Expression And Significance Of Immunotherapy-linked Protein PD 1,PD-L1 In Lung Adenocarcinoma

ObjectiveLung cancer is one of the leading cause of morbidity and mortality worldwide, and has the highest mortality rate in China. Adenocarcinoma is the most common pathology type in lung cancer. After the target therapy, immunotherapy has become the new hot spot in the field of lung cancer therapy. immunotherapy-related protein PD-1(programmed cell death-1) / PD-L1(programmed cell death-ligand 1) is one of the hotspot researching protein, yet its expression features and its correlations with targeted therapy-linked driver mutation EGFR/BRAF/ALK remains no definite conclusion.This study is taking 109 cases of invasive surgical specimens of patients with lung adenocarcinoma as the research object, taking expressions of PD-1/ PD-L1 in lung adenocarcinoma as the breakthrough point, and using immunohistochemistry and image analysis technology to systematically research and analyze expression features and correlations between them. Besides, our study will analyze the correlations between PD-1/ PD-L1 expression and clinicopathological parameters in adenocarcinoma with different EGFR status, and will explore the intrinsic relation between EGFR pathway and PD-1/PD-L1 pathway preliminary. This study is aimed at providing the clinical application of targeted therapy and immunotherapy of theoretical basis.Content1. To detect the protein expression of PD-1 and PD-L1 in lung adenocarcinoma, then to analyze the correlation between PD-1 and PD-L1 expression, especially to analyze the correlation between them in different EGFR mutation status.2. To research the correlation between PD-1/PD-L1 expression and clinicopathological parameters, such as gender, age, tumor histological type, stage, lymph node metastasis.3. To research the correlation between PD-1/PD-L1 expression and EGFR, BRAF, ALK mutation status.4. To quantitatively detect the expression of PD-L1 in lung adenocarcinoma by image analysis technology. To analyze the correlation between PD-L1 expression and clinicopathological parameters, and compare the result between image analysis and manual counting.Material and Method109 cases of lung adenocarcinoma was collected during the period of Aug, 2010 ~ Jan, 2016, including 51 cases of EGFR wild type and 58 cases of EGFR mutation type. Of the 109 cases, 51 cases were male and 58 were female, age range from 41 to 81, the average age is 62.2, the median age is 62. Histology types including 57 cases of acinar predominant adenocarcinoma, 25 of solid predominant adenocarcinoma,14 of lepidic predominant adenocarcinoma, 11 of papillary predominant adenocarcinoma, 2 of micropapillary predominant adenocarcinoma. 44 cases with solid pattern and 30 with micropapillary pattern, 34 with lymph node metastasis. Tumor sizes are range from 0.4 to 10 cm, median maximum tumor diameters is 2.5cm. TNM stage: 43 cases of stage IA, 15 of stage IB, 14 of stage IIA, 5 of stage IIB, 22 of stage IIIA, 2 of stage IIIB, 8 of stage IV.Immunohistochemistry EnVison method was used to detect PD-L1 protein expression. PD-1 antibody, PD-L1 antibody and secondary antibody(goat anti-mouse and anti-rabbit IgG, enzyme labeled) were purchased from ZSGB-BIO, and DAB+ CHROMOGEN Kit was purchased from DAKO. Semi-quantitative method was used to evaluate the expression of PD-1 and PD-L1.Detection of EGFR, BRAF and ALK used ARMS method. DNA and RNA extraction kits were purchased from QIAGEN, mutant detection kits were purchased from Amoy-Dx. The interpretation of detection results referred to kit instructions.Image analysis technology on IHC section was used to quantitatively detect PD-L1 expression. At high magnification, 5 fields were picked randomly from each IHC section and processed image collection and analysis. The value of stain degree was calculated by using the formula [(brown area surface density/blue area surface density) × brown area mean optical density]. Stain degree value≥0.05 was defined as a positive result.All statistical analyses were run by SAS 9.1 software. Chi-square test was used to analyze the correlation between PD-1 and PD-L1 expression, also the correlation between those expression and clinicopathological parameters and lung adenocarcinoma driver mutation status. The differences of stain degree values