PD-L1 Expression (CTCs or Serum)

PD-L1 Expression Overview

Programmed death-ligand 1 (PD-L1), also known as CD274 or B7-H1, is a transmembrane protein that plays a crucial role in immune evasion by cancer cells. Its interaction with programmed death-1 (PD-1) on T cells inhibits T cell activity, allowing tumors to escape immune surveillance. In the context of cancer immunotherapy, particularly with immune checkpoint inhibitors targeting the PD-1/PD-L1 pathway, assessing PD-L1 expression has become a vital biomarker.

Traditionally, PD-L1 expression is evaluated on tumor tissue biopsies using immunohistochemistry (IHC). However, the invasive nature of biopsies, tumor heterogeneity, and the dynamic nature of PD-L1 expression have led to growing interest in less invasive "liquid biopsy" approaches. These include the assessment of PD-L1 on circulating tumor cells (CTCs) and soluble PD-L1 (sPD-L1) in serum.

PD-1/PD-L1 Pathway Biology

The PD-1/PD-L1 pathway is a critical immune checkpoint that regulates T-cell activation and peripheral tolerance, preventing autoimmune responses. In the tumor microenvironment, this pathway is often hijacked by cancer cells to evade detection and destruction by the immune system.

• PD-L1: Expressed on antigen-presenting cells (APCs) and various tumor cells, as well as some non-malignant cells within the tumor microenvironment. Its expression can be constitutive or induced by inflammatory cytokines like interferon-gamma (IFN-γ).
• PD-1: A receptor primarily found on activated T cells, B cells, and myeloid cells. When PD-1 binds to PD-L1 (or PD-L2), it delivers an inhibitory signal, leading to T-cell anergy, exhaustion, and apoptosis. This effectively "turns off" the immune response against the tumor.

Therapeutic blockade of the PD-1/PD-L1 interaction aims to restore anti-tumor T-cell activity, leading to tumor regression in a subset of patients across various cancer types.

PD-L1 on Circulating Tumor Cells (CTCs)

Circulating tumor cells (CTCs) are cancer cells that have detached from the primary or metastatic tumor and entered the bloodstream. Their detection and characterization, including PD-L1 expression, represent a non-invasive "liquid biopsy" approach.

• Source of Information: CTCs offer a real-time snapshot of the tumor, which can be particularly valuable in cases of inaccessible tumor sites, heterogeneous tumors, or when repeat biopsies are not feasible.
• Dynamic Expression: PD-L1 expression on CTCs can be dynamic, changing during disease progression or in response to treatment. This allows for monitoring of potential changes that might influence treatment decisions.
• Prognostic and Predictive Role: Studies are investigating the utility of PD-L1-positive CTCs as a prognostic marker for disease progression and as a predictive marker for response to anti-PD-1/PD-L1 therapies in various cancers, including lung, breast, and prostate cancer.
• Advantages: Minimally invasive, repeatable sampling, potential to reflect overall tumor heterogeneity better than a single tissue biopsy.
• Challenges: Rarity of CTCs in blood, lack of standardized isolation and detection methods, technical variability in PD-L1 staining and interpretation.

Soluble PD-L1 (sPD-L1) in Serum

Soluble PD-L1 (sPD-L1) refers to the circulating, non-membrane-bound form of the PD-L1 protein found in bodily fluids, such as serum or plasma. It is believed to be shed from tumor cells or immune cells, or to be a product of alternative splicing.

• Mechanism of Action: sPD-L1 can potentially act as a decoy, binding to PD-1 on T cells and thereby preventing the binding of membrane-bound PD-L1. This could theoretically either promote or inhibit immune responses, making its exact biological role complex and debated.
• Prognostic Value: Elevated levels of sPD-L1 have been observed in several cancer types, often correlating with advanced disease stage, higher tumor burden, and poorer prognosis.
• Predictive Biomarker: Research is ongoing to determine if sPD-L1 can predict response to immune checkpoint inhibitors. Some studies suggest that higher baseline sPD-L1 levels might be associated with a worse response to PD-1/PD-L1 blockade, while others show no clear correlation or even a positive association in specific contexts.
• Monitoring Treatment: Changes in sPD-L1 levels during treatment could potentially serve as a dynamic marker for monitoring therapeutic efficacy or disease progression.
• Advantages: Highly non-invasive (simple blood draw), relatively easy to measure using ELISA or other immunoassays.
• Challenges: Lack of standardization in detection assays, varying reported cut-off values, and inconsistent clinical correlations across different cancer types and studies.

Clinical Utility in Cancer Management

The assessment of PD-L1 expression, whether on tissue, CTCs, or in serum, is aimed at guiding treatment decisions for patients receiving immune checkpoint inhibitors.

