Solid histological component of adenocarcinoma might play an important role in PD‐L1 expression of lung adenocarcinoma

Abstract Background In this study we aimed to clarify the PD‐L1 positive expression in lung adenocarcinoma, including various adenocarcinoma subtypes paying particular attention to its component. Methods A total of 307 lung adenocarcinoma patients who underwent lobectomy or segmentectomy, as well as systematic lymph node dissection (ND2a), from February 2008 to March 2020 at our hospital, were enrolled into the study. A final diagnosis of adenocarcinoma was obtained from the resected lung specimens of all 307 patients to determine the histological type, adenocarcinoma subtype, and component of adenocarcinoma by ethics of 5%. PD‐L1 was immunohistochemically stained using the murine monoclonal antibody clone 22C3. Results When PD‐L1 expression‐positive was defined by tumor proportion score (TPS) ≥1%, the positive cases were 6/33 in adenocarcinoma (Ad) in situ (AIS), 2/26 in minimally invasive Ad (MIA), 12/60 in lepidic predominant Ad (LPA), 44/91 in papillary predominant Ad (PPA), 24/49 in acinar predominant Ad (APA), 23/28 in solid predominant Ad (SPA), 4/7 in micropapillary predominant Ad (MPA), and 0/13 in invasive mucinous Ad (IMA). In the high proportion group (APA, PPA, SPA, and MPA) of PD‐L1 expression, SPA was the only subtype which was statistically significant when both PD‐L1 expression‐positive was defined by TPS ≥ 1% (p < 0.0001) and TPS ≧ 50% (p < 0.0001). We then considered the solid component. We investigated 279 cases of the other subtype group excluding SPA. The group containing a solid component (≥5%) tended to be PD‐L1 expression‐positive both when defined by TPS ≥1% (p < 0.0001) and TPS ≧50% (p = 0.0049). Conclusions The PD‐L1 expression tended to be positive when a solid component was confirmed (≥5%) in specimens of lung adenocarcinoma patients.


INTRODUCTION
Lung cancer is the leading cause of cancer deaths in most developed countries worldwide. 1 Despite multidisciplinary therapies that have been used for patients with advanced non-small cell lung cancer (NSCLC), the overall survival rates are still unsatisfactory, especially when genetic mutation is not detected. Recently, several humanized monoclonal antibodies to block immune checkpoints have been developed which have proven to be useful in selected patients with unresectable NSCLCs. 2,3 The association between programmed cell death 1 (PD-1) and programmed death-ligand 1 (PD-L1) can target these monoclonal antibodies. Inhibition of the PD-1/PD-L1 axis enhances antitumor immunity in order to prevent tumor cells escaping from host immune responses, thereby providing a promising strategy for effective tumor immunotherapy. 4 Previous clinical trials have revealed treatment effect predictor of immune checkpoint inhibitors (ICIs), such as PD-L1 expression [5][6][7] and tumor mutation burden 8 are positively correlated, and SKT11 and KEAP1 mutations are negatively correlated. 9 Therefore limited and controversial debates still continue today. On the other hand, pembrolizumab is an ICI which can be used for single agent administration in the first treatment of patients with advanced NSCLC. The tumor proportion score (TPS) has been found to be the most correlated effect predictor of pembrolizumab. 5,7,10 ICIs have been approved as consolidation therapy after radiation chemotherapy for locally advanced stage disease, 11 and expected to be expanding the indication in the region of treatment for more early stage lung cancer, 12,13 such as radiation combination therapy, 14 pre-and postoperative therapy. In particular, with regard to early stage lung cancer, in the near future, we may be able to judge adaptation of preand postoperative ICI therapy according to the pathological and genetic findings obtained from specimens at diagnosis. To date, preoperative biopsy using bronchoscopy is used to diagnose histological subtypes of lung adenocarcinoma. 15 To clarify the adenocarcinoma subtype which tends to express PD-L1 is important. We have previously reported on the relationship between PD-L1 expression and adenocarcinoma subtypes using 78 resected cases. 16 In order to obtain more precise knowledge, in this study we used a greater number of resected lung cancer specimens to investigate the histological component of adenocarcinoma.

Patients and pathological specimens
A total of 307 lung adenocarcinoma patients (148 men and 159 women; age range = 42-90 years; mean age = 69.2 years) who underwent lobectomy or segmentectomy, together with systematic lymph node dissection (ND2a), from February 2008 to March 2020 at our hospital were enrolled into this study. The exclusion criteria were patients who had undergone preoperative induction therapy. The mean postoperative follow-up period was 26.8 months (range = 0-121 months A final diagnosis of adenocarcinoma was obtained from resected lung specimens of all 307 patients by two pathologists in order to determine the histological type, adenocarcinoma subtype, and component of adenocarcinoma by ethics of 5% based on the World Health Organization (WHO) pathological classification published in 2015. 17

