Relationship between PD-L1 expression and 18F-FDG uptake in gastric cancer

Purpose: Immunotherapy has been successfully utilized for treatment of gastric cancer, so the identification of clinicopathologic features that are predictive of response to this therapy is crucial. 18F-FDG PET/CT can provide information on the molecular phenotype of many malignant tumors. The correlation between 18F-FDG accumulation and PD-L1/PD-L1-TILs status in gastric cancer patients has not been investigated. The aim of the current study is to assess whether 18F-FDG accumulation is associated with PD-L1/PD-L1-TILs status, and whether 18F-FDG PET/CT may be useful for predicting PD-L1/PD-L1-TILs expression of gastric cancer. Results: Tumors with positive PD-L1 expression had higher SUVmax than in tumors with negative PD-L1 expression (15.0 ± 8.0 vs. 7.2 ± 4.2, respectively; P = 0.004). Tumors with positive PD-L1-TILs expression also had higher SUVmax than in tumors with negative PD-L1-TILs expression (10.3 ± 6.5 vs. 6.6 ± 3.7, respectively; P = 0.034). Multivariate analysis suggested that SUVmax remained significantly correlated with the status of PD-L1 (P = 0.043) and PD-L1-TILs (P = 0.016). PD-L1 expression was predicted with an accuracy of 67.2% when a SUVmax value of 8.55 was used as a cutoff point for analysis. Similarly, PD-L1-TILs expression was predicted with an accuracy of 64.2%, when a SUVmax value of 7.9 was used as the threshold for analysis. Conclusion: Higher 18F-FDG accumulation in gastric cancers is correlated with positive PD-L1/PD-L1-TILs expression. 18F-FDG PET/CT may be used to predict the status of PD-L1/PD-L1-TILs and thus aid in optimal treatment decision. Methods: A retrospective analysis was conducted on 64 patients with gastric cancer who underwent 18F-FDG PET/CT. SUVmax was calculated from the 18F-FDG accumulation of the primary tumor. The relationship between SUVmax and PD-L1/PD-L1-TILs status was analyzed.


INTRODUCTION
Gastric cancer is the fifth most frequent malignant tumors worldwide [1]. Surgery is still the major treatment for gastric cancer. However, gastric cancer patients are often diagnosed with inoperable or metastatic disease, and treatment outcomes for such patients remain poor. Therefore, it is essential to identify and develop effective therapeutic regimens for these patients. Despite the use of Trastuzumab for patients with positive HER2 expression and targeting VEGFR2 leading to improved survival [2][3][4], there is still a considerable number of patients who are unresponsive to treatment. Immunotherapy has become interesting in many malignant tumors, and the PD-1/PD-L1 pathway is the main mechanism underlying immunotherapy [5,6]. Immunotherapy with anti-PD-L1 blocker is seen as an effective therapeutic approach for many malignant tumors. Recently, pembrolizumab was approved to use in gastric cancer patients with metastatic or recurrent locally lesions and high PD-L1 expression in the USA [7]. Many studies have suggested that gastric cancer patients with high PD-L1 expression show an elevated overall response rate than those lacking PD-L1 expression [8]. Teng MW et al. showed that TIL positive/PD-L1 positive pattern and TIL positive/PD-L1 negative pattern can be regarded as "Hot tumor" which can expect therapeutic effect from PD-1 targeted therapy combined with or without another chemotherapy [9]. Recent Phase 3 clinical trials (KEYNOTE-061) concluded the case with PD-L1 combined positive score (CPS) >10 had clinically significant results by the first line PD-L1 targeted therapy in a patient with unresectable advanced gastric cancer or recurrent gastric cancer [10]. It is therefore meaningful to identify useful clinicopathologic feature in gastric cancer patients to predict PD-L1 expression. However, so far there are no validated clinicopathologic characteristics to select a priori patients who may benefit from immunotherapy in gastric cancer. 18 F-FDG PET/CT is a noninvasive method to detect malignant tumors [11][12][13]. Our previous studies suggested that 18 F-FDG PET/CT could be useful for predicting molecular phenotype in several malignant tumors, including LDHA in lung cancer and FBP1 expression in hepatocellular carcinoma [14,15]. However, the relationship between 18 F-FDG accumulation and PD-L1 status and the underlying molecular mechanisms are still unclear in gastric cancer patients.
In current study we assessed whether the PD-L1 status of tumor cells (PD-L1) or PD-L1 status of tumor infiltrating lymphocytes (PD-L1-TILs) is correlated with 18 F-FDG accumulation. We also assessed whether 18 F-FDG PET/CT has the potential to predict PD-L1/PD-L1-TILs status in gastric cancer. So far, our study is the first to deliver data of 18 F-FDG PET/CT for predicting PD-L1/PD-L1-TILs expression, as well as to demonstrate that 18 F-FDG PET/CT has a great effect on determining optimal treatment methods by predicting response to immunotherapy in gastric cancer patients.

