Multicenter Evaluation of an Amplicon-Based NGS Panel for Comprehensive Molecular Profiling

Multicenter, pan-cancer evaluation

The study was designed as a multicenter, in-house analytical evaluation conducted across five academic and clinical research laboratories in Europe. This design allowed the investigators to assess not only analytical accuracy but also reproducibility across independent laboratories, an essential requirement for translational cancer research.

A total of 193 pan-cancer research samples were included in the analysis, representing more than 13 distinct tumor types. These included common and rare malignancies such as lung cancer, colorectal cancer, ovarian cancer, breast cancer, central nervous system (CNS) tumors, gastrointestinal stromal tumors (GIST), and cancers of unknown primary (CUP), among others.

DNA and RNA sample composition

The cohort consisted of both DNA and RNA samples:

• 125 DNA samples
• 68 RNA samples

This dual-analyte design enabled the evaluation of a broad range of variant classes, including DNA-based alterations (SNVs, indels, CNVs, HRD, TMB, MSI) and RNA-based fusions.

Pre-characterized reference samples

All samples were pre-characterized using orthogonal reference methods, providing a robust benchmark against which the performance of the Oncomine Comprehensive Assay Plus could be assessed. These reference data included known:

• SNVs and indels
• CNVs
• Gene fusions
• HRD status
• TMB status
• MSI status

Amplicon-based NGS for CGP

The Oncomine Comprehensive Assay Plus is an amplicon-based NGS panel designed to enable comprehensive genomic profiling from a highly automated workflow. The assay covers more than 500 cancer-relevant genes, allowing simultaneous interrogation of multiple variant classes and genomic signatures.

Amplicon-based workflows are particularly well suited for research settings where sample quantity and quality may be limited. Each targeted NGS method has unique advantages, but a key benefit of using an amplicon-based sequencing method to interrogate solid tumor samples is that it requires less DNA and/or RNA than a hybrid capture–based method. The lower input requirement for amplicon-based sequencing is due to PCR-based amplification of short targeted sequences, which affords greater depth of coverage and high accuracy.

Tumor cell content variability

One of the most notable aspects of the study cohort was the wide variability in tumor cell content. Samples ranged from as low as 10–20% tumor cell content to near-pure tumor samples, reflecting real-world research conditions rather than idealized sample sets.

Low tumor purity is a well-recognized challenge in molecular profiling, as it can significantly impact variant detection sensitivity, particularly for low-allele-fraction variants.

SNV and indel detection performance

Copy number variation detection

Gene fusion detection

Gene fusions represent an essential class of actionable alterations, particularly in solid tumors. In this study, fusion detection achieved an overall concordance of 94.2% when compared with orthogonal methods.

Consistency across five laboratories

A major strength of the study was its assessment of reproducibility across five independent laboratories. The Oncomine Comprehensive Assay Plus demonstrated a median reproducibility of 94.0% for SNV and indel detection across sites.

In addition to variant detection, reproducibility was also observed for HRD, TMB, and MSI assessments when replicate samples were tested across laboratories. This consistency is essential for collaborative research efforts and multicenter translational studies.

Reproducibility of genomic signatures

In addition to variant detection, reproducibility was also observed for HRD, TMB, and MSI assessments when replicate samples were tested across laboratories. This consistency is essential for collaborative research efforts and multicenter translational studies.

The findings from this multicenter evaluation highlight the potential of the Oncomine Comprehensive Assay Plus as a sensitive, specific, and reproducible platform for comprehensive molecular profiling in research settings.

Key advantages demonstrated by the study include:

• Broad genomic coverage (>500 genes)
• High sequencing success in low-quality and low-tumor-content samples
• Strong concordance with orthogonal methods across multiple variant classes
• Reliable detection of clinically relevant genomic signatures
• High reproducibility across laboratories

Based on this multicenter evaluation, the Oncomine Comprehensive Assay Plus demonstrated excellent analytical performance for comprehensive genomic profiling. High concordance rates were observed for SNVs, indels, CNVs, fusions, and genomic signatures such as HRD, TMB, and MSI, even in challenging samples with low tumor content.

The study’s results underscore the assay’s potential to support advanced cancer research by enabling robust, scalable, and reproducible molecular profiling across diverse tumor types.