Diagnostic next-generation sequencing to detect MYD88 L265P in Lymphoplasmacytic lymphoma compared to ddPCR

Area of Science:

• Hematology
• Molecular Diagnostics
• Oncology

Background:

• Lymphoplasmacytic lymphoma (LPL) is a B-cell neoplasm characterized by bone marrow infiltration.
• The MYD88 L265P mutation is a key diagnostic marker for LPL.
• Previous studies questioned the sensitivity of routine next-generation sequencing (NGS) for detecting MYD88 L265P.

Purpose of the Study:

• To evaluate the diagnostic utility and sensitivity of NGS for MYD88 L265P detection.
• To compare NGS performance against droplet digital PCR (ddPCR) for MYD88 L265P detection.
• To assess the impact of tumor content on NGS-based mutation detection.

Main Methods:

• Analyzed 34 lymphoid neoplasm cases using routine NGS and ddPCR.
• Assessed MYD88 L265P detection in NGS data using manual review and BAMtools.
• Determined the limit of detection for ddPCR (0.4% VAF with 10 ng DNA).

Main Results:

• NGS and ddPCR showed comparable MYD88 L265P variant allele frequency (VAF) detection down to 0.5% VAF (R² = 0.968).
• Implementing a ddPCR-informed threshold eliminated false positives in NGS.
• Low tumor content did not impede MYD88 L265P detection by NGS.

Conclusions:

• NGS is a sensitive and reliable method for detecting the MYD88 L265P mutation in lymphoid neoplasms.
• Adequate sequencing coverage and specific assessment parameters are crucial for accurate NGS detection.
• NGS can be effectively utilized in clinical settings for MYD88 L265P variant identification.