Relationship between SP142 PD-L1 Expression and 18F-FDG Uptake in Non-Small-Cell Lung Cancer

Objectives Immune checkpoint blockers constitute the first-line treatment for advanced non-small-cell lung cancer (NSCLC) with ≥50% PD-L1 expression. In NSCLC, PD-L1 positivity is correlated with high 18F-fluorodeoxyglucose (18F-FDG) uptake. However, these studies only included patients undergoing surgical resection, almost all in their early stages. Moreover, differences in 18F-FDG uptake between NSCLC with high (≥50%) and low (49%) PD-L1 expression remain unknown. We aimed to investigate the association between metabolic parameter 18F-FDG uptake and PD-L1 expression status in NSCLC patients. Methods From February 2017 to June 2018, 428 consecutive NSCLC patients who underwent 18F-FDG positron emission tomography/computed tomography (PET/CT) and SP142 PD-L1 expression analysis were retrospectively assessed. The association between clinicopathological characteristics and PD-L1 expression was examined. Results The frequency of PD-L1-positive tumors was 38.1% (163/428), 28.5% (91/319), and 64.2% (61/95) for NSCLC, adenocarcinoma (ADC), and squamous cell carcinoma (SCC), respectively. Maximal standard uptake (SUVmax) was significantly higher in PD-L1-positive than in PD-L1-negative NSCLC (p < 0.0001), ADC (p < 0.0001), and SCC (p=0.006). SUVmax was significantly higher in NSCLC (p=0.001) and ADC (p=0.003) with high rather than low PD-L1 expression. The receiver operating characteristic curve yielded area under the curve values of 0.726 (95% CI, 0.679–0.774, p < 0.0001), 0.694 (95% CI, 0.634–0.755, p < 0.0001), and 0.625 (95% CI, 0.513–0.738, p=0.044) for NSCLC, ADC, and SCC, respectively. Conclusion 18F-FDG tumor uptake is strongly, positively correlated with PD-L1 expression in NSCLC and significantly differs between high and low PD-L1-expressing individuals.


Introduction
During the past decades, owing to heavy smoking and air pollution, lung cancer has become the most common cancer and the leading cause of cancer death in China [1][2][3][4]. e 5year survival rate of lung cancer is 20.2%, and that of patients with advanced disease is significantly worse [5]. Nonetheless, in the last decade, immunotherapy has emerged oncotherapeutic modality. Positive results have been obtained in the treatment of non-small-cell lung cancer (NSCLC) [6][7][8], which have drastically revolutionized cancer therapy. e immune checkpoint status (programmed death ligand-1, PD-L1) test is an effective method to identify patients who can benefit from immunotherapy. PD-L1-positive patients with advanced NSCLC, specifically patients with high PD-L1 expression, have better survival [7,8]. Analysis of the immune checkpoint status has become a standard of care, and tumor tissues aspirated during biopsy or intraoperatively resected are usually obtained from patients with tumors. However, owing to the inherent limitations of invasive procedures in patients with advanced tumors, tissues are not always readily available or may be difficult to access, resulting in sampling errors or insufficient material. erefore, a complementary noninvasive biomarker for predicting PD-L1 expression in advanced NSCLC would be highly useful.
Recent studies have reported the predictive value of 18 Ffluorodeoxyglucose positron emission tomography/ computed tomography ( 18 F-FDG PET/CT) to determine the PD-L1 expression status in cancer patients; however, their results are not consistent [9][10][11][12][13][14][15][16][17][18][19][20][21][22]. Hence, tumor FDG uptake is generally higher in PD-L1-positive malignancies [9-15, 19, 22]. However, Chen et al. reported contrasting findings, reporting an inverse correlation between metabolic tumor volume (MTV) and total lesion glycolysis (TLG) with PD-L1 expression [20]. ree other studies have reported the absence of an association between the maximal standard uptake value (SUVmax) and PD-L1 expression status [16,17,21]. For clinical purposes, it is important to know if the SUVmax can predict the PD-L1 expression status and identify patients who could potentially benefit from PD-L1/ PD-1-targeted therapies. In addition, patients with NSCLC undergoing surgical resection have been previously assessed in these studies, almost all in their early stages. However, clinical studies have reported that antibodies targeting PD-L1 are an efficacious treatment alternative for patients with advanced NSCLC, who have ≥1% PD-L1 expression [6][7][8]; studies including more patients with advanced disease would be persuasive and clinically significant in assessing the association between 18 F-FDG uptake and PD-L1 expression in NSCLC. Furthermore, pembrolizumab has been approved as a single agent for the first-line therapy for patients with advanced NSCLC and high PD-L1 expression (≥50%). To our knowledge, no studies have investigated the differences in 18 F-FDG uptake between NSCLC patients with high PD-L1 expression (≥50%) and low PD-L1 expression (1%-49%). In this study, we retrospectively assessed patients with NSCLC, including more patients with advanced disease, and investigated the association between 18 F-FDG PET/CT metrics and PD-L1 expression status.

