Fructose 1,6-Bisphosphatase 1 Expression Reduces 18F-FDG Uptake in Clear Cell Renal Cell Carcinoma

Purpose To determine the relationship between fructose 1,6-bisphosphatase 1 (FBP1) expression and fluorine 18 (18F) fluorodeoxyglucose (FDG) uptake in patients with clear cell renal cell carcinoma (ccRCC), and to investigate how 18F-FDG uptake and FBP1 expression are related to tumor metabolism and tumor differentiation grade. Materials and Methods A total of 54 patients with ccRCC underwent 18F-FDG combined positron emission tomography and computed tomography (PET/CT) before tumor resection. The maximum standardized uptake value (SUVmax) for the primary tumor was calculated from the 18F-FDG uptake. The relationship between SUVmax of primary tumor and the expression of FBP1, hexokinase 2 (HK2), and glucose transporter 1 (GLUT1) was analyzed via immunohistochemical analysis. Results We identified an inverse relationship between FBP1 expression and SUVmax (P=0.031). SUVmax was higher in patients with high-grade ccRCC (mean, 11.6 ± 5.0) than in those with low-grade ccRCC (mean, 3.8 ± 1.6, P < 0.001). FBP1 expression was significantly lower in patients with high-grade ccRCC (mean, 0.23 ± 0.1) than in those with low-grade ccRCC (mean, 0.57 ± 0.08; P=0.018). FBP1 status could be predicted with an accuracy of 66.7% when a SUVmax cutoff value of 3.55 was used. GLUT1 expression in ccRCC was positively correlated with 18F-FDG uptake and FBP1 status, whereas HK2 expression was not. Conclusion SUVmax in patients with ccRCC is inversely associated with the expression of FBP1, and FBP1 may inhibit 18F-FDG uptake via regulating GLUT1. SUVmax is higher in patients with high-grade ccRCC than in those