The solid subtype, according to the IASLC/ATS/ERS classication of lung adenocarcinoma, as a predictive marker for PD-L1 expression

Background: PD-L1 expression in tumor cells can predict the ecacy of PD-1/PD-L1 inhibitors and prognosis in patients. However, the correlation between the PD-L1 expression and the novel lung adenocarcinoma classication are obscure. Methods: 126 lung adenocarcinoma cases were reviewed in the Third Aliated Hospital of Soochow University from Jan. to Dec. 2019. PD-L1 (DAKO 22C3) was used to test the PD-L1 expression in lung cancer tissue. Result: TPS was used to interpret the PD-L1 expression. The negative, low positive and high positive of PD-L1 were 72 cases (57.14%), 39 cases (30.95%) and 15 cases (11.90%). PD-L1 TPS in solid structure was signicantly higher than that in acinar structure, lepidic structure and papillary structure (P<0.001, respectively). The results of c 2 test showed the PD-L1 expression had the signicant difference with gender (P = 0.005), age (P = 0.030), smoking history (P = 0.024), lymph node metastasis (P <0.001), TNM stage (P = 0.001), acinar structure (P = 0.003) and solid structure (P < 0.001). Multi-factor linear regression results suggested that solid structure, TNM stage and smoking history were associated with PD-L1 expression (P < 0.05). The solid structure showed more capability to PD-L1 expression (β = 0.428). Conclusion: PD-L1 expression was heterogeneity in lung adenocarcinoma. The solid structure, TNM stage and smoking history were correlation to up-regulation of PD-L1 expression, and solid structure was the most importance factor.


Background
Due to the highest incidence and mortality rate in kinds of malignant tumor, lung cancer is a major public health problem (1,2). Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, with lung adenocarcinoma (ADC) being most frequent (3)(4)(5). According to the new classi cation criteria of ADC released by International association for the study of lung cancer (IASLC), American thoracic society (AST) and European respiratory society (ERS) in 2011, lung ADC can be classi ed into acinar subtype, lepidic subtype, papillary subtype, micropapillary subtype, and solid subtype based on morphological characteristics (6). The recently-emerged targeted therapies and immunotherapies have good therapeutic effects on NSCLC. Among them, the immune checkpoint inhibitors represented by PD-1/PD-L1 have achieved signi cant therapeutic e cacy in clinical practice. According to KEYNOTE-024 study, PD-L1 expression in tumor cells can predict the e cacy of PD-1/PD-L1 inhibitors and prognosis in patients (7).
At present, there is limited study on the correlation between lung cancer, especially the new pathological classi cation, and the expression of PD-L1. To this end, in the present study, we aimed to explore the expression of PD-L1 in lung cancer tissues and to investigate its relationship with the new classi cation of lung ADC.

Immunohistochemistry staining
All samples were xed with neutral formaldehyde solution and embedded into para n. The para nembedded sections were serially cut into 4 μm-thick slices for PD-L1 staining (DAKO PD-L1 22C3) on Autostainer Link 48 staining platform. The interpretation of PD-L1 was performed by two pathologists using a double-blind method. Tumor cells with PD-L1 staining on the cell membrane were positive cells.

Statistical analysis
Statistical analyses were carried out using the IBM SPSS statistical software V24.0 (IBM, Armonk, NY). χ 2 test, one-way anova and multivariate linear regression model were used as appropriate. All statistical analyses were two-sided test and the statistical signi cance was de ned as P <0.05.

Comparison of PD-L1 expression between the ve subtypes of the novel lung adenocarcinoma classi cation
The details of PD-L1 expression in the ve subtypes of the novel lung adenocarcinoma classi cation are shown in Table 1. The result of Mann-Whiteny U test suggested that the patients with the acinar or lepidic structure frequently had low TPS of PD-L1 (P=0.019; P=0.009, Fig.1 A, B). The PD-L1 TPS of patients with papillary or micropapillary structure was no different from that of patients without those structure (P=0.169; P=0.201, Fig.1 C, D). However, there was a signi cant association between solid structure and high TPS of PD-L1 (P<0.001, Fig.1 E). The result of one-way anova showed that PD-L1 TPS in solid structure was signi cantly higher than that in acinar structure, lepidic structure and papillary structure (P<0.001, respectively, Fig.1 F).
2. The correlation of PD-L1 expression with clinicopathological parameters and new classi cation of ADC IHC was used to detect PD-L1 expression in 126 cases of ADC tissues, revealing negative in 72 cases (57.14%), low expression in 39 cases (30.95%) and high expression in 15 cases (11.90%). The expression of PD-L1 was signi cantly correlated with gender, age, smoking history, lymph node metastasis, TNM stage, acinar structure and solid structure of patients (P < 0.05 Table 2). The high expression of PD-L1 was instructive in decision-making of clinical drug regimen for ADC patients. Therefore, patients were further divided into PD-L1 non/low expression (111 cases) and PD-L1 high expression groups (15 cases).
As a result, high expression of PD-L1 was correlated with gender, smoking history, lymph node metastasis, TNM stage, acinar structure and solid structure of patients (P < 0.05 Table 2).

In uencing factors of PD-L1 expression
In this study, we aimed to investigate the following pathological parameters, including patient's age, gender, smoking history, lymph node metastasis, TNM stage, and lung morphological subtypes. First, a univariate linear regression equation was constructed to screen the independent variables (Table 3). Subsequently, the patient's age, gender, smoking history, lymph node metastasis, TNM stage, acinar structure and solid structure were included in the multivariate linear analysis in a stepwise method. The  Table 3, Fig.2), PD-L1 expression was increased by times when the lung ADC progressed from TNM stage I-II to TNM stage III-IV (B = 20.866, P = 0.001, Table 3), PD-L1 expression was increased by 18.836 times in the case of smoking history (B = 18.836, P = 0.001, Table 3). The comparison of the standardized regression coe cients of the three parameters indicated that the effect of solid structure (β = 0.428) was stronger than TNM stage (β = 0.345) and smoking history (β = 0.275) on PD-L1 expression.

Discussion
In addition to traditional surgical resection and adjuvant chemoradiotherapy, immunotherapy has become an important novel approach for tumor treatment. PD-1 / PD-L1 inhibitors, as representative drugs, have signi cant therapeutic effects on various tumors(8). PD-L1 expression is heterogeneity in different types of cancer, along with the uniformed detection techniques and scoring standards(9,10). In this study, DAKO PD-L1 22C3 was used to detect PD-L1 expression (11), The positive rate of PD-L1 in ADC tumor tissue was 42.86% (54/126), and the high expression rate was 11.90% (15/126). The expression of PD-L1 also varied between different gender and age, suggesting that more attention should be paid to PD-L1 detection and the possibility of clinical medication on women and patients no more than 60 years. PD-L1 is highly expressed in the tissues of patients with advanced lung cancer (with lymph node metastasis, TNM III-IV), which is consistent with previous studies (12,13) and also provides an objective basis for the application of PD-L1 inhibitors in patients with advanced cancer. In addition, the high expression of PD-L1 in ADC tissues of smoking patients suggests that smoking patients are more likely to bene t from PD-L1 inhibitors, which might be associated with the immunosuppressive state in the tumor microenvironment caused by smoking.
The 2011 new classi cation of lung ADC has recommended a detailed description of invasive ADCs based on different histological subtypes (6). In this study, we analyzed the correlation between PD-L1 expression and ve common histological subtypes of lung ADC (acinous subtype, lepidic subtype, papillary subtype, micropapillary subtype, and solid subtype). Previous studies have revealed the signi cant correlation between solid subtypes and poor prognosis of patients (14,15). In this study, we found that the appearance of solid structures in cancer tissues was signi cantly associated with upregulated expression of PD-L1 expression(16), suggesting that the solid structure, as a histological subtype correlated with poor prognosis, may be related to the immunosuppressive state in ADC tumor immune microenvironment. And these patients might bene t more from PD-L1 inhibitors. Multiple linear regression equations evaluating the in uencing factors of PD-L1 expression suggested that solid structure, TNM stage and smoking history were signi cantly correlated with up-regulated expression of PD-L1, and solid structure was the most signi cant factor affecting PD-L1 expression. Here, we investigated the correlation between ADC histological subtypes and the expression of PD-L1. The percentage of ADC histological subtypes need to be calculated in further studies.

Conclusion
The novel lung adenocarcinoma classi cation showed a superior correlation with patient's prognosis. The patient's selection for PD-L1 immunological therapy based on the PD-L1 expression. However, PD-L1 expression was heterogeneity in ADC. There was a signi cant association between solid structure and higher expression of PD-L1. The solid subtype is an important predictor of PD-L1 expression. The patients with solid subtype might bene t more from PD-L1 inhibitors.

Declarations
Ethical approval and consent to participate This study protocol was approved by the Ethics Committee of Soochow University and conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from patients before enrollment in this study.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Funding
This study was supported in part by the Young Talent Development Plan of Changzhou Health Commission (CZQM2020040).

Competing interests
These authors declare that they have no competing interests.   Figure 1 The comparison of PD-L1 expression between the ve subtypes of the novel lung adenocarcinoma classi cation. The box charts were used to show the difference of PD-L1 TPS between the patients with and without acinar structure (A), lepidic structure (B), papillary structure (C), micropapillary structure (D) and solid structure (E). The result of the comparison between groups showed that PD-L1 TPS in solid structure was signi cantly higher than that in acinar structure, lepidic structure and papillary structure (F).