PD-L1, FGFR1, PIK3CA, PTEN, and p16 expression in pulmonary emphysema and chronic obstructive pulmonary disease with resected lung squamous cell carcinoma

Background Emphysema and chronic obstructive pulmonary disease (COPD) are well known independent risk factors for lung cancer. However, the developmental mechanisms between emphysema/COPD and lung cancer remain unknown. The purpose of this study was to evaluate PD-L1, FGFR1, PIK3CA, PTEN, and p16 expression in squamous cell carcinoma (SCC) associated with emphysema/COPD. Methods A total of 59 patients with squamous cell lung carcinoma (SCC) resected between 2008 and 2012 were retrospectively reviewed. Emphysema was assessed according to the Goddard score. Total severity was divided into none-mild (0–7), moderate (8–15), and severe (≥ 16). Local severity around the existing tumor was divided into no emphysema (0) and presence of emphysema (1–4). COPD severity was based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria. PD-L1, FGFR1, PIK3CA, PTEN, and p16 expression were evaluated by immunohistochemistry (IHC). Expression level was classified as tumor cells (TC) 3 (≥ 50%), TC2 (5–49%), TC1 (1–4%), or TC0 (< 1%), and as tumor-infiltrating immune cells (IC) 3 (≥ 50%), IC2 (5–49%), IC1 (1–4%), or IC0 (< 1%) for PD-L1. Expression level was compared between none-mild/moderate-severe total emphysema, no/presence of local emphysema, no COPD/COPD, and GOLD 1/GOLD 2, 3. Results PD-L1 expression was significantly correlated with severity of emphysema in TC0, 1, 2 vs. TC3 (P = 0.012). PD-L1 was significantly higher inversely in none-mild emphysema compared to moderate-severe (95% CI, 0.061–5.852, P = 0.045). There were no other significant associations between PD-L1, FGFR1, PIK3CA, PTEN, and p16 expression and total/local severity of emphysema or presence of COPD/GOLD stage. Conclusions PD-L1 expression in SCC was correlated with severity of emphysema in TC0, 1, 2 vs. TC3 and more frequent in none-mild emphysema than moderate-severe emphysema.


Background
Lung cancer is the primary cause of cancer-related death worldwide. It is well known that smoking is a major risk factor for lung cancer [1]. Smoking causes emphysema and chronic obstructive pulmonary disease (COPD) [2,3]. Several studies have found that emphysema and COPD are independent risk factors for lung cancer [4][5][6], especially squamous cell lung carcinoma (SCC) [7,8].
The genetic causes of emphysema and COPD are largely unknown [18,19]. Furthermore, the developmental pathway between COPD and non-small cell lung cancer (NSCLC) remains elusive [20][21][22]. The purpose of this study was to evaluate PD-L1, FGFR1, PIK3CA, PTEN, and p16 expression in SCC associated with emphysema and COPD.

Ethical considerations
This was a retrospective study approved by the institutional review board (Date of approval: Dec 27, 2012, approval number: 2693) of Tokyo Women's Medical University Hospital. Written informed consent was obtained from all patients before tumors were resected and additional consent was waived.

Patients population and samples
This study included 59 patients who underwent surgery for SCC between February 2008 and December 2012 at Tokyo Women's Medical University, Yachiyo Medical Center. All patients had computerized tomography (CT) and respiratory function tests to assess the suspected malignancy and determine eligibility for surgical intervention prior to surgery. Resected tumors were immediately fixed with 10% formalin, stained with hematoxylin and eosin (HE) and prepared for immunohistochemistry (IHC) staining.

Data analysis
Each expression of IHC was compared between none-mild (0-7) and mild-severe (8-24) emphysema groups of total Goddard scoring, no-emphysema (0) and presence of emphysema (1-4) tumor area groups, non-COPD and COPD groups, and GOLD 1 and GOLD 2, 3 groups. Data analysis was carried out using Statistical Analysis System (SAS institute, Cary, NC) and Graph Pad PRISM (Graph-Pad Software, La Jolla, CA). P values < 0.05 were regarded as statistically significant. The t-test was used to compare between each expression and Goddard score. Chi-squared test was used to compare between each expression and severity of emphysema, presence of COPD/Gold staging. Groups were compared using 95% confidence intervals (CI).

