Clinical Signicance of Circulating Exosomal PD-L1 and Soluble PD-L1 in Nasal-type NK/T-cell Lymphoma Patients Receiving VIPD Containing Chemotherapy

Background: Several studies showed that circulating exosomal PD-L1 was associated with immunosuppression and prognosis in cancer patients. However, its prognostic value in extranodal NK/T cell lymphoma (ENKTCL) was poorly understood. Methods: We retrospectively evaluated the prognostic value of pretreatment circulating PD-L1+ exosomes and soluble PD-L1 in the plasma of ENKTCL patients treated with VIPD-containing chemotherapy. Results: A total of 107 ENKTCL patients, including 101 early stage and 6 advanced stage patients were enrolled in our study. Most patients were middle-aged and young male adults, with a male to female ratio of approximately 3.7:1. The 5-year progression-free survival (PFS) and 5-year overall survival (OS) rates were 65.2% and 85.7% for the whole cohort, respectively. For early-stage patients, the L/P-VIPD group had better 5-year PFS than the VIPD group (73.6% vs. 53.7%, P=0.031). Compared with the healthy individuals (n=16), the patients with ENKTCL exhibited signicantly elevated exoPD-L1 and sPD-L1 levels in the blood. Five-year PFS and OS rates were signicantly lower in the high exoPD-L1 and sPD-L1 group than in the low exoPD-L1 and sPD-L1group. However, no correlation was found between circulating exoPDL1 and sPD-L1 levels (P=0.12, r=0.45). Conclusions: Our results demonstrated that VIPD-containing chemotherapy combined with radiotherapy was a promising regimen in ENKTCL patients. Circulating exoPD-L1 and sPD-L1 levels were signicantly elevated in ENKTCL and might be potential biomarkers for predicting the survival outcomes of ENKTCL patients. The Simoa HD-1 Analyzer is a digital immunoassay technology used for the detection of various proteins that has signicantly higher sensitivity than the ELISA technique[20]. CD63 is one of the most important tetraspanins enriched on the exosomal surface and is considered a specic marker for exosomes[21]. Here, we used two markers, CD63 and PD-L1, to detect PD-L1 expressed on exosomes in the plasma by Simoa. antibody for Simoa setup Detection of PD-L1-positive performed on a HD-1 an

This study evaluated the prognostic value of plasma exoPD-L1 and soluble PD-L1 (sPD-L1) levels in ENKTCL patients and was conducted at the Fudan University Shanghai Cancer Center. The inclusion criteria of the current study were as follows: 1) de nite diagnosis of ENKTCL according to the World Health Organization (WHO) classi cation of lymphoid neoplasms; 2) the primary tumor site localized in the upper aerodigestive tract; 3) no prior treatment with detailed clinical information and follow-up data available; and 4) an Eastern Cooperative Oncology Group performance status (ECOG PS) score between 0-2. Patients with concomitant malignant tumors or severe organ dysfunction were excluded. This study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committees of the Fudan University Shanghai Cancer Center, and all the participants provided written informed consent.

Evaluation and treatment
The patients in our cohort were staged based on the Ann Arbor staging system. VIPD was administered in a three-week VIPD or L/P-VIPD combined with involved-eld radiotherapy (IFRT) was delivered as the rst-line treatment in early-stage patients. All advanced-stage patients received VIPD chemotherapy, and IFRT was a palliative or salvage therapy after failure of primary chemotherapy. Patient responses were evaluated according to the revised response criteria for malignant lymphoma [17]. All patients in our cohort underwent positron emission tomography-computed tomography (PET/CT) or magnetic resonance imaging (MRI) and/or computed tomography (CT) before treatment, after every two cycles of chemotherapy and after treatment.
Exosome and peripheral blood mononuclear cell (PBMC) isolation from the blood Plasma samples of 500 µl was thawed on ice. Exosomes was then isolated by using exoRNeasy Plasma Midi Kit (Qiagen, #77044). PBMCs were isolated from whole blood with density gradient medium according to the manufacturer's instructions (Lymphoprep™, STEMCELL Technologies, #07851).
