The potential prognostic and predictive roles of programmed cell death protein 1 expressed by tumor-infiltrating lymphocytes in solid tumors : a meta-analysis

Aim: Several previous studies have evaluated the potential role of programmed cell death protein 1 (PD-1) expressed by tumor-infiltrating lymphocytes (TILs) in various solid tumors and performed its prognosis role in patients’ survival with inconsistent results. This study aims to further systematically evaluate the association of PD-1 by TILs with clinicopathological parameters and clinical outcomes in solid tumor patients. Methods: A comprehensive search was conducted in PubMed, Embase, Web of Science, CNKI and Wanfang databases for relevant studies. The potential prognostic and predictive roles of PD-1 were assessed by pooled hazard ratio (HR), odds ratio (OR) and 95% confidence intervals (CI). A total of 1863 patients were selected for in-depth analysis. Results: The results demonstrated that PD-1 by TILs was correlated to overall survival for ovarian cancer (HR = 0.40, 95% CI: 0.26-0.61, P < 0.00001). Higher PD-1 expression was associated with lymph node metastasis (OR = 2.55, 95% CI: 1.22-5.29, P = 0.01) and tumor grade (OR = 3.08, 95% CI: 2.07-4.57, P < 0.00001). Conclusion: The prognostic role of PD-1 by TILs is variant in different tumor types, which highlights the role of PD-1 by TILs as a potential predictive and prognostic biomarker and the development of strategies against the PD-L1/PD-1 axis would be a promising therapeutic target for some solid tumors.


INTRODUCTION
Programmed cell death protein 1 (PD-1), a member of the CD28 receptor family, is expressed by activated lymphocytes and inhibits their proliferation functions after binding to PD-1 ligands such as PD-L1 [1] .The interactions with PD-1/PD-L1 signaling has been shown to improve clinical outcome and restore functional T-cell responses in several cancers [2] .
Although PD-1 has generated increasing interest as a target for immune modulation in cancers, the prognostic values of PD-1 expressed by tumor-infiltrating lymphocytes (TILs) in solid tumors were still unclear [3] .Several previous studies have reported the PD-1 by TILs is more than a predictive biomarker but as a worse prognosis marker in multiple solid tumors such as gastric cancer [4] , non-small cell lung cancer (NSCLC) [5] , renal cell cancer [6] and nasopharyngeal cancer [7] .Another studies showed that PD-1 expression is associated with favorable survival in breast cancer [8] , glioblastoma [9] , metastatic melanoma [10] , ovarian cancer [11] and primary human papillomavirus-positive head and neck cancers [12] .Furthermore, one study displayed that the positive expression of PD-1 expression is not correlated with overall survival (OS) for esophageal squamous cell carcinoma (ESCC) [13] .The different of tissue samples, detection methods and evaluation criterions might be partly responsible for the inconsistent results.
And with the development of PD-L1/PD-1 targeted therapy, some predictive and prognostic biomarkers are crucial to be identified for the option of individualized anti-PD-1 targeted treatment [14] .Therefore, we conducted this meta-analysis to comprehensively evaluate the prognostic value of PD-1 by TILs in solid tumors, which will further facilitate the development of PD-L1/PD-1 immune check-point targeted therapy and identify novel strategies targeting PD-1.

Publication searching
The eligible studies published in PubMed, Embase, Web of Science, CNKI and Wanfang databases were searched using the following keywords: "programmed cell death 1 receptor" or "PD-1" or "programmed death 1" or "CD279 antigen" and "cancer" or "tumor" or "neoplasm" or "carcinoma" and "prognosis" or "outcome" or "survival".In addition, we also manually screened the reference lists derived from randomized controlled trials and systematic review to avoid omitting related publications.The search language was limited to English and Chinese.

Inclusion and exclusion criteria
Inclusion criteria for this meta-analysis are: (1) full text available; (2) study focus on the association of PD-1 with clinicopathological parameters and OS; (3) cohort study, cross-sectional study or case-control study; (4) sufficient data or higher dots per inch of K-M survival curves.In addition, the exclusion criteria are as follows: (1) cell or animal studies; (2) case reports or review; (3) conference abstracts or comments; (4) repeated articles.

Data extraction and quality assessment
Two investigators (Liu RZ and Ku JW) independently extracted the data from the relevant studies.The disagreements were resolved by consensus.The extracted data are as follows: first author name, publication year, patient source, cancer type, number of patients, detection method, clinicopathological parameters, effect size, hazard ratio (HR) and 95% confidence intervals (CI).The quality of eligible studies were assessed through the Newcastle-Ottawa scale (NOS) method [15] .Study with NOS scores above to 6 point were usually considered to be higher quality.

Statistical analysis
All statistical analysis were conducted using the RevMan5.2 and STATA version 12.0 (STATA Corporation, College Station, TX, USA).HR and 95% CI were combined to assess the survival impact of PD-1 in solid tumors.For studies that offered only Kaplan-Meier curves, Engauge Digitizer (version 4.1) was performed to extract the survival data and calculate the estimated HRs and 95% CIs according to Tierney's method [16] .Additionally, pooled odds ratio (OR) and 95% CI were used to determine the association of PD-1 and clinicopathological features.

Characteristics of included studies
A total of 701 studies were identified by electronic search and 388 studies were excluded because of duplication.After reading the titles and abstracts, 221 studies were excluded and 92 possible full text studies were carefully reviewed.Finally, 10 manuscripts containing 12 retrospective cohort studies were included for quantitative analysis in the meta-analysis [Figure 1].The patients were diagnosed with various solid cancers including: ESCC, NSCLC, hepatocellular carcinoma, pancreatic cancer, breast cancer and ovarian cancer.The features of included studies were presented in Table 1.
To detect the expression of PD-1 by TILs, all studies used immunohistochemistry, except for 2 studies [22,23] , which used quantitative immunofluorescence, but the proportion of PD-1 expression was consistent with the others in that study.The detailed methodologies used to detect PD-1 are summarized in Table 2. Furthermore, 2 cohorts of patients were reported by Harter et al. [24] and Webb et al. [25] , respectively.PD-1 by TILs was assessed and the survival curves were reported independently, so they have been statistically analyzed as 4 individual studies.

PD-1 by TILs and overall analysis
A total of 12 studies with 1863 patients were enrolled in survival analysis.Seven studies with data on PD-1 positive expression and OS in solid tumors.There are 2 studies provided OS for breast cancer (2 cohort

PD-1 by TILs and clinicopathological parameters
The average positive expression rates of PD-1 by TILs were 31.35% in all of the studies.There were the higher PD-1 overexpression in NSCLC, ESCC and pancreatic cancer, with accounting for 35.71%, 61.23% and 50.01%, respectively.And PD-1 expression levels in melanoma, breast cancer and ovarian cancer ranged from 8.59% to 22.97%.
Four studies including 1209 tissue samples investigated the association of PD-1 overexpression with status of lymph node.With significant heterogeneity (P = 0.0008, I 2 = 82%), a random-effects model showed a    5B].We did not find the significant association of PD-1 with age, TNM stage or tumor invasion depth in solid tumor [Table 3].

Publication bias
Begg's and Egger's test were applied to evaluate the publication bias of the included studies.No obvious asymmetry was presented through the visual assessment of the Begg's funnel plots [Figure 6].Furthermore, the formal evaluation of Egger's test also failed to find the significant bias (P = 0.723).

Sensitivity analysis
Sensitivity analysis was conducted to justify the influence of individual study on the synthetic results of OS.The pooled HR was not significantly influenced after omitting any singly study for the effect of PD-1 expression on OS in our study [Figure 7].

DISCUSSION
PD-1, as one of the co-inhibitory receptors, plays an important role in cancer immunity equilibrium and immunity escape stages [26] .In the present study, we comprehensively assessed the association of PD-1   With respect to the tumor type, when we performed the subgroup meta-analysis stratified by tumor types, ovarian cancer was correlated with better survival for patients with high PD-1 levels rather than other solid tumor.Although PD-1 by TILs was not associated with OS for all of included studies in the metaanalysis [12,13,[22][23][24][25][26][27][28][29][30] .However, the results of studies using different clone to PD-1 antibodies were controversy in breast cancer [27,29] [Supplementary Figure 1] in our meta-analysis.One recent study reported the opposite results using variant PD-L1 antibodies in melanoma and lung cancer [14] .The difference of antibody clones, limited specificity, or distinct IHC protocols used may be partly explain the contradictory results [31] .Further studies are urgent to clarify the impact of antibodies on the results of studies.Another important finding in the present study is that patients with lymph node metastasis and tumor grade 3/4 have higher PD-1 by TILs than patients with non-lymph node metastasis and 1/2 tumor grade.
It is known that tumor grade and lymph node metastasis are usually major barriers to cancer treatment.And patients developed lymph node metastasis and tumor grade 3/4 have lower survival rates.To a certain extent, PD-1 by TILs may be contributed to the immunosuppression to aggravate the tumor growth and carcinogenesis, and further negatively affecting patients' survival.One study in clinical trials showed that PD-1-positive tumors tend to be more responsive to anti-PD-1 or anti-PD-L1 therapies [32] .It is reasonable to suggest that patients with lymph node metastasis and tumor grade 3/4 seem to be more sensitive to anti-PD-1 or anti-PD-L1 antibodies-based therapies.
Besides, PD-L1 expression state is another key point of PD-1/PD-L1-mediated tumor immune escape.In tumor tissues, PD-1 was mainly expressed by TILs, and PD-L1 was detected by both tumor cells and TILs [33] .PD-1 by TILs was significantly correlated with PD-L1 expressed by tumor cells [34,35] .Furthermore, the findings that PD-L1-positive TILs in cancer provides a suitable microenvironment for the development of tumor growth and treatment resistance, which was known to be mediated by the induction of activated IL-6 signaling [36,37] .Although immunotherapy using recombinant antibodies and vaccines, such as the therapies targeting PD-L1/PD-1, have been linked with prognosis and treatment response for a few solid tumors including a number of GI malignancies [38,39] , the expression of PD-L1 by CIK cells, TILs, and tumor cells within the tumor microenvironment remains to be elucidated.
Although the quality assessment of included studies is higher, there are still some limitations in the study.First of all, the quality of included studies is with selection bias due to the deletion of some unqualified literatures.Secondly, the screening of language is only English and Chinese and could not represent the whole population.Thirdly, the research objects are mainly cancerous tissues and the potential role of PD-1 in blood specimen remains unclear.Finally, the sample size in some of studies is small and further studies with larger sample size are still needed.
In conclusion, this meta-analysis demonstrates that PD-1 expressed by TILs is associated with lymph node metastasis and tumor grade in solid tumor.And more importantly, the prognostic role of PD-1 is variant in different solid tumors, which assumed that PD-1 by TILs seems to be a potential predictive biomarker and the development of strategies against the PD-L1/PD-1 axis would be a promising therapeutic target for some solid tumors.

Figure 2 .
Figure 2. Forest plots of PD-1 expression and OS in solid tumor patients.The squares and horizontal lines correspond to the study-specific HR and 95% CI.The area of the square reflects the study-specific weight.The diamonds represents the pooled OR and 95% CI.The solid vertical line is at the null value (HR = 1).The associations between positive or negative expression of PD-1 (A) and strong or moderate positive expression of PD-1 (B) with OS are shown.PD-1: programmed cell death protein 1; OS: overall survival; HR: hazard ratio; OR: odds ratio; CI: confidence interval

Figure 3 .
Figure 3. Forest plots for subgroup meta-analysis by patient source.The relationships between PD-1 overexpression and OS in Asia patients (A) and in non-Asia patients (B) are shown.PD-1: programmed cell death protein 1; OS: overall survival; CI: confidence interval

Figure 4 .
Figure 4. Forest plots for subgroup meta-analysis stratified by cancer type.The relationships between PD-1 expression and OS in breast cancer (A), ESCC (B) and ovarian cancer (C) are shown.PD-1: programmed cell death protein 1; OS: overall survival; ESCC: esophageal squamous cell carcinoma; CI: confidence interval

Figure 5 .
Figure 5. Forest plots of PD-1 expression and the clinical pathological parameters of patients with solid tumors.The squares and horizontal lines correspond to the study-specific OR and 95% CI.The area of the square reflects the study-specific weight.The diamonds represents the pooled OR and 95% CI.The solid vertical line is at the null value (OR = 1).The associations of PD-1 expression with lymph node status (A) and tumor grade (B) are shown.PD-1: programmed cell death protein 1; OR: odds ratio; CI: confidence interval expressed by TILs with OS in solid tumor and revealed that the prognostic role of PD-1 by TILs is variant in different solid tumor types.This study included 10 eligible publications with 12 cohort studies and a total of 1863 patients.To the best of our knowledge, this is the first systematic assessment of the association of PD-1 by TILs with OS in solid tumor.