Baseline characteristics and molecular classification by staining
A total of 59 patients with surgically resected SCC were eligible for this study. Patient baseline characteristics included sex, median age, smoking history, histology, pathological stage, total severity of emphysema, local severity of emphysema, presence of COPD, and GOLD stage (Table 1). There were no significant differences between total emphysema status, local emphysema status, COPD status and GOLD stage based on sex, smoking history and stage ( Table 2). Classifications of TC0-3, IC0-3 for PD-L1 ( Fig. 1) and of TC0-3 for FGFR1, PIK3CA, PTEN, and p16 expression were recorded (Table 3).
Comparison between each classification of expression and severity of emphysema IHC expression of each molecular antibody was compared to total/local severity of emphysema (Table 4). PD-L1 expression was significantly correlated with total severity of emphysema in TC0, 1, 2 vs. TC3 (P = 0.012). However, there was no other significant association between classification of expression and total/local severity of emphysema. Since there was significant association in total severity of emphysema, the results between TC0, 1, 2 (PD-L1 0-49) and TC3 (PD-L1 50-100) for total severity of emphysema were compared. There was significant difference between groups (95% CI: 0.061-5.852, P = 0.045) (Fig. 2).

Comparison between each classification of expression and presence of COPD/ GOLD stage
The results of each classification of expression and presence of COPD/Gold stage was compared (Table 4). There was no significant association between classification of expression and presence of COPD/GOLD stage.

Discussion
Few reports have investigated the associations between TC, PD-L1, emphysema, mutational analysis, and COPD in NSCLC [28,29]. One study on lung adenocarcinoma found that patients with emphysema had more PD-L1 positive cells than patients without COPD chronic obstructive pulmonary disease, GOLD Global Initiative for Chronic Obstructive Lung Disease emphysema [28]. Another study on NSCLC showed that PIK3CA expression was significantly associated with COPD [29].
In this study, PD-L1 expression was significantly correlated with severity of total emphysema in TC0, 1, 2 vs. TC3 (P = 0.012). Furthermore, there was a significant difference inversely in total severity of emphysema between TC0, 1, 2 (PD-L1 0-49) and TC3 (PD-L1 50-100) (95% CI: 0.061-5.852, P = 0.045). There were no other significant associations between classification of expression and total/local severity of emphysema, and between classification of expression and presence of COPD/ GOLD stage. These results differ from previous studies on the associations between PD-L1 expression and emphysema in patients with lung adenocarcinoma [28], and between PIK3CA gene mutation and COPD in patients with NSCLC [29]. One explanation for this discrepancy is that our study focused on SCC while other studies focused on lung adenocarcinoma or NSCLC. Another is that our study focused on protein expression while other study focused on the gene mutation. These differences may have influenced the results and may    This study identified for the first time a significant association between high expression of PD-L1 (≥ 50%) and none-mild emphysema in SCC. However, the mechanism behind this association remains unknown. Previous studies have shown an increased proportion of PD-1 positive CD8 T-cells in the lungs of patients with COPD lung such as lung emphysema. This appeared to contribute to dysfunctional of CD8 T-cells [30,31]. Our findings suggest that tumor need not escape from CD8 T-cell attack, particularly given the low expression of PD-L1 when emphysema is moderate-severe. On the contrary, if a tumor escapes CD8 T-cell attack, it might be attributed to high expression of PD-L1 in none-mild emphysema.
This study found that none-mild emphysema was an independent predictive factor of high PD-L1 expression (≥ 50%). This result suggests the utility of non-invasive ways of predicting high expression of PD-L1 (≥ 50%), such as CT imaging. CT imaging may also be an effective way of deciding to use immune checkpoint inhibitors [9,10] in patients with SCC before determining PD-L1 expression with IHC. This might prevent significant life threatening events associated with surgery and bronchoscopy, such as decreasing respiratory function, embolism, bleeding, and pneumothorax. In addition, this might lead to insights regarding the mechanistic correlation between PD-L1 expression and emphysema.
This study had several limitations. First, this was a retrospective study performed at a single institution, which may have influenced the results. A prospective multicenter randomized control trial is needed to confirm our results. Second, although this study shows an association between each expression and Goddard, COPD, and GOLD stage, the associations between those and overall survival rates remain unknown. An evaluation of overall survival is needed. Third, although LAA was evaluated with Goddard criteria, it is unclear if the automated evaluation for LAA [32] would provide the same results. Additional studies are needed to determine what methods are most appropriate for evaluating LAA.

Conclusion
In conclusion, there was a significant association between TC0, 1, 2 vs. TC3 groups and none-mild vs. moderate-severe emphysema groups in patients who underwent surgical resection of lung SCC. PD-L1 expression was significant higher in none-mild emphysema than in moderate-severe emphysema. There was no significant association between any other classification of expression for PD-L1, FGFR1, PIK3CA, PTEN, p16 and total/local severity of emphysema, between classification of expression for PD-L1, FGFR1, PIK3CA, PTEN, p16 and presence of COPD/ GOLD staging. Further studies are needed to evaluate overall survival between classification of each expression and total/local severity of emphysema, presence of COPD/ GOLD stage.