Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy

Abstract

With recent approvals for multiple therapeutic antibodies that block cytotoxic T lymphocyte associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) in melanoma, non-small-cell lung cancer and kidney cancer, and additional immune checkpoints being targeted clinically, many questions still remain regarding the optimal use of drugs that block these checkpoint pathways. Defining biomarkers that predict therapeutic effects and adverse events is a crucial mandate, highlighted by recent approvals for two PDL1 diagnostic tests. Here, we discuss biomarkers for anti-PD1 therapy based on immunological, genetic and virological criteria. The unique biology of the CTLA4 immune checkpoint, compared with PD1, requires a different approach to biomarker development. Mechanism-based insights from such studies may guide the design of synergistic treatment combinations based on immune checkpoint blockade.

Figure 1. Opportunities for biomarker development based on mechanistic nodes in immune checkpoint pathways

The nature of recognized tumour antigens, immune cell migration into tumours and the expression of immune checkpoint receptors and ligands all provide opportunities for biomarker development. CD8⁺ T effector (T_eff) cells are thought to be the major type of immune cell affected by the programmed cell death protein 1 (PD1) immunosuppressive checkpoint pathway. In contrast, cytotoxic T lymphocyte associated antigen 4 (CTLA4) predominantly regulates the activity of CD4⁺ T cells, both effector and regulatory (T_reg) subtypes. Priming of T cells requires their recognition of processed tumour antigens presented by antigen presenting cells (APCs) such as monocytes and dendritic cells, through a unique T cell receptor (TCR) that binds to a major histocompatibility complex (MHC) molecule and tumour-derived peptide antigens. Such antigens may be generated from mutant or non-mutated tumour-associated proteins. Priming of T cells generally occurs in lymphoid tissue and CD4⁺ T cells provide help for CD8⁺ T cell priming in the form of cytokines. Both CD4⁺ and CD8⁺ T cells are then activated through co-stimulatory pathways such as CD28 B7-1 (also known as CD80) and CD28 B7-2 (also known as CD86), causing them to proliferate, secrete inflammatory cytokines, acquire cytolytic properties and migrate to sites of antigen display, that is, tumour deposits. CTLA4 has a major role in regulating the amplitude of CD4⁺ T cell priming and CD4⁺ T cell help for CD8⁺ T cell priming in lymphoid tissue. Within hours to days, activated T cells also begin to express the co-inhibitory receptor PD1. CD4⁺ T helper 1 (T_H1) cells and CD8⁺ T cells in the tumour microenvironment (TME) produce interferon-γ (IFNγ), which, on the one hand, activates tumour killing by macrophages and antigen display by tumour cells, but, on the other hand, induces PDL1 expression by these same macrophages and tumour cells. Tumour-specific PD1⁺ CD8⁺ T cells encountering PDL1⁺ cells within the TME (tumour or stromal cells) will be functionally disabled. Additionally, CTLA4 expressed by T_reg cells in the TME enhances their ability to suppress CD8⁺ T cell-dependent cytokine production and direct tumour cell killing. Drugs blocking the immune checkpoints CTLA4, PD1 and PDL1 interrupt these immunosuppressive interactions and restore the ability of T cells to eliminate antigen-expressing cancer cells.

Figure 2. Mechanisms for intratumoural PDL1 expression

Constitutive broad (innate) expression of membranous programmed cell death 1 ligand 1 (PDL1) by tumour cells is thought to be driven by dysregulated signalling pathways such as PI3K AKT, or chromosomal alterations and amplifications such as are found in Hodgkin lymphoma. In contrast, adaptive focal expression of PDL1 by tumour cells and macrophages occurs at the interface of tumour cell nests with immune infiltrates secreting pro-inflammatory factors such as interferon-γ (IFNγ; the ‘immune front’). The ligation of PDL1 with programmed cell death protein 1 (PD1) molecules will down-modulate T cell function, essentially creating a negative feedback loop that dampens antitumour immunity. The innate and adaptive mechanisms for PDL1 induction are not mutually exclusive: constitutive oncogene-driven PDL1 expression may be further upregulated by inflammatory cytokines. In boxed insets, tumour cells are shown as blue, macrophages are purple and T cells are orange; black outlining of cells indicates PDL1 protein expression, such as would be demonstrated with immunohistochemistry.

Figure 3. PDL1 expression patterns in different types of cancer

In all panels, brown staining indicates programmed cell death 1 ligand 1 (PDL1) protein expression, detected with immunohistochemistry. Upper left panel: a melanoma specimen shows broad PDL1 expression on all malignant cells, independent of an immune infiltrate. This pattern, seen in approximately 1% of melanocytic lesions, suggests oncogene-driven constitutive PDL1 expression. Upper right panel: a squamous cell carcinoma of the head and neck (SCCHN) shows broad areas of low-level PDL1 expression on tumour cells (blue star), with heightened expression at the boundary of tumour nests (red star) with immune infiltrates (black stars); these features suggest a combination of oncogene-driven PDL1 expression and adaptive immune resistance. Lower left panel: a breast carcinoma shows PDL1 expression on malignant cells (blue star) confined to areas immediately adjacent to immune cells (T cells and associated macrophages, black star), many of which also express PDL1. This pattern is seen in approximately 20% of breast cancers. Lower right panel: a gastric carcinoma shows PDL1 expression on infiltrating immune cells (black stars) immediately adjacent to tumour cells, but not on the surface of tumour cells themselves (blue stars). A similar pattern of PDL1 expression has also been described in colorectal cancers. Distinct patterns of PDL1 expression may be observed not only in different tumour types, but also in individual cases within the same tumour type. Scale bar, approximately 50 μm.

Figure 4. Multifactorial biomarkers of clinical response to PD1 pathway blockade

Tumour mutational load, the intensity of intratumoural CD8⁺ T cell infiltrates and tumour programmed cell death ligand 1 (PDL1) expression have each been proposed as distinct biomarkers of response to anti-programmed cell death protein 1 (PD1) PDL1 therapies. However, these factors are functionally interrelated and are often found coordinately in individual tumour specimens. Multifactorial biomarker panels incorporating these and other variables may provide stronger predictive value than individual markers for therapeutic outcomes.