PD-1 Expression Status on CD8+ Tumour Infiltrating Lymphocytes Associates With Survival in Cervical Cancer

Despite the expansion of PD-1 checkpoint blockade to multiple types of cancer, whether the programmed cell death 1 (PD-1) expression status on CD8+ tumour infiltrating lymphocytes (TILs) could be a prognostic factor in cervical cancer is still unclear. In this study, we performed ex vivo phenotypic analysis of PD-1 expression on CD8+ TILs by flow cytometry from 47 treatment-naïve cervical cancer patients. With a median follow-up of 26.1 months (95% confidence interval [CI], 24-28.2 months), we then linked the quantitative cellular expression results to progression-free survival and overall survival. Based on the intensity of PD-1 expression, we further categorised the cervical cancer patients into PD-1high expressers (29.8%, 14/47) and PD-1low expressers (70.2%, 33/47). Multivariate analysis revealed that PD-1high expressers are correlated with early recurrence (HR, 5.91; 95% CI, 1.03-33.82; P= 0.046). Univariate analysis also demonstrated that PD-1high expressers are associated with poor overall survival in cervical cancer (HR, 5.365; 95% CI, 1.55-18.6; P=0.008). Moreover, our study also demonstrated that CD8+/CD4+ TIL ratio and HPV infection status are risk factors for early relapse and mortality in cervical cancer patients. In conclusion, this study confirms that PD-1 expression status is an independent prognostic factor for progression free survival in cervical cancer. These findings could be important in predicting the relapse of cervical cancer as a cellular diagnosis method and could be important knowledge for the selection of prospective PD-1 blockade candidates.


INTRODUCTION
Targeting the pathways of immune checkpoint receptors (ICRs) on tumour infiltrating lymphocytes (TILs) and their ligands in the tumour microenvironment revolutionized the way we treat advanced stage cancers (1). With the approval of ipilimumab by FDA, representing the first checkpoint inhibitor for metastatic melanoma, the era of checkpoint blockade in cancer treatment has officially started (2). However, the application of CTLA-4 blockade is very limited due to the high grade adverse effects and non-specific immune activation it causes (3,4). It is thought that the benefits CTLA-4 inhibitor brings are proportional to the magnitude of immune tolerance it releases (5). PD-1/PD-L1 axis blockades demonstrate noteworthy benefits in treating multiple types of cancer including melanoma, head and neck cancer, bladder cancer, lung cancer and triple negative breast cancer with manageable adverse effects (6)(7)(8)(9)(10). Currently there are 3 approved PD-1 inhibitors, namely nivolumab, pembrolizumab and cemiplimab; and 3 approved PD-L1 inhibitors, atezolizumab durvalumab and avelumab for treating multiple types of cancer (11,12).
The research of PD-1/PD-L1 blockades in cervical cancer initiated from 2015 onwards, of which 4 studies publicized their results with 2 phase I trials, 1 phase II trial and 1 phase I-II trial. The overall response rate (ORR) reported from those checkpoint blockade studies targeting PD-1/PD-L1 axis ranged from 17% to 27% (13)(14)(15)(16). In 2018, FDA subsequently approved pembrolizumab for the treatment of advanced cervical cancer with disease progression during or after chemotherapy (11).
PD-1 and other ICRs expression on T-cells have contradictory roles in the immune regulation. On the one hand, it is shown that the increased PD-1 expression on T cells represents a more exhausted phenotype with impaired T-cell functionality against cancers or chronic virus infection (17,18). On the other hand, the upregulation of PD-1 and other ICRs is also linked to antigen experience, T-cell activation and T-cell differentiation (19,20). Moreover, PD-1 positive TILs has been suggested to be a favourable prognostic factor in HPV-associated head and neck cancer (21). Therefore, it is crucial to further explore the role of ICRs on T-cells in immune modulation and their potential application as biomarkers.
Little is known about the association between the expression of key ICRs on CD8+ TILs and prognosis in cervical cancer. We have previously described the dominant expression patterns among multiple ICRs on TILs and shortlisted PD-1 and Tim-3 as the key ICRs on TILs which may associate with clinical outcomes in multiple types of cancer (22). With the identification and characterization of PD-1 high and PD-1 low T cell subpopulations in the tumour from multiple cancer types including lung cancer, hepatocellular carcinoma and nasopharyngeal carcinoma, we believe that it is important to evaluate if the PD-1 expression status on CD8+ TILs associates with the recurrence or the overall survival of cervical cancer patients (23)(24)(25). Therefore, in this study, we hypothesize that (1) either the intensity of PD-1 expression (PD-1high VS PD-1low) or the frequency of key ICRs (PD-1 and Tim-3) on CD8+ TILs may have a prognostic value as a cellular biomarker in cervical cancer patients; (2) CD8+ and CD4+ TIL ratio is linked to the outcome of cervical cancer. Therefore, together with all the available clinical characteristics, we will conduct a systemic approach to determine what clinical factors or cellular markers on TILs can be used to predict the prognosis of cervical cancer patients.
Multichromatic Flow Cytometry Staining 6-color panels were designed for ex vivo phenotypic analysis. After the dissociation of tumour samples, TILs were each initially stained with LIVE/DEAD ® Fixable Aqua Dead Cell Stain Kit (ThermoFischer Scientific) for 20 mins before surface staining with conjugated antibodies in Fluorescence-activated cell sorting (FACS) washing buffer (Phosphate-buffered saline (PBS) with 0.5M Ethylenediaminetetraacetic acid (EDTA) and 7.5% Bovine serum albumin (BSA) solution) for another 20 mins and fixed with 1x CellFix solution (BD Biosciences). Commercial conjugated antibodies used include CD3-Alexa Fluor 700 (344822, Biolegend), CD4-FITC (345768, BD Biosciences), CD8-APC-Cy7 (560179, BD Biosciences), PD-1-BV650 (564104, BD Biosciences), Tim3-BV421 (345008, Biolegend). The antibody cocktails were tested in advance with or without the use of tumour dissociation solution to ensure proper function. Fluorescence minus one (FMO) controls were applied accordingly in order to properly position gates. Flow cytometry in this study was conducted using a 4-laser BD LSR Fortessa flow cytometer. Quality check of the cytometer lasers and fluidic system was conducted on a daily basis prior to experiments. After excluding dead cells and doublets, we then selected CD3+ TILs and further gated CD4+ and CD8+ TILs, respectively. Fluorescent minus One (FMO) was applied to facilitate proper gating. Antibodies were titrated by PBMCs from healthy donors in Weatherall Institute of Molecular Medicine, University of Oxford prior to the experiment. In order to ensure the quality of FACS data, we ruled out any tumour samples in which the viable CD3+ TILs were lower than 10,000 cells. Full gating strategy of the 6-color FACS panel was attached in Supplementary Table 1.

