Immunotherapy-Related Testing: Biomarkers, Methods, and Clinical Applications

Immunotherapy-related testing encompasses a comprehensive suite of laboratory assays used to evaluate predictive biomarkers, monitor immune responses, and manage immune-related toxicities in patients undergoing cancer immunotherapy. Accurate detection of key indicators such as PD-L1 expression, tumor mutational burden (TMB), and microsatellite instability (MSI) is essential for patient stratification and treatment optimization. This article provides a technical overview of the sample types, testing items, analytical methods, and quality requirements for immunotherapy-related testing performed in accredited third-party laboratories.

Technical Overview

Immunotherapy has transformed the treatment landscape for multiple malignancies through immune checkpoint inhibitors (such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies), adoptive cell therapies, therapeutic vaccines, and cytokine-based regimens. However, only a subset of patients derives durable clinical benefit, and a meaningful proportion experiences immune-related adverse events (irAEs). Immunotherapy-related testing therefore serves three core purposes: predicting therapeutic response through biomarker assessment, monitoring pharmacodynamic and dynamic immune changes during treatment, and supporting the early detection and management of immune toxicity.

Predictive biomarker testing is typically performed under a companion diagnostic or complementary diagnostic framework, in which analytical validity, clinical validity, and clinical utility must be demonstrated before results are used for therapeutic decision-making. Laboratories conducting immunotherapy-related testing should operate under recognized quality management systems, including ISO 15189 for medical laboratories and ISO/IEC 17025 for general testing and calibration laboratories. Specimen handling, pre-analytical controls, assay validation parameters (accuracy, precision, limit of detection, reportable range), and post-analytical interpretation all follow documented standard operating procedures aligned with applicable national and international standards.

Testing Samples

Formalin-Fixed Paraffin-Embedded (FFPE) Tissue

FFPE tumor tissue is the most widely used specimen for immunohistochemical and sequencing-based biomarker assessment. Archived diagnostic blocks or core needle biopsies are acceptable provided that fixation is standardized (typically 6–72 hours in 10% neutral buffered formalin) and tissue morphology is preserved. Tumor cellularity, necrosis, and fixation quality are evaluated during pathologist review before testing, and macrodissection may be performed to enrich tumor content above validated thresholds.

Fresh or Frozen Tumor Tissue

Fresh or flash-frozen tissue is preferred for certain high-complexity applications, including RNA-based immune gene signature profiling and single-cell immune phenotyping, where formalin-induced fragmentation can compromise analytical performance. Samples should be transported under validated cold-chain conditions and processed within stability windows established during method validation.

Peripheral Blood

Whole blood, plasma, and serum specimens support liquid biopsy and immunomonitoring workflows. Plasma is used for circulating tumor DNA (ctDNA) extraction and molecular residual disease (MRD) assessment; whole blood is used for lymphocyte subset analysis by flow cytometry and germline controls; and serum is used for cytokine panels, autoantibody screening, and general inflammatory markers associated with irAE surveillance.

Bone Marrow and Other Body Fluids

Bone marrow aspirates, cerebrospinal fluid, pleural effusions, and ascites may be tested for hematologic malignancies, central nervous system involvement, or when tissue biopsy is contraindicated. Cell blocks prepared from effusions can undergo immunohistochemistry, while cell-free DNA from body fluids can be analyzed by targeted sequencing when validated for that specimen type.

Testing Items

Immunotherapy-related testing panels are structured around predictive, pharmacodynamic, and safety-related biomarkers:

• PD-L1 expression: Membranous staining scored as Tumor Proportion Score (TPS) or Combined Positive Score (CPS) to guide checkpoint inhibitor eligibility across multiple tumor types.
• Tumor mutational burden (TMB): Quantification of somatic mutations per megabase of coding sequence, reported as mut/Mb, to identify patients with high mutation loads who may respond to checkpoint blockade.
• Microsatellite instability (MSI) and mismatch repair (MMR) status: MSI-H or dMMR status is a pan-tumor predictive marker for immune checkpoint inhibitor responsiveness.
• Tumor-infiltrating lymphocytes (TILs): Histopathologic and immunophenotypic quantification of intratumoral and stromal lymphocytes as an indicator of pre-existing antitumor immunity.
• Immune gene expression signatures: Interferon-gamma signaling and T-cell-inflamed gene signatures measured by multiplex gene expression assays.
• Circulating tumor DNA and molecular residual disease: Variant tracking in plasma for treatment response assessment, progression detection, and post-surgical monitoring.
• Driver and resistance alterations: Targeted genotyping for EGFR, ALK, KRAS, BRAF, STK11, KEAP1, and other genes that influence immunotherapy benefit-risk decisions.
• HLA genotyping: Human leukocyte antigen typing to support neoantigen prediction and adoptive cell therapy development.
• T-cell receptor (TCR) repertoire diversity: Clonality assessment to characterize T-cell expansion and immune reconstitution during therapy.
• Lymphocyte subsets and cellular immunity: Flow cytometric enumeration of CD3+, CD4+, CD8+ T cells, B cells, NK cells, and regulatory T cells.
• Cytokine and soluble immune markers: Quantification of IFN-gamma, IL-2, IL-6, IL-10, TNF-alpha, and related mediators for immune activation profiling.
• Autoantibodies and irAE-related markers: Organ-specific autoantibody panels, complement components, immunoglobulins, and inflammatory markers supporting immune toxicity surveillance.

