NGS Panels vs Hotspot Testing: Breadth Versus Focus

Hotspot Assays

Hotspot tests, often PCR-based, interrogate a defined list of recurrent mutation positions, for example EGFR exon 19 deletions, L858R and T790M, or KRAS codon 12 and 13 changes. They are fast, cheap, sensitive at low input and work on small samples.

Their limitation is that they only find what is on the list. An unusual or novel variant in the same gene, or a fusion, will not be detected.

Broad NGS Panels

Large panels sequence the coding regions of many genes and can detect substitutions, insertions and deletions, copy-number changes, and often fusions and complex biomarkers such as tumour mutational burden and microsatellite instability.

They need more tissue and time and cost more, but a single test can cover current and emerging targets, reducing the chance of tissue exhaustion from sequential testing.

The Negative-Result Problem

A negative hotspot result is common and can be misleading. For example, many EGFR exon 20 insertions and uncommon EGFR mutations are missed by assays designed around the classical variants.

When a targeted therapy exists for a mutation type that the assay does not fully cover, a negative result should be treated as incomplete rather than definitive.

Choosing an Approach

In settings where rapid results drive urgent decisions, a hotspot test for the most actionable alterations followed by reflex broad sequencing is a common compromise.

Guidelines increasingly recommend broad panel testing up front in cancers such as advanced non-small-cell lung cancer, where multiple biomarkers each have approved drugs.

Interpretation Notes

A report should list which genes and regions were covered. Read a negative result against that scope, not as a blanket absence.

If an initial limited test is negative and clinical suspicion of a driver is high, escalating to a broader assay is appropriate.

Key Takeaways

• ·Hotspot assays are fast and sensitive but only detect listed variants.
• ·Broad NGS panels cover many alteration types and biomarkers in one test.
• ·A negative hotspot result can miss exon 20 insertions, uncommon mutations and fusions.
• ·Guidelines increasingly favour broad panel testing up front where multiple targets exist.