Defining High CD8+/CD4+ TIL Ratio Group and Low CD8+/CD4+ TILS Ratio Group
The ratio of CD8+ versus CD4+ TILs was analysed for all patients. Considering the sample size of this study, we ranked the CD8+/CD4+ ratio of all patients in the cohort and used the median CD8+/CD4+ ratio value as the cut-off to define the CD8+/CD4+ high ratio group and CD8+/CD4+ low ratio group. The range of CD8+/CD4+ TIL ratios in patients from this cohort is 0.72 ± 0.80.

HPV Detection and Genotyping
Clinical kit appointed by the Cervical Cancer Prevention Program in China was used for HPV detection and genotyping in our study. Cervical swab samples were collected during vaginal or colposcopy examination. Hybribio Female Sample Collection Kit (HBCK-F) was used to collect liquid based cytology specimens. 21 HPV GenoArray Diagnostic Kit (HBGA-21PKG) was applied to detect HPV and genotyping. Hybribio diagnostic kit is a Polymerase Chain Reaction (PCR) based test, which amplifies extracted HPV DNA from cervical samples. Amplicons are then hybridized with specific HPV probes in the kit following by immunoassay method to achieve colourimetric results. HPV genotyping in the kit includes 15 high risk types: HPV 16,18,31,33,35,39,45, 51, 52, 53, 56, 58, 59, 66 and 68 and 6 low risk types: HPV 6, 11, 42, 43, 44 and 81. 10 patients in our cohort were negative according to this kit test (negative for all 21 genotypes) when recruited in this study. 37 patients tested positive for high risk genotypes in our cohort. All patients were treatment-naïve prior to HPV testing.

Statistical Analysis
The PRISM 8 and SPSS 26 software were used for statistical analyses and graph plotting. Categorical variables were presented as absolute numbers and percentages, which were compared by Chi-square exact test for significance. Numeric variables were expressed as mean and standard deviation, which were compared by t test for significance. Overall survival was defined from the time of diagnosis in their first visit in Xinjiang Tumour Hospital to the recorded date of death. Progression free survival was defined from the date of receiving clinical treatments against cancer to the date of local or distant relapse confirmed by radiological examination. Survival rates were analysed by Kaplan-Meier method. The significance (0.05) of Kaplan-Meier curves was compared by log-rank test. Univariate and multivariate cox models were applied to analyse the prognostic factors (PD-1 status, CD8 VS CD4 TIL ratio, PD-1 expression level on CD8 TILs and Tim-3 expression level on CD8 TILs, tumour stage, tumour size, histological classification, HPV infection status and lymph node metastasis). Hazard ratios with a 95% confidence internal were calculated for the estimation of risk.