Study population
Patients' clinicopathologic features are shown in Table 1. Among the 64 cases, 50 were treated with total or subtotal gastrectomy with lymphadenectomy, and 14 were treated with chemotherapy. 12 patients had well/ moderately differentiated adenocarcinoma, 39 patients had poorly differentiated adenocarcinoma, 9 patients had signet-ring cell carcinoma, the other 4 patients were

Correlation between clinicopathologic characteristics and PD-L1/PD-L1-TILs status
Patients were separated into two groups on the basis of PD-L1/PD-L1-TILs status. The relationship between clinicopathologic characteristics in patients treated with gastrectomy and PD-L1/PD-L1-TILs expression were evaluated (n=50) ( Table 2). No significant differences in gender, tumor location and size, vascular invasion, T stage, N stage， distant metastasis， histologic type were observed between PD-L1-positive and PD-L1-negative groups. Whereas, the SUVmax of the primary tumors was significantly different between these two groups. Similar correlations were also observed for PD-L1-TILs expression ( Table 2).
In the multivariate analysis including factors with a P value of 0.2 or less, only the SUVmax of primary tumors remained significantly associated with PD-L1 status [

DISCUSSION
Immune checkpoint blocker has been widely used for treatment of metastatic or recurrent advanced gastric cancer [7]. The status of PD-L1 is being explored as a predictive marker for response to anti-PD-L1 blocker [8,16]. Detecting PD-L1 expression is now common in the management of gastric cancer. 18 F-FDG PET/CT is a noninvasive diagnostic tool to detect malignant tumors. Though several studies have suggested the association between SUVmax and PD-L1 status in lung cancer [17,18], and our previous study have demonstrated the association between SUVmax and the status of PD-L1 in bladder cancer [19], but possible underlying mechanisms are still unclear. In the current study we demonstrate that gastric cancers with positive expression of PD-L1/PD-L1-TILs had higher SUVmax compared with those lacking PD-L1/PD-L1-TILs expression. To our knowledge, this is the first study that analyzes the correlation between 18 F-FDG accumulation and PD-L1/PD-L1-TILs status in gastric cancer patients.
Immunotherapy was widely used for treating malignant tumors [7,20,21]. However, the clinicopathologic characteristics of patients correlated with response to immune checkpoint blocker are still unknown, and selecting the patients who are possible to achieve response from targeting PD-L1 and excluding those who are unresponsive to the immunotherapy is still an important question. The status of PD-L1/PD-L1-TILs was often assessed by immunohistochemistry analysis [22]. Whereas, tumor tissue obtained by gastroscopy or surgical resection are invasive. For these reasons, other noninvasive methods, such as 18 F-FDG PET/CT, which could predict the expression of PD-L1/PD-L1-TILs and inform optimal treatment decision with anti-PD-L1 antibodies would be of important clinical value in gastric cancer patients.
In our study we discovered a positive association between SUVmax and PD-L1/PD-L1-TILs status in gastric cancers. The ROC curves analysis demonstrated that 18 F-FDG accumulation of primary tumors could be useful for predicting PD-L1/PD-L1-TILs status. Multivariate analysis revealed that SUVmax was the only significant predictor of PD-L1/PD-L1-TILs status in gastric cancers. However, the molecular mechanism of association between 18 F-FDG accumulation and PD-L1/PD-L1-TILs status is still unclear. HIF1α played a key role in regulating 18 F-FDG accumulation of tumor cells [15,23]. In addition, HIF-1α was a transcription factor of PD-L1 and could upregulate PD-L1 expression [24]. These studies demonstrated that the positive association between 18 F-FDG accumulation and PD-L1/PD-L1-TILs expression may be a reflex of the HIF-1α activation. Pearce EL et.al [25] show that PD-L1 blockade by the PD-L1 antibody could significantly inhibit the AKT pathway, leading to the suppressed translation of glycolytic related enzymes, demonstrating that PD-L1 was the regulation factor of 18 F-FDG accumulation in tumor cells. In addition， previous studies show that peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been implicated in regulating 18 F-FDG [26] and the PD-L1 expression [27].
So the association between 18 F-FDG and the PD-L1 expression may also be a reflex of the PPAR-gamma pathway activation.
Novel immunotherapeutic methods are being advanced to suppress the expression of PD-L1. For these reasons, noninvasive strategies, including molecular imaging tools, which could be used for predicting the status of PD-L1/PD-L1-TILs, are of important clinical value, and have good prediction effect on the response to anti-PD-L1 blocker.
This study is limited by its small sample and retrospective design. Though 18 F-FDG PET/CT could have a good predictive value, it is not feasible to obtain an optimal cutoff for SUVmax in the clinical setting, and 18 F-FDG PET/CT cannot supersede immunohistochemistry analysis for detecting PD-L1/PD-L1-TILs expression. In addition, because of the frequency of physiological 18 F-FDG uptake and inflammation induced 18 F-FDG uptake, sometimes it is hard to identify the 18 F-FDG uptake from the gastric tumor itself. And there is a partial overlap between positive and negative PD-L1/PD-L1-TILs expression cases in 18 F-FDG uptake. However, this study can promote the advancement of noninvasive methods to infer PD-L1/PD-L1-TILs status. Progress in new radiotracers may improve the accuracy of this technique.

