Comprehensive Immune Profiling via 5 Test Modes


to semi-quantitatively measure transcript levels of 43 genes related to T-cell receptor signaling (TCRS) and other components of the immune cycle, as well as 11 genes associated with tumor infiltrating lymphocytes (TILs)

  • Next Generation Sequencing (NGS) Assay
  • Amplicon-based targeted NGS for digital gene expression detection
  • 395 genes representing immune-related gene functions.
  • 54 of these 395 genes that can be divided into multiple functional Immune Phenotypes
    • T-cell Receptor Signaling (TCRS)
    • Tumor Infiltrating Lymphocytes (TILs)
  • 10 genes are used as control genes
  • Remaining genes in the assay have not been fully validated and are not included in IRC results


to assess microsatellite instability (MSI)

  • 5 markers
    • 2 mononucleotide repeat markers (BAT-25, BAT-26)
    • 3 dinucleotide repeat markers (D2S123, D5S346 and D17S250)
  • Reported as
    • MSI-high
    • MSI-low
    • MSS (microsatellite stable)
  • MSI typically used as a prognostic marker of survival in the setting of Lynch syndrome/hereditary nonpolyposis colorectal cancer (HNPCC)
  • Marker of response to CPIs in colorectal cancer (Le, PMID:26028255 – 2016)


Immunohistochemistry (IHC) to measure PD-L1 protein expression and patterns of lymphocyte infiltration. 

  • Uses automated stainer platforms and commercially available antibodies
    • PD-L1 (22C3, 28-8, SP142, all FDA-approved)
    • CD3 (Laboratory Developed Test)
    • CD8 (Laboratory Developed Test)
  • PD-L1 scoring follows FDA guidelines for:
    • Non-small cell lung cancer
    • Stomach and gastroesophageal junction adenocarcinoma
    • Melanoma
    • Head and neck squamous cell carcinoma
    • Urothelial carcinoma
  • CD3 and CD8 scoring pattern:
    • Infiltrating pattern
      • TILs within groups of neoplastic cells
    • Non-infiltrating
      • TILs present but no consistent pattern of infiltrating groups of neoplastic cells
      • includes cases with an abundance of TILs at the advancing edge of the tumor, but without infiltration of the neoplastic cells
    • Minimal to Absent
      • minimal to no TILs present within any part of the tumor


to detect copy number gain of PD-L1 and PD-L2

  • Measures copy number of PD-L1 (CD274) & PD-L2 (PDCD1LG2):
    • PD-1 ligands that when amplified are associated with PD-L1 expression
    • Located 40kb apart on 9p24.1
    • Detected using a pool of fluorescently labeled BAC clones that map to the gene region
  • Reported using ASCO–CAP HER2 Test Guideline Recommendations for determining copy number:
    • Amplified
      • Ratio of PD-L1/2 to CEP9 is equal to or greater than 2.0 for any copy number value of PD-L1/2
      • Ratio of PD-L1/2 to CEP9 is less than 2.0 and the copy number value of PD-L1/2 is equal to or greater than 6.0
    • Equivocal
      • Ratio of PD-L1/2 to CEP9 is less than 2.0 and copy number value of PD-L1/2 is equal to or greater than 4.0 and less than 6.0
    • Not Amplified
      • Ratio of PD-L1/2 to CEP9 is less than 2.0 and the copy number value of PD-L1/2 is less than 4.0.


to estimate mutational burden (MuB)

  • 1.16 Mb AmpliSeq capture of 6,602 exons
  • 409 oncogenes
  • Full exon coverage
  • Reported as mutational burden (MuB), mutations per Mb exonic sequence
  • Specific mutations are not reported

3 Legs of the Stool

Mutational Burden (MuB)

  • MuB is reported as number of mutations per megabase (Mb) of exonic DNA
  • MuB was calibrated against 4 clinically relevant peer-reviewed publications reporting a correlation of high mutational burden with response to checkpoint inhibitors:
    • Melanoma
      • Snyder, PMID:25409260 – 2016
      • Van Allen, PMID:26359337 – 2015
    • NSCLC
      • Rizvi, PMID:25765070 – 2016
    • Bladder (urothelial) cancer
      • Rosenberg, PMID:26952546 – 2016
  • Using an equivalent whole exome and these 4 references as a calibrator the cut-off values for MuB as measured by number of mutations per Mb DNA was established using an internal reference population of 167 subjects.
  • MuB is classified as “very high”, “high”, “intermediate”, “low”, and “very low”.

T Cell Receptor Signaling (TCRS)

  • May be expressed on:
    • Immune infiltrating cells
    • Neoplastic cells
    • Other cells of the tumor microenvironment
  • Further subdivided by multiple immune phenotypes.
  • Directly involved in checkpoint blockade
  • Functions that relate to TCRS

Tumor Infiltrating Lymphocytes (TILs)

  • 11 genes to classify subsets of infiltrating immune cells
    • Galon et. al. (Galon, PMID:24122236 – 2014)
    • Emphasis on CD3 and CD8
      • Associated IHC patterns of expression
  • TILs as a predictive immune biomarker/prognostic marker of survival/associated response to CPIs:
    • Positive
      • CD3+CD8+ cytotoxic lymphocytes
    • Negative
      • FOXP3+ Tregs


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