Testing Methods

Immunohistochemistry (IHC)

IHC using validated PD-L1 clones and standardized detection systems is the reference method for PD-L1 scoring. Stained slides are interpreted by qualified pathologists using tumor-type-specific scoring algorithms (TPS, CPS, immune cell score), with established cut-offs. MMR protein testing for MLH1, MSH2, MSH6, and PMS2 is likewise performed by IHC as a functional surrogate for MSI status.

Next-Generation Sequencing (NGS)

Targeted panel sequencing, whole-exome sequencing, and comprehensive genomic profiling are used for TMB calculation, MSI inference from sequencing data, driver mutation detection, and ctDNA analysis. Bioinformatics pipelines incorporate validated mutation callers, germline filtering, and TMB normalization against reference standards. Sequencing workflows follow internal quality metrics for depth, uniformity, on-target rate, and variant-level limit of detection.

Polymerase Chain Reaction (PCR)

Multiplex fluorescent PCR followed by capillary electrophoresis remains a validated method for MSI determination using a standard panel of mononucleotide and dinucleotide markers. Real-time quantitative PCR supports gene expression assays and MRD tracking of known tumor variants, with allele-specific assays achieving high analytical sensitivity.

Flow Cytometry

Multi-color flow cytometry enables quantitative immunophenotyping of peripheral blood mononuclear cells, lymphocyte subset enumeration, activation marker analysis, and functional assays such as intracellular cytokine staining. Instrument setup, compensation, and gating strategies are controlled through daily quality assurance and standardized reference controls.

Immunoassays

ELISA and multiplex bead-based immunoassays quantify serum and plasma cytokines, autoantibodies, immunoglobulins, and complement proteins. Calibration curves, internal quality controls, and lot-to-lot bridging ensure traceable and reproducible quantification across analytical runs.

Fluorescence In Situ Hybridization (FISH)

FISH is applied for selected gene amplification or structural alterations relevant to therapeutic stratification when tissue-based confirmation of sequencing findings is required.

Applications

Immunotherapy-related testing supports clinical and translational decision-making across multiple settings:

• Patient selection: Identifying individuals most likely to benefit from checkpoint inhibitors based on PD-L1, TMB, MSI/MMR, and integrated biomarker profiles.
• Treatment response monitoring: Serial ctDNA and immunomonitoring assessments to detect response, pseudoprogression, hyperprogression, or acquired resistance.
• Immune-related toxicity management: Laboratory surveillance of organ function, autoantibodies, and inflammatory markers to support early irAE recognition and grading.
• Adoptive cell therapy support: HLA typing, TCR repertoire analysis, and cellular product characterization for engineered T-cell programs.
• Clinical trial support: Biomarker-enriched enrollment, pharmacodynamic endpoint testing, and exploratory correlative studies conducted under validated laboratory conditions.
• Translational research: Immune microenvironment characterization, neoantigen discovery, and resistance mechanism investigation.

FAQ

What is the difference between MSI testing and MMR testing?

MSI testing detects the phenotypic consequence of mismatch repair deficiency by analyzing microsatellite marker length changes using PCR or NGS, whereas MMR testing evaluates the expression of MLH1, MSH2, MSH6, and PMS6 proteins by immunohistochemistry. The two approaches are highly concordant and are frequently performed as complementary methods.

Can blood samples replace tumor tissue for immunotherapy testing?

Liquid biopsy using plasma ctDNA is a validated alternative for genotyping and TMB estimation when tissue is unavailable or exhausted, but PD-L1 immunohistochemistry requires tissue with preserved morphology. Blood-based immunomonitoring complements, rather than replaces, tissue-based predictive biomarker testing.

How is PD-L1 expression reported?

PD-L1 results are reported using tumor-type-appropriate scoring algorithms. TPS represents the percentage of viable tumor cells with partial or complete membranous staining, while CPS includes tumor cells, lymphocytes, and macrophages within the same field. Reports state the antibody clone, scoring method, and applicable threshold interpretation.

What quality standards apply to immunotherapy-related testing?

Testing should be performed in laboratories accredited to ISO 15189 or ISO/IEC 17025, with documented assay validation, participation in external quality assessment and proficiency testing programs, competency-qualified personnel, and traceable reference materials throughout the analytical workflow.

What is the typical turnaround time?

IHC-based assays such as PD-L1 and MMR are generally completed within a few working days, while comprehensive NGS panels, TMB calculation, and bioinformatics interpretation typically require one to two weeks depending on panel size and specimen quality.

How are immune-related adverse events monitored in the laboratory?

Baseline and on-treatment monitoring includes complete blood counts, liver and renal function panels, thyroid function, cortisol and adrenal markers, glucose, creatine kinase, cytokine levels, and organ-specific autoantibodies, interpreted in the context of the patient's clinical presentation and treatment timeline.