Programmed death ligand‐1 expression in gastrointestinal cancer: Clinical significance and future challenges

Abstract Cancer immunotherapy has caused a paradigm shift from conventional therapies that directly target cancer cells to innovative therapies that utilize the host immune system. In particular, programmed cell death‐1 (PD‐1)/programmed death ligand‐1 (PD‐L1) inhibitors have achieved an impressive breakthrough and been approved for clinical use in several types of cancer including gastrointestinal (GI) cancer. To identify and develop predictive biomarkers for PD‐1 inhibitors is of great concern in clinical practice. Although PD‐L1 expression is considered a logical biomarker as PD‐L1 is a substantial target of the immune checkpoint inhibitors, its clinical significance in GI cancer remains unclear. In this review, we summarize the current evidence for PD‐L1 expression as a prognostic and predictive biomarker for PD‐1/PD‐L1 inhibitors in GI cancer from recent publications, and emerging evidence from recent key clinical trials on the efficacy of PD‐1/PD‐L1 inhibitors. Challenging clinical issues for PD‐L1 assessment are then discussed from the viewpoint of the methodology for PD‐L1 evaluation including the differences in PD‐L1 detection assays and evaluation criteria for PD‐L1 positivity. Moreover, we highlight the biological features of PD‐L1 expression in terms of tumor spatial and temporal heterogeneity, which suggests important implications for biomarker analysis. Finally, we describe future perspectives using liquid biopsy for better assessment of PD‐L1 status. This new information should improve our understanding of the clinical significance of PD‐L1 in GI cancer, leading to optimal patient selection and treatment strategy for the clinical use of PD‐1/PD‐L1 inhibitors in patients with GI cancer.

axis has demonstrated favorable antitumor effects and achieved an impressive breakthrough in cancer immune therapy for several types of cancer including melanoma, non-small cell lung cancer (NSCLC), and gastrointestinal (GI) cancer. [5][6][7][8] To identify and develop predictive markers for ICIs is of great concern in clinical practice. To date, several predictive markers for PD-1/PD-L1 inhibitors, such as tumor mutation burden (TMB) and mismatch repair deficiency (dMMR)/microsatellite instability (MSI), have been reported. [9][10][11] Above all, PD-L1 expression is considered a logical biomarker because PD-L1 is a substantial target of the ICIs. An initial phase I study on the use of nivolumab, one of the PD-1 inhibitors, supported a potential role for assessment of PD-L1 expression on tumor cells in patients with several types of solid tumor including melanoma, NSCLC, renal cell carcinoma, and colorectal cancer (CRC). 12 However, in contrast to the clinical use of PD-L1 assessment in patients with melanoma and NSCLC, [13][14][15] the clinical significance of PD-L1 expression in GI cancer remains unclear from contradictory outcomes in multiple studies on the correlation between PD-L1 expression and the ICI response or prognosis.
In this review, we focus on PD-L1 expression in GI cancer and summarize its clinical significance as a prognostic biomarker and as a predictive biomarker for PD-1 inhibitors. In addition, we discuss challenging issues for PD-L1 assessment from the viewpoint of methodology for PD-L1 evaluation, and biological features of PD-L1 expression that display spatial and temporal heterogeneity, with future perspectives using liquid biopsy for better assessment of PD-L1 status. This new information should improve our understanding of the clinical significance of PD-L1 in GI cancer, leading to optimal patient selection and treatment strategy for the clinical use of PD-1/ PD-L1 inhibitors in patients with GI cancer.

