Closing the Diagnostic Gap: Overcoming False Negatives via Integrated Liquid Biopsy and Reflex Next Generation Sequencing

Key Takeaways:

• Use concurrent plasma and tissue next-generation sequencing (NGS) to capture tumor heterogeneity and reduce false negatives.
• Implement "plasma-first" workflows to initiate targeted therapy faster when tissue samples are insufficient.
• Treat negative liquid results as non-informative; confirm with tissue NGS to ensure no driver is missed.

In the era of precision oncology, the "wait and see" approach is no longer tenable for patients with advanced non-small cell lung cancer (NSCLC). With the number of actionable targets – including EGFR, ALK, ROS1, BRAF, RET, MET, and KRAS G12C – expanding, the speed and accuracy of molecular profiling have become the primary determinants of first-line success. However, clinicians frequently encounter a significant hurdle: the diagnostic gap.¹

Up to 25 percent of tissue biopsies yield insufficient material for comprehensive genomic profiling (CGP), and even when tissue is available, the inherent spatial heterogeneity of tumors can lead to false-negative results. To bridge this gap, an integrated diagnostic workflow utilizing liquid biopsy and reflex Next-Generation Sequencing (NGS) is becoming a new potential standard of care for certain lung cancers.¹

The challenge of tissue limitations and false negatives

Tissue remains the gold standard, but it is often fraught with logistical and biological limitations. Tissue-based NGS can fail due to inadequate tumor cell content or DNA degradation during processing. Furthermore, a single-site biopsy may fail to capture the full clonal architecture of a metastatic disease, potentially missing resistance mutations or heterogeneous drivers.^1,2

Recent data suggests that relying solely on tissue-based testing may under-identify actionable alterations in nearly 10 to 15 percent of patients. This "false negative" risk in tissue is often addressed through liquid biopsy, which analyzes circulating tumor DNA (ctDNA) shed into the plasma.³

The power of "liquid-first" and concurrent testing

Integrating liquid biopsy into the initial diagnostic workup significantly reduces the time to treatment. The NILE study demonstrated that adding liquid biopsy to the standard of care increased the detection rate of guideline-recommended biomarkers by 48 percent compared to tissue alone.^1,4,5

For the clinician, the "plasma first" or "concurrent" approach – where blood is drawn at the same time the tissue biopsy is ordered – minimizes the risk of missing a driver mutation due to tissue insufficiency. When ctDNA identifies a validated driver, the clinician can initiate targeted therapy immediately, often weeks before the tissue NGS results would have been available.^6,7

Ensuring no patient is left behind

A "reflex" NGS protocol ensures that if the initial liquid biopsy is negative, the laboratory automatically proceeds to tissue NGS, or vice versa. This is critical because liquid biopsy, while highly specific, has lower sensitivity compared to tissue, particularly in patients with low tumor burden or isolated CNS progression.⁷

Studies have shown that while liquid biopsy has a high positive predictive value, its negative predictive value is not absolute. As noted in the updated IASLC Statement on Liquid Biopsy, a negative liquid biopsy result should be considered a "non-informative" result rather than a true negative, potentially necessitating reflex tissue-based testing to confirm the absence of a driver.⁷

Clinical integration: A multi-modal framework

To optimize outcomes, clinicians should adopt a complementary testing framework:

• Concurrent testing – Order plasma and tissue NGS simultaneously for certain patients with suspected advanced NSCLC.
• Reflexive transition – If plasma is negative for drivers, consider reflexing immediately to tissue CGP. If tissue is "quantity not sufficient," consider reflexing immediately to plasma.
• Resistance monitoring – Consider use of liquid biopsy at the time of progression to identify acquired resistance mechanisms, such as the EGFR T790M or C797S mutations.

A unified diagnostic front

Closing the diagnostic gap in lung cancer requires a shift from a sequential testing mindset to an integrated, multi-modal strategy. By leveraging the speed of liquid biopsy and the depth of reflex NGS, clinicians can overcome the pitfalls of false negatives and tissue insufficiency.

This integrated approach ensures that every patient is matched with the most effective therapy at the earliest possible moment, ultimately improving survival and quality of life in the face of advanced disease.