Quantitative spatial profiling of PD-1/PD-L1 interaction in patients with cancer

Immunotherapy (ITx) has emerged in less than a decade as one of the most important treatment modalities for patients with cancer. Immune checkpoint inhibitors, represented by anti-programmed death receptor-1 (PD-1)/programmed death ligand-1 (PD-L1) antibodies, are now widely used as a standard of care across primary organs and histologic subtypes (1-3). Although the prognosis of the overall population with advanced cancer has been improved by anti-PD-1/PD-L1 antibody-based regimens, their tailored prescriptions have not yet been achieved. In other words, ITx still lacks satisfactory biomarkers to predict its efficacy, unlike molecularly targeted agents for which companion biomarkers with high predictive accuracy (i.e., the presence of driver alterations) are available. Although tissue PD-L1 expression assessed by immunohistochemistry is an approved method for selecting patients for anti-PD-1/PD-L1 antibody-based regimens, better outcomes are not necessarily guaranteed by PD-L1 positivity.

PD-L1 expression, calculated as a tumor proportion score (TPS), is a biomarker available for routine use in the selection of a treatment regimen for an individual patient with advanced non-small cell lung cancer (NSCLC) (4). PD-L1 positivity is crucial in selecting anti-PD-1/PD-L1 antibody monotherapy or other regimens in the first-line setting. However, even in patients with high PD-L1 expression, objective responses to anti-PD-1/PD-L1 antibody monotherapy are achieved in up to 40–50% of the population (5). Additionally, TPS has further limited utility as a biomarker for combination regimens with cytotoxic agents or anti-cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibodies used independently of TPS in advanced NSCLC. Such a limitation of the approach based solely on tissue PD-L1 expression would still drive researchers to diligently search for novel biomarkers with a more predictive ability (6).

Gavrielatou et al. investigated the association between “PD-1/PD-L1 colocation score” and the efficacy of ITx in 154 patients with advanced NSCLC (7). Patients with a high PD-1/PD-L1 colocation score in the overall cohort had a significant benefit from ITx compared to those with a low score in an objective response, progression-free survival (PFS), and overall survival (OS). The predictive ability of the PD-1/PD-L1 colocation score was marked in the subgroup analysis stratified by line of treatment in the cohort receiving ITx as a second or subsequent line of treatment (n=85). At the same time, no similar dissociation was observed in the first-line setting (n=69). It was also shown that the colocation score did not correlate with the tissue PD-L1 expression levels. Thus, a key message from the analysis was the potential utility of the PD-1/PD-L1 colocation score as a novel biomarker distinct from conventional PD-L1 expression assessment, particularly for those receiving ITx in the second or subsequent line of treatment.

“PD-1/PD-L1 colocation score” may be an unfamiliar term. However, the basic idea behind it is simple and in line with the mechanism of action of anti-PD-1/PD-L1 antibodies. In short, the PD-1/PD-L1 colocation score reflects the spatial proximity between PD-1 and PD-L1 in formalin-fixed paraffin-embedded tumor samples, which is quantified by the algorithm using multiplexed quantitative immunofluorescence (QIF). The present method assesses receptor/ligand interactions, in contrast to conventional PD-L1 assays focusing solely on the ligand. Thus, it attempts to reflect the interplay between tumor and immune cells within the tumor microenvironment in predicting ITx results. In addition to the report by Gavrielatou et al., the association between PD-1/PD-L1 colocation and ITx outcomes in patients with advanced cancer treated with anti-PD-1 antibodies has been addressed by several previous studies (Table 1).

Table 1. Studies on PD-1/PD-L1 colocation and anti-PD-1 antibody efficacy among patients with cancer.

Authors	Subjects	ITx regimens	Methods	Key results
Gavrielatou et al. [2022] (7)	NSCLC (n=124)	aPD-1/PD-L1 monotherapy, aPD-1/PD-L1 + aCTLA-4, aPD-1/PD-L1 + chemo	Multiplexed QIF	A high PD-1/PD-L1 colocation score was associated with better response, PFS, and OS
Giraldo et al. [2018] (8)	Meckel cell carcinoma (n=16)	aPD-1 monotherapy	Multiplexed QIF	The density of PD-1+ cells adjacent to a PD-L1+ cell was higher in the responder than non-responder
Johnson et al. [2018] (9)	Melanoma (n=166)	aPD-1 monotherapy	Multiplexed QIF	High PD-1/PD-L1 colocation score ± IDO-1/HLA-DR co-expression were associated with better response, PFS, and OS
Sánchez-Magraner et al. [2020] (10)	NSCLC (n=40)	aPD-1 monotherapy	FRET	Patients with the highest 40% of FRET efficiency had a better OS than those with the lowest 60%
Sánchez-Magraner et al. [2020] (10)	Melanoma (n=176)	aPD-1 monotherapy	FRET	Patients with the highest 20% of FRET efficiency had a better OS than those with the lowest 80%

