Clinical validation of the next-generation sequencing-based Extended RAS Panel assay using metastatic colorectal...

Area of Science:

• Molecular diagnostics
• Oncology
• Genomics

Background:

• Metastatic colorectal cancer (mCRC) treatment decisions are influenced by RAS mutation status.
• Accurate and comprehensive RAS mutation detection is crucial for personalized therapy selection.
• Next-generation sequencing (NGS) offers a powerful tool for genomic analysis.

Purpose of the Study:

• To validate an NGS-based companion diagnostic assay for simultaneous detection of 56 RAS mutations.
• To assess the assay's ability to identify mCRC patients benefiting from panitumumab treatment.
• To evaluate the assay using DNA from formalin-fixed paraffin-embedded tumor samples.

Main Methods:

• Utilized samples from the PRIME study comparing panitumumab + FOLFOX4 with FOLFOX4.
• Employed a multiplex assay for DNA qualification, library preparation, and sequencing on the MiSeqDx instrument.
• Performed NGS mutational analysis of KRAS and NRAS exons 2, 3, and 4, comparing results with Sanger sequencing.

Main Results:

• The Extended RAS Panel demonstrated high agreement with Sanger sequencing (98.7% positive, 97.6% negative).
• In clinical validation, panitumumab + FOLFOX4 significantly improved progression-free survival (PFS) in RAS-negative patients.
• Treatment effect differed significantly between RAS-negative and RAS-positive patients for both PFS and overall survival (OS).

Conclusions:

• The NGS-based Extended RAS Panel is a validated companion diagnostic for panitumumab selection in mCRC.
• The assay enables broad, rapid, and specific genomic analyses, aligning with clinical guidelines.
• The Extended RAS Panel identifies approximately 13% more patients with RAS mutations compared to KRAS exon 2 testing alone.