Detection of exoPD-L1 in the plasma based on a single-molecule array (Simoa) The Simoa HD-1 Analyzer is a digital immunoassay technology used for the detection of various proteins that has signi cantly higher sensitivity than the ELISA technique [20]. CD63 is one of the most important tetraspanins enriched on the exosomal surface and is considered a speci c marker for exosomes [21]. Here, we used two markers, CD63 and PD-L1, to detect PD-L1 expressed on exosomes in the plasma by Simoa.
Preparation of the Simoa Simoa homebrew kits for the the detection of extracellular vesicles (EVs) were prepared according to the manufacturer's guidelines. In brief, a capture anti-PD-L1 antibody was diluted to a concentration of 0.2 mg/mL with Bead Conjugation Buffer (Quanterix), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Thermo Fisher Scienti c, Waltham, MA, USA) was used to activate paramagnetic carboxylated microparticles (Quanterix). Then, 3 µL of biotin solution (5.2 µg/µl) was added to a detection antibody solution (100 µL, 1.0 mg/mL) to start the biotinylation reaction. The recovered anti-CD63 antibody was adjusted to a concentration of 0.2 mg/mL, and the beads were stored at 4 °C. Finally, an assay for detecting PD-L1-positive exosomes via PD-L1-CD63 was developed.
Simoa setup Detection of PD-L1-positive exosomes was performed on a novel automated Simoa HD-1 Analyzer (Quanterix). Microscopic magnetic beads coated with an anti-PD-L1 capture antibody were diluted to 500,000/test, and an anti-CD63 detection antibody was adjusted to a working concentration of 0.3 µg/mL. Streptavidin-β-galactosidase (SBG) was diluted to a working concentration of 150 pM with SBG Diluent (Quanterix). The detection process was a three-step protocol. First, 25 µL of microscopic bead (coated with the anti-PD-L1 capture antibody) solution was incubated in a 100-µL sample for 45 min, followed by three wash steps. Then, 100 µL of anti-PD-L1 detection antibody was incubated with the microscopic beads for 5 min and 15 s. Finally, 100 µL of SBG was added and incubated for 5 min and 15 s. An RGP substrate solution (20 µL) was mixed with the microscopic beads and loaded into the Simoa disk array. The array was sealed with oil after loading, and the microscopic beads were imaged. Automatic analysis was performed with HD-1 Analyzer software (Quanterix), and the Simoa signal was present in AEB.
Detection of sPD-L1 and exoPD-L1 sPD-L1 in the blood was detected using a commercial ELISA kit (R&D Systems, # DB7H10). Serum samples were available for 96 ENKTCL patients and were measured in accordance with the manufacturer's instructions. For the detection of exoPD-L1, a total of 100 µl of isolated exosomes was added to each well and then tested following the instructions. The minimum detectable level of sPD-L1 was 1.51 pg/ml. Standards and all the samples were tested in duplicate.

Statistical analysis
Overall survival (OS) was de ned as the time from the date of diagnosis to the day of last follow-up or death. Progression-free survival (PFS) was de ned as the time from the day of diagnosis to the time of rst progression, last follow-up or death. Survival analysis was performed using the Kaplan-Meier method, and the log-rank test was used to compare survival curves. Signi cant factors (P value less than 0.05) in univariate analyses were further examined by multivariate analysis with Cox regression. The prognostic cutoff points of plasma exoPD-L1, sPD-L1 and PET/CT SUVmax were determined by X-tile, and the most discriminant threshold for PFS was selected. Spearman correlation analysis was employed to identify correlations between variables. Table 1 summarizes patient clinical characteristics. A total of 107 patients with newly diagnosed nasal-type ENKTCL including 101 early-stage and 6 advanced-stage patients were enrolled in our study. Most patients in our cohort were middle-aged and young male adults, with a median age of 44 years (range, 17-76 years) and a male to female ratio of approximately 3.7:1. A majority of our patients had normal LDH levels (n = 91), low International Prognostic Index (IPI) scores (≤ 1, n = 97), low prognostic index of natural killer cell lymphoma (PINK) scores (≤ 1, n = 96) and early-stage disease (n = 96). B symptoms were present in over half of the patients (n = 55) in our cohort. The primary tumor sites included the nasal cavity (n = 103) and nasopharynx (n = 4). Pretreatment PET/CT was performed in 51 patients, with a median SUVmax value of 9.7 (range 3.2-25.1). Ki-67 was highly expressed in most patients, with a wide range of distribution from 5-99% and a median value of 60%. For early stage patients, there is no obvious difference between the baseline clinical characteristics for VIPD group and L/P-VIPD group. Evaluation of e cacy and survival outcomes

Patient characteristics
Of the 101 patients with early-stage ENKTCL, VIPD was used to treat 34 patients, and L/P-VIPD was used to treat 67 patients. All the advanced-stage patients (n = 6) received VIPD chemotherapy. The complete response (CR) rate and objective response rate (ORR) for the whole cohort after treatment were 76.2% and 95.2%, respectively, whereas three patients developed progressive disease (PD). Thirty patients (30.8%) relapsed (n = 30, 28.0%) or developed PD (n = 3, 2.8%) during the follow-up period.

Pretreatment exoPD-L1 levels and their correlations with clinical features
Exosomes from ENKTCL patient were isolated by exoRNeasy Plasma Midi Kit. As shown in Fig. 2A, the TEM image revealed that exosomes were 30-100 nm intact vesicles. Flow NanoAnalyzer showed that the isolated exosomes were approximately 50-250 nm in diameter, with a median value of around 70 nm (Fig. 2B). Western blotting demonstrated that some speci c biomarkers for exosomes, such as TSG101, ALIX, Hsp90α/β and CD63, were enriched in the isolated exosomes (Fig. 2C). Western blot analysis established that plasma exoPD-L1 level was signi cantly higher in ENKTCL patients than in healthy subjects (Fig. 2D). In accordance with the above result, the exoPD-L1 level detected by simoa was also obviously higher in the ENKTCL patients (Fig. 2E).
To validate the speci city of simoa, the plasma exoPD-L1 level was tested by both simoa and commercial ELISA method. A relatively good linear relationship between the simoa signal and the exoPD-L1 concentration detected by ELISA was obtained (Fig. 2F). Therefore, 99 ENKTCL patients and 16 healthy subjects with available plasma was further tested by simoa. In our study, we found that pretreatment plasma exoPD-L1 levels were signi cantly higher in our patients than in healthy subjects (P < 0.001) (Fig. 2G). Receiver operating characteristic curve (ROC) showed that plasma exoPD-L1 provides an excellent diagnosis accuracy (AUC = 0.9968) to differentiate the ENKTCL patient (n = 99) and healthy control groups (n = 16) (Fig. 2H). The optimal prognostic cutoff point for the pretreatment plasma exoPD-L1 level measured by simoa was 1.2 (around 43.0 pg/ml) (X-tile, Supplementary Fig. 1) and our cohort was classi ed into two groups based on this value. To investigate the correlation between plasma exoPD-L1 and prognostic factors, pretreatment exoPD-L1 levels were compared based on the clinical parameters of our cohort (Table 2). Compared with those in the low plasma exoPD-L1 group (simoa signal < 1.2), the patients in the high plasma exoPD-L1 group were more likely to have a high SUVmax level and a higher recurrence rate (Table 2). However, no correlations were observed between the plasma exoPD-L1 level and age, sex, disease stage, the B symptom status, the LDH level, the IPI score or the PINK score. The pretreatment plasma exoPD-L1 levels of patients with a higher SUVmax were signi cantly higher than those of patients with a lower SUVmax level (P = 0.003) (Fig. 2I). Prognostic value of plasma exoPD-L1 in ENKTCL The prognostic signi cance of plasma exoPD-L1 was evaluated in our cohort. Patients with a high pretreatment plasma exoPD-L1 level had worse 5-year PFS (35.7% vs. 86.1%, P = 0.007) and OS (56.0% vs. 88.1%, P = 0.012) than those with a low pretreatment exoPD-L1 level (Fig. 2J, 2K).
