Seq-ing the SINEs of central nervous system tumors in cerebrospinal fluid (CSF)
Authors:
Christopher Douville, Samuel Curtis, Mahmoud Summers, Tej D. Azad, Jordina Rincon-Torroella, Yuxuan Wang, Austin Mattox, Bracha Avigdor, Jonathan Dudley, Joshua Materi, Divyaansh Raj, Sumil Nair, Debarati Bhanja, Kyle Tuohy, Lisa Dobbyn, Maria Popoli, Janine Ptak, Nadine Nehme, Natalie Silliman, Cherie Blair, Kathy Judge, Gary L. Gallia, Mari Groves, Christopher M. Jackson, Eric M. Jackson, John Laterra, Michael Lim, Debraj Mukherjee, Jon Weingart, Jarushka Naidoo, Carl Koschmann, Natalya Smith, Karisa C. Schreck, Carlos A. Pardo, Michael Glantz, Matthias Holdhoff, Kenneth W. Kinzler, Nickolas Papadopoulos, Bert Vogelstein, Chetan Bettegowda
Journal:
Cell Reports Medicine, Volume 4, Issue 8, 15 August 2023, 101148.
Abstract:
It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique called Real-CSF (repetitive element aneuploidy sequencing in CSF) to detect cancers of the central nervous system from evaluation of DNA in the cerebrospinal fluid (CSF). Short interspersed nuclear elements (SINEs) are PCR amplified with a single primer pair, and the PCR products are evaluated by next-generation sequencing. Real-CSF assesses genome-wide copy-number alterations as well as focal amplifications of selected oncogenes. Real-CSF was applied to 280 CSF samples and correctly identified 67% of 184 cancerous and 96% of 96 non-cancerous brain lesions. CSF analysis was considerably more sensitive than standard-of-care cytology and plasma cell-free DNA analysis in the same patients. Real-CSF therefore has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain.
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.