Tumor immunity is related to 18F‐FDG uptake in thymic epithelial tumor

Abstract Background 2‐deoxy‐2‐[fluorine‐18] fluoro‐d‐glucose (18F‐FDG) positron emission tomography (18F‐FDG‐PET) is a convenient modality to assess the metabolic activity within tumor cells. However, there is no consensus regarding the relationship between 18F‐FDG uptake and the immune environment in thymic epithelial tumors (TETs). We conducted a clinicopathological study to elucidate the relationship between 18F‐FDG uptake and programmed death ligands 1 and 2 (PD‐L1/PD‐L2) expression in patients with TETs. Methods: A total of 108 patients with histologically confirmed TETs classified as thymomas or thymic carcinomas who underwent surgical resection or biopsy or needle biopsy and 18F‐FDG PET before any treatment between August 2007 and March 2020 were enrolled in this study. Tumor specimens underwent immunohistochemical staining for PD‐L1, PD‐L2, GLUT1, HIF‐1α, VEGFR2, VEGF‐C, and β2 adrenergic receptor. Results: High uptakes of SUVmax, SUVmean, MTV, and TLG were identified in 28 (25.9%), 61 (56.5%), 55 (50.9%), and 55 (50.9%) of 108 patients, respectively. High uptake of SUVmax significantly correlated with PS (performance status) of 1–2, thymic carcinoma, and advanced stage, and SUVmax on 18F‐FDG uptake displayed a close association with PD‐L1 and PD‐L2 expressions, but not with MTV and TLG. Our analysis revealed that SUVmax was identified as being significant relationship for positive PD‐L1/PD‐L2 expression. GLUT1, HIF‐1α, and VEGFR2 were significantly associated with the expression of PD‐L1/PD‐L2 from the biological viewpoint. Conclusion 18F‐FDG accumulation was closely associated with the expression of PD‐L1/PD‐L2, which, in turn, was correlated with glucose metabolism and hypoxia. PD‐L1/PD‐L2 could affect the glucose metabolism and hypoxia in thymic tumor cells.


| BACKGROUND
Thymic epithelial tumors (TETs), which are generally classified as thymomas and thymic carcinomas, are uncommon neoplasms present in less than 2.0% of all malignancies. 1 In particular, thymic carcinoma is a rare cancer with a dismal outcome and no available therapeutic agents for its advanced form. Thus, the identification of new targets that can serve as predictive and prognostic markers for the development of an optimal treatment plan is essential.
2-deoxy-2-[fluorine -18] fluoro-d-glucose ( 18 F-FDG) positron emission tomography ( 18 F-FDG-PET) is a convenient modality to assess the metabolic activity within tumor cells, although it shows some limitations such as false-positive findings. 2 Although it has been already known as one of the main biological mechanisms, glucose metabolism, hypoxia, and angiogenesis are closely linked to the accumulation of 18 F-FDG within tumor cells. In particular, several studies have demonstrated that the expression levels of glucose transporter 1 (GLUT1) and hypoxia-inducible factor-1α (HIF-1α) are correlated with 18 F-FDG uptake in thoracic tumors. 3 The 18 F-FDG uptake level can help to predict the grade of malignancy in TETs, allowing staging of the extent of the disease, prognosis, and therapeutic sensitivity. 3 Programmed death ligand-1 (PD-L1) has been recently shown to be expressed in patients with TETs and is closely correlated with the grade of malignancy and survival. 4,5 Immune checkpoint inhibitors (ICIs) targeting programmed death-1 (PD-1) or PD-L1 have been identified as effective therapeutic agents for patients with various human cancers. In particular, PD-L1 expression within tumor cells is thought to be a predictor of response to and outcome of therapy in patients with advanced lung cancer who received anti-PD-1 antibody. 6 Therefore, ICIs could serve as a potential optimal treatment option for neoplasms with PD-L1 expression.
Several recent studies have shown that PD-L1 expression within tumor cells is closely related to 18 F-FDG uptake. [7][8][9][10] In patients with non-small cell lung cancer (NSCLC), PD-L1 expression is linked to 18 F-FDG uptake, GLUT1, and HIF-1α. Also, GLUT1 and HIF-1α have been described to be closely associated with angiogenesis such as vascular endothelial growth factor (VEGF). 2 A recent investigation indicated that the increased expression of HIF-1α is associated with enhanced expression of PD-L1, and contributes to the activation of T-cell function and mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways. 11 Furthermore, HIF-1α directly binds to the hypoxia response element in the proximal promoter of PD-L1 and controls its expression under hypoxia. 12 Thus, our hypothesis is that the percentage of glucose metabolism determined by HIF-1α is suggestive of an immune reaction according to PD-L1 expression. However, little is known about the relationship between 18 F-FDG uptake and PD-L1 expression in patients with TETs. Moreover, anti-PD-1 antibody has been already known to provide an optimal blockade of PD-L1 and PD-L2, and some reports have shown that the expression of PD-L2 may be a potential prognostic marker in lung cancer. 13,14 Nevertheless, it remains unclear whether PD-L2 expression is associated with 18 F-FDG uptake and tumor aggressiveness in patients with TETs. Although maximal standardized uptake value (SUV max ) has been generally used as a measurement of 18 F-FDG uptake, it remains unknown about the correlation between PD-L1 expression and metabolic tumor volume (MTV) or total lesion glycolysis (TLG) on 18 F-FDG uptake. Thus, not only SUV max but also MTV or TLG should be investigated for the association of PD-L1 expression with 18 F-FDG uptake.
To address this gap in the literature, we conducted a clinicopathological study to elucidate the relationship between 18 F-FDG uptake and PD-L1/PD-L2 expression in patients with TETs and correlated the findings with GLUT1 and HIF-1α expression.

| Patients
A total of 118 consecutive patients with histologically confirmed TETs classified as thymomas or thymic carcinomas who underwent surgical resection or biopsy or needle biopsy and 18 F-FDG PET before any treatment at our institution between August 2007 and March 2020 were enrolled in this study. Of them, 10 patients were excluded and hypoxia. PD-L1/PD-L2 could affect the glucose metabolism and hypoxia in thymic tumor cells. 18 F-FDG uptake, GLUT1, HIF-1α, immunohistochemistry, PD-L1, PD-L2, thymic epithelial tumor because of inadequate tumor specimens and radiographic information, therefore, a total of 108 patients were enrolled in this study. Pathological diagnosis and tumor subtyping were performed according to the 2015 WHO histological classification of TETs and the TNM staging system. 15 The diagnoses were confirmed using light microscopy and immunohistochemistry. Surgically resected or biopsied primary tumors (n = 108) were included in this study in accordance with the institutional guidelines and the Helsinki Declaration. Ninety-four patients received surgical resection, and biopsy was performed in 14 patients. This study was approved by the institutional ethics committee. The requirement for written informed consent was waived by the ethics committee of our institution because of the retrospective nature of the study.

| PET imaging and data analysis
Patients fasted for at least 6 h before PET imaging, which was performed using a PET/CT scanner (Biograph 6 or 16, Siemens Healthineers K.K.) with a 585-mm field of view. Three-dimensional data acquisition was initiated for 60 min after injecting 3.7 MBq/kg of FDG. We acquired eight bed positions (2-min acquisition per bed position) according to the range of imaging. Attenuation-corrected transverse images obtained with 18 F-FDG were reconstructed with the ordered subset expectation maximization algorithm, based on the point spread function into 168 × 168 matrices with a slice thickness of 2.00 mm.
For the semiquantitative analysis, functional images of the standardized uptake value (SUV) were produced using attenuation-corrected transaxial images with the injected dosage of 18 F-FDG, patient's body weight, and the crosscalibration factor between PET and the dose calibrator. The SUV was defined as follows: CT scanning for initial staging was performed with intravenous contrast medium, and the CT images were interpreted by board-certified radiologists. We used RAVAT software (Nihon Medi-physics Co. Ltd.) on a Windows workstation to semi-automatically calculate the maximum of SUV (SUV max ) and metabolic tumor volume (MTV), total lesion glycolysis (TLG), defined as MTV multiplied by SUV mean , of each lesion using SUV thresholds obtained by the SUV in the liver VOI. Each threshold was defined as average of SUV (SUV mean ) plus 1.5×S.D. of SUV in the liver. These SUV thresholds were the optimum values to generate a 3D volume of interest (VOI) in which the whole tumor mass is completely enclosed in all cases, with CT image as the reference. In case of the activity other than tumors, including myocardium, gastro-intestinal tracts, kidneys, and urinary tracts, were eliminated by manually according to the diagnosis by the board-certified nuclear medicine physician.

