Clinical impact of programmed cell death ligand 1 expression in colorectal cancer

https://doi.org/10.1016/j.ejca.2013.02.015Get rights and content

Abstract

Background

Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed.

Materials and methods

A tissue microarray (n = 1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression.

Results

Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8+ lymphocytes (P = 0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P = 0.0001) and validation (P = 0.03) sets. A similar trend (P = 0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n = 42) were found to be significantly associated (r = 0.33, P = 0.03).

Conclusion

PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.

Introduction

Tumour-infiltrating lymphocytes (TILs) are widely considered to reflect primary host immune response against solid tumours. Recent reports have demonstrated a direct correlation between colorectal cancer (CRC) patient survival and tumour infiltration by cluster of differentiation 8 (CD8) positive T lymphocytes expressing typical activation markers.1, 2 However, the immune system is characterised by the presence of a number of inhibitory mechanisms preventing ‘excessive’ lymphocyte activation.3 In particular, programmed cell death receptor 1 (PD-1; CD279) is typically expressed by activated lymphocytes.4 Its engagement by specific ligands, including PD ligand 1 (PD-L1; B7-H1; CD274) and PD ligand 2 (PD-L2; B7-DC; CD273), induces down-regulation of antigen-stimulated lymphocyte proliferation5, 6 and cytokine production,6, 7 ultimately resulting in lymphocyte ‘exhaustion’ and in the induction of immunological tolerance.6, 8, 9, 10

PD-L1 is constitutively expressed by T and B cells, macrophages and dendritic cells (DC) and is up-regulated upon activation by interferons (IFN).8, 9 PD-L1 is also expressed on additional cell types including endothelial, pancreatic and muscle cells.4 In contrast, PD-L2 expression is much more restricted and typically detectable in activated DC and macrophages.9 Importantly, up-regulation of the expression of PD-1 ligands in malignant cells has been suggested to play a central role in tumour-immune system interaction5, 11 since, by triggering PD-1, cancer cells might shut down specific immune responses. Indeed, the expression of PD ligands on tumour cells was shown to suppress the cytolytic activity of CD8+ T-cells.12, 13

PD-L1 and, to a lesser extent, PD-L2, have been reported to be expressed by tumour cells of different origins, including glioblastoma, ovarian and renal cell carcinomas, squamous cell carcinoma of the head and neck, oesophageal and non-small cell lung cancers.5, 14, 15, 16, 17, 18 A strong correlation between expression of PD ligands on tumour cells and severe prognosis has been observed in oesophageal cancer and in renal cell carcinoma.15, 17 Capitalising on this background, PD-1/PD-L1 blockade by anti PD-1 or anti PD-L1 monoclonal antibodies has been envisaged as an appealing option to activate the host immune system to eradicate tumours. Recently, promising results of phase I clinical trials involving patients bearing a variety of malignancies have been published.19, 20, 21

Expression of PD-L1 in human CRC has not been addressed so far. In this study we used a tissue microarray (TMA)22 including 1420 well documented, clinically annotated CRC specimens23 to investigate the expression of PD-L1 in CRC and its clinical significance.

Section snippets

Tissue microarray construction

The TMA used for this study includes 1420 unselected, non-consecutive, primary, sporadic CRCs treated between 1987 and 1996, and 71 normal mucosa specimens from the Institute of Pathology of the University of Basel (Switzerland), the Institute of Clinical Pathology, Basel (Switzerland) and the Institute of Pathology of the Stadtspital Triemli, Zürich (Switzerland). TMA was constructed with materials collected from the Tissue Biobank of the Institute of Pathology, University Hospital Basel. This

Immunohistochemical detection of PD-L1

Representative stainings of the tissues under investigation, as observed upon incubation with 27A2 mAb specific for PD-L1, are shown in Fig. 1. PD-L1 was detectable in epithelial cells from normal colonic mucosa (Fig. 1A), and, importantly, in cancer cells (Fig. 1C–E).

In 433 MMR-proficient CRC (36%) a strong positivity (Fig. 1D) was observed, whereas in 723 and 41 cases, respectively, PD-L1 expression was low (Fig. 1C), or absent (Fig. 1B). Among the 223 MMR-deficient cases a strong positivity

Discussion

The aim of this study was to analyse the expression of PD-L1 in a large series of CRC samples and to evaluate its clinical relevance. Here we report that untransformed normal epithelial cells of colon mucosa do express PD-L1. More importantly, we have observed that PD-L1 expression is markedly enhanced in tumour cells in over 30% of CRC.

Unexpectedly, strong PD-L1 expression in MMR-proficient CRC was found to be associated with early tumour stage, absence of lymph node metastases, lower tumour

Conflict of interest statement

None declared.

Acknowledgements

Financial support was provided by the Swiss National Fund for scientific research (SNF) Grant No. PP00P3-133699 and 31003A-122235 and by the Italian Association for Cancer Research (AIRC) IG Grant No. 10555.

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    These authors contributed equally to this work.

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