Correlations between clinical features and pretreatment sPD-L1 levels sPD-L1 concentrations in the serum were measured in ENKTCL patients and healthy subjects. The sPD-L1 level was signi cantly higher in the ENKTCL patients than in the healthy individuals (P = 0.16, r = 0.02). The optimal cutoff point for the pretreatment sPD-L1 level was 219.0 pg/ml (X-tile, Supplementary Fig. 1).
The correlation between plasma exoPD-L1 and sPD-L1 levels was also explored in our cohort. Interestingly, our results demonstrated that the baseline exoPD-L1 level was not associated with the sPD-L1 level in the blood (P = 0.12, r = 0.45) (Fig. 3H).
For stage I patients (n = 47), subgroup analysis demonstrated that a high sPD-L1 level was an adverse factor affecting the survival outcome. The 5-year PFS and OS rates were 71.6% and 93.9%, respectively, for patients with a low level of sPD-L1 (< 219 pg/mL, n = 8), while the rates were 31.3% (P = .0022) and 65.6% (P = 0.0082), respectively, for patients with a high pretreatment sPD-L1 level (≥ 219 pg/mL, n = 39).

Discussion
ENKTCL is a distinct subtype of lymphoma characterized by frequent EBV infection and a propensity for midline facial tissue involvement, with a higher rate of incidence in Asia than in Western countries [1]. VIPD is one of the standard treatments recommended by the National Comprehensive Cancer Network (NCCN) guidelines for the treatment of earlystage ENKTCL. In the present study, we reported the clinical e cacy of VIPD-containing regimens in ENKTCL with the largest cohort to date. In addition, we demonstrated for the rst time that plasma exoPDL1 was an independent prognostic factor in ENKTCL patients. We also found that exoPD-L1 was not correlated with sPD-L1 in the blood of ENKTCL patients. The above results indicate that exoPD-L1 may contribute to immune evasion and cancer development.
VIPD is an effective and well-tolerated treatment regimen for early-stage ENKTCL patients [8,22]. A phase II study consisting of 30 stage I/II ENKTCL patients demonstrated that 100% of patients achieved a response to VIPD chemotherapy, with 3-year PFS and OS rates of 85.19% and 86.28%, respectively [22]. The response rate of VIPD in our cohort was similar to the rate found in the above study, whereas survival outcomes were even better in our study of earlystage ENKTCL patients, which reported a 5 year PFS of 53.7% and 5 year OS of 82.5%. The relatively small sample size and the majority of the enrolled patients in the VIPD group being low risk may partly explain these results.
As lymphoma cells lack L-asparagine synthetase and are unable to synthesize the essential amino acid asparagine, Lasparaginase and Peg can exert anticancer effects by hydrolyzing serum asparagines in ENKTCL patients. Some Lasp/Peg based regimens such as GELOXD, SMILE and DDGP have been developed in the last decade and have been proven to be effective in ENKTCL patients [1,7,23]. In this study, L/P-VIPD resulted in excellent survival outcomes, with 5year PFS and 5-year OS rates of 73.6% and 88.7%, respectively, consistent with the results of a previous study [8]. Compared with VIPD, the combination of L-asp/Peg could improve PFS in early-stage patients, indicating that L-asp/Peg treatment may play a key role in the prevention of recurrence.
Blockade of the PD1/PD-L1 pathway is considered a promising therapeutic approach in non-Hodgkin lymphomas, such as ENKTCL and peripheral T cell lymphoma [24]. Clinical trials have reported that PD-1 inhibitors are effective in patients with relapsed/refractory NK/T cell lymphoma and have achieved an overall response rate of 57%-100% [25,26]. However, previous work predominantly focused on the expression of PD-L1 on tumor cells, and the role of exoPD-L1 has not been determined. In our study, we found that the level of PD-L1 + exosomes was signi cantly higher in the plasma of ENKTCL patients than in that of healthy individuals. Consistent with previous reports [27,28], our data showed that the detection of exoPD-L1 could also differentiate ENKTCL patients from a healthy population with high sensitivity and speci city, indicating that the plasma exoPD-L1 level is a potential marker for cancer screening and diagnosis of patients with suspected malignancy.
