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A comparative study of four cell-free DNA assays for detecting circulating tumor DNA in early breast cancer.

Author(s): Mulder CV, Jongbloed EM, Liefaard MC, Makrodimitris S, Hazelaar DM, Beaufort CM, de Weerd V, Mulder L, van Deurzen CHM, Sonke GS, Jager A, Wilting SM, Martens JWM, Jansen MPHM, Lips EH

Publication: Breast Cancer Res, 2025, Vol. 27, Page 120

PubMed ID: 40598388 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the detection of circulating tumor DNA (ctDNA) using four different next-generation sequencing (NGS) assays and investigated whether patient age or tumor characteristics influenced detection.  Blood was collected from 56 patients with tri­ple negative (TN) or luminal B type breast cancer into EDTA, CellSave or Streck tubes (number of each not specified). Plasma was obtained by centrifugation at 1,711 g for 10 min at room tempera­ture, followed by 12,000 g for 10 min at 4⁰C within 4 h (EDTA) or 72 h (CellSave/Streck) of venipuncture. Plasma was stored at -80°C until cfDNA isolation with the QiaAmp Kit. cfDNA was quantified using the Quant-IT dsDNA high-sensitivity Assay and stored at -30°C until analysis. Single-nucleotide variants were detected in 24 specimens by Next-Generation sequencing (NGS) using the Oncomine Breast cfDNA panel. Copy number variants (CNVs) were detected by NGS in 40 specimens using the mFAST-seqS method and were considered aneuploid if the aneuploid score was ≥5. Methylation was assessed in 40 specimens by NGS. Methylated reads within 1,000 bp of the transcription start site were counted, differentially methylated regions were identified, Z-scores were calculated and positivity was defined as a z-score > than the 95% confidence interval. CNVs were detected in 40 specimens by shallow genome-wide NGS using the ThruPLEX DNA-Seq Kit and a Illumina NEXTsq2000 instrument. Tumor fraction and CNV were calculated from shallow sequencing data using the IchorCNA package.

   Summary of Findings:

   There were no significant differences in patient age or tumor characteristics (distributions by subtype, clinical T stage, clinical N stage, grade or pathological response) between the 40 specimens chosen for analysis (sufficient DNA for ≥3 assays) and the 16 specimens excluded. Among the 24 specimens assayed with the Oncomine NGS assay, 3 specimens had mutations: a TP53 mutation with a VAF of 28.5%, a TP53 mutation with a VAF of 1.1% and a PIK3CA mutation with a VAF of 7.5%. The specimen with a VAF of 28.5% for TP53 was also found to have an elevated aneuploidy score using the mFAST-SeqS assay, a tumor fraction >0 in shallow whole genome sequencing (sWGS) and a positive (z-score above the 95% confidence interval for differentially methylated genes) genome-wide NGS methylation profiling (MeD-Seq). In a specimen with a VAF of 7.5% for PIK3CA in the Oncomine assay, a mutation with a tumor fraction >0 was found by sWGS but only after analysis was restricted to fragments of 90-150 bp and the specimen was negative using the MeD-Seq Assay and the genome-wide NGS CNV assay (mFAST-Seq). The remaining specimen that was positive in the Oncomine assay for TP53 mutation (VAF 1.1%), was negative in the other three assays. An elevated aneuploidy score (mFAST-SeqS) was found in a total of five specimens including the aforementioned specimen positive in all four assays, three specimens that were negative (one was not assessed) in Oncomine but positive using MeD-Seq, and one that had a tumor fraction > 0 using sWGS but was negative in the other assays.  There weren’t any specimens that were positive using sWGS but negative in Oncomine and mFAST-Seq assays. In addition to the four aforementioned specimens, 19 specimens were positive in the MeD-Seq assay and negative using all other assays. Positivity (a positive in ≥1 assay) in one or more assays occurred in 65% of specimens but was not affected by patient age, tumor characteristics (subtype, clinical T stage, clinical N stage, grade or pathological response) or cfDNA concentration.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • Streck/BCT

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	Unspecified Range
   Next generation sequencing Specific	Technology platform	Oncomine mFAST-SeqS sWGS MeD-Seq
   Preaquisition	Prognostic factor	Stage T1/T2 Stage T3/T4 N Stage negative N Stage positive Low grade Intermediate grade High grade Complete response Partial response
   Preaquisition	Diagnosis/ patient condition	Triple negative breast cancer Luminal B type breast cancer

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