Expression of Programmed Death-Ligand 1 in Cutaneous Squamous Cell Carcinoma Arising in Sun-Exposed and Nonsun-Exposed Skin.

BACKGROUND: A recent clinical trial has shown the efficacy of an anti-programmed death-1 (PD-1) antibody against advanced squamous cell carcinoma (SCC). The expression of PD-ligand 1 (PD-L1) in tumor cells correlates with a favorable response to anti-PD-1 therapy in various malignancies. In recent studies, it has been shown that SCC frequently expresses PD-L1. However, there has been no previous study focusing on the difference in PD-L1 expression between SCC in sun-exposed skin and that in nonsun-exposed areas. AIMS: The purpose of this study was to investigate the relationship between sun-exposure status and PD-L1 expression in patients with SCC.
MATERIALS AND METHODS: We investigated 80 patients with SCC (40 patients with SCC in sun-exposed skin and 40 patients with SCC in nonsun-exposed skin) by immunohistochemical staining for PD-L1. Fisher's exact test was used for statistical analyses of the differences between the two groups.
RESULTS: Patients with SCC in sun-exposed skin showed a significantly higher expression level of PD-L1 in tumor cells than did patients with SCC in nonsun-exposed skin (P = 0.0133).
CONCLUSIONS: We found that the expression level of PD-L1 in patients with SCC in sun-exposed skin was higher than in patients with SCC in nonsun-exposed skin. Practical data are needed for appropriate applications of new therapeutic options for SCC. Copyright:

Introduction

Cutaneous squamous cell carcinoma (SCC) is a relatively common nonmelanoma skin cancer that develops in association with ultraviolet (UV) ray exposure, irradiation, scar tissue after a trauma or burn, Bowen's disease, or a chronic wound such as osteomyelitis or decubitus ulcer.[1] Cases of SCC have recently been increasing due to increase in the frequency of UV ray exposure, ozone depletion, and increase in longevity.[2] Although the mortality rate from SCC is quite low, some patients with SCC have metastatic lesions, and effective chemotherapy with valid evidence has not been established for such patients.[23] Recently, treatment with an anti-programmed death-1 (PD-1) antibody has resulted in a dramatic improvement in the prognosis of several malignancies, including melanoma and lung cancer, and the results of a clinical trial for unresected SCC have recently been reported.[345] Recent studies have suggested that the expression of programmed death-ligand 1 (PD-L1) in tumor cells might correlate with a favorable response to anti-PD-1 therapy.[6] Some studies have shown that SCC often express PD-L1.[78910111213]

Although chronic sun exposure is the primary cause of most cases of SCC, it may also occur as a complication of burn scars where skin is not damaged by UV light exposure.[14] Bowen's disease can also occur on nonsun-exposed skin. This evidence prompted us to investigate whether PD-L1 expression is associated with sun exposure. However, the influence of sun exposure on PD-L1 in SCC has not been discussed in detail. In this study, we evaluated the difference between expression of PD-L1 in SCC over sun-exposed and nonsun-exposed skin.

Materials and Methods

Patients

Eighty patients (40 patients with SCC in sun-exposed skin and 40 patients with SCC in nonsun-exposed skin) who were diagnosed as having SCC and who received surgical treatment in Tottori University were included in this study. This study was carried out in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Tottori University, Faculty of Medicine, Japan (No. 17A076). Informed consent was obtained from each patient enrolled in this study.

Immunohistochemistry

Formalin-fixed and paraffin-embedded 4-μm-thick sections of specimens were used for immunohistochemistry. Immunohistochemical staining for PD-L1 in tumor cells was performed as reported previously using Roche Biomedical Ventana antibodies (PD-L1; SP263).[11] PD-L1 assays were performed according to the manufacturer's specifications using the Ventana Ultraview System (Roche Diagnostics, Basel, Switzerland). The percentage of PD-L1-positive cells was calculated by light microscopy. At first, we randomly chose three areas in each lesion and counted the number of tumor cells in those three areas. Next, we counted the number of tumor cells expressing PD-L1 in those three areas. Finally, we calculated the percentage of tumor cells expressing PD-L1. Necrotic, keratinized, and inflammatory cells were excluded. Membrane staining of tumor cells was defined as positive, and cytoplasmic staining of tumor cells was excluded. Normal lymph nodes were stained with PD-L1 antibodies as a positive control [Figure 1a and b]. PD-L1 was not expressed on normal skin in both sun-exposed and nonsun-exposed areas. We defined a specimen containing at least 1% of tumor cell-expressing PD-L1 as a positive case and a specimen containing <1% of tumor cell-expressing PD-L1 as a negative case, as previously reported.[812]

