Mutational profiling of micro-dissected pre-malignant lesions from archived specimens

Area of Science:

• Genomic Medicine
• Cancer Genomics
• Molecular Pathology

Background:

• Increased detection of pre-malignant lesions (PMLs) necessitates reliable prognostic markers.
• Over- and under-treatment of PMLs stem from a lack of prognostic information.
• Previous mutational profiling studies of PMLs were limited by specimen quality and lack of outcome data.

Purpose of the Study:

• To develop and validate robust DNA sequencing strategies for analyzing degraded, formalin-fixed, paraffin-embedded (FFPE) pre-malignant lesion specimens.
• To enable comprehensive molecular characterization of early-stage cancer lesions for biomarker discovery.

Main Methods:

• Evaluation of targeted sequencing approaches using varying DNA input, library preparation (Blunt-End, Single-Strand, A-Tailing), and target sizes (whole exome vs. gene panel).
• Development of PML-specific variant calling algorithms using high-input FFPE and frozen DNA.
• Application of the optimized approach to micro-dissected regions from breast ductal carcinoma in situ (DCIS) specimens.

Main Results:

• Blunt-End and Single-Strand library preparation significantly increased whole exome coverage compared to A-Tailing.
• PML-specific variant filtering enhanced both recall and precision of mutation detection.
• The approach successfully identified candidate driver events and measured intra-lesion genetic heterogeneity in DCIS samples.

Conclusions:

• Optimized experimental and analytical strategies improve DNA sequencing accuracy from archived micro-dissected PMLs.
• This facilitates deeper molecular characterization of early cancer lesions.
• Achieves a milestone towards developing prognostic markers and precision chemo-prevention strategies.