| Statistical analysis
Statistical analyses were performed using Student's ttest, and the χ 2 test was performed for continuous and categorical variables, respectively. A p value < 0.05 was considered to be statistically significant. Univariate and multivariate analyses of the relationship between PD-L1 expression and different variables were performed by logistic regression analysis. Receiver operating characteristic (ROC) curve analyses were used to evaluate the potential for 18 F-FDG uptake on PET (SUV max , SUV mean , MTV, and TLG) to discriminate high from low PD-L1 expression, and the sensitivity and specificity were calculated to determine the optimal cut-off value differentiating positive from negative PD-L1 expression by the ROC curve. SUV values were used as a continuous variable and ROC analysis was performed. The correlations between SUVmax, MTV, and TLG on 18 F-FDG uptake were analyzed using Spearman's correlation coefficient test. All statistical analyses were performed using GraphPad Prism software (v.8.0; GraphPad Software) and JMP 14.0 (SAS Institute Inc.).
Representative 18 F-FDG PET images are shown in Figure A1 and A2 (online only). The different variables according to 18 F-FDG uptake by SUV max , SUV mean , MTV, and TLG were statistically compared ( Table 1). The patient with more than above cut-off values in each 18 F-FDG accumulation was defined as high uptake. High uptakes of SUV max , SUV mean , MTV, and TLG were identified in 28 (25.9%), 61 (56.5%), 55 (50.9%), and 55 (50.9%) patients, respectively. High uptake of SUV max and SUV mean was significantly correlated with PS (performance status) of 1-2, thymic carcinoma, advanced stage, and high MTV and TLG were closely associated with histology and disease stage.

| Univariate and multivariate analyses according to value of 18 F-FDG uptake on PET
Next, multivariate analysis was performed using different variables with significance of p < 0.05 on the univariate stage in the in the SUV max (Table 3). By multivariate analysis, disease stage, histology, GLUT1, and HIF-1α were identified as independent predictors for SUV max on 18 F-FDG uptake.