CONCLUSIONS
Gastric cancer with positive PD-L1/PD-L1-TILs expression have elevated 18 F-FDG accumulation. 18 F-FDG PET/CT has the ability to become a useful method to assess the molecular phenotypic information of gastric cancer, and have good prediction effect on the response to anti-PD-L1 blocker in gastric cancers. Additional prospective and large studies are required to verify our results and evaluate if molecular imaging can be useful for predicting the status of PD-L1 in gastric cancers, as well as for assisting the treatment decision making on when to employ anti-PD-L1 blocker therapies.

F-FDG PET/CT scan
Gastric cancer patients had been asked to fast for more than six hours before 18 F-FDG was injected. Patients' glucose levels were measured before 18 F-FDG administration, and in this study there were no patients whose blood glucose level exceeded 140 mg/dL. The mean accumulation time was approximately 60 minutes. PET was carried out with a combined PET/CT. The CT was used for attenuation correction.
Two board-certified nuclear medicine physicians assessed the 18 F-FDG accumulation. ROIs were placed on the tumor uptake lesion of PET imaging for semiquantitative analysis. The following formula was used to calculating SUVmax of the primary tumor: decaycorrected tracer tissue concentration /(injected 18 F-FDG dose /patients' weight).

Immunohistochemical analysis
Tumor tissues were paraffin-embedded and used for immunohistochemical analysis. The markers CK (cytokeratin) and LCA (the lymphocyte common antigen) were used to differentiate tumor cells and tumor infiltrating lymphocytes. Positivity for PD-L1 (indicating tumor cells) or PD-L1-TILs (indicating tumor infiltrating lymphocytes) was assessed by one board-certified pathologist. The positive percentage of stained cells covered by PD-L1 and PD-L1-TILs was quantified. Cases with >1% positive percentage of PD-L1/PD-L1-TILs were considered as high expression.

Statistical analysis
All values are demonstrated as mean ± SD. The statistical differences between different groups were compared using Mann-Whitney U test or chi-square test. SPSS software was used for statistical analysis.