What tests are required for targeted immunotherapy in cancer treatment, including MMR (Mismatch Repair) testing, Tumour Mutational Burden (TMB), Liquid Testing (such as Guardant360), and extended gene panel - genomic testing?

Tests for Targeted Immunotherapy in Cancer Treatment

Comprehensive 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, 2

Mismatch Repair (MMR) Testing

Recommended Testing Methods

• Immunohistochemistry (IHC):

  • First-line test for assessing MMR status 1
  • Requires a four-antibody panel detecting MLH1, MSH2, MSH6, and PMS2 proteins
  • Loss of nuclear staining indicates deficient MMR (dMMR)
  • Strong consensus recommendation (8.7/10) from ESMO 1

• PCR-based MSI Testing:

  • Recommended when IHC results are equivocal or not available 1
  • Two validated panels:
    1. Five poly-A mononucleotide repeats panel (BAT-25, BAT-26, NR-21, NR-24, NR-27) - preferred due to higher sensitivity and specificity
    2. Panel with two mononucleotide (BAT-25, BAT-26) and three dinucleotide repeats (D5S346, D2S123, D17S250)
  • MSI is present when two or more markers show repeat length alterations

Clinical Implications

• For colorectal cancer, ESMO recommends using both MMR-IHC and MSI-PCR to avoid false positives (which occur in ~10% of cases) 1
• MSI-H/dMMR status is associated with high response rates to PD-1/PD-L1 inhibitors across multiple tumor types 1, 3

Tumor Mutational Burden (TMB)

Testing Approach

• Defined as the total number of somatic mutations per megabase of DNA
• Can be assessed through:
  • Whole exome sequencing (WES)
  • Targeted gene panels (most clinically practical)
  • FDA-approved companion diagnostics like FoundationOne CDx 3

Clinical Relevance

• High TMB (≥10 mutations/Mb) is associated with improved response to immunotherapy 4
• Real-world data shows median PFS of 24.2 months for immunotherapy vs. 6.75 months for chemotherapy in TMB-high patients 4
• TMB and MSI status are often correlated but not always concordant, with some tumors being TMB-high but MSI-stable 1

Liquid Biopsy (Guardant Testing)

• Allows detection of genomic alterations from circulating tumor DNA (ctDNA) in blood
• Advantages:
  • Minimally invasive
  • Can be performed when tissue is insufficient or inaccessible
  • Useful for monitoring treatment response and resistance mechanisms
• Limitations:
  • Lower sensitivity compared to tissue testing, particularly for MSI assessment
  • May miss alterations present at low levels in circulation

Extended Gene Panel - Genomic Testing

Comprehensive NGS Panels

• NGS represents the most efficient approach for comprehensive biomarker assessment 1
• Key advantages:
  • Simultaneous assessment of MSI, TMB, and targetable gene alterations
  • Can identify other actionable mutations beyond immunotherapy targets (EGFR, ALK, ROS1, BRAF, etc.)
  • Strong consensus recommendation (9/10) from ESMO 1

Specific Biomarkers for Targeted Therapies

• Beyond immunotherapy markers, panels should include:
  • Oncogenic drivers (EGFR, ALK, ROS1, NTRK fusions)
  • Tumor-specific targets (HER2, BRAF, KRAS, etc.)
  • Emerging biomarkers (CLDN18.2 for gastric cancer) 1

Patient Selection for Testing

• High priority for testing:

  • All patients with advanced (unresectable or metastatic) solid tumors with high incidence of MSI/dMMR (colorectal, endometrial, gastric) 1
  • Patients without other actionable driver mutations 1
  • Tumors highly likely to harbor NTRK fusions 1

• Consider testing in:

  • Advanced solid tumors with low incidence of MSI/dMMR 1
  • Patients who have progressed on standard therapies 1

Common Pitfalls and Considerations

1. Testing discordance: Significant discordance (37-43%) has been observed between local and central MSI/dMMR testing 3, highlighting the importance of validated assays

2. Turnaround time: Median turnaround time for NGS can be lengthy (73 days in real-world studies) 4, which may delay treatment decisions

3. Tissue requirements: Adequate tissue quantity and quality are essential for reliable results; consider liquid biopsy when tissue is insufficient

4. Interpretation challenges:

   • TMB thresholds vary by tumor type and assay platform
   • Different NGS panels may yield different TMB estimates 5, 6
   • Some MSH6 mutations in endometrial cancers may cause minimal microsatellite shifts that standard MSI testing might miss 2

5. Heterogeneity: Significant heterogeneity in TMB and immune cell infiltration can exist between multiple primary tumors in the same patient 7

By implementing comprehensive molecular profiling with NGS, clinicians can identify patients most likely to benefit from immunotherapy while also detecting other actionable alterations for targeted therapies, ultimately improving patient outcomes through precision medicine approaches.