Study Design and Patient
Cohort. From February 2017 to June 2018, we retrospectively reviewed all NSCLC patients who underwent PET/CT by searching our hospital's database at the Department of Nuclear Medicine. is study was approved by the Institutional Review Board of our hospital. Patients with NSCLC were included in accordance with the following criteria: (1) available pathology reports with newly diagnosed NSCLC, (2) patients not receiving previous chemotherapy or radiotherapy before PET/CT and sampling for PD-L1 analysis, (3) no history of malignancy, (4) an interval between sampling and PET/CT shorter than 1 month, and (5) presence of a well-defined lesion. We retrospectively reviewed clinical data of these patients, including sex, age at diagnosis, pathological tumor-lymph node-metastasis stage (in accordance with the International Association for the Study of Lung Cancer, 8th TNM Lung Cancer Staging System), and smoking status. Smoking status was divided into two categories: nonsmoker and smokers (including current and previous smokers).

18 F-FDG PET/CT Acquisition and Analysis.
At the time of diagnosis, all patients underwent 18 F-FDG PET/CT scans (Biograph Mct64, Siemens, Erlangen, Germany). Before the examination, the patients were fasted for 4-6 h to maintain serum glucose levels below <11.1 mmol/L. e 18 F-FDG injection volume was 0.10～0.15 mCi/kg. After injection, all patients rested for 1 h and were additionally administered at least 500 mL of water. Imaging was carried out at six or seven bed positions from the base of the skull to the midthighs [15]. Visual analysis and PET/CT measurements were independently performed by two experienced nuclear medicine physicians (W. HQ and L.Q.) blinded to the PD-L1 expression status and the clinical data, and interpretations were reached through consensus. e SUVmax values of the primary tumors were determined.

PD-L1 Histopathological and Mutational Analyses.
Hematoxylin and eosin-stained slides and immunostained slides upon primary diagnosis (performed at the Department of Pathology in our hospital) were available for all patients. For immunostaining for SP142 (Ventana Medical Systems Inc.), the specimens were formalin-fixed, paraffinembedded, sectioned, and stained in accordance with standard clinical operating procedures. e proportion of PD-L1-positive tumor cells was determined by calculating the percentage of stained tumor cells. Positivity was defined as a proportion of PD-L1-positive tumor cells >1%. Proportions of PD-L1-positive tumor cells ≥50% and 1-49% were defined as indicative of high and low expression, respectively.
We retrospectively reviewed the mutational status of the epidermal growth factor receptor (EGFR, exons [18][19][20][21][22] gene in all patients. Tumor samples were obtained through surgical resection or biopsy. EGFR mutations were analyzed using the amplification-refractory mutation system. Cycle sequencing of the purified PCR products was performed using the ADx Mutation Detection Kits (Amory, Xiamen, China) in accordance with the manufacturer's instructions.

