What tests are used to determine the effectiveness of targeted immunotherapy for cancer treatment, including MMR (Mismatch Repair) testing, Tumor Mutational Burden (TMB) testing, and liquid testing such as Guardant testing?

Tests for Targeted Immunotherapy in Cancer Treatment

Molecular profiling using Next-Generation Sequencing (NGS) is the optimal approach for identifying patients who may benefit from targeted immunotherapy, as it can simultaneously assess multiple biomarkers including MSI, TMB, and gene alterations in a single test. 1

Key Biomarker Tests for Immunotherapy

Mismatch Repair (MMR) Testing

• Purpose: Identifies defects in DNA repair mechanisms that lead to microsatellite instability (MSI)
• Testing methods:

  • Immunohistochemistry (IHC): First-line test that uses a four-antibody panel detecting MLH1, MSH2, MSH6, and PMS2 proteins

    • Loss of nuclear staining indicates deficient MMR (dMMR)
    • Advantages: Widely available, cost-effective
    • Limitations: Can have false positives in approximately 10% of cases 1

  • PCR-based MSI Testing: Recommended when IHC results are equivocal

    • Uses validated panels of mononucleotide and dinucleotide repeats
    • Classified as MSI-High, MSI-Low, or microsatellite stable (MSS)
    • For colorectal cancer, ESMO recommends using both MMR-IHC and MSI-PCR to avoid false positives 1

Tumor Mutational Burden (TMB)

• Definition: Total number of somatic mutations per megabase of DNA
• Testing methods:
  • Whole exome sequencing (WES)
  • Targeted gene panels
  • FDA-approved companion diagnostics
• Clinical significance:
  • High TMB (≥10 mutations/Mb) is associated with improved response to immunotherapy 1
  • TMB-high tumors without MSI may still respond to immunotherapy 2
  • The optimal threshold for predicting MSI status is approximately 17 mutations/Mb 3

Liquid Biopsy (Guardant Testing)

• Purpose: Detects genomic alterations from circulating tumor DNA (ctDNA) in blood
• Advantages:
  • Minimally invasive (simple blood draw)
  • Can monitor treatment response in real-time
  • Useful when tissue biopsy is not feasible or insufficient
  • Can detect emerging resistance mechanisms 1
• Limitations:
  • Lower sensitivity compared to tissue testing, particularly for MSI assessment
  • May miss alterations present at low levels in circulation

Extended Gene Panel Testing (Genomic Testing)

• Purpose: Comprehensive molecular profiling beyond basic biomarkers
• Components:
  • Core immunotherapy markers (MSI, TMB, PD-L1)
  • Oncogenic drivers (EGFR, ALK, ROS1, NTRK fusions)
  • Tumor-specific targets (HER2, BRAF, KRAS)
  • Emerging biomarkers (CLDN18.2 for gastric cancer) 1
• Benefits: Single test can identify multiple treatment options and clinical trial eligibility

Relationship Between Biomarkers

• MSI-high tumors typically have high TMB, though the relationship varies by tumor type 2
• PD-L1 expression shows great variability across cancer types and can be expressed in MSI-negative and TMB-low cases 2
• For MSI-high tumors, further analysis for TMB or PD-L1 expression is not required to receive immunotherapy 2

Testing Recommendations

• Priority patients for testing:
  • All patients with advanced (unresectable or metastatic) solid tumors with high incidence of MSI/dMMR (colorectal, endometrial, gastric)
  • Consider testing in advanced solid tumors with low incidence of MSI/dMMR
  • Patients who have progressed on standard therapies 1

Clinical Impact

• MSI-H/dMMR status is associated with high response rates to PD-1/PD-L1 inhibitors across multiple tumor types, with objective response rates of 39.6-55.9% 1
• Patients with high TMB may benefit from immunotherapy even without MSI-H status 2
• Comprehensive genomic profiling can identify actionable alterations in approximately 21% of cancer patients 3

Common Pitfalls and Considerations

• Discordance between testing methods may occur, with a small fraction of tumors losing MMR protein expression while remaining microsatellite stable, and vice versa 1
• Some high TMB tumors may be microsatellite stable due to mutations in genes like POLE 3
• The prevalence of MSI-H varies significantly across different cancer types and populations 4
• NGS is emerging as the preferred method for simultaneously assessing multiple biomarkers, but tissue availability and quality remain important considerations 1

By understanding these biomarker tests and their relationships, oncologists can better select appropriate immunotherapy options for cancer patients, potentially leading to improved outcomes and quality of life.