Identification of PD-1 high and PD-1 low Expressers
When analysing the frequencies and the expression pattern of PD-1 on CD8+ lymphocytes, subpopulations of PD-1+ CD8 TILs (PD-1 bright and PD-1 dim ) from tumour samples were observed in the FACS data from cervical cancer patients. We categorized all the cervical cancer patients in our cohort into 2 subgroups based on their distinct PD-1 expression status. PD-1 high expressers are defined as patients with higher frequency of PD-1 bright subpopulation on CD8+ TILs when compared to that of PD-1 dim subpopulation on CD8+ TILs ( Figure 1A). PD-1 low expressers are defined as patients with lower frequency of PD-1 bright subpopulation than that of PD-1 dim subpopulation on CD8+ TILs ( Figure 1B). The detailed PD-1 expression profiling on CD8 TILs in all cervical patients from our cohort is listed in Supplementary Figure 3, where the frequencies of PD-1-CD8 TILs, PD-1 dim CD8 TILs and PD-1 bright CD8 TILs are compared in each individual patient.

PD-1 high Expression Status Positively
Correlates With Lymph Node Metastasis 14 out of 47 cervical cancer patients are PD-1 high expressers, accounting for 29.8% of patients in our cohort. In contrast, 70.2% (33/47) of patients in the cohort are PD-1 low expressers. It is noticeable that among all the clinical characteristics listed in Table 1, it is more frequent for PD-1 high expressers (**P=0.004) to be diagnosed with lymph node metastasis (78.6%) compared to PD-1 low expressers (30.3%). The average age of cervical cancer patients in our study is 54.9, with no statistical difference (p=0.73) between PD-1 high expressers and PD-1 low expressers on the basis of age. The proportions of patients in our cohort with stage I, stage II and stage III cervical cancer are 14.9% (7/ 47), 40.4%(19/47) and 44.7%(21/47), respectively. The majority of patients (66%, 31/47) in our study were diagnosed with large tumours above 4cm in diameter, relative to 34% of patients with tumours less than 4cm in diameter. No statistical difference in cancer stage or tumour size was observed between PD-1 high expressers and PD-1 low expressers, although PD-1 high expressers have higher proportions of late tumour stage (64.3%) and large tumour size (85.7%) when compared to that of PD-1 low expressers (36.4% and 57.6%, respectively). In the category of histological stratification, no patients were diagnosed with welldifferentiated cancer type in our cohort. However, more than half of the patients were diagnosed with moderately-differentiated cancer type (59.6%, 28/47) compared to 40.4% (19/47) of patients with poorly-differentiated cancer type. The majority patients in our study have elevated squamous cell carcinoma antigen (SCC) level (83%, 39/47), of which no statistical difference was observed between PD-1 high expresser and PD-1 low expresser groups.

PD-1 high Expressers Have Significantly Higher Risk for Disease Relapse
The independent effect of PD-1 expression status (PD-1 high VS PD-1 low ) of CD8+ TILs on progression-free survival and overall survival was evaluated by Cox proportional hazards regression models adjusted for tumour stage, tumour size, lymph node metastasis, HPV infection status, tumour pathohistological   2 and 3). The 2-years overall survival was only 50% for patients with PD-1high expression status but was 90.9% for patients with PD-1 low expression status ( Figure 2B). Therefore, PD-1 expression status has a prognostic value to predict relapse in cervical cancer patients.