Statistical Analysis.
e clinicopathological characteristics and PET/CT findings are presented as continuous variables. Univariate and multivariate logistic regression analyses were performed to assess the association between the PD-L1 expression status and each of the considered factors, i.e., age, sex, smoking status, tumor stage, lymph node metastases, and EGFR status. e association between quantitative continuous variables (including SUVmax and maximum diameter) and PD-L1 expression status was also investigated using the Mann-Whitney U test. A p value <0.05 was considered statistically significant. e cutoff value of SUVmax was determined through receiver operating characteristic (ROC) curve analysis. All data were statistically analyzed using a software program (SPSS version 21.0; SPSS, Chicago, IL, USA).
Among ADC patients (Table 2), PD-L1 positivity was significantly more frequent in male patients (p � 0.027), smokers (p � 0.027), stage II/III/IV patients (p � 0.008), patients with lymph node metastasis (p < 0.0001), carriers of wild-type EGFR (p � 0.004), and at higher SUVmax values (p < 0.0001). On multivariate analysis, higher SUVmax and the presence of lymph node metastasis and wild-type EGFR were independent predictors of PD-L1 positivity. In the SCC group (Table 3), SUVmax was significantly higher in PD-L1-positive than in PD-L1-negative patients (p � 0.006). No significant association between PD-L1 expression and additional clinicopathological characteristics was observed.
e results are shown in Tables 1-3. Furthermore, significant differences were observed between groups with high PD-L1 expression and low PD-L1 expression levels for NSCLC (15.52 ± 7.72 vs. 11.63 ± 6.18; p � 0.001) and ADC (12.91 ± 6.41 vs. 9.24 ± 5.25; p � 0.003), respectively. In the SCC group, no significant association between groups with high PD-L1 expression and low PD-L1 expression levels was observed. e quantitative differences are shown in Figures 2  and 3(a)-3(c).