were analyzed by Kruskal-Wallis test. The corrsltion between qualitative result of PD-L1 expression by image analysis and clinical pathological parameters used Chi-square test. Consistency between image analysis results and manual counting results were analyzed by Kappa test.Result 1. PD-1 and PD-L1 expression in lung adenocarcinoma and the correlation between themThe positive rates of PD-1 and PD-L1 expression are 68.807%(75/109) and 27.523%(30/109), the correlation between them has statistical significance(P<0.05). PD-1 and PD-L1 expression are higher in 51 cases with EGFR wild type(74.510%, 39.216%) than in 58 cases with EGFR mutation type(63.793%, 17.241%). The correlation between PD-1/PD-L1 expression has statistical significance in cases without EGFR mutaion(P<0.05), while has no statistical significance in cases with EGFR mutation(P>0.05). 2. EGFR, BRAF, ALK status51 cases are EGFR wild type and 58 cases are EGFR mutation type in the 109 cases. EGFR mutant type including 22 cases of 19-Del, 1 of both 19-Del and T790 M, 27 of L858 R, 1 of both L858 R and S768 I, 2 of G719 X, 2 of S768 I, 1 of both G719 X and S768 I, 1 of L861 Q and 1 of 20-Ins. BRAF mutation rate is 1.835%(2/109), while ALK fusion rate is 3.670%(4/109) over all and 7.843% in EGFR wild types. 3. Relationship between PD-1/PD-L1 expression and clinicopathological featuresPD-1 expression were positively associated with age(P<0.05, and the expression of PD-L1 is closely related with histological type, tumor size, lymph node metastasis(P<0.05). Positive rate of PD-L1 in different histological types from high to low as micropapillary predominant adenocarcinoma(100%), solid predominant adenocarcinoma(56%), acinar predominant adenocarcinoma(22.8%), papillary predominant adenocarcinoma(9.1%), lepidic predominant adenocarcinoma(0%). Although there is no statistical significance between PD-L1 expression and stage(P=0.08), we found that the later the clinical stage was, the higher the positive rate is. Positve rate of StageⅠ, Ⅱ, Ⅲ or Ⅳ are respectiv ely 18.966%, 31.579% and 40.625%. There is no correalation difference of PD-1/PD-L1 expression and clinicopathological parameters between EGFR mutant cases and EGFR wild type. 4. The correalation between PD-1/PD-L1 expression and EGFR, BRAF, ALK statusNone of these correalation between PD-1 expression and EGFR, BRAF, ALK status has statistical significance. PD-L1 expression is only associated with EGFR status, but except BRAF and ALK status. Patients with EGFR wild type has a higher PD-L1 expression rate of 39.216% compared to those with EGFR mutant type(17.241%). 5. Comparision of PD-L1 expression results between image analysis and manual countingThe value of stain degree have difference in different gender(P<0.05). Qualitative result of PD-L1 by image analysis is associate with histological type(P<0.05). The consistent rate between image analysis and manual counting is 88.073%.Conclusion1. The features of PD-1 and PD-L1 expression and the correlation between them in adenocarcinoma with mutant EGFR are differ from that with native EGFR. PD-1 and PD-L1 expression are higher in EGFR wild type than EGFR mutation type. The correlation between PD-1/PD-L1 expression has statistical significance in cases without EGFR mutaio, while has no statistical significance in cases with EGFR mutation.2. The correlation between PD-1 expression and age and micropapillary pattern has statistical significance(P<0.05).3. PD-L1 positive expression is associated with histological type, tumor size, lymph node metastasis and solid patterns(P<0.05). Positive rate of PD-L1 in different histological types from high to low as micropapillary predominant adenocarcinoma, solid predominant adenocarcinoma, acinar predominant adenocarcinoma, papillary predominant adenocarcinoma, lepidic predominant adenocarcinoma.4. Analysis result of PD-L1 IHC images by calculating stain degree value have a good consistency compare with manual counting results(Consistent rate =88.073%, K=0.7157). PD-L1 expression correlated significantly with histological types(P<0.05), may correlated with age(P=0.0573). there is no statistically significant correlation between other clinical pathological parameters and PD-L1 expression(P>0.05).Quantitative results for PD-L1 protein expression may have more objective significance.