Immunohistochemistry
Immunohistochemistry was performed using the PD-L1 kit (PD-L1 IHC 22C3 pharmDX; Dako) according to the manufacturer's instructions. This antibody was selected since the Food and Drug Administration (FDA) approved this system as a companion diagnostic test to determine the applicability of treatment using pembrolizumab. In brief, serial 3 μmthick tissue sections were cut from formalin-fixed, paraffinembedded blocks. Sections were deparaffinized in xylene and rehydrated through a graded series of ethanol concentrations. Antigen retrieval was carried out using a 97 C water bath for 20 min in Envitio FLEX Target Retrieval solution (Dako). Intrinsic peroxidase activity was blocked using hydrogen peroxide for 5 min. After washing the section with a wash buffer (Dako), primary antibodies were applied to cover the specimen. Sections were incubated at room temperature for 30 min. After three washes in the wash buffer for 5 min each, slides were incubated with antimouse linker antibody specific to the host species of the primary antibody, and were then incubated with a ready-to-use visualization reagent consisting of secondary antibody molecules and horseradish peroxidase molecules coupled to a dextran polymer backbone. Specimens were then counterstained with hematoxylin for 5 min and cover-slipped ( Figure 1).

Assessment of PD-L1 expression
We followed the instructions given in the PD-L1 Immunohistochemistry Testing in Lung Cancer manual of the IASLC (https://www.iaslc.org/sites/default/files/wysiwyg-assets/pd-l1_atlas_book_lo-res.pdf). Briefly, the authors counted PD-L1-positive tumor cells, defined as complete circumferential or partial cell membrane staining. Cytoplasmic staining and tumor-associated immune cells, such as macrophages, were excluded from scoring. The tumor proportion score (TPS) was calculated as follows: TPS(%) = (PDÀL1Àpositive tumor cells / Total number of tumor cells) Â 100 The TPS was used to categorize the staining status as follows: <1% (negative staining), ≥1% and <50% (weakly positive staining), and ≥50% (highly positive staining).

Statistical analysis
Statistical analysis was performed using JMP software. Clinicopathological characteristics by categorical variables were evaluated using the chi-square test. p < 0.05 was considered statistically significant. The overall survival (OS) and recurrence-free survival (RFS) were analyzed using the Kaplan-Meier method.

DISCUSSION
Lung cancer management is becoming more individualized for age, comorbidities, cancer type, stage, and patient preference. 18 Immunotherapy is one of the most expected fields of individualized management for NSCLC. Many study results regarding PD-L1 protein expression in NSCLC have been reported. However, there are very few studies which refer to adenocarcinoma subtypes. To the best of our knowledge, this is the first study concerning the relationship between PD-L1 expression and adenocarcinoma component. With regard to immunotherapy, preoperative neoadjuvant immunotherapy may become one of the options for NSCLC management in the future. 19,20 The technical progression of bronchoscopic biopsy may support this trend. 21 Today, adenocarcinoma subtypes can be diagnosed by examining the specimen obtained by bronchoscopic biopsy. Matsuzawa et al., who analyzed the concordance rate of histological subtypes between small biopsy samples and surgically-resected specimens, stated concordance rate of PPA and SPA were relatively high. 15 Therefore, solid compartment of biopsybased analysis may also have more reliability than the other components. This fact gives more importance to this report. Regarding the frequency of PD-L1 expression in adenocarcinoma subtypes, when cutoff TPS was ≧ 1%, the highest positive rate of PD-L1 expression was observed in the SPA (73.7%), followed by the APA (49.2%), PPA (43.3%), and MPA (40.0%). This result is almost concordant with 404 cases reported by Song et al. 22 Peng reported when cutoff TPS was ≧ 25%, the highest positive rate of PD-L1 expression was observed in the SPA (44.7%), followed by APA (25.1%), MPA (20%), and PPA (10.9%). When cutoff TPS was ≧ 50%, the highest positive rate of PD-L1 expression was also observed in the SPA (53.6%) in this study. In any case, SPA tend to contribute positive PD-L1 expression.
With regard to subtypes, we previously reported that adenocarcinoma subtypes are divided into three groups by prognosis 23 24 In the era of individualized therapy for lung cancer, when specimens including those with a solid component can be obtained by bronchoscopic biopsy before surgery, preoperative neoadjuvant immunotherapy might become one of the options in the treatment of adenocarcinoma. Further, in inoperable cases, when specimens including a solid component can be obtained, PD-L1 expression may be positive.
In conclusion, there was only a significant difference in PD-L1 expression in SPA both in TPS ≧ 1%, and in TPS ≧ 50%. Even when a solid component was confirmed (≥5%) in the lung adenocarcinoma patient specimens, PD-L1 expression tended to be positive. Therefore, a solid histological component of adenocarcinoma might play an important role in PD-L1 expression of lung adenocarcinoma. There are several limitations in this study. First, subtype diagnosis in adenocarcinoma is not always coincident between pathologists. 25,26 Second, this was not a prospective study, and therefore bias might exist. Third, the postoperative observation period was relatively short and the number of cases relatively small. Further studies including larger numbers of patients are necessary to confirm the present results. Fourth, this study includes old specimens, and the accuracy of PD-L1 staining results might therefore be insufficient.

ACKNOWLEDGMENTS
This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.