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Validation of a DNA-Based Next-Generation Sequencing Test for Molecular Diagnostic Variant and Fusion Detection in Formalin-Fixed, Paraffin-Embedded Tissue Specimens and Liquid Biopsy Plasma/Cell-Free DNA Samples.

Author(s): Werner TV, Kock S, Weber I, Kayser G, Werner M, Lassmann S

Publication: J Mol Diagn, 2022, Vol. 24, Page 784-802

PubMed ID: 35787794 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the yield and integrity of DNA isolated from FFPE tissue specimens using two different extraction methods and evaluated the suitability of the AVENIO Targeted and Surveillance Panels for genotyping FFPE tissue specimens and plasma cfDNA.  FFPE specimens from 26 cancer patients (diagnosed with lung, colorectal, pancreas, ovary, breast, prostate, melanoma, or soft tissue cancer) were collected during surgical resection (14 specimens) and/or biopsy (14 specimens). All specimens were obtained from the archive (further details not provided).  The effect of DNA extraction method was investigated by extracting DNA from four FFPE specimens (a NSCLC/adenocarcinoma biopsy, a duodenal carcinoma/adenocarcinoma biopsy, an inflammatory myofibroblastic tumor resection specimen, and a desmoid fibromatosis resection specimen) using both the Maxwell RSC FFPE Plus DNA Kit and the AVENIO Tumor Tissue Kit. NGS was conducted using DNA extracted from 22 FFPE specimens: DNA from 20 FFPE specimens was extracted using the Maxwell RSC FFPE Plus DNA Kit and DNA from 2 FFPE specimens was extracted using the AVENIO Tumor Tissue Kit. cfDNA was extracted from 40 plasma samples and one pleural effusion from a total of 29 patients diagnosed with lung (25 patients), colon (3 patients), and salivary gland (1 patient) tumors (no further details provided). DNA was extracted from plasma using the AVENIO cfDNA Isolation Kit. DNA integrity was assessed using the Qubit 1X dsDNA HS Assay Kit and the Genomic DNA ScreenTape on the 4200 TapeStation System. Sequencing libraries were constructed using the AVENIO Tumor Tissue Library Prep Kit and the AVENIO Tumor Enrichment Kit and were enriched using the AVENIO Tumor Surveillance Panel and the AVENIO ctDNA Targeted Panel. Samples were sequenced using the NextSeq Mid Output Kit (Targeted and Surveillance) or the NextSeq High Output Kit (Surveillance).

   Summary of Findings:

   DNA concentrations, DNA integrity numbers (DINs), and Q-scores from all four FFPE specimens were higher when extraction was with the Maxwell RSC FFPE Plus DNA Kit (Maxwell) than with the AVENIO Tumor Tissue Kit (AVENIO). There was a strong correlation between DIN value and the normalized Q-score (R² =0.7948, P < 0.0001). Sequencing depth was highly variable among FFPE tissue specimens, with a mean sequencing depth of 2052x and a range of 825x to 3380x. Using the AVENIO Tumor Tissue workflow, the same changes (one duplication, one fusion, and one SNV) were found in each of three specimens when extraction was with the Maxwell or the AVENIO Kit; however, the duplication was only detected by manual inspection of the sequencing data. In a second cohort of 22 FFPE specimens, clinically relevant SNVs were identified and all five wildtype tissue specimens were correctly identified, although the workflow failed to properly identify a duplication that was apparent upon manual inspection.  

   cfDNA yield from plasma was highly variable (0.17-4.8 ng/mL), which the authors attributed to variability in input volume (1.5-5 mL), although the cfDNA isolated was in the recommended size range of 200 to 700 bp.  The mean sequencing depth of plasma cfDNA was 4619x with the AVENIO cfDNA Targeted Panel libraries and 4275x with the AVENIO cfDNA Surveillance Panel libraries.  Wildtype status was confirmed in all nine wildtype cfDNA plasma specimens (7 specimens with the AVENIO ctDNA Targeted Panel and 2 specimens with the AVENIO ctDNA Surveillance Panel), and the expected mutations were also detected with the exception of some complex mutations that were detected by manual inspection but not by the AVENIO workflow.  Nevertheless, the AVENIO workflow for cfDNA isolation and sequencing properly identified variants in nine plasma specimens with artificial cfDNA that was spiked-in as part of the certification process.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Pleural Fluid
   • Tissue - Colorectal
   • Tissue - Skin
   • Tissue - Breast
   • Tissue - Pancreas
   • Tissue - Prostate
   • Tissue - Ovary

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Melanoma
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing
   DNA	PCR
   DNA	Fluorometry
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Maxwell RSC FFPE Plus DNA Kit AVENIO Tumor Tissue Kit
   Next generation sequencing Specific	Technology platform	AVENIO cfDNA Surveillance Panel libraries AVENIO cfDNA Targeted Panel libraries

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