Serial Testing of Tumor-Derived DNA in Cerebrospinal Fluid (CSF) to Monitor Leptomeningeal Disease and Assess Therapeutic Response in Lung Cancer Metastasis to the Central Nervous System – A Case Report
Authors:
George Bobustuc, Alexandra Larson, Vindhya Udane, Kala F. Schilter, Qian Nie, Honey V. Reddi
Journal:
Annals of Clinical Case Reports. 2025.
Abstract:
Metastatic Leptomeningeal disease (LMD) most often seen in lung cancer, poses unique diagnostic and therapeutic challenges due to limited sensitivity of standard cerebrospinal fluid (CSF) workup and genetic divergence from the primary tumor associated with resistance to central nervous system (CNS) penetrating treatments. Among CNS complications, LMD is associated with poor outcomes and average survival following diagnosis is three to six months. Routine methods for diagnosing LMD currently fall short, making it difficult to investigate therapeutic options for this type of disease progression. Sequencing of tumor-derived DNA (tDNA) found in CSF is emerging as a useful clinical tool to inform diagnosis and monitoring of CNS malignancies with the potential to guide treatment. The present case of metastatic lung cancer illustrates the use of serial CSF tDNA analysis via Belay Summit™, a novel liquid biopsy assay, to interrogate LMD driver mutations and guide therapeutic decisions alongside tumor profiling.
Analytical validation of the Belay Vantage™ assay for evaluation of MGMT promoter methylation using enzymatically converted tumorDNA from cerebrospinal fluid (CSF)
Authors:
Kala F Schilter, Qian Nie, Jennifer N Adams, Rakshitha Jagadish, Anthony Acevedo, Alexandra Larson, Samantha A Vo, Brett A Domagala, Kyle M Hernandez, Christopher Douville, Yuxuan Wang, Brian Coe, Chetan Bettegowda, Honey V Reddi
Journal:
Cancer Genetics. Volumes 294–295, June 2025, Pages 94-98
Abstract:
MGMT promoter methylation status (hypermethylation) is one of the strongest prognostic and predictive biomarkers in glioblastoma (GBM) and is associated with a more favorable response to alkylating chemotherapies such as Temozolomide (TMZ). Additionally, it is associated with pseudo progression in GBM, a phenomenon in which early radiographic changes after treatment are indicative of possible tumor recurrence though on histological examination it is consistent with treatment effect. Current methods for evaluation of MGMT promoter methylation status are limited to tumor tissue, requiring invasive biopsy or surgery, prompting the need for a liquid biopsy-based assay to expand and manage therapeutic interventions. The Belay Vantage™ assay evaluates MGMT promoter methylation status in cerebrospinal fluid (CSF) of individuals with known or suspected central nervous system tumors using low input DNA. The assay uses quantitative polymerase chain reaction (qPCR) on DNA extracted from CSF after enzymatic conversion and has an analytical sensitivity of 95.5 % and specificity of 100 %.
Analytical Validation and Clinical Sensitivity of the Belay Summit assay for the detection of DNA variants in cerebrospinal fluid (CSF) of primary and metastatic CNS cancer
Authors:
Qian Nie, Kala F. Schilter, Kyle M. Hernandez, Jennifer N. Adams, Rakshitha Jagadish, Anthony Acevedo, Alexandra Larson, Brett A. Domagala, Samantha A. Vo, Sakshi Khurana, Kathleen Mitchell, Dean Ellis, Baymuhammet Muhammedov, Yuxuan Wang, Christopher Douville, Brian Coe, Chetan Bettegowda, Honey V. Reddi
Journal:
Journal of Molecular Diagnostics, 2025;27(7):615‑629.
Abstract:
In contrast to most solid tumors, cancers of the central nervous system (CNS) pose a unique challenge for effective detection and tracking via plasma because of the blood-brain barrier. Informed diagnosis of primary and metastatic CNS tumors can be facilitated using a liquid biopsy assay that evaluates tumor-derived DNA from the cerebrospinal fluid (CSF), potentially increasing the efficacy of diagnosis and reducing the uncertainty and morbidities associated with the current standard of care that involves neurosurgical procedures. The Belay Summit assay involves tumor-derived DNA–based genomic profiling of CSF to inform diagnosis of CNS tumors. The analytical sensitivity of Summit for single-nucleotide/multinucleotide variants and insertions/deletions is 96% at a 95% limit of detection of 0.30% variant allele fraction. Analytical sensitivity for chromosomal arm-level aneuploidy is 91% at abs(log2r) of 0.09 limit of detection. Clinical sensitivity across a cohort of 124 specimens, including primary and metastatic CNS tumors, was demonstrated to be 90% with a specificity of 95%, supporting the potential for positive clinical utility. These results demonstrate that the Belay Summit assay can accurately and reproducibly be used to inform the diagnosis of primary and metastatic CNS tumors using CSF.
Detection of low-frequency DNA variants by targeted sequencing of the Watson and Crick strands
Authors:
Joshua D Cohen, Christopher Douville, Jonathan C Dudley, Brian J Mog, Maria Popoli, Janine Ptak, Lisa Dobbyn, Natalie Silliman, Joy Schaefer, Jeanne Tie, Peter Gibbs, Cristian Tomasetti, Nickolas Papadopoulos, Kenneth W Kinzler, Bert Vogelstein
Journal:
Nat Biotechnol. 2021 Oct;39(10):1220-1227. doi: 10.1038/s41587-021-00900-z.Epub 2021 May 3.
Abstract:
Identification and quantification of low-frequency mutations remain challenging despite improvements in the baseline error rate of next-generation sequencing technologies. Here, we describe a method, termed SaferSeqS, that addresses these challenges by (1) efficiently introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching target sequences with strand-specific PCR. The method achieves high sensitivity and specificity and detects variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of <5 × 10-7 mutants per base pair (bp). We demonstrate that it can evaluate mutations in a single amplicon or simultaneously in multiple amplicons, assess limited quantities of cell-free DNA with high recovery of both strands and reduce the error rate of existing PCR-based molecular barcoding approaches by >100-fold.
Detection of circulating tumor DNA in early- and late-stage human malignancies
Authors:
Chetan Bettegowda, Mark Sausen, Rebecca J Leary, Isaac Kinde, Yuxuan Wang, Nishant Agrawal, Bjarne R Bartlett, Hao Wang, Brandon Luber, Rhoda M Alani, Emmanuel S Antonarakis, Nilofer S Azad, Alberto Bardelli, Henry Brem, John L Cameron, Clarence C Lee, Leslie A Fecher, Gary L Gallia, Peter Gibbs, Dung Le, Robert L Giuntoli, Michael Goggins, Michael D Hogarty, Matthias Holdhoff, Seung-Mo Hong, Yuchen Jiao, Hartmut H Juhl, Jenny J Kim, Giulia Siravegna, Daniel A Laheru, Calogero Lauricella, Michael Lim, Evan J Lipson, Suely Kazue Nagahashi Marie, George J Netto, Kelly S Oliner, Alessandro Olivi, Louise Olsson, Gregory J Riggins, Andrea Sartore-Bianchi, Kerstin Schmidt, le-Ming Shih, Sueli Mieko Oba-Shinjo, Salvatore Siena, Dan Theodorescu, Jeanne Tie, Timothy T Harkins, Silvio Veronese, Tian-Li Wang, Jon D Weingart, Christopher L Wolfgang, Laura D Wood, Dongmei Xing, Ralph H Hruban, Jian Wu, Peter J Allen, C Max Schmidt, Michael A Choti, Victor E Velculescu, Kenneth W Kinzler, Bert Vogelstein, Nickolas Papadopoulos, Luis A Diaz Jr
Journal:
Science Translational Medicine, 2014;6(224):224ra24.
Abstract:
The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction-based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.
TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal
Authors:
Patrick J Killela, Zachary J Reitman, Yuchen Jiao, Chetan Bettegowda, Nishant Agrawal, Luis A Diaz Jr, Allan H Friedman, Henry Friedman, Gary L Gallia, Beppino C Giovanella, Arthur P Grollman, Tong-Chuan He, Yiping He, Ralph H Hruban, George I Jallo, Nils Mandahl, Alan K Meeker, Fredrik Mertens, George J Netto, B Ahmed Rasheed, Gregory J Riggins, Thomas A Rosenquist, Mark Schiffman, Ie-Ming Shih, Dan Theodorescu, Michael S Torbenson, Victor E Velculescu, Tian-Li Wang, Nicolas Wentzensen, Laura D Wood, Ming Zhang, Roger E McLendon, Darell D Bigner, Kenneth W Kinzler, Bert Vogelstein, Nickolas Papadopoulos, Hai Yan
Journal:
PNAS, 2013;110(15):6021–6026.
Abstract:
Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein(DAXX) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.
Molecular Analysis of Cerebrospinal Fluid Tumor-Derived DNA to Aid in the Diagnosis and Targeted Treatment of Breast Cancer Brain Metastasis
Authors:
Michael Youssef, Alexandra Larson, Vindhya Udhane, Viriya Keo, Kala F. Schilter, Qian Nie, Honey V. Reddi
Journal:
Diseases, 2025, 13(10), 336.
Abstract:
A woman in her 40s with a history of ER/PR+, HER2-negative breast cancer presented with a seizure three years after mastectomy. Magnetic resonance imaging (MRI) revealed a right caudate head mass, which was concerning for either high-grade glioma or metastatic disease, but biopsy was deemed too high risk. Cerebrospinal fluid (CSF) tumor-derived DNA (tDNA) analysis by next-generation sequencing (NGS) was ordered, revealing a gain-of-function variant in PIK3CA, ERBB2 copy number gain, and high aneuploidy, findings consistent with breast cancer brain metastasis. Based on these results, the patient was treated with stereotactic radiosurgery (SRS) followed by trastuzumab deruxtecan, a HER2-targeted therapy. This case highlights the diagnostic and therapeutic value of CSF tDNA analysis in central nervous system (CNS) lesions when biopsy is not feasible. The report also illustrates how clonal evolution, such as acquired ERBB2 amplification, can occur in metastatic disease and influence treatment decisions.
Analysis of cerebrospinal fluid (CSF) tumor-derived DNA to obviate biopsy of IDH-mutant brainstem glioma in an adult
Authors:
Michael Youssef, Alexandra Larson, Vindhya Udhane, Kala F. Schilter, Qian Nie, Honey V. Reddi
Journal:
The Journal of Liquid Biopsy, Volume 9100318, September 2025.
Abstract:
Adult brainstem gliomas are rare and present unique diagnostic and therapeutic challenges due to their critical location and limited biopsy feasibility. Molecular profiling of tumor-derived DNA (t-DNA) isolated from cerebrospinal fluid (CSF) is emerging as a minimally invasive alternative for characterizing these tumors and guiding targeted therapy. A 34-year-old woman with brainstem glioma was treated with a standard course of radiation and temozolomide (TMZ) and remained stable for several years. After surveillance imaging revealed disease progression and raised suspicion of IDH-mutant disease on MRI spectroscopy, molecular profiling of CSF was ordered. The Belay Summit test, a novel NGS-based liquid biopsy assay for central nervous system (CNS) tumors, identified variants in IDH1 and TP53 as well as loss of CDKN2A/CDKN2B. Based on these findings, the patient received a short course of radiation and was started on the IDH inhibitor vorasidenib. This case demonstrates the use of t-DNA from CSF for molecular profiling of adult brainstem glioma to identify actionable genomic alterations without surgical risk and allow patients to receive targeted therapy without tissue diagnosis.
Detection and quantification of rare mutations with massively parallel sequencing
Authors:
Isaac Kinde, Jian Wu, Nick Papadopoulos, Kenneth W Kinzler, Bert Vogelstein
Journal:
The Proceedings of the National Academy of Sciences, 2011 Jun 7;108(23):9530-5.
Abstract:
The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System (“Safe-SeqS”), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant (“supermutants”) only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.
Detection of rare mutations, copy number alterations, and methylation in the same template DNA molecules
Authors:
Yuxuan Wang, Christopher Douville, Joshua D Cohen, Austin Mattox , Sam Curtis, Natalie Silliman, Maria Popoli, Janine Ptak, Lisa Dobbyn, Nadine Nehme, Jonathan C Dudley, Mahmoud Summers, Ming Zhang, Lan T Ho-Pham, Bich N H Tran, Thach S Tran, Tuan V Nguyen, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W Kinzler, Bert Vogelstein
Journal:
The Proceedings of the National Academy of Sciences, August 2023
Abstract:
The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To further increase the sensitivity of such assays with limited amounts of sample, it would be useful to be able to evaluate the same template molecules for all these changes. Here, we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The epigenetic and genetic alterations present in the DNA molecules can then be obtained from the original and copied strands, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung, and colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics.