For a patient with advanced solid tumor, when should tumor mutation burden be tested and what TMB cutoff indicates eligibility for pembrolizumab (Keytruda) immunotherapy?

Tumor Mutational Burden Testing and Pembrolizumab Eligibility

Pembrolizumab is FDA-approved for patients with unresectable or metastatic TMB-high solid tumors (≥10 mutations/megabase by an FDA-approved test) who have progressed after prior treatment and have no satisfactory alternative treatment options. 1

When to Test TMB

Test TMB only after exhausting other biomarker-guided therapies and when considering pembrolizumab as a last-line option for advanced solid tumors. 2, 1

Testing Algorithm:

• First priority: Test for MSI-H/dMMR status, as this provides stronger predictive value for immunotherapy response than TMB alone 2, 3
• Second priority: Test for PD-L1 expression if tumor-type specific (e.g., cervical cancer requires CPS ≥1) 2
• Third priority: Test for actionable driver mutations (EGFR, ALK, BRAF, NTRK, RET fusions) that may have targeted therapy options 2
• Last resort: Consider TMB testing only when the above biomarkers are negative or therapies have failed, and no other satisfactory treatment options exist 1, 4

Critical Tumor-Specific Exceptions:

Do NOT test TMB for colorectal cancer outside clinical trials, as NCCN explicitly recommends against it due to poor clinical activity (only 11% objective response rate and 31% disease control rate in TMB-H metastatic colorectal cancer) 2

TMB-High Definition and Cutoff

The FDA-approved cutoff is ≥10 mutations/megabase (mut/Mb) as measured by an FDA-approved test. 1, 2

FDA-Approved Testing Method:

• FoundationOne CDx assay is the FDA-approved companion diagnostic for determining TMB-H status for pembrolizumab treatment 2
• This is a 324-gene NGS panel that measures TMB across the coding area 2
• The assay demonstrated good concordance with whole exome sequencing in retrospective validation 2

Important Nuances About the 10 mut/Mb Threshold:

While the FDA approval uses ≥10 mut/Mb, higher TMB cutoffs may better predict response in certain contexts. The MyPathway trial showed that patients with TMB ≥16 mut/Mb had a 38.1% objective response rate to atezolizumab, compared to only 2.1% for TMB <16 mut/Mb 2. However, the FDA-approved indication remains ≥10 mut/Mb 1.

Expected Clinical Outcomes

Patients with TMB-H tumors (≥10 mut/Mb) treated with pembrolizumab achieved a 29% objective response rate compared to 6% in non-TMB-H tumors in the KEYNOTE-158 trial. 2, 3, 4

Response Characteristics:

• Median follow-up in KEYNOTE-158 was 37.1 months 4
• The benefit was observed across multiple tumor histologies, though none were colorectal cancer 2
• This approval is under accelerated approval status based on tumor response rate and durability of response, with continued approval contingent on confirmatory trials 1

Critical Caveats and Pitfalls

TMB Alone Is Insufficient:

TMB-H status does not universally predict immunotherapy response across all tumor types. 2, 3 Several mechanisms can prevent effective anti-tumor response even with high TMB:

• Somatic mutations may generate neoantigens with insufficient immunogenicity 2
• MHC silencing can prevent neoantigen presentation 2
• Resistance to interferon signaling may occur 2
• T cell exclusion from the tumor microenvironment can limit immune response 2

Specimen Requirements:

• TMB assessment requires adequate tumor tissue with sufficient tumor content 2
• Blood-based TMB (bTMB) from circulating tumor DNA requires ≥5% tumor fraction for accurate genome-wide copy number profiling 2
• Low circulating tumor DNA fractions (<1-2%) significantly limit accuracy of TMB calling from blood samples 2, 3

Relationship to Other Biomarkers:

MSI-H tumors frequently demonstrate high TMB, but TMB-H can occur independently of MSI status. 3 Prioritize MSI/MMR testing first, as MSI-H/dMMR tumors have superior response rates to immunotherapy compared to MSS/TMB-H tumors 2, 3.

Age-Related Confounders:

In patients >50 years old, low variant allele frequency mutations detected in cell-free DNA may represent clonal hematopoiesis (CHIP) rather than tumor-derived mutations, potentially inflating TMB estimates 2

Practical Implementation

Order TMB testing only when:

1. Patient has unresectable or metastatic solid tumor 1
2. Disease has progressed after prior treatment 1
3. No satisfactory alternative treatment options exist 1
4. Tumor is NOT colorectal cancer (unless in clinical trial) 2
5. Other biomarkers (MSI-H/dMMR, PD-L1, actionable mutations) have been evaluated first 2, 3

Use FoundationOne CDx assay as the FDA-approved companion diagnostic to ensure results are interpretable for pembrolizumab eligibility 2, 1