• Patient Selection: For certain cancers (e.g., non-small cell lung cancer), PD-L1 expression on tumor cells (via IHC) is an approved companion diagnostic to select patients most likely to benefit from anti-PD-1/PD-L1 therapies.
• Overcoming Limitations of Tissue Biopsy:
  • Tumor Heterogeneity: Liquid biopsies (CTCs, sPD-L1) can potentially capture the overall PD-L1 status of the tumor and its metastases more comprehensively than a single tumor biopsy, addressing inter- and intra-tumor heterogeneity.
  • Accessibility: Valuable for patients with inaccessible tumors or when repeated biopsies are not clinically feasible or safe.
  • Dynamic Changes: PD-L1 expression can change over time, under selective pressure from treatment, or during disease progression. Liquid biopsies allow for dynamic monitoring.
• Prognostic Information: Both PD-L1 on CTCs and sPD-L1 have shown potential as prognostic markers, providing insights into disease aggression and patient outcomes.
• Future Role: While tissue PD-L1 IHC remains the standard, liquid biopsy approaches are evolving as complementary tools to refine patient selection, monitor therapy, and predict resistance or recurrence, moving towards truly personalized immunotherapy.

Detection Methods and Challenges

The accurate and standardized detection of PD-L1 on CTCs and sPD-L1 in serum presents several technical and clinical challenges.

• For CTCs:
  • Isolation: Various methods are used, including antibody-based enrichment (e.g., CellSearch system, which captures EpCAM-positive cells), size-based filtration, or density gradient centrifugation. Each method has its own biases and recovery rates.
  • Detection: Immunofluorescence or immunohistochemistry is typically used on isolated CTCs. Staining protocols, antibody clones, and interpretation criteria for "positive" PD-L1 expression on CTCs are not yet standardized, leading to variability across studies.
  • Rarity: The extremely low number of CTCs in peripheral blood makes their isolation and characterization technically demanding.
• For sPD-L1:
  • Immunoassays: Enzyme-linked immunosorbent assays (ELISAs) are the most common method for quantifying sPD-L1 in serum or plasma. However, different commercial kits use different antibodies and standards, leading to inconsistent results and lack of comparability between studies.
  • Pre-analytical Variables: Sample collection, processing, and storage conditions can influence sPD-L1 levels and assay reproducibility.
  • Biological Variation: sPD-L1 levels can be influenced by various factors beyond the tumor, including inflammation and other systemic conditions, making interpretation challenging.

Standardization of pre-analytical, analytical, and post-analytical phases is crucial for the clinical adoption of these liquid biopsy biomarkers.

Future Directions and Considerations

The field of PD-L1 liquid biopsy is rapidly evolving, with several key areas of ongoing research and development:

• Standardization: Efforts to standardize CTC isolation, PD-L1 staining protocols, and sPD-L1 immunoassay kits are paramount to ensure robust and reproducible results across clinical settings.
• Integration with Other Biomarkers: Combining PD-L1 on CTCs or sPD-L1 with other liquid biopsy markers (e.g., circulating tumor DNA (ctDNA) for mutational analysis, tumor mutational burden, or microsatellite instability) may provide a more comprehensive picture of tumor biology and predict treatment response more accurately.
• Clinical Trials: Prospective clinical trials are needed to validate the prognostic and predictive utility of these markers in large, diverse patient cohorts across different cancer types and treatment regimens.
• Understanding Biological Role: Further research is required to fully elucidate the biological significance of sPD-L1 and the mechanisms by which it is shed, as well as the dynamic changes in PD-L1 expression on CTCs.
• Accessibility and Cost: Developing cost-effective and accessible platforms for PD-L1 liquid biopsy will be essential for widespread clinical implementation, especially in resource-limited settings.

As immunotherapy becomes a cornerstone of cancer treatment, liquid biopsy approaches for PD-L1 expression offer a promising avenue to refine patient selection, monitor treatment efficacy, and predict outcomes in a less invasive and more dynamic manner.

Frequently Asked Questions (FAQ)

References

1. Gandara, D.R., et al. (2018). PD-L1 expression in circulating tumor cells (CTCs): A novel biomarker for treatment selection in advanced NSCLC. Clinical Cancer Research, 24(20), 4732-4740.
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3. Salgia, R., et al. (2019). Soluble PD-L1 in cancer: a prognostic and predictive biomarker. Oncotarget, 10(49), 5092-5103.
4. Aggarwal, C., et al. (2020). Clinical implications of PD-L1 expression in circulating tumor cells in NSCLC patients. Journal of Thoracic Oncology, 15(7), 1146-1154.
5. Ilie, M., et al. (2017). Soluble PD-L1 as a new biomarker in cancer: a review. Journal of Thoracic Disease, 9(Suppl 15), S1380-S1385.
6. Reck, M., et al. (2021). PD-L1 testing for immunotherapy in lung cancer: a new consensus on standard operating procedures for the quantification of PD-L1. Annals of Oncology, 32(3), 329-338.