Published OnlineFirst June 15, 2017; DOI: 10.1158/2326-6066.CIR-16-0381 Title: A Four-Factor Immunoscore System that Predicts Clinical Outcome for Stage II/III Gastric Cancer Running Title: Immunoscore as Prognostic Model for Gastric Cancer Ti Wen1†PhD., Zhenning Wang2†MD., Yi Li3 MD., Zhi Li1 MD., Xiaofang Che1 PhD., Yibo Fan1 MD., Shuo Wang1 MD., Jinglei Qu1 MD., Xianghong Yang5 MD., Kezuo Hou1BS., Wenyang Zhou5 MS., Ling Xu1 MD., Ce Li1 MS., Jin Wang1 MD., Jing Liu1 MD., Liqun Chen3BS., Jingdong Zhang4 MD., Xiujuan Qu1* MD., Yunpeng Liu1* MD. 1 Department of Medical Oncology, Key Laboratory of Anticancer Drugs and Biotherapy of Liaoning Province 2 Department of Surgical Oncology and General Surgery, The First Hospital of China Medical University, No.155, Nanjing North Street, Heping District, Shenyang, 110001, China 3 Department of Pathology, 4 Department of Medical Oncology, Cancer Hospital of Liaoning Province, No.44, Xiaoheyan Road, Dadong District, Shenyang, 110042, China 5 Department of Pathology, Shengjing Hospital of China Medical University, No.36, Sanhao Street, Heping District, Shenyang, 110004, China *Corresponding Author: Yunpeng Liu, MD. PhD., Tel/Fax: +86-(0)24-83282312; E-mail: [email protected] and Xiujuan Qu, MD. PhD. Tel/Fax: +86-(0)24-83282312; E-mail:[email protected]; Department of Medical Oncology, The First Hospital of China Medical University, No.155, Nanjing North Street, Heping District, Shenyang 110001, China; †These two authors contributed equally to this work. Keywords: gastric cancer, immunoscore system, PD-L1, PD-1, CD8, prognostic model Grant Support This work was supported by Key National Science and Technology Major Project of China (No. 2013ZX09303002, to YP Liu), National Natural Science Foundation of China (NSFC) (No. 31300743 to T Wen, 81673025 to L Xu, 81602098 to YB Fan), Science and Technology Plan Project of Liaoning Province (No. 2015020734 to T Wen, 2014225013, 2014226033, to YP Liu), the Key Laboratory Project of the Education Department of Liaoning Province (No.LZ2014037, to YP Liu) and Health and Family Planning Commission of Liaoning Province (No. LNCCC-D07-2015, to T Wen). Author Contributions: Ti Wen, Xiujuan Qu, and Yunpeng Liu were responsible for study design, data collection, data analysis and manuscript writing. Yi Li, Zhi Li, Xiaofang Che, Yibo Fan, Shuo Wang, Jinglei Qu, Xianghong Yang, Kezuo Hou, and Wenyang Zhou were responsible for data collection, data analysis. Zhenning Wang, Ling Xu, Ce Li, Jin Wang, Jing Liu, Liqun Chen, and Jingdong Zhang were responsible for data collection. 1 Downloaded from cancerimmunolres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 15, 2017; DOI: 10.1158/2326-6066.CIR-16-0381 Abstract The American Joint Committee on Cancer (AJCC) staging system is insufficiently prognostic for operable gastric cancer (GC) patients, therefore complementary factors are under intense investigation. Although the focus is on immune markers, the prognostic impact of a single immune factor is minimal, due to complex antitumor immune responses. A more comprehensive evaluation may engender more accurate predictions. We analyzed immune factors by immunohistochemical staining in two independent cohorts. The association with patients’ survival was analyzed by the Kaplan-Meier method. Our immunoscore system was constructed using Cox proportional hazard analysis. PD-L1+ immune cells (ICs), PD-L1+ tumor cells (TCs), PD-1hi, and CD8More were found among 33.33%, 31.37%, 33.33%, and 49%, respectively, of patients from the discovery cohort, and 41.74%, 17.4%, 38.26%, and 30.43% from the validation cohort. PD-L1+ ICs and PD-1hi ICs correlated with poorer overall survival (OS), but PD-L1+ TCs correlated with better OS and clinical outcomes and infiltration of more CD8+ T cells. These four factors were independently prognostic after tumor/lymph nodes/metastasis (TNM) stage adjustment. An immunoscore system based on hazard ratios of the four factors further separated GC patients with similar TNM staging into low-, medium-, or high-risk groups, with significantly different survival. Our prognostic model yielded an area under the receiver operating characteristic curve (AUC) of 0.856 for prediction of mortality at 5 years, superior to that of TNM staging (AUC of 0.676). Thus, this more comprehensive immunoscore system can provide more accurate prognoses and is an essential complement to the AJCC staging system for operable GC patients. 2 Downloaded from cancerimmunolres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 15, 2017; DOI: 10.1158/2326-6066.CIR-16-0381 Introduction Gastric cancer (GC) is one of the most common malignancies worldwide [1]. Conventionally, tumor/lymphnode/metastasis (TNM) staging has been used to assess the prognosis of GC patients, however, the clinical prognosis may vary significantly among patients with the same TNM stage due to its highly heterogeneous nature [2]. Thus, researchers are intensely searching for complementary factors such as molecular signatures, or genetic features [3]. In addition to tumor-intrinsic factors, ongoing work is revealing extrinsic immune factors that affect tumor prognosis [4-7]. However, due to the complicated characteristics of tumor immune response, the prognostic value of single immune factors are unreliable and have limitations when applied to large-scale populations. Thus, a comprehensive immunoscore system is needed to make more accurate prognoses for GC patients. A designated TNM-Immune (TNM-I) system has been defined in colorectal cancer by assessment of intratumor T cells, which is a prognostic factor superior to the AJCC/UICC TNM classification [8]. However, an immunoscore system with prognostic significance for GC patients in clinical settings has not been identified so far. The TNM-I system for colorectal cancer is based on density and location of CD3/CD8-positive cells, rather than the function of T cells. However, the latter has an important impact on the prognosis of GC. A hallmark of tumor progression is evasion of immune destruction , and PD-L1/PD-1 is one of the most important signaling pathways mediating tumor immune escape [9, 10]. Pembrolizumab and nivolumab, monoclonal antibodies that block PD-L1/PD-1 interactions, are now used therapeutically for advanced melanoma and non-small-cell lung cancer (NSCLC) [11, 12]. A completed phase Ib trial (Keynote-012) also showed an overall response rate (ORR) to pembrolizumab treatment was 33.3% among patients with GC [13]. Because blockade of 3 Downloaded from cancerimmunolres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 15, 2017; DOI: 10.1158/2326-6066.CIR-16-0381 PD-L1/PD-1 can be effective in GC therapy, expression of PD-L1 and PD-1 were thought to have prognostic significance. However, the prognostic value of PD-L1/PD-1 remains controversial [14-23]. One reason is that PD-L1 can be expressed by many cell types, such as tumor cells or immune cells; whether PD-L1 expressed by different cells has different prognostic values is unclear [14, 15, 18]. Additionally, PD-L1 is regulated by intrinsic and extrinsic mechanisms; whether this has a different impact on the prognostic value of PD-L1 is not known [24]. GC is closely associated with inflammation, large numbers of pre-existing CD8+ T cells are found at the tumor sites of GC patients. However, the prognostic effect of increased CD8+ T cells infiltration in GC is still under debate [6, 7, 14, 25]. These controversial viewpoints may be attributed to the complicated characteristics of tumor immune response. Therefore, a more reliable model incorporating multiple immune effectors is necessary and may provide better prognosis compared with single-factor predictors. In the current study, expression of PD-L1 in tumor cells and immune cells, expression of PD-1 in immune cells, and infiltration of CD8+ T cells were evaluated in two independent patient cohorts with stage II-III GC and analyzed for clinical characteristics. We developed an immunoscore system based on the hazard ratios (HRs) derived from the multivariable analyses. This approach can further divide patients with the same TNM staging into subgroups who are at low, moderate, or high risk, thereby providing more accurate prognoses and clues for the selection of immunotherapy options for GC patients. 4 Downloaded from cancerimmunolres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 15, 2017; DOI: 10.1158/2326-6066.CIR-16-0381 Materials and Methods Patients This study was conducted on two independent cohorts of GC patients with stage II-III, who were followed up at the First Hospital of China Medical University. Patients were enrolled into two cohorts if they met the following criteria: Gastric cancer with stage 2-3; D2 lymphadenectomy; no neoadjuvant therapy before surgery; received 5-flurouracil-based standardized combination chemotherapy if relapsed. Newly collected specimen with more than 2 years of follow-up (from February 2012 to September 2012) were assigned to the discovery cohort (n = 51) and patients with 5 years of follow-up data (between April 2006 and July 2011) were selected according to the random number generated by the R/sample function to form validation cohort (n = 115). The median follow-up period was 31 months (range 3 ~ 100 months), and 73 patients (43.98%) died during this period, with a 5-year overall survival (OS) probability of 50.1%. The study was approved by the Ethics Committee of China Medical University, and all procedures were conducted in accordance with ethical principles. Clinical information of all patients was retrieved from the Hospital Information System (characteristics are listed in Supplementary Table S1). Immunohistochemistry (IHC) Standard indirect immunoperoxidase protocols were used for the immunohistochemistry assay. Briefly, embedded tumor tissues were sectioned to 4-μm thickness, dewaxed and rehydrated. Heat-induced antigen retrieval in citrate buffer was conducted followed by 3% hydrogen peroxide.