Figure 1

(a and b) H and E, and Positive control for programmed death ligand 1 staining of normal lymph node with IHC showing positive lymphocytes staining brown (×40); (c and d) tumor cells of squamous cell carcinoma in sun exposed skin (H and E, ×40) and high concentration of PD-L1 positive cells (IHC, ×40); (e and f) tumor cells of squamous cell carcinoma in non-sun exposed skin (H and E, ×100) with less than normal PD-L1 positive cells (IHC, ×100). IHC-Immunohistochemistry

Statistical analysis

Fisher's exact test was used for statistical analysis of differences between the two groups. A P <0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, USA).

Results

Patients' background

Patients' data are presented in Table 1. The face was the most frequent site in the group of SCC in sun-exposed skin and the leg was the most frequent site in the group of SCC in nonsun-exposed skin.

Table 1

Clinical summary of patients enrolled in this study

Characteristic	Patients with SCC (n=80)
	Sun-exposed skin (n=40)	Nonsun-exposed skin (n=40)
Age (year)
Average	84.4	78.4
Median	84	80
Range	65-99	46-97
Sex
Male	30	21
Female	10	19
Primary lesion
Head	5	0
Face	31	0
Neck	4	0
Trunk	0	10
Leg	0	26
Arm	0	4

SCC: Squamous cell carcinoma

Expression of programmed death-ligand 1 in tumor cells

The results of analysis of PD-L1 expression are shown in Table 2. Twenty-seven patients (67.5%) in the group of SCC in sun-exposed skin had 1% or more of tumor cell-expressing PD-L1 and 15 patients (37.5%) in the group of SCC in nonsun-exposed skin had 1% or more of tumor cell-expressing PD-L1 (P = 0.0133) [Figure 1c-f].

Table 2

Results of analysis of programmed death-ligand 1 expression

Location	Total	PD-L1- positive (%)	PD-L1- negative (%)	P
Sun-exposed	40	27 (67.5)	13 (32.5)	0.0133
Nonsun-exposed	40	15 (37.5)	25 (62.5)

PD-L1: Programmed death-ligand 1

Discussion

In reported studies, 20%–70% of the patients with SCC had PD-L1 expression in tumor cells.[78910111213] In the present study, 52.5% of the patients had PD-L1 expression in tumor cells. Although the expression of PD-L1 in sun-exposed skin and that in nonsun-exposed skin were evaluated in some previous studies, no statistically significant difference was found.[1213] However, the number of patients with SCC in nonsun-exposed skin in those studies was relatively small.[1213] In our study, a significant correlation was found between sun exposure and expression of PD-L1 in tumor cells. According to a previous study, SCC has a high mutation burden because of the damage caused by UV light.[15] In patients with Merkel cell carcinoma, another type of nonmelanoma skin cancer, Wong et al. reported that Merkel cell polyoma virus-negative Merkel cell carcinoma was associated with UV-induced DNA damage and that UV-induced DNA damage caused a high mutation burden and high expression level of PD-L1 in tumor cells.[16] Therefore, in patients with SCC, the reason for the expression level of PD-L1 in sun-exposed skin being higher than that in nonsun-exposed skin may be the mutation burden due to sun exposure. SCC has various precursor lesions (Bowen's disease, actinic keratosis, scar tissue, and chronic wound). The precursor lesion may affect the expression of PD-L1, and further research is needed.

A recent clinical trial has shown that cemiplimab, a new agent of an anti-PD-1 antibody, induced a response in about half of the patients with advanced SCC.[3] In that trial, although the expression of PD-L1 in tumor cells was not evaluated, it may be possible to use expression of PD-L1 for prediction of the response to treatment. Another study showed that expression of PD-L1 in other tumors, such as malignant melanoma, correlated with the efficacy of anti-PD-L1 treatment.[17] Thus, differential expression of PD-L1 between sun-exposed SCC and nonsun-exposed SCC would impact on immune responses during anti-PD-L1 treatment, potentially resulting in the differential therapeutic responses to PD-L1 blockade. In the present study, we showed that the expression level of PD-L1 in patients with SCC in sun-exposed skin is significantly higher than that in patients with SCC in nonsun-exposed skin. Practical data are needed for appropriate applications of new therapeutic options for SCC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.