Quality control and quantification in IG/TR next-generation sequencing marker identification: protocols and...

Area of Science:

• Molecular diagnostics
• Next-generation sequencing (NGS)
• Immunogenetics

Background:

• Next-generation sequencing (NGS) is crucial for assessing clonality, marker identification, and minimal residual disease (MRD) in lymphoid neoplasms.
• A significant gap exists in standardized quality control (QC) metrics for robust clinical application of NGS-based immunoglobulin (IG) and T cell receptor (TR) gene rearrangement assays.
• Lack of standardization hinders reliable diagnostic use of these powerful molecular techniques.

Purpose of the Study:

• To establish and validate quality control (QC) options for standardized NGS-based IG/TR gene rearrangement analysis.
• To ensure robust clinical diagnostics for lymphoid malignancies through improved accuracy and reproducibility.
• To provide a complete protocol for reliable marker identification and MRD quantification.

Main Methods:

• Development of two types of quality controls (QCs) by the EuroClonality-NGS Working Group.
• A central polytarget QC (cPT-QC) to monitor primer performance in multiplex NGS assays.
• A standardized human cell line-based DNA control (cIT-QC) for in-tube QC and MRD calibration, integrated into the ARResT/Interrogate bioinformatic platform.

Main Results:

• Successful integration of cPT-QC and cIT-QC into the ARResT/Interrogate platform.
• The developed QCs enable monitoring of primer performance and provide a calibrator for MRD quantification.
• The EuroClonality-NGS protocol demonstrates high reproducibility, accuracy, and precision for IG/TR gene rearrangement analysis.

Conclusions:

• The EuroClonality-NGS Working Group has established a complete, standardized protocol for IG/TR gene rearrangement analysis using NGS.
• The implemented QCs ensure robust marker identification and MRD quantification, crucial for diagnosing lymphoid malignancies.
• This standardized approach enhances the clinical utility and reliability of NGS in hematologic diagnostics.