| DISCUSSION
To the best of our knowledge, this is the first study to evaluate the relationship between PD-L1/PD-L2 expression and 18 F-FDG uptake on PET in patients with TETs. We found that high expression of PD-L1 and PD-L2 was closely associated with high accumulation of 18 F-FDG; in particular, PD-L1/PD-L2 expression levels were significantly correlated with those of glucose metabolism and hypoxia. As angiogenetic markers, VEGFR2 and β2-AR were associated with the expression of PD-L1/PD-L2. Moreover, we confirmed that the expression of PD-L1 and PD-L2 was closely associated with not MTV or TLG but F I G U R E 1 Cut-off values for SUV max , SUV mean , MTV, and TLG were determined by receiver operating characteristic (ROC) curve analyses. Optimal 18 F-FDG uptake cut-offs for SUV max , SUV mean , MTV, and TLG as determined by ROC curve analysis, were 7.0 (sensitivity: 44.0%, specificity: 72.2%, p = 0.015), 3 SUV max on 18 F-FDG uptake, confirmed by multivariate analysis. Overall, PD-L1 and PD-L2 expressions indicated a strong correlation with glucose metabolism, as determined by GLUT1. Although SUV max is closely correlated with MTV and TLG on 18 F-FDG uptake, the upregulation of PD-L1 and PD-L2 may play a crucial role in the pathogenesis of tumor glucose metabolism in patients with TETs. Further studies with an experimental approach using thymic tumor cell lines are warranted to elucidate the results of our study. Several researchers have described that PD-L1 is frequently expressed in TETs, and a WHO classification is closely related to positive PD-L1 expression, but there was some discrepancy regarding the trend for worsened survival. 4,[16][17][18][19] Padda et al. reported that the high expression of PD-L1 could predict a significantly worse OS, which was correlated with more aggressive histology. 16 However, Yokoyama et al. described that the low PD-L1 expression and a high number of PD-1-positive tumor infiltrative lymphocytes (TILs) were significant predictors of worse survival in patients with thymic carcinoma. 18 Considering the evidence from previous studies, it is debatable whether PD-L1 could absolutely predict a worse outcome for patients with TETs. As our study also indicated that the expression of PD-L1 was higher in thymic carcinoma than in thymoma, PD-L1 may highly express in human neoplasms with malignant phenotype.
PD-L1 is an important target for PD-1 blockade, whereas PD-L2, as another PD-1 ligand, may also play a crucial role in the inhibition of PD-1 in human neoplasms. The prevalence of PD-L2 was significantly correlated with PD-L1, and PD-L2 status was also a significant predictor of PFS with pembrolizumab, independent of PD-L1 status. 20 A previous study reported that GLUT1 expression is associated with better clinical outcomes in advancedstage classical Hodgkin's lymphoma and is significantly associated with PD-L1 and PD-L2 expressions. 21 This study supports the hypothesis that GLUT1-related signaling pathways play an important role in the PD-L1 or PD-L2 pathway. Furthermore, a previous article reported that PD-L2-positive pheochromocytoma and paraganglioma were characterized by higher HIF-1α expression. That study reported the enrichment of transcripts involved in the hypoxic response in relation to PD-L2, but not PD-L1 expression. 22 When the researchers considered a broader subset of 200 genes involved in the hypoxic response, PD-L2 upregulation strikingly emerged as a stronger and more substantial determinant of tumor hypoxia than PD-L1, suggesting a potential mechanistic relationship between hypoxia and PD-L2-mediated antitumor immune control. Their data suggest that PD-L2 has a more predominant role than PD-L1 in shaping the immunetolerogenic environment, given the highly significant association with key pathways involved in innate, adaptive immunity, and inflammation in pheochromocytomas and paragangliomas. Recently, Rouquette et al. reported that the PD-L2 antibody stained no tumor epithelial cells in TETs. 19 Although we also performed PD-L2 staining using the same antibody, no staining was observed in our study, corresponding to their results. 19 Previous investigations have supported the potential of PD-L1 as an alternative target of HIF-1α and suggested that the distribution of glucose metabolism determined by HIF-1α could reflect the immune response reflected by the expression of PD-L1. 11,12 In addition, direct blockade of PD-L1 within cancer cells has been reported to diminish glycolysis by inhibiting the mTOR pathway and the expression of glycolysis enzymes. 23 Takada et al. reported the radiological features of PD-L2 expression in 222 patients with lung adenocarcinoma. 24 In their study, the SUV max for 18 F-FDG uptake was found to be significantly higher in PD-L2-positive than in PD-L2-negative cases. 24 It remains unknown why the expression level of PD-L2 is closely related to 18 F-FDG uptake. PD-L2 seemed to be more strongly correlated with glucose metabolism, hypoxia, and angiogenesis, compared with PD-L1. Further investigation should be conducted to elucidate the relationship between PD-L2 and 18 F-FDG uptake from the perspective of basic science. HIF-1α, hypoxia-inducible factor-1α; MTV, metabolic tumor volume; OR, odds ratio; PD-L1, programmed death ligand-1; PD-L2, programmed death ligand-2; SUV mean , mean standardized uptake value; TLG, total lesion glycolysis; UV max , maximum standardized uptake value; VEGF-C, vascular endothelial growth factor-C; VEGFR2, vascular endothelial growth factor receptor 2; β2-AR, beta-2 adrenergic receptor.
Bold values mean statistically significant difference. Our study is a first investigation to evaluate whether MTV or TLG could be correlated with the expression of PD-L1, thus, it remains unclear why SUV max was chosen as a better marker for the close correlation of PD-L1 expression than TLG or MTV. Considering that PD-L1 was not identified as independent predictor for the 18 F-FDG uptake by SUV max , we feel the possibility of weak association between 18 F-FDG uptake and PD-L1 expression in patients with TETs.
There are several limitations to our study. First, our study had a small sample size, which may have biased the results of our study. Since thymic cancer is a rare neoplasm, only limited numbers of samples were collected. Second, we tried to examine PD-L1 staining using clone 28-8; however, there are several kinds of PD-L1 clones. An additional investigation using other clones of PD-L1 may be needed to confirm the results of our study. Moreover, the AUC for determining cut-off value of 18 F-FDG uptake and Spearman correlation is relatively low, having some limitations of statistical analysis, thus, this limitation also may bias the results of our conclusion. But, there is controversial issue which cut-off value is optimal to dichotomize the uptake value of 18 F-FDG on PET. Finally, the results of our study were not confirmed by experimental investigations. In the level of tumor cell lines, little is known about any data elucidating the association between PD-L1 expression and 18 F-FDG uptake. Further examination is needed to approach some basic mechanism.
In conclusion, the relevance and distribution of 18 F-FDG uptake on PET were significantly associated with the expression of PD-L1 and PD-L2 in patients with TETs, and PD-L2 seemed to be more correlated with 18 F-FDG uptake than PD-L1. In particular, PD-L1 and PD-L2 exhibited a close relationship with upregulation of tumor glucose metabolism (GLUT1) and hypoxia (HIF-1α), which play essential roles in the mechanism of 18 F-FDG uptake within tumor cells.