Discussion
Immune checkpoint-targeted therapies have changed the therapeutic landscape of NSCLC. Among patients with advanced NSCLC and PD-L1 positivity, antibodies that block the PD-L1 protein improve survival [7,8]. In clinical practice, however, high-quality tissues from these patients with advanced diseases are not always available for analyzing PD-L1 expression. erefore, in clinical practice, the presence of a complementary noninvasive biomarker that can predict the PD-L1 expression status would be valuable for patients with advanced NSCLC. In the present cohort, the SUVmax was significantly higher in PD-L1-positive than in PD-L1-negative NSCLC patients. Furthermore, the SUVmax was significantly higher among patients with high PD-L1 expression levels than among those with low PD-L1 expression levels in the NSCLC (p � 0.001) and the ADC (p � 0.003) groups. In addition, we examined the association between PD-L1 expression and various clinicopathological features. PD-L1 was expressed in 28.5% of lung adenocarcinomas and 64.2% of lung squamous cell cancers.
In ADC patients, PD-L1 positivity was associated with a wild-type EGFR status (p � 0.046) and with the presence of lymph node metastasis (p � 0.022). ese results are consistent with those of previous studies on NSCLC patients [9-15, 23, 24]. In malignant tumors, the association between PD-L1 expression and 18 F-FDG uptake was not consistent. Chen et al. [20] reported that MTV and TLG are negative predictors of PD-L1 expression in squamous cell carcinoma of the head and neck, whereas the SUVmax did not differ upon stratification based on the PD-L1 expression status. de Heer et al. [21] reported that the PD-L1 expression status in melanoma was not correlated with any of the PET   Contrast Media & Molecular Imaging parameters. Moreover, two studies reported that the PET parameters were significantly associated with PD-L1 expression in both oral squamous cell carcinoma and bladder cancer [19,22]; however, their cohorts were relatively small, with less than 100 patients. erefore, their results warrant careful confirmation through studies with larger patient cohorts. Furthermore, the results were inconsistent with those of NSCLC. Two different studies failed to identify an association between the SUVmax and PD-L1 expression [16,17]. However, their patient cohorts were small, with 55 and 49 lung cancer patients [16,17], respectively. Other studies have reported that the SUVmax was higher in PD-L1-positive NSCLC patients [9][10][11][12][13][14][15]. Only one of these studies has a larger cohort size than the present cohort [11]. Finally, a meta-analysis identified only a weak correlation between SUVmax and PD-L1 expression in lung cancer, concluding that the SUVmax cannot be considered a surrogate marker for the PD-L1 status [18]. To our knowledge, most of these studies only included patients undergoing surgical resection [9][10][11][12][13][14][15][16]. In these studies, the cohort of  Contrast Media & Molecular Imaging advanced NSCLC patients was very small. Only Jreige et al. [17] obtained tumor tissues either via biopsy or through surgical resection from 49 patients with NSCLC, representing a small sample size. e present cohort included a larger number of patients with advanced NSCLC (123 patients with III/IV stage tumors). e present results reveal a significant correlation between 18 F-FDG uptake and PD-L1 expression levels. We believe that our data are reliable, as they were obtained with a relatively large sample size (428 patients) and from a higher number of advanced NSCLC patients, relative to previous studies. Several studies have reported that in NSCLC, PD-L1 positivity is correlated with high 18 F-FDG uptake [9][10][11][12][13][14][15]. However, in those studies, PD-L1 expression in tumor cells was assessed through different PD-L1 IHC assays, and different cutoff values were defined for PD-L1 positivity. Zhang et al. [9] reported that in SCC, PD-L1 expression levels (cutoff, 5%), assessed through immunostaining with the 28-8 antibody, are significantly correlated with the SUVmax. Moreover, 18 F-FDG uptake was significantly correlated with PD-L1 expression (cutoff, 10%) (as assessed by using 28-8 and E1L3N antibodies) in ADC patients [13]. Furthermore, Kasahara et al. [14] reported that in SCC, a high SUVmax on 18 F-FDG PET is associated with PD-L1 positivity (cutoff, 11%), as revealed through the E1L3N assay. ree other studies reported that the SUVmax was an independent predictor for PD-L1 positivity (cutoff, 5%), as revealed through the SP142 assay [10][11][12]. Furthermore, Wu et al. [15] reported that among patients with pulmonary sarcomatoid carcinoma, the SUVmax was markedly higher when PD-L1 positivity was ≥50% rather than <50%. No studies have investigated the differences in 18 F-FDG uptake between patients with high PD-L1 expression (≥50%) and low PD-L1 expression (1-49%) levels. e present results show that the SUVmax of the PD-L1 high-expression group was significantly higher than that of the low-expression group in both the NSCLC (p � 0.001) and the ADC (p � 0.003) group. It may hence be possible to provide more information regarding the first-line immunotherapy decisions for patients with advanced NSCLC. us far, four PD-L1 assays have been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for clinical use among urothelial carcinoma patients, i.e., based on the Dako 28-8 and 22C3 