Relationship Between Programmed Death-ligand 1 and Clinicopathological Characteristics in Non-small Cell Lung Cancer Patients

Yan-yan Chen,Liu-bo Wang,Hui-li Zhu,Xiang-yang Li,Yan-ping Zhu,Yu-lei Yin,Fan-zhen Lü ,Zi-li Wang,and Jie-ming Qu*

1Department of Respiratory Medicine,2Department of Pathology,

3Department of Thoracic Surgery,Huadong Hospital,Fudan University,Shanghai 200040,China

MORE than 1 500 000 lung cancer patients are diagnosed every year,and non-small cell lung cancer (NSCLC) accounts for 80 percent of all lung cancer cases.

Programmed death-ligand 1 (PD-L1),known as B7-H1 or CD274,was the fourth member of ultra-family of immunoglobulin,which was firstly identified in 1999.1,2Human PD-L1 gene,located at 9p24-2,encodes I-type transmembrane protein including 290 amino acids.PD-L1 in combination with its receptor,programmed death-1(PD-1),mediates important regulation of immunological responses through immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic region of PD-1 molecule.PD-L1 is widely expressed on immune and nonimmune cells,including T cells,B cells,macrophages and dendritic cells,which induces T-cell anergy or apoptosisviaPD-1 expressed on activated T cells.Gaoet al3,4found that high expression of PD-L1 on T cells and hepatocellular carcinoma cells is associated with poorer prognosis.Meanwhile,the correlation between tumor PD-L1 expression and poor prognosis has also been revealed in variety of human cancers,such as urothelial carcinoma and ovarian cancer.5Chenet al6found that up-regulated PD-L1 expression in NSCLC is related to the degree of tumor cell differentiation and TNM stage.Therefore in this retrospective study,we aimed to evaluate the relationship between clinicopathologic characteristics and PD-L1 expressed in tumor cells as well as tumor associated macrophages (TAM),which present in the micro-environment of tumors have immunosuppressive effect and tumorpromoting action,7in NSCLC patients.

PATIENTS AND METHODS

Patients

From 2008 to 2010,208 NSCLC patients (116 men,age 35-82 years) who underwent surgery or CT-guided biopsy,were recruited from Huadong Hospital,Fudan University.The clinical data of 208 patients were retrospectively analyzed.The pathological staging was performed according to UICC 2009 criterion.

Immunohistochemical staining

Anti-PD-L1 rabbit polyclonal antibody and anti-human PD-1 antibody were purchased from Abcam (Cambridge,UK).Anti-CD68 antibody,which is the specific biomarker of macrophages was purchased from Santa Cruz Biotechnology(CA,USA).

Immunohistochemical staining was performed as previously described.3,4Briefly,NSCLC samples embedded in paraffin were sliced sequentially,then slices were de-paraffined routinely and incubated in 3% H2O2at the room temperature for 10 minutes to inactivate androgenic peroxides.Then the slices were washed with distilled water and soaked in PBS three times,5 minutes each time.Then the slices were sealed in 10% normal goat serum,incubated at the room temperature for 10 minutes and then serum was poured out.The slices were incubated with the first antibodies overnight at 4°C.Then the slices were washed with PBS three times,5 minutes each time.Two-stage immunohistochemical kit (ChemMate™ Envision Detection Kit,Peroxidase/DAB,DAKO,Denmark) was used.The slices were added with a proper amount of ChemMate™ Envision Detection+HRP,incubated for 30 minutes at room temperature,then washed with PBS three times,5 minutes each time.The slice was developed with DAB for 2-5 minutes,then washed with tap water,re-stained with HE,dehydrated,hyalinized,and sealed.In case yellow-brown granules of PD-L1 appeared in membrane or cytoplasm,regarded as PD-L1 positive.

PD-L1 expression in tumor cells

Slices were evaluated by two independent pathologists,using a two-grade scoring system.The staining intensity was divided into 0-3 scores∶not stained,0 score;yellowish,1 score;yellow-brown,2 scores;brown,3 scores.Slices were divided into 4 categories according to percentage of positive cells that were scored as 0,1,2,and 3,respectively.0 score,positive cells ≤2%;1 score,positive cells 3%-20%;2 scores,positive cells 21%-60%;3 scores,positive cells＞60%.The final score was the product of the staining intensity with the corresponding percentage,arranged from 0 to 9.The final score ＞3 is considered as positive expression.7,8

PD-L1 expression in TAM

The paraffin-embedded samples were sliced consecutively into slice series at 4 μm in thickness.The first slice was used for identification of macrophages by CD68 antibody.Then the second slice incubated with PD-L1 antibody was used to identify PD-L1 expression in macrophagesviathe aid of the location for macrophages by the first slice.7,8

Statistical analysis

SPSS,version 13.0,was used for all statistical analysis.The relationship between PD-L1 expression and the clinical pathology was evaluated usingχ2test.Spearman’s rank correlations were used to determine the correlation between PD-L1 expression in tumor cells or macrophages.AP-value＜0.05 was considered to be statistically significant.

