Rapid multiplexed qPCR analysis of cerebrospinal fluid and blood for highly prevalent somatic alterations present in central nervous system tumors

doi:10.21203/rs.3.pex-2017/v1

Method Article

Rapid multiplexed qPCR analysis of cerebrospinal fluid and blood for highly prevalent somatic alterations present in central nervous system tumors

https://doi.org/10.21203/rs.3.pex-2017/v1

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This protocol describes a qPCR-based assay that is designed to detect twelve potential mutations from clinical specimens that are significant to CNS neoplasms. The assay is optimized for plasma and cerebrospinal fluid (CSF), where both cell-free DNA (cfDNA) and DNA derived from circulating tumor cells (CTCs) can be analyzed.

Molecular biology

Genetics

Biomarkers

qPCR

cfDNA

mutations

neoplasms

biomarkers

The low yields of cerebrospinal fluid (CSF) studies and serologies in current diagnostic workflows for suspected neoplasms often lead to surgical biopsies of high-risk lesions, particularly in the central nervous system (CNS). To address this need, we developed an assay to rapidly identify hallmark mutations of neoplasms in CSF and plasma that have the potential to facilitate diagnosis, treatment monitoring, and detection of recurrence (Gupta et al. Blood 2021). Here we describe the qPCR-based approach to rapidly detect hallmark point mutations in TERT promoter, IDH1/2, BRAF, MYD88, and H3F3A genes. The reaction protocol is adapted from previous descriptions (Gupta et al. Blood 2021; Shankar et al. PNAS 2018, Shankar et al. JAMA Oncol 2015), with further updates for improved sensitivity and specificity. This assay can be performed on plasma or CSF to analyze both cfDNA as well as DNA derived from circulating tumor cells (CTCs). Variant alleles can be detected from the liquid biopsies within 80 minutes.

Platinum TAG DNA Polymerase (Fisher Scientific, Catalog # 10966034)

HotStart Polymerase with DNTPs (Fisher Scientific, Catalog # 501965265)

Applied Biosystems TaqMan Gene Expression Master Mix (Fisher Scientific, Catalog # 4369016)

Reference Dye (Aligent, Catalog # 600530-53)

Custom DNA, PNA, and LNA oligos (Figure 6) - (Integrated DNA Technologies)

Nuclease Free Water

BD Vacutainer PPT Plasma Preparation Tubes (Fisher Scientific, Catalog # 22204418)

QIAamp MinElute ccfDNA Midi Kit (Qiagen, Catalog # 55284)

DNeasy Blood and Tissue Kit (Qiagen, Catalog #69504)

QuantStudio™ 7 Pro Real-Time PCR System (Fisher Scientific, Catalog # A43183), or another compatible system

MicroAmp Fast 96-Well Reaction Plates 0.1ml (Fisher Scientific, Catalog # 4346907)

Applied biosystems MicroAmp Adhesive PCR films (Fisher Scientific, Catalog #4311971)

Corning Falcon® 15 mL High Clarity PP Centrifuge Tube (Westnet, Catalog #352096)

1.5ml DNA/RNA LoBind Tubes PCR Clean Eppendorf (Genesee Scientific Corporation, Catalog #86-923)

Collect CSF in 15mL Falcon tube. Collect blood in BD Vacutainer PPT Plasma Preparation Tube.
Centrifuge CSF at 600 xg, 10 minutes at 4^oC. Centrifuge blood at 1100 xg, 10 minutes at 20^oC.
Gently transfer the CSF supernatant, cell pellet, and plasma fraction to separate 1.5 mL Eppendorf tubes.
Perform DNA extractions using the respective Qiagen extraction kits (Figure 5) using manufacturer-provided instructions and reagents. Extract from a minimum of 2mL of plasma and 5mL of CSF to obtain optimal DNA yield.
Modification to manufacturer-provided kit protocols: elute the final cell-free DNA (from plasma or CSF) into 35 µL of Ultraclean water and cellular pellet DNA (from CSF) into 50 µL of Buffer AE.
Determine the appropriate quantities and concentrations for the necessary reagents (Figure 7).
Prepare reaction master mixes with the required ingredients (Figure 1).
Add 7 µL of master mix to each reaction well in a 96-well plate - positive controls, negative controls, no template controls, and samples (Figure 2)
Add 5 ng of control DNA to designated wells and fill those control wells to a final volume of 10 µL.
Add 3 µL of DNA extracts to the wells designated for samples.
Cover qPCR plate with Optical Adhesive PCR film.
Centrifuge plate for 5-10 seconds on a tabletop centrifuge and keep on ice, away from light.
Place plate in qPCR machine and run protocol (Figure 3).
Analyze data to assess the significance of the findings (Figure 4). Data analysis software may vary.

Gupta, M. et al. A rapid genotyping panel for detection of primary central nervous system lymphoma. Blood. 138, 382–386 (2021).
Shankar, G. M. et al. Rapid intraoperative molecular characterization of glioma. JAMA Oncology, 1, 662 (2015).
Shankar, G. M. et al. . Genotype-targeted local therapy of glioma. PNAS. , 115 (2018)

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Rapid multiplexed qPCR analysis of cerebrospinal fluid and blood for highly prevalent somatic alterations present in central nervous system tumors

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Rapid multiplexed qPCR analysis of cerebrospinal fluid and blood for highly prevalent somatic alterations present in central nervous system tumors

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Associated Publications

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