PD-1, programmed death receptor-1; PD-L1, programmed death ligand-1; ITx, immunotherapy; NSCLC, non-small cell lung cancer; aPD-1/PD-L1, anti-programmed death receptor-1/programmed death ligand-1 antibody; aCTLA-4, anti-cytotoxic T-lymphocyte-associated protein-4 antibody; QIF, quantitative immunofluorescence; PFS, progression-free survival; OS, overall survival; aPD-1, anti-programmed death receptor-1 antibody; IDO-1, indoleamine 2;3-dioxygenase-1; HLA-DR, human leukocyte antigen-D related; FRET, fluorescence resonance energy transfer.

Giraldo et al. showed that the density of PD-1⁺ cells adjacent to a PD-L1⁺ cell was higher in responder than non-responder in a cohort of Meckel cell carcinoma (n=16) (8). Johnson et al. showed that a high PD-1/PD-L1 colocation score was associated with better response, PFS, and OS on ITx in the larger melanoma cohort (n=166) using a similar approach using multiplexed QIF (9). As another approach more focused on PD-1/PD-L1 functional interaction over their simple spatial proximity, Sánchez-Magraner et al. applied the fluorescence resonance energy transfer (FRET) method in quantifying PD-1/PD-L1 colocation (10). There, higher FRET efficiency was associated with better OS in the cohort of NSCLC (n=40) and melanoma (n=176). Gavrielatou et al. added data with a specific value. It provided additional supporting data on this issue by examining the largest samples from patients with NSCLC under diverse clinical scenarios.

Despite accumulating positive signals on the quantitative spatial profiling of PD-1/PD-L1 interaction, several points need to be solved before clinical application as a predictive biomarker for ITx. First, it is not yet clear in which clinical setting (e.g., ITx regimen, treatment regimen) the novel parameter can be used to guide clinical decisions. Gavrielatou et al. found that the PD-1/PD-L1 colocation score may be most useful in the second or later-line setting. On the other hand, the score failed to stratify survival for those who received ITx in the first-line setting. On this point, we must be careful of the cohort characteristics in that one-third of patients received ITx as a combination regimen with cytotoxic agents (n=33) or other (n=18), as shown in the Tab. S1. Today, most patients with advanced NSCLC receive ITx primarily in the first-line setting. In contrast, the same populations received ITx as an anti-PD-1/PD-L1 monotherapy in the second or later-line setting. Combination regimens would be more likely used in the first-line setting or in patients with low or negative PD-L1 expression under such circumstances. In other words, the difference in the predictive ability of the colocation score may also be due to differences in the ITx regimens and not just the treatment lines.

Second, the available data have not yet demonstrated the superiority of the colocation score as a predictive biomarker for ITx over conventional PD-L1 expression levels. Although conventional PD-L1 assessment failed to stratify ITx outcomes in their cohort, it would still be considered a practical biomarker, at least for anti-PD-1/PD-L1 monotherapy in advanced NSCLC. Head-to-head comparisons are necessary to establish the colocation score as a new standard for this population. As an alternative, the combination of the conventional PD-L1-based approach and the quantitative spatial profiling of PD-1/PD-L1 interaction may be worth exploring. Future studies may also need to investigate this novel parameter as a biomarker for combination regimens that are standard options for broader populations, regardless of PD-L1 expression, to address unmet clinical needs. A standardized protocol for the quantitative spatial profiling of PD-1/PD-L1 interaction would also be needed. Further validation by a unified approach in the larger clinical scenario-based cohorts and optimization of the cutoff value for the PD-1/PD-L1 colocation score are still warranted based on the results obtained in small cohorts using different assays (multiplexed QIF, FRET, etc.). Future research should consider these points in light of the updated standard of care, not only in NSCLC but also in other cancers for which ITx regimens are available.

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