Relying solely on Next-Generation Sequencing (NGS) is insufficient for HER2 testing in biliary tract cancers. Data shows NGS misses up to 15% of patients with HER2 overexpression detected by immunohistochemistry (IHC). Performing both tests is essential to avoid denying patients effective targeted therapies.

While liquid biopsies are a valuable, less invasive tool, a negative result is inconclusive for ruling out actionable mutations in NSCLC. It may simply mean the tumor isn't shedding enough DNA. Therefore, a negative liquid biopsy should never be the final word; it must be followed by a tissue biopsy to ensure patients don't miss out on targeted therapies.

Despite immunotherapy's success in other lung cancers, it is completely ineffective for patients with ROS1 fusions. A large global data series showed a 0% response rate. These genetically-driven cancers are "cold tumors," making upfront genomic testing critical to avoid futile and toxic treatment with checkpoint inhibitors.

In community SCLC care, molecular strategies are not monolithic. Genomic alteration testing (NGS) is ready for immediate use and can identify targets today. In contrast, neuroendocrine subtyping is still investigational and not yet clinically actionable, pending results from research studies.

DNA-based NGS can fail to detect clinically actionable fusions in NSCLC due to assay design limitations (low sensitivity). It can also report fusions of unclear significance (low specificity). Integrating RNA-based NGS is critical to reliably identify true driver fusions and clarify ambiguous DNA findings.

Clinicians ordering "NGS for lung" often misunderstand that Next-Generation Sequencing alone does not cover all actionable biomarkers, such as PD-L1 or HER2. This requires pathologists to interpret the clinician's intent and order a more comprehensive and appropriate test panel.

While both are used, some oncologists consider Next-Generation Sequencing (NGS) the gold standard over Immunohistochemistry (IHC) for MSI testing. NGS provides tumor mutation burden (TMB) data, which can identify rare, ultra-hypermutated but technically microsatellite-stable (MSS) tumors that are likely to respond to immunotherapy but would be missed by IHC alone.

To integrate RNA sequencing, labs can use a sequential workflow (DNA-NGS first, then RNA-NGS on driver-negative cases), which is cost-effective but slower. Alternatively, upfront co-testing is faster and decision-free but more expensive and may be unnecessary for patients with common DNA-level drivers.

For post-progression biopsies, which are often small and contain necrotic tissue, institutions may prioritize DNA-based NGS panels. This strategy is based on the rationale that most resistance mechanisms are genetic mutations detectable by DNA sequencing, reserving RNA panels primarily for identifying less common fusion events.