Characterization of Chronic Lymphocytic Leukemia Immunoglobulin Rearrangements from Partial Read Sequencing

Area of Science:

• Hematology
• Oncology
• Genetics

Background:

• Immunoglobulin variable region mutational status is a prognostic biomarker in chronic lymphocytic leukemia (CLL).
• Next-generation sequencing (NGS) of rearranged VDJ sequences presents challenges for B-cell clone characterization due to length and variability.
• Standardization is required to align NGS procedures with current clinical guidelines for CLL management.

Purpose of the Study:

• To develop a simple, low-cost, and efficient strategy for sequencing the immunoglobulin heavy chain gene (IGH) variable domain.
• To enable faster results through shorter reads (MiSeq 150 × 2) suitable for clinical application.
• To integrate clonality and mutational status determination into a single analysis pipeline.

Main Methods:

• A novel sequencing strategy was developed for the IGH variable domain.
• The method was validated using 319 CLL patients and 47 healthy donors.
• A clone-centered consensus sequence strategy was employed for B-cell clone identification and clonal threshold determination.

Main Results:

• The developed method allows for efficient sequencing of the IGH variable domain using shorter reads.
• Clonality and mutational status are determined within a single analysis pipeline.
• The method was validated against Sanger sequencing, the current gold standard for IGHV mutational status determination.

Conclusions:

• The new strategy provides a standardized, efficient, and cost-effective approach for IGHV mutational status determination in CLL.
• This method overcomes limitations of Sanger sequencing and facilitates B-cell clone characterization.
• The findings support the adaptation of this NGS-based procedure to current clinical guidelines for CLL prognosis.