VEEVA APPROVED EQA webinar EGFR Liquid Biopsy Results Review Webinar video recordings

This video provides an in-depth exploration of the results from the 2024 External Quality Assessment (EQA) scheme focused on lung cancer testing using liquid biopsy. Organized by EMQN and GenQA and funded by AstraZeneca and MSD, the webinar aims to assess the accuracy of testing and clinical reporting for EGFR and KRAZ gene variants. Dr. Simon Patton and Prof. Sandi Deans guide the audience through the EQA's objectives, methodology, and key findings, highlighting common errors and best practices in molecular pathology. The discussion emphasizes the critical need for sensitive testing methods, standardized reporting, and accurate clinical interpretation in the context of circulating tumor DNA (ctDNA).

The EQA scheme's primary goal is to improve clinical service by evaluating laboratories' ability to correctly determine genotypes, interpret results within a clinical context, use internationally accepted nomenclature (HGVS), and provide accurate patient information. The webinar details the global participation in the EQA, noting a shift towards Next Generation Sequencing (NGS) as the dominant testing methodology over targeted assays. The samples used are custom artificial plasma, rigorously validated by multiple independent laboratories using diverse methods, including Roche cobas, droplet digital PCR (ddPCR), NGS panels, and the Guardant 360 assay, ensuring their suitability for various testing strategies.

Three distinct clinical cases are presented to illustrate common genotyping and interpretation errors. Case one involved a patient with non-small cell lung carcinoma, where laboratories were expected to detect a pathogenic KRAZ variant but no actionable EGFR variants. Errors included false positives/negatives for KRAZ, sample swaps, and over-interpretation of the absence of EGFR variants. Case two focused on a patient with metastatic lung adenocarcinoma, previously treated with TKIs, requiring detection of both a sensitizing EGFR L858R variant and a resistance T790M variant. Errors here involved false positives (e.g., ROS1 fusion), false negatives for either variant, and critical interpretation errors regarding third-generation TKI sensitivity. Case three, involving a patient with insufficient tissue for molecular testing, required detection of an EGFR G719S variant. This case saw a high number of genotyping errors, including false positives, false negatives, sample swaps, and clerical errors like incorrect patient names or conflicting report sections.

General observations from the EQA highlight several recurring challenges. A significant point is the necessity for highly sensitive methods capable of detecting variant allelic frequencies (VAFs) as low as 0.01-0.1% in ctDNA, given its lower concentration compared to solid tumor tissue. Laboratories must balance the scope of variants tested with the required sensitivity and understand their assay's limitations. While HGVS nomenclature and the use of single, standardized gene reference sequences (like MAIN Select/Plus Clinical) have shown improvement, deductions still occur for incorrect or absent usage. Finally, the webinar strongly advises against reporting benign or likely benign variants to avoid clinician confusion and cautions against over-interpreting the absence of actionable variants in ctDNA, emphasizing the need to recommend repeat sampling or tissue biopsy due to inherent sensitivity limitations.

Key Takeaways:

• Importance of External Quality Assessment (EQA): EQA schemes like those run by EMQN and GenQA are crucial for assessing and improving the accuracy of molecular testing and clinical reporting in the life sciences, particularly for complex areas like liquid biopsy in lung cancer.
• Global Shift to NGS: There's a clear trend towards Next Generation Sequencing (NGS) as the predominant technology for ctDNA testing in lung cancer, indicating its growing adoption and perceived utility over more targeted assays.
• Sensitivity for ctDNA Testing: Laboratories must employ highly sensitive methods capable of detecting variant allelic frequencies (VAFs) as low as 0.01-0.1% for circulating tumor DNA (ctDNA), as its concentration is significantly lower than in solid tissue. Understanding and clearly stating the assay's limit of detection is paramount.
• Common Genotyping Errors: Recurring errors include false positive and false negative variant detections, incorrect variant reporting (e.g., reporting a deletion when a different variant is present), and critical sample handling issues like sample swaps between cases.
• Critical Interpretation Errors: Over-interpretation of results, especially the absence of an actionable variant in ctDNA, is a significant pitfall. Laboratories should avoid definitive statements about treatment response based solely on negative ctDNA findings and instead recommend further testing (e.g., tissue biopsy).
• Standardized Nomenclature and Reference Sequences: Adherence to HGVS nomenclature for describing variants (both nucleic acid and predicted amino acid changes) and using a single, standardized gene reference sequence (e.g., MAIN Select/Plus Clinical) per report is essential for clarity, data sharing, and minimizing misinterpretation.
• Comprehensive Reporting Practices: Clinical reports should avoid listing benign or likely benign variants to prevent confusion. Furthermore, if multiple genes are tested (e.g., KRAZ alongside EGFR), results for all tested genes should be reported, even if no actionable variants are found.
• Contextual Interpretation: Results must be interpreted within the full clinical context, including prior molecular testing and treatment history. Providing separate, unintegrated interpretations for different variants within the same report can lead to confusion for the end-user.
• Understanding Resistance Variants: When resistance variants (e.g., EGFR T790M) are detected, the interpretation must clearly explain their implications for treatment options, such as sensitivity to third-generation TKIs, to guide appropriate patient management.
• Methodology Limitations: Reports should always include sufficient information regarding the methodology used and the limitations of the assay performed. This transparency is critical for clinicians to understand the scope and reliability of the test results.
• Impact of Regular Participation: Consistent and regular participation in EQA schemes is shown to drive improvements in laboratory performance over time, reducing critical genotyping errors and exposing laboratories to a broader range of challenging clinical scenarios and rare variants.
• Challenges of Low VAF: The dilemma of detecting extremely low VAFs in ctDNA presents a continuous challenge for EQA providers and laboratories, balancing the need for clinical relevance with the practical limitations of current technologies and the educational goals of EQA.

Tools/Resources Mentioned:

• HGVS Nomenclature: Standardized human gene variation society nomenclature for describing genetic variants.
• MAIN Select and MAIN Plus Clinical: Gene reference sequence initiatives supported by EQA providers for standardization.
• HQ estimator: A tool mentioned for indicating when nucleic acid change cannot be determined.
• Roche cobas EGFR mutation test: A widely used diagnostic assay for EGFR mutations.
• Bio-Rad primers: Used for droplet digital PCR (ddPCR) validation.
• TrueSight Oncology CTDNA specific panel: An NGS panel used for validation.
• Guardant 360 diagnostic assay: A commercial liquid biopsy assay used for validation.

Key Concepts:

• EQA (External Quality Assessment): A system for objectively checking the performance of laboratories by an external agency, often involving blinded sample testing.
• Liquid Biopsy: A non-invasive method to detect tumor-derived material (like ctDNA) from bodily fluids, typically blood plasma.
• ctDNA (circulating tumor DNA): Fragments of DNA released from tumor cells into the bloodstream, used for cancer detection, monitoring, and treatment selection.
• EGFR (Epidermal Growth Factor Receptor) and KRAZ (Kirsten rat sarcoma viral oncogene homolog) gene variants: Common oncogenes whose mutations are crucial biomarkers in lung cancer for guiding targeted therapies.
• VAF (Variant Allelic Frequency): The proportion of DNA reads containing a specific variant compared to the total number of reads at that genomic position, indicating the abundance of a mutation.
• NGS (Next Generation Sequencing): High-throughput sequencing technologies that can sequence millions of DNA fragments simultaneously, enabling comprehensive genomic profiling.
• ddPCR (droplet digital PCR): A highly sensitive PCR method that partitions a sample into thousands of individual droplets, allowing for absolute quantification of nucleic acids and detection of low-frequency variants.
• TKIs (Tyrosine Kinase Inhibitors): A class of targeted cancer drugs that block the activity of tyrosine kinase enzymes, often used in lung cancer with specific EGFR mutations.