Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Storage Time and DNA Quality Determine BRCA1/2 Sequencing Success in Prostate Cancer: A Multicentre Analysis with Therapeutic Implications.

Author(s): Vescovo M, Raspollini MR, Nibid L, Castiglione F, Nardi E, de Biase D, Massari F, Giunchi F, Pepe F, Troncone G, Malapelle U, Carosi M, Casini B, Melucci E, Fassan M, Toffolatti L, Guerini-Rocco E, Conversano F, Rappa A, Tommasi S, Coppola CA, Zeppa P, Caputo A, Gaeta S, Pagni F, Seminati D, Vecchione A, Scarpino S, Righi D, Taffon C, Prata F, Perrone G

Publication: Cancers (Basel), 2025, Vol. 17, Page

PubMed ID: 40427202 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the BRCA1/2 sequencing success rate, concentration and fragmentation of DNA from FFPE prostate cancer biopsy and resection specimens that had been stored as FFPE blocks for <1 year, 1-2 years, or > 2 years. The effect of biopsy site on NGS success rate was also investigated. This study used 559 FFPE prostate cancer biopsies (495 from the primary tumor and 64 from metastatic sites, including lymph, bone, liver, bladder, lung, colon, abdominal wall, and meninges) and 395 FFPE prostate cancer surgical resections. The specimens were collected at 11 different centers between May 1997 and March 2024 (no further details on specimen procurement or processing were provided). Storage durations were classified as stored short-term (<1 year; 223 biopsies and 89 surgical specimens), middle-term (1–2 years; 86 biopsies and 55 surgical specimens) or long-term (>2 years; 250 biopsies and 251 surgical specimens) but no other details of storage conditions were provided. DNA was extracted and sequenced using center-specific workflows. Extraction methods used included the QIAGEN EZ1 & EZ2 DNA Tissue Kit, MagCore genomic DNA FFPE Kit, MagMAX FFPE DNA/RNA Ultra Kit, MAXWELL CSC DNA FFPE Kit, QIAGEN Mini Amp Kit, QIAamp DNA FFPE Tissue Kit, or the QuickExtract FFPE DNA Extraction Kit. The BRCA1 and BRCA2 genes were sequenced using MiSeq or Ion GeneStudio s5 instruments and the Myriapod, AmoyDX Focus or Oncomine Tumor-Specific Panel Kits.

   Summary of Findings:

   Overall, BRCA1/2 sequencing was successful in 77.1% of the FFPE prostate cancer specimens, but the success rate depended on the storage duration of the FFPE block. The sequencing success rate was significantly higher in FFPE specimens that were stored < 1 year than >2 years (87.8% versus 69.1%, P<0.001) and stored 1-2 years versus >2 years (82.3% versus 69.1%, P<0.001). The median storage duration was shorter for specimens for which sequencing was successful than for those in which sequencing failed (657 days versus 1626.5 days, P<0.001). Further, storage for > 2 years was associated with a significant decrease in NGS success rate [Odds ratio (OR)= 0.36; 95% confidence interval (CI)= 0.26–0.50; P<0.001] and storage for <1 year was associated with a higher success rate (OR=2.8, 95% CI=1.92–4.11, P<0.001). Importantly, when the specimens were binned by 12-month intervals, the NGS success rate remained >80% for FFPE specimens stored <4 years, but 71.6% for specimens stored for 4-5 years, 64.5% for specimens stored for 5-6 years and 59.5 for specimens stored for >6 years. Interestingly, two of the centers had low (33.3% and 35.7%, respectively) NGS success rates for FFPE specimens stored < 1 year, but high success rates for specimens stored 1-2 years (91.70% and 100%, respectively) and five of the centers had equivalent or higher success rates for specimens stored < 1 year and 1-2 years.

   The NGS success rate was higher for surgical prostate specimens than biopsies (83.3% versus 72.8%, P<0.001) but was not affected by biopsy site. When broken down by FFPE block storage duration, the NGS success rate was higher for surgical prostate specimens than biopsies stored < 1 year (96.6% versus 84.3%, P=0.003), stored 1-2 years (96.4% versus 73.3%, P<0.001) and stored > 2 years (75.7% versus 62.4%, P=0.001). Further, DNA from biopsies had a significantly lower hazard ratio (HR) for sequencing success compared with DNA from surgical specimens (HR = 0.7362; P= 0.011). Surgical specimens also yielded a higher concentration of DNA than biopsies (13.05 ng/μL versus 2.2 ng/μL), but the authors attribute this difference, in part, to the larger tissue volume. The median DNA fragmentation rate was higher (more intact) for DNA from surgical specimens than biopsies (1.2 versus 0.41). The DNA concentration and DNA fragmentation index were also associated with a 0.35% (per unit) and 26.6% increase in the sequencing success rate (hazard ratio (HR)=1.0035; 95% CI=1.0024–1.0046; P<0.001 and HR=1.2661; 95% CI=1.1479–1.3965; P< 0.001, respectively).

   Biospecimens

   • Tissue - Prostate
   • Tissue - Lymph Node
   • Tissue - Liver
   • Tissue - Bone
   • Tissue - Other
   • Tissue - Lung
   • Tissue - Colorectal
   • Tissue - Muscle (Skeletal)
   • Tissue - Bladder

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Prostate Lymph node Bone Liver Bladder Lung Colon Abdominal wall Meninges
   Storage	Storage duration	<1 year 1-2 years >2 years
   Biospecimen Acquisition	Method of tissue acquisition	Biopsy Surgical resection

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