| Prognostic significance of PD-L1 expression in GI cancer
Multiple studies on the relationship between PD-L1 expression and patient survival in GI cancer have been reported. Recent studies involving large numbers of patients are summarized in Table 1. [16][17][18][19][20][21][22][23][24][25][26][27][28][29] Although the differences in patient background and assessment methods of PD-L1 expression were major limitations, the prognostic significance of PD-L1 expression in GI cancer was highly heterogeneous in each study. Most of the studies demonstrated that PD-L1 positivity is a poor prognostic biomarker as PD-L1 contributes to immune evasion. However, other recent studies did not confirm this; indeed, several studies indicated that PD-L1 positivity is a better prognostic factor. The latter studies discussed that PD-L1 expression does not necessarily represent an immunosuppressive state in the tumor microenvironment, but rather acts as a surrogate marker of immune activation because PD-L1 is upregulated by some inflammatory cytokines such as interferon-γ, which is secreted from activated immune effector cells. 30 In fact, high PD-L1 expression was associated with a high density of tumor-infiltrating lymphocytes (TILs), which is regarded as the preferred state of the host immune response. 24 In addition, some specific situations such as Epstein-Barr virus infection and dMMR in GI cancer are also reported to be associated with high PD-L1 status. 31,32 Although these molecular subtypes and genetic profiles themselves have been reported to be promising predictive biomarkers for PD-1 inhibitors, 33,34 prognostic prediction for such cases by assessment of PD-L1 alone apparently remains difficult given the contradictory results from several studies. [35][36][37] Taken together, the prognostic significance of PD-L1 expression in GI cancer is still unknown. It may be challenging to consider PD-L1 expression as the result of preferable host immune response or as a predisposition to cause immune evasion. Therefore, comprehensive analysis with other immune markers and immune profiles will be required to better assess the role of PD-L1 expression as a prognostic factor.

| Predictive role of PD-L1 expression for PD-1 inhibitors in GI cancer
To date, PD-1/PD-L1 inhibitors, such as nivolumab and pembrolizumab, for clinical use in GI cancer have been approved based on the results of several important clinical trials. The recent key trials on the efficacy of the PD-1/PD-L1 inhibitors in GI cancer are summarized in Table 2. [38][39][40][41][42][43][44][45][46][47][48][49] Here, we introduce the current clinical evidence and the significance of PD-L1 expression as a predictive biomarker for the ICIs in each clinical trial. Several clinical trials on the efficacy of combination therapy of ICIs and PD-L1 inhibitors are ongoing and are not discussed in detail here.

| Gastric and GEJ cancer
For gastric cancer (GC) and gastroesophageal junction (GEJ) cancer, the initial phase Ib trial KEYNOTE-012 showed 22% ORR in patient with PD-L1 + advanced GC and triggered the initiation of further trials. 43 In this trial, there was no association between the response to pembrolizumab and higher PD-L1 expression on tumor cells, while a weak association between high PD-L1 + mononuclear TA B L E 1 Recent studies on the relationship between PD-L1 expression and patient survival in GI cancer

| Colorectal cancer (dMMR/MSI-H colorectal cancer)
Although ICIs had been expected to be less effective for CRC,  We discuss the clinical issues concerning PD-L1 evaluation in terms of their technical and biological aspects.

| PD-L1 IHC detecting assay
Various monoclonal primary antibodies against PD-L1 with laboratory-developed or standardized assays have been used in experimental studies and clinical trials (  Although some reports directly compared stainability between the two assays using the same tissue samples of NSCLC, 55 few studies made such a comparison using the same samples of GI cancer. Further comparative studies will be required for accurate diagnosis of PD-L1 expression.  (Table 3). Although it is not established which of these is better for the assessment of PD-L1 expression, several studies have indicated the usefulness of CPS. 56 We recently demonstrated the utility of CPS as a prognostic biomarker in GC. 26 Interestingly, Herbst et al reported that the PD-1/PD-L1 pathway was inhibited, especially when PD-L1 was expressed by tumor-infiltrating immune cells. 57 From these considerations and the results of recent clinical trials, CPS may become the standard method for PD-L1 evaluation as a prognostic and predictive biomarker.

| Assessment methods for PD-L1 expression
Elucidation of the optimal cut-off value is another concern. A meta-analysis in solid tumors demonstrated a positive dose-response relationship between PD-L1 positivity (with cut-off value of 1, 5, and 10) and survival benefit. 58 As with NSCLC, in which TPS with cut-off value of 50% is adopted for clinical use of PD-1 inhibitors, future studies will determine optimal organ-specific cut-off values of PD-L1 expression in GI cancer.