ExoPD-L1 can mediate immune evasion in various cancers, such as melanoma and head and neck cancer [14,28]. Our study proved hat abundant exoPD-L1 was present in the plasma of ENKTCL patients and that lymphoma cells might actively release these exosomes to create an immunosuppressive microenvironment to support cancer development. A high level of exoPD-L1 has been demonstrated to be associated with adverse clinicopathologic features, including the disease stage, in various cancers [14]. Our data demonstrated that a high level of plasma exoPD-L1 was associated with an elevated baseline SUVmax. PD-L1 expression is associated with glucose metabolism in various cancers, such as lung cancer and cervical cancer [29,30]. The SUVmax in PET/CT was signi cantly higher in the high exoPD-L1 group and high sPD-L1 group, possibly because lymphoma cells may release PD-L1 to promote glycometabolism.
ExoPD-L1 can also serve as a biomarker for response evaluation and prognosis assessment in some cancers, such as melanoma and head and neck cancer [14,28]. Our research also showed that a high level of plasma exoPD-L1 could predict patient response and prognosis. High expression of plasma exoPD-L1 in patients was associated with a poor response and relatively poor prognosis. These studies indicate that exoPD-L1 may be a promising biomarker for tumor progression.
Previous studies have reported the clinical signi cance of sPD-L1 in non-Hodgkin lymphoma, such as diffuse large B cell lymphoma and NK/T cell lymphoma [24,31,32]. The correlation between the pretreatment sPD-L1 level and the clinical features are controversial [24,31]. Our study demonstrated that a high baseline sPD-L1 level was correlated with some adverse clinical characteristics, including an advanced stage, an elevated LDH level, B symptoms, a high IPI score and a high PINK score.
The predictive value of pretreatment sPD-L1 has been assessed in previous studies [24,31]. Patients with a high concentration (> 3.23 ng/L, n = 34) of sPD-L1 were found to have signi cantly worse PFS (3-year PFS 85.0 vs. 25.6%, P < 0.001) and OS (3-year OS 51.7 vs. 91.3%, P < 0.001) than those with a low concentration (≤ 3.23 ng/L, n = 63) [31]. Consistent with this study, our research found that a high pretreatment level of sPD-L1 was an important prognostic factor for PFS and OS, indicating that the baseline sPD-L1 level is a potential prognostic biomarker in ENKTCL patients.
The correlation between exoPD-L1 and sPD-L1 levels in the blood was investigated in a previous study [14]. In this study, no correlation between the exoPD-L1 and sPD-L1 levels in the plasma was observed in the head and neck cancer patients. Although both plasma exoPD-L1 and sPD-L1 were associated with a relatively poor prognosis in our study, there was no relationship between these two variables in our patients. The following reasons may explain the above results: 1) PD-L1 on exomes was more stable than sPD-L1 in the plasma; 2) the cell sources of exoPD-L1 and sPD-L1 might be different, and 3) the production and secretion of sPD-L1 by cells was independent of the process of exosome biogenesis.
Several other risk factors were also identi ed in our study, including the LDH level, baseline SUVmax, IPI and PINK. 18 F-FDG PET/CT is a standard imaging technology used to stage disease, evaluate responses and monitor for relapse in lymphoma patients [33]. The baseline SUVmax in PET/CT is an important prognostic factor in lymphoma [34,35]. Our results support previous observations that the baseline SUVmax correlates with prognosis in ENKTCL [35]. The PINK score represents a new model used to predict outcomes in ENKTCL patients treated with non-anthracycline-based therapies, and this model was con rmed in subsequent studies [7,36]. Our research assessed the impact of the PINK on prognosis and found that a high PINK score was associated with a worse outcome.
In conclusion, our results revealed that VIPD-containing chemotherapy was an effective regimen for patients with nasaltype ENKTCL and that abundant PD-L1 was enriched on exosomes derived from patient plasma. In addition, high levels of circulating exoPD-L1 and sPD-L1 were important predictors of adverse survival outcomes, and they could serve as valuable diagnostic and prognostic biomarkers for ENKTCL patients.