddersfield, UK.
¹³ Department of Physiology, Michigan State University, East Lansing, MI, USA.
¹⁴ Division of Neuro-Oncology, Department of Neurosurgery, University of California, San Francisco, California, USA.
¹⁵ Department of Neurology& Rehabilitation, University of Illinois at Chicago, Chicago, Illinois, USA.
¹⁶ Department of Research and Innovations, Datar Cancer Genetics, Nasik, India.
¹⁷ Brain Tumor Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands.
¹⁸ Department of Oncology, Mayo Clinic, Rochester, Minnesota, USA.
¹⁹ NORLUX Neuro-Oncology Laboratory, Department of Cancer Research, Luxembourg Institute of Health, Luxembourg, Luxembourg.
²⁰ Division of Cancer Sciences, University of Manchester, Manchester, UK.
²¹ Peninsula Medical School, University of Plymouth, Plymouth, UK.
²² Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland, USA.
²³ Departments of Pathology and Translational Molecular Pathology, University of Texas, MD Anderson Cancer Center, Houston, Texas, USA.
²⁴ Clinical Relationships, Focused Ultrasound Foundation, Charlottesville, Virginia, USA.
²⁵ Department of Diagnostics and Intervention Oncology, Umeå University, Umeå Sweden.
²⁶ Cancer Research UK National Biomarker Centre, University of Manchester, UK.
²⁷ Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
²⁸ Department of Pediatrics, McGill University, Montreal, Quebec, Canada.
²⁹ Apollo Hospitals and Exsegen Genomics Research, Hyderabad, India.
³⁰ Department of Biochemistry and Biomedicine, University of Sussex, Brighton, UK.
³¹ Division of Neuroscience, University of Sheffield and Department of Neurosurgery, Sheffield Teaching Hospitals, Sheffield, UK.
³² Division of Neuroscience, University of Manchester, Manchester, UK.
³³ Division of Neuro-Oncology, Department Neuroscience 'Rita Levi Montalcini', University of Turin, Turin, Italy.
³⁴ Vall d'Hebron Institute of Oncology (VHIO) and University, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.
³⁵ Department Neuro-Oncology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands.
³⁶ Department of Neuro-Oncology, Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.
³⁷ Department of Neurology, University of Zurich, Zurich, Switzerland.
³⁸ Departments of Neurosurgery and Neurology, Wayne State University, Detroit, Michigan, USA.
³⁹ Department of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK.
⁴⁰ Center for Neuro-Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, USA.
⁴¹ Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK.
⁴² Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
⁴³ Leeds Institute of Medical Research, The University of Leeds, Leeds, UK.
⁴⁴ Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy.

PMID: 40884415
PMCID: PMC12833549 (available on 2026-08-30)
DOI: 10.1093/neuonc/noaf140

Review

Preanalytical variables and analytes in liquid biopsy approach for brain tumors: A comprehensive review and recommendations from the RANO Group and the Brain Liquid Biopsy Consortium

Chetan Bettegowda et al. Neuro Oncol. 2025.

. 2025 Oct 1;27(10):2496-2513.

doi: 10.1093/neuonc/noaf140.