and the Vantana SP142 and SP263 monoclonal antibodies. We performed immunostaining to evaluate PD-L1 expression in 428 NSCLC patients, using the SP142 monoclonal antibody. However, in previous studies, PD-L1 expression in lung cancer cells was assessed through different PD-L1 immunostaining assays. ree studies have used the SP142 monoclonal antibody [10][11][12]. Herein, the frequency of PD-L1-positive tumors was higher than those reported previously using the SP142 antibody, most probably owing to the fact that while in the latter studies, a 5% threshold was applied to define PD-L1 positivity, and we used a cutoff value of 1%. Rittmeyer et al. [25] reported that atezolizumab provides survival benefits in previously treated NSCLC patients in accordance with the 1% cutoff value, and this improvement was associated with PD-L1 expression in tumor cells, as assessed via the SP142 PD-L1 immunostaining assay. Takada et al. [26] reported that surgically resected, PD-L1-positive ADC and NSCLC patients had a worse prognosis than their PD-L1-negative counterparts at the 1% cutoff value. Moreover, the 1% cutoff value for assessing PD-L1 expression via the SP142 PD-L1 immunostaining assay was a sensitive indicator to postoperatively predict the patient prognosis [27]. erefore, we considered that a 1% cutoff value would be acceptable for dichotomizing PD-L1 expression in the present study. We demonstrated that when the SP142 PD-L1 immunostaining assay was used, a high SUVmax was significantly associated with PD-L1 positivity in patients at the 1% cutoff value, as well as at the 5% cutoff value.
is study shows that different clinicopathological parameters were differently associated with PD-L1 expression; as determined via univariate and multivariate analyses, the SUVmax had the highest odds ratio (OR) among all other parameters (5.15 in NSCLC, 9.07 in ADC, and 8.4 in SCC). A high negative-predictive value was obtained in NSCLC and ADC, and a high positive-predictive value was obtained in SCC. However, herein, considering only the SUVmax, the accuracy in predicting PD-L1 expression levels was not very high, and the AUC was 0.726, 0.694, and 0.625 for NSCLC, ADC, and SCC, respectively. In further studies, we intend to combine the SUVmax and other parameters with significant ORs in a combined predictive model to increase the accuracy, e.g., the presence of lymph node metastases and the morphological findings on CT. Furthermore, the cutoff values of SUVmax obtained through ROC curve analysis were 6.06, 4.07, and 20.72 for NSCLC, ADC, and SCC patients, respectively, which varied with the pathological subtype. Moreover, previous studies have suggested that in lung cancer, FDG uptake is influenced by the tumor histologic subtype [28,29]. We reasoned that the application of different cutoffs for different pathological subtypes may be appropriate. ese values were higher than those reported previously in the same three patient subgroups [9][10][11][12][13][14], probably owing to the use of different methods for PD-L1 immunostaining. e underlying reason was speculated to be the large cohort of advanced NSCLC patients in this study. Our data showed that SUVmax was strongly correlated with PD-L1 expression. However, since the cutoff for SUVmax was not optimized, further studies with a more rigorous and detailed design are warranted.
is study has several limitations. First, this was a retrospective, single-center study. Second, only the SP142 PD-L1 immunostaining assay was used to assess PD-L1 expression in tumor cells. Büttner et al. [30] reported that the highest concordance and interobserver reproducibility were achieved with 28-8, 22C3, and SP263 clinical assays, whereas a lower PD-L1 expression levels were detected via the SP142 assay. Further studies are required to establish the comparability among various PD-L1 immunostaining analyses.
ird, NSCLC is a heterogeneous condition comprising various histopathologic patterns. Different pathological NSCLC subtypes exhibit distinct FDG metabolic activities, Contrast Media & Molecular Imaging which may partly account for the contradictory results reported by previous studies. erefore, subgroup analyses are necessary to establish specific cutoff values in accordance with different pathological subtypes, stage, and to the use of different assays.

Conclusions
In conclusion, this study shows that in NSCLC, the SUVmax for 18 F-FDG PET/CT is significantly associated with that of PD-L1 expression, at the 1% cutoff value for assessing through immunostaining with the SP142 antibody. Unlike previous studies, which mostly investigated patients with early-stage disease, this study is the first to assess patients with early and substantially advanced NSCLC. Moreover, this study shows that the SUVmax was higher in the group with high PD-L1 expression than in the low PD-L1 expression group of NSCLC and ADC patients. We speculated that the SUVmax is increased upon PD-L1 upregulation.
us, determination of the SUVmax may help in identifying patients potentially benefiting from treatment with PD-L1 inhibitors.
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional Research Committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. is study has been approved by the Shanghai pulmonary hospital Ethics Committee (number: K19-033Y).

Consent
e requirement for patient informed consent was waived.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.