Statistical and Evolutionary Analysis of Sequenced DNA from Breast Cancer FFPE Specimens

Area of Science:

• Oncology
• Genetics
• Bioinformatics

Background:

• Formalin-fixed paraffin-embedded (FFPE) tissue blocks remain crucial for cancer genetic epidemiology, despite the availability of instant freezing.
• FFPE samples present challenges due to DNA artifacts that can affect variant calls in genetic analyses.
• Understanding the information obtainable from FFPE samples is vital for retrospective cancer research.

Purpose of the Study:

• To assess the utility of mid-depth whole-exome bulk DNA sequencing for extracting genetic information from FFPE breast tumor samples.
• To evaluate the effectiveness of bioinformatics tools in mitigating FFPE-related artifacts.
• To investigate the molecular evolution, clonal structure, and primary-metastasis relationships in breast cancer using FFPE data.

Main Methods:

• Collected 15 paired breast tumors (primary and lymph node metastases) of diverse molecular subtypes.
• Employed multiple bioinformatics tools to identify and remove FFPE-induced DNA variant artifacts.
• Performed whole-exome sequencing and analyzed data using commercial and in-house computational methods, including copy number and clonal structure analyses.

Main Results:

• Bioinformatics tools showed variable effectiveness in artifact removal, necessitating careful calibration.
• Copy number analysis revealed ploidy levels and a positive association between oncogene frequency and DNA copy number.
• Clonal structure analysis provided insights into early tumor expansion and the interdependence of primary tumors and lymph node metastases.

Conclusions:

• Significant molecular evolutionary features of tumor DNA can be successfully recovered from routine FFPE clinical samples.
• Advanced computational analysis can overcome FFPE data imperfections for valuable genetic insights.
• FFPE-based sequencing remains a viable approach for retrospective studies in cancer genetics and epidemiology.