Authors

Affiliations

¹ Department of Neurosurgery, Johns Hopkins Khatib Brain Tumor Center, Johns Hopkins University, Baltimore, USA.
² Department of Neurosurgery, Henry Ford Health System, Detroit, Michigan, USA.
³ Department of Molecular Medicine and Medical Biotechnology, Federico II University, Napoli, Italy.
⁴ Miami Cancer Institute, and Florida International University, Miami, Florida, USA.
⁵ Neuropathology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
⁶ Department of Neurosurgery, Duke University, Durham, North Carolina, USA.
⁷ University of Pennsylvania, Philadelphia, Pennsylvania, USA.
⁸ Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Maryland, USA.
⁹ Department of Neurosurgery, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
¹⁰ Department of Neuro-Oncology, Netherlands Cancer Institute, Amsterdam, The Netherlands.
¹¹ Faculty of Life Sciences and the Institute of Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat-Gan, Israel.
¹² School of Applied Sciences, University of Huddersfield, Huddersfield, UK.
¹³ Department of Physiology, Michigan State University, East Lansing, MI, USA.
¹⁴ Division of Neuro-Oncology, Department of Neurosurgery, University of California, San Francisco, California, USA.
¹⁵ Department of Neurology& Rehabilitation, University of Illinois at Chicago, Chicago, Illinois, USA.
¹⁶ Department of Research and Innovations, Datar Cancer Genetics, Nasik, India.
¹⁷ Brain Tumor Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands.
¹⁸ Department of Oncology, Mayo Clinic, Rochester, Minnesota, USA.
¹⁹ NORLUX Neuro-Oncology Laboratory, Department of Cancer Research, Luxembourg Institute of Health, Luxembourg, Luxembourg.
²⁰ Division of Cancer Sciences, University of Manchester, Manchester, UK.
²¹ Peninsula Medical School, University of Plymouth, Plymouth, UK.
²² Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland, USA.
²³ Departments of Pathology and Translational Molecular Pathology, University of Texas, MD Anderson Cancer Center, Houston, Texas, USA.
²⁴ Clinical Relationships, Focused Ultrasound Foundation, Charlottesville, Virginia, USA.
²⁵ Department of Diagnostics and Intervention Oncology, Umeå University, Umeå Sweden.
²⁶ Cancer Research UK National Biomarker Centre, University of Manchester, UK.
²⁷ Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
²⁸ Department of Pediatrics, McGill University, Montreal, Quebec, Canada.
²⁹ Apollo Hospitals and Exsegen Genomics Research, Hyderabad, India.
³⁰ Department of Biochemistry and Biomedicine, University of Sussex, Brighton, UK.
³¹ Division of Neuroscience, University of Sheffield and Department of Neurosurgery, Sheffield Teaching Hospitals, Sheffield, UK.
³² Division of Neuroscience, University of Manchester, Manchester, UK.
³³ Division of Neuro-Oncology, Department Neuroscience 'Rita Levi Montalcini', University of Turin, Turin, Italy.
³⁴ Vall d'Hebron Institute of Oncology (VHIO) and University, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain.
³⁵ Department Neuro-Oncology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands.
³⁶ Department of Neuro-Oncology, Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.
³⁷ Department of Neurology, University of Zurich, Zurich, Switzerland.
³⁸ Departments of Neurosurgery and Neurology, Wayne State University, Detroit, Michigan, USA.
³⁹ Department of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK.
⁴⁰ Center for Neuro-Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, USA.
⁴¹ Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK.
⁴² Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
⁴³ Leeds Institute of Medical Research, The University of Leeds, Leeds, UK.
⁴⁴ Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy.

PMID: 40884415
PMCID: PMC12833549 (available on 2026-08-30)
DOI: 10.1093/neuonc/noaf140

Abstract

This review explores the pivotal role of preanalytical variables in bringing liquid biopsy approaches into the clinic for brain tumors. Preanalytical variables encompass a range of critical issues, from blood sample collection and handling to the impact of tumor heterogeneity and patient-specific factors. These variables introduce challenges such as false positives, false negatives, and variability in the analysis of tumor signals, which can hinder the diagnostic and prognostic utility of liquid biopsies. Understanding the nuances of preanalytical variables is essential for the successful implementation of liquid biopsy in clinical settings. This paper delves into strategies aimed at mitigating the influence of preanalytical variables by emphasizing the importance of standardized sample collection protocols, optimized sample processing and storage, quality control measures, and the integration of multiple liquid biopsy modalities.

Keywords: cfDNA; circulating tumor cells; clinical trials; extracellular vesicles; liquid biopsy; metabolites; microRNA; preanalytical variables; proteins; strategies.

© The Author(s) 2025. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.

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Conflict of interest statement

Chetan Bettegowda: CB is a consultant for Bionaut Labs, Haystack Oncology, and Privo Technologies and is a co-founder of Belay Diagnostics and OrisDx. Houtan Noushmehr: No conflict of interest. Alessandra Affinito: No conflict of interest. Manmeet S. Ahluwalia: MSA is a consultant for Allovir, Anheart Therapeutics, Apollomics, Autem Therapeutics, Bayer, Cairn Therapeutics, EquilliumBio, GT Medical Technologies, Incyte, Menarini Ricerche, QV Bioelectronics, Recordati, Servier Pharmaceuticals, Sumitomo Pharma Oncology, Theraguix, Viewray, and Xoft; is on the data safety and monitoring committee for VBI Vaccines; is on the Scientific Advisory Board for Modifibiosciences and Bugworks; and is a shareholder of Cytodyn, MedInnovateAdvisors LLC, Mimivax, and Trisalus Lifesciences. MSA has also received the following funding: 1R01CA277728-01A1, 1R01 CA264017-01A1. Olaf Ansorge: Co-Founder and consultant for EviCure, Ltd. Katayoun Ayasoufi: No conflict of interest. Stephen Bagley: SB has consulted for Bayer, Modifi Bio, Servier, and Telix and has received research grants (to institution) from GSK, Incyte, Kite, Lilly, and Novocure. Jill Barnholtz-Sloan: No conflict of interest. Myron Best: No conflict of interest. Dieta Brandsma: No conflict of interest. Chaya Brodie: No conflict of interest. Anke Brüning-Richardson: No conflict of interest. Ana valeria Castro: No conflict of interest. Gerolama Condorelli: No conflict of interest. Ahmad Daher: No conflict of interest. Vineet Datta: No conflict of interest. John De Groot: JDG is on the advisory boards of Alpha Pharmaceuticals, CapitalOne, Carthera, DSP Pharma, Insightec, Kazia, Kintara Pharmaceuticals, Monteris, Mundipharma, Nerviano, Samus, Sapience, Servier, and Telix; is on the data safety and monitoring board for Chimerix and VBI; and consults for Carthera, Deciphera, Insightec, Kazia, Kintara, MundiPharma, and Nerviano. Pim French: No conflict of interest. Evanthia Galanis: EG is a past advisory board member of Boston Scientific (DMC, compensation to employer <$10 000), Karyopharm Therapeutics, Inc (DSMB, compensation to employer <$10 000), and Kiyatec, Inc (personal compensation <$10 000); is a current advisory board member of Boehringer Ingelheim (compensation to employer <$10 000), Modifi Biosciences (compensation to employer <$10 000), and Servier Pharmaceuticals (compensation to employer <$10 000); is on the Education Steering Committee of Servier Pharmaceuticals (compensation to employer <$10 000/yr), and has received Grant/Research/Clinical Trial Funding (to Mayo) from Servier Pharmaceuticals LLC and Denovo Biopharma. Anna Golebiewska: No conflict of interest. Petra Hamerlik: No conflict of interest. C. Oliver Hanemann: No conflict of interest. Matthias Holdoff: No conflict of interest. Jason Huse: No conflict of interest. Mustafa Khasraw: MK has received grants paid to his institution or contracts from AbbVie, BioNTech, BMS, CNS Pharmaceuticals, Daiichi Sankyo Inc, Immorna Therapeutics, Immvira Therapeutics, JAX lab for genomic research, and Personalis Inc. MK also received consulting fees from AnHeart Therapeutics, George Clinical, Manarini Stemline, and Servier and is on a data safety monitoring board for BPG Bio. Suzanne LeBlang: No conflict of interest. Beatrice Melin: No conflict of interest. Florent Mouliere: FM is co-inventor on patents related to cell-free DNA sequencing; has consulted for Roche Dx; and has received material support from Biomodal. Claire O’Leary: No conflict of interest. Janusz Rak: No conflict of interest. Amitava Ray: AR is an employee of Exsegen Genomics, a for-profit organization that is currently working on liquid biopsy for brain tumor diagnostics. Stephen Robinson: No conflict of interest. Ola Rominiyi: No conflict of interest. Federico Roncaroli: No conflict of interest. Roberta Rudà: No conflict of interest. Joan Seoane: No conflict of interest. Nik Sol: No conflict of interest. Martin J.van den Bent: No conflict of interest. Michael A. Vogelbaum: MAV has received clinical trial funding for his institution from DeNovo, Infuseon, and Oncosynergy and has received honoraria from Alexion, Biodexa, and Servier. Tobias Walbert: No conflict of interest. Colin Watts: No conflict of interest. Tobias Weiss: TW received honoraria from Philogen S.p.A. Michael Weller: MW has received research grants from Novartis, Quercis, and Versameb and honoraria for lectures, advisory board participation, or consulting from Anheart, Bayer, Curevac, Medac, Neurosense, Novartis, Novocure, Orbus, Pfizer, Philogen, Roche, and Servier. Patrick Y. Wen: PYW has received research support from Astra Zeneca, Black Diamond, Bristol Meyers Squibb, Chimerix, Eli Lily, Erasca, Global Coalition For Adaptive Research, Kazia, MediciNova, Merck, Nerviano, Novartis, Philogen, Quadriga, Servier, and VBI Vaccines and has served on the advisory board or as a consultant to Anheart, Alexion/Astra Zeneca, Black Diamond, Chimerix, Day One Bio, Fore Biotherapeutics, Genenta, Glaxo Smith Kline, Kintara, Merck, Mundipharma, Novartis, Novocure, Nuvation Bio, Prelude Therapeutics, Sapience, Servier, Symbio, Tango, Telix, and VBI Vaccines. Victoria Wykes: No conflict of interest. Stephen Yip: SY is an advisory board member and has received honoraria from Amgen, AstraZeneca, Bayer, Pfizer, Servier, and Roche. Susan Short: No conflict of interest. Riccardo Soffietti: No conflict of interest.

References

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Preanalytical variables and analytes in liquid biopsy approach for brain tumors: A comprehensive review and recommendations from the RANO Group and the Brain Liquid Biopsy Consortium

Affiliations

Preanalytical variables and analytes in liquid biopsy approach for brain tumors: A comprehensive review and recommendations from the RANO Group and the Brain Liquid Biopsy Consortium

Authors

Affiliations

Abstract

Conflict of interest statement

References

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Medical