DISCUSSION
Several HPV-negative cervical cancer studies demonstrated that HPV-negativity in cervical cancer correlates with poor diseasefree survival and overall survival when compared to that of HPVpositive cervical cancer (26)(27)(28). However, these studies are based on Caucasian populations; little information between HPV infection status and prognosis of cervical cancer in Chinese population is known. Our study confirmed that HPVnegative cervical cancer patients have a higher risk for recurrence and shorter overall survival when compared to HPV-positive cervical cancer patients in China. Of note, in our cohort we did not observe increased frequency of adenocarcinoma in HPVnegative cervical cancer patients when compared to HPVpositive cervical cancer patients, which has been shown in previous research (29). In fact, the histological classification of all the HPV negative cervical cancer patients is squamous cell carcinoma and only 2 HPV positive patients are classified as adenocarcinoma in our cohort ( Table 1).
Through ex vivo flow cytometric data obtained prior to clinical interventions of all the patients in the cohort, we have also investigated if the CD8+/CD4+ TIL ratio is a risk factor for cervical cancer. Our results indicated that increasing CD8+/CD4+ TIL ratio is a favourable prognostic factor, correlating with improved overall survival and delayed recurrence in cervical cancer. This result is in line with multiple previous studies in breast cancer, nasopharyngeal cancer, ovarian cancer and cervical cancer (30)(31)(32)(33). It implicates that CD8+ TILs have a prominent role in controlling the progression of disease, with positive impact on overall survival, whereas the high percentage CD4+TILs in the tumour microenvironment may contribute to poor prognosis. Therefore, CD8+/CD4+ TIL ratio through biopsy can be a useful marker in predicting the outcome of cervical cancer and tuning the ratio of CD8+/CD4+ TILs by clinical intervention such as chemotherapy or radiotherapy may improve survival for patients. It is reported that in a colorectal cancer study that Naïve Tregs (CD3+CD4+FOXP3 low CD45RA + ) and effector Tregs (CD3+ CD4+OXP3 high CD45RA − ) have immunosuppressive activity whereas non-Tregs (CD3+CD4+FOXP3 low CD45RA − ) have antitumour activity (34). However, our study only compared the bulk CD8+/CD4+ TILs in cervical cancer and is unable to distinguish Fr-III from bulk CD4+ TILs.
In the application of immune checkpoint receptor blockades, initially, it was thought that the increased frequency of ICRs such as PD-1 on TILs may be useful markers to grade the magnitude of T-exhaustion (35). However, the upregulation of PD-1 expression level is not just linked to T-cell exhaustion but is also associated with T-cell activation and T-cell differentiation in the cancer microenvironment and in the presence of chronic virus infection (36)(37)(38)(39). This may partially explain why there is no association between the frequency of Tim-3 or PD-1 on CD8+ TILs and the recurrence or mortality of cervical cancer. Furthermore, we also investigated whether PD-1 expression has The age, tumour stage, tumour size, cancer differentiation stage, histological classification, HPV status, lymph node metastasis and treatment options are compared between PD-1 high expressers and PD-1 low expressers. P value of numeric variable (age) was calculated by t test and P value of categorical variable was calculated by chi square test. In the comparison of age between 2 groups of patients (PD-1 high and PD-1 low expressers), mean and standard deviation were calculated.   the potential to serve as a prognostic or a predictive biomarker for the recurrence and mortality of cervical cancer patients since no previous research on PD-1 expression status on T cells and its correlation with survival was reported in cervical cancer studies. Most of the PD-1 expression-related cancer research was conducted on paraffin-embedded samples, which may obscure its potential correlation with disease relapse or overall survival of cancer patients due to the difficulties of PD-1 expression quantification on paraffin blocks (40,41). In a liver cancer study, PD-1 high CD8+ TILs have been reported to exhibit a differential gene expression profile with a more exhausted T-cell functionality when compared to PD-1 low CD8+ TILs. However, PD-1 high CD8+ TILs are more functionally restored with a favourable response to PD-1 blockade compared to PD-1 low CD8+ TILs (23). In an HPV-associated nasopharyngeal cancer study, it is suggested that PD-1 high CD8+ TILs are associated with worse-disease free survival, and that HPV infection status may be a factor to determine the percentage of PD-1 high CD8+ TILs in NPC; a higher frequency of PD-1 high CD8+TILs with impaired anti-tumour functionality in HPV-tumours was detected when compared to that of HPV+ tumours (24). Based on PD-1 expression status on peripheral CD8 T cells, a recent/newly-published study in cervical cancer sub-divided patients into PD-1 high , PD-1 int and PD-1 low subgroups and found significantly increased frequencies of PD-1 high and PD-1 int but not PD-1 low on CD8 T cells from cervical cancer patients when compared to healthy donors (42). These previous results in combination with our findings that PD-1 high expressers associate with early relapse in cervical cancer may indicate PD-1 expression status on CD8+ TILs not only has a prognostic value in disease recurrence but also may serve as a criterion to select patients for PD-1 blockade and cellular diagnosis.
To our knowledge, this is the first study analysing the correlation between PD-1 expression status on CD8 TILs and survival rates in cervical cancer patients. Indeed, our study demonstrated that PD-1 expression status, but not the frequency of PD-1 on CD8+ TILs, is an independent prognostic factor for progression free survival. PD-1 expression status may potentially be an independent prognostic factor for overall survival in cervical cancer as well since we observed that PD-1 high expressers have significantly higher mortality risk via the univariate analysis but not multivariate analysis.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by ethics committee of the Third Affiliated Hospital of Xinjiang Medical University. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
TD, XL, and RZW: study design. PWF, YNF, XL, MMM, YPG, DND, HCC, YCP, and XY: data acquisition. XL, PWF, DND, and YPG: data analysis. XL, TD, and RZW: paper writing and edition. All authors contributed to the article and approved the submitted version.