RESULTS

Clinicopathological characteristic of NSCLC patients

Totally,208 NSCLC patients were collected in this study including 130 squamous cancers,46 adenocarcinoma,6 adenosquamous carcinoma,12 large cell lung cancers,4 sarcoid carcinoma,and 10 non-or low-differentiated carcinoma.TNM staging was as follows∶114 cases at TNM stage I,30 cases stage II,48 cases stage III,and 16 cases stage IV (Table 1).

Relationship between the PD-L1 expression in tumor cells and the clinicopathological factors

Of 208 patients,136 (65.3%) were PD-L1 positive in the tumor cells,which is expressed either in cytoplasm or membrane (Fig.1).Significantly increased expression of PD-L1 was also found in non-smoking patients (P=0.036,Table 2).In patients with lymphatic metastasis,only 35.5% were PD-L1 positive,compared to 58.3% in non-metastatic patients (P=0.009).

Relationship between PD-L1 expression in TAM and the clinicopathological factors

Macrophages,which were identified by CD68 antibody,were successfully observed in 206 cases.We found 116 patients with PD-L1 positive macrophages,including 84 cases both in tumor cells and macrophages (Fig.1).And the expression of PD-L1 in macrophages did not relate to sex,age,pathological type,pleural infiltration,tumor size and staging (Table 3).Significant correlation between PD-L1 expression in tumor cells and in macrophages (r=0.228,P=0.021) were also found.Increased expression of PD-L1 in macrophages was found in non-smoking,non-lymphatic metastasis and well differentiated cancer patients (allPvalues ＜ 0.05).

Table 1.Clinicopathological characteristics of 208 NSCLC patients

Figure 1.Results of immunohistochemical staining of PD-L1 (A,C,E) and CD68 (B,D,F) in NSCLC cells and TAM.HE staining × 200

Table 2.Relationship between the expression of PD-L1 in tumor cells and the clinicopathological factors of NSCLC patients

DISCUSSION

PD-L1 is expressed widely in both lymphatic and nonlymphatic tissues for example heart,spleen,kidneys,liver,skin,muscle and placenta in addition to lung.High expression of PD-L1 was found in CD8-positive T cells,leading to less secretion,poorer proliferation and subsequent apoptosis.While the CD8-positive T cells may restore the secretion and proliferation function after PD-L1 was blocked by monoclonal antibodies.9

Furthermore,it is reported that increased expression of PD-L1 was also found in various human tumors comparedto normal tissues,including thymus tumor,breast cancer,hepatic cancer,colonic cancer,cystic cancer and tongue squamous cancer,as well as lung cancer.10,11However,the predictive role of PD-L1 in NSCLC patients remains controversial.Muet al12reported that high expression of PD-L1 in the primary foci of NSCLC was an independent predictor of poor prognosis,whereas Konishiet al13also reviewed that PD-L1 and PD-L2 did not correlate to the prognosis of NSCLC,nor did they relate to other clinically pathological factors.In our study,65.3% of total NSCLC patients were found to be PD-L1 positive in tumor cells,which was negatively correlated with lymph node metastasis.Therefore,we think that PD-L1 may promote tumor cell apoptosis and in turn reduce metastasis.

Table 3.Relationship between expression of PD-L1 in TAM and the clinicopathological factors of NSCLC patients

Furthermore,we also detected the expression of PD-L1 in the TAM.We observed that PD-L1 was expressed not only in NSCLC cells,but also in TAM.Interestingly,increased expression of PD-L1 in TAM was negatively correlated to lymphatic metastasis.Based on our results,patients with PD-L1 high expression TAM would had less lymph node metastasis (P=0.0096).

Our researches revealed that TAM may promote metastasis of tumors through inhibiting the function of T cells.TAM with PD-L1 high expression was more liable to development apoptosis,thereby inhibiting the pro-metastatic potency of TAM and in turn reduced lymph node metastasis.However,the specific mechanism remains to be confirmed by more pre-clinical and clinical experiments.

Nearly 50% of patients who undergo surgery would develop metastatic disease and less than 25% of them would obtain treatment and benefit from chemotherapy.Thus new therapeutic targets for lung cancer treatment are urgently needed.And several targets have recently been found,such as Recombinant Human Epidermal Growth Factor.If we regard tumors as the product in the microenvironment of tumors and may conclude that molecular signals participate in the information exchange between tumors and their hosts,then the top key abnormal pathway in the research of lung cancer may include receptor of tyrosine kinase,epithelial-mesenchymal transition,tumor angiogenesis and stem cell labeling;the research on the targeted therapy aiming at the above links is under way;14mesenchymal therapy may be used to prevent and interfere with the development of tumors.Mesenchymal therapy is an early,dynamic and targeted therapy;therefore it may be used in multiple links in the therapeutic stage,including chemical prophylactic therapy,therapy of recurred tumors and therapy of metastatic tumors.Mesenchymal therapy of low density may reverse the signal transfer between the tumor and its hosts while the tumor is small and in a critically unbalanced state,thereby reducing as much as possible its toxic action on other tissues.The result of our researches presented in this paper offered a certain experimental basis for the targeted therapy based on the mesenchyma of tumors.

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