| Tumor heterogeneity of PD-L1 expression
Tumor heterogeneity is one of the most crucial hallmarks of cancer. 59 As a result of this heterogeneity, the bulk tumor may attain a diversity of distinct molecular features with differential levels of sensitivity to treatment. 60 Moreover, tumor heterogeneity often poses substantial issues in biomarker analysis, and thus a better understanding of this issue should have important implications for clinical practice.  the heterogeneity, the optimal number of biopsies and the need for biopsies for metastatic lesions are pivotal issues to be considered.

| Temporal heterogeneity of PD-L1 expression
Temporal heterogeneity is described as the dynamic variation in the genetic diversity during the course of disease. In addition to genetic and phenotypic alterations in the process of tumor progression, the effect of cancer therapies on PD-L1 expression should be considered (Figure 1). Several reports have demonstrated that some cytotoxic agents including fluorouracil, paclitaxel, and radiation therapy can upregulate PD-L1 expression via cell signaling pathways in GI cancer. [67][68][69] Notably, Yang et al reported that GC patients with a preferable response to chemotherapy displayed PD-L1 downregulation and showed better RFS, whereas pretreatment PD-L1 status was not associated with survival. 70 Ogura et al also demonstrated equivalent results in patients with rectal cancer who received CRT. 71 These impressive results suggest the importance of temporal assessment of PD-L1 expression during the course of treatment.
Nevertheless, previous clinical trials on the efficacy of PD-1/PD-L1 inhibitors for patients who received prior therapies used pretreatment samples for PD-L1 assessment. Therefore, a re-biopsy strategy should be established to account for the current PD-L1 status after treatment, leading to a better appreciation of the true significance of PD-L1 expression as a biomarker.

| FUTURE PER S PEC TIVE S
To overcome the clinical issues described above for the assessment of PD-L1 expression, several strategies have been considered, including the development of companion diagnostics, as well as multiple and repeated biopsies from both primary and metastatic tumors. However, this may not be feasible in clinical practice, as tissue biopsies are limited to very few sampling points and accessible metastatic sites. Given this situation, liquid biopsy (LB) is a promising option to solve these problems because LB is less invasive, more systemic, and can be obtained at multiple time points during the treatment course ( Figure 1). 72 LB includes a variety of analytes such as circulating tumor DNA, cell-free RNA, and circulating tumor cells (CTCs). Among them, PD-L1 expression on CTCs can be a potential predictive biomarker for PD-1 inhibitors. In fact, several studies have demonstrated that PD-L1 status in CTCs correlated with PD-L1 status in tumor tissue and helps to predict the therapeutic effect of PD-1 inhibitors in NSCLC and melanoma. 73,74 For GI cancer, Yue et al reported that the abundance of PD-L1 high CTCs was a predictive biomarker of PD-1/PD-L1 inhibitors and the dynamic changes of PD-L1 high CTCs correlated with disease outcomes. 75 In addition, assessment of systemic PD-L1 status using other analytes as LB may be applicable. Chen et al reported the utility of exosomal PD-L1 on extracellular vesicules as a predictive biomarker for PD-1 inhibitors in melanoma. 76 Moreover, soluble PD-L1 is also reportedly an available analyte for survival analysis in GI cancer. [77][78][79] Interestingly, a functional analysis described by Takeuch et al demonstrated glycosylation of soluble PD-L1 plays an important role in its binding to PD-1 receptor. 80 Such functional analysis may be help for biomarker analysis of PD-L1 using LB.
Although LB has some limitations such as low detection rate and unestablished standard protocols for clinical use, 81 further studies will reveal clinical utilities of LB in assessment of PD-L1 expression in GI cancer.

| CON CLUS IONS
We reviewed the current evidence for PD-L1 expression as a prognostic and predictive biomarker for PD-1/PD-L1 inhibitors. Although PD-L1 is a promising biomarker, some associated clinical issues remain to be addressed. Accurate assessment of PD-L1 expression will reveal its true clinical significance and lead to the establishment of more effective strategies for the clinical use of ICIs.

D I SCLOS U R E
Conflict of Interest: The authors declare no conflict of interest.
Funding: This work was supported in part by the following grant and foundation: Japan Society for the Promotion of Science, Grantin-Aid for Scientific Research; Grant no. 20K16308.