Validation of targeted next-generation sequencing of cell-free DNA from archival cerebrospinal fluid specimens for the detection of somatic variants in cancer involving the leptomeninges: Cytopathologic and radiographic correlation.
Author(s): Neil AJ, Chukwueke UN, Hoover N, Marris SRN, Rojas-Rudilla V, Manning DK, Mito JK, Cibas ES, Sholl LM
Publication: Cancer Cytopathol, 2024, Vol. 132, Page 214-223
PubMed ID: 37812603 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to assess potential effects of prolonged storage of archived cerebrospinal fluid (CSF) specimens at ≤ 4°C (1-125 ml) and CSF aliquot volume (1-8 mL) on the concentration of cell-free DNA (cfDNA), percentage of gDNA, and the mean target coverage (MTC) of the OncoPanel targeted next-generation sequencing (NGS).
Conclusion of Paper
Neither the duration of CSF storage at ≤4°C (1-125 d) nor the volume of CSF aliquots (1-8 mL) were correlated with DNA concentration (R2=0.02 and 0.003, respectively) or mean target coverage (R2=0.01 and 0.001, respectively) in archived CSF specimens from patients with suspected leptomeningeal metastasis. The ability to detect tumor variants in archival CSF specimens via the OncoPanel was also not affected by the volume of the CSF specimen or by storage duration. The authors note that DNA concentration and MTC were strong predictors of successful tumor variant detection in cfDNA from CSF, as the median DNA concentration of CSF specimens with a detected variant was significantly higher than CSF specimens with no variant detected (1.38 versus 0.342 ng/µl; p<0.005), and 77% (10/13 specimens) of CSF specimens with a detected variant had a DNA concentration >0.5 ng/µl. The median MTC was also significantly higher in CSF specimens with a detected variant than those with no variant detected (147 versus 20 reads; p<0.005), and 87% (13/15 specimens) of CSF specimens with a detected variant had a MTC >50 reads.
Total DNA concentration was strongly correlated with “nucleated cell count” in CSF specimens (R2=0.746). The median percentage of gDNA fragments detected in CSF specimens was not correlated with the duration of CSF storage (R2=0.054), the volume of the CSF sample (R2=0.323), or “nucleated cell count” (R2=0.130).
Studies
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Study Purpose
The purpose of this study was to assess potential effects of prolonged storage of archived cerebrospinal fluid (CSF) specimens at ≤ 4°C (1-125 d) and CSF aliquot volume (1-8 m) on the concentration of cell-free DNA (cfDNA), the percentage of gDNA, and the mean target coverage of the OncoPanel targeted next-generation sequencing (NGS). Specimens included in the study were limited to those from patients (suspected of a leptomenigeal metastasis, had biomarker testing of a tumor that revealed a somatic variant captured by the OncoPanel assay, and had ≥1 ml of an archived CSF specimen that was stored at ≤4°C for 1-125 d in the standard tube associated with lumbar puncture kits. Remnant, archived CSF specimens collected from patients (age range: 27-82 y) diagnosed with non-small cell lung cancer (7 patients), breast cancer ( 4 patients), acute lymphocytic leukemia (3 patients), central nervous system diffuse large B-cell lymphoma (3 patients), blastic plasmacytoid dendritic cell neoplasm (2 patients); and melanoma, glioblastoma, neuroendocrine carcinoma, leiomyosarcoma, cervical small cell carcinoma, and ependymoma (1 patient each) were used in the study. No additional details on collection or storage were provided. Cell counts from CSF specimens were either located on the patient’s electronic medical records or were determined with a GloCyte Automated CSF Cell Counter. For cfDNA isolation, CSF specimens were centrifuged at 1600 g for 10 min, the supernatant was transferred to a new tube, centrifuged at 3000 g for 10 min, and cfDNA was isolated using the QIAmp Circulating Nucleic Acid Kit. DNA concentration was quantified with a Qubit fluorometer, and DNA fragment sizes were determined with a LabChip GX analyzer. Hybrid capture libraries were prepared and sequenced using the OncoPanel assay. Somatic variant detection in cfDNA from CSF was considered to be successful if ≥1 previously identified variant was detected by OncoPanel. DNA fragment sizes were assessed with a LabChip machine in a subset of 12 CSF specimens. Additional details on cfDNA analysis and sequencing were not provided.
Summary of Findings:
Neither the duration of CSF storage at ≤4°C (1-125 d) nor the volume of CSF aliquots (1-8 mL) were correlated with DNA concentration (R2=0.02 and 0.003, respectively) or mean target coverage (R2=0.01 and 0.001, respectively) in archived CSF specimens from patients with suspected leptomeningeal metastasis. The ability to detect tumor variants in archival CSF specimens via the OncoPanel was also not affected CSF volume or storage duration. Notably, the median DNA concentration was significantly higher in CSF specimens with a detected variant than those without (1.38 versus 0.342 ng/µl; p<0.005); further, 77% (10/13 specimens) of CSF specimens with a detected variant had a DNA concentration >0.5 ng/µl. The median MTC was also significantly higher in CSF specimens with a detected variant than those without (147 versus 20 reads; p<0.005), and 87% (13/15 specimens) of CSF specimens with a detected variant had a MTC >50 reads. Total DNA concentration was strongly correlated with “nucleated cell count” in CSF specimens (R2=0.746). Of the 12 CSF specimens analyzed by LabChip, 8 had detectable DNA fragments, 6 of which displayed genomic DNA (gDNA) contamination. The median percentage of gDNA fragments detected in CSF specimens was not correlated with the duration of CSF storage (R2=0.054), the volume of the CSF sample (R2=0.323), or “nucleated cell count” (R2=0.130).
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Leukemia
- Neoplastic - Lymphoma
- Neoplastic - Melanoma
- Neoplastic - Mixed type
- Neoplastic - Sarcoma
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Next generation sequencing DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Aliquot size/volume 1-8 mL
Storage Storage duration 1-125 d