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.

Experts overwhelmingly favor tissue-based testing (IHC or NGS) to determine MSI status in localized colorectal cancer. They express concern that liquid biopsies (ctDNA) may produce false negatives because smaller, localized tumors might not shed enough DNA into the bloodstream for reliable detection.

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.

Unlike colorectal cancer, where MSI is often clonal, MSI in gastric cancer is typically sporadic. This can lead to regional heterogeneity within a single tumor, with some parts being MSI-high and others MSS (microsatellite stable), which has significant implications for immunotherapy efficacy.

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.

For certain therapies like Enhertu, eligibility is based on immunohistochemistry (IHC), not NGS. Labs must run HER2 IHC in parallel because NGS, as a population-based test, can miss intratumoral heterogeneity (small clusters of positive cells) that IHC can detect, thus identifying more eligible patients for targeted therapy.

While Next-Gen Sequencing (NGS) provides genetic data, IHC directly measures the protein, is faster, cheaper, and requires less tissue. This makes it more scalable for routine clinical use, especially with small biopsy samples. High-level IHC loss correlates well with genetic loss seen on NGS.

Experts indicate that Tumor Mutational Burden (TMB) is losing relevance for guiding immunotherapy in GI cancers. The TMB cutoff of 10 is not considered reliable across tumor types, and clinicians still prefer combination chemo-immunotherapy even in TMB-high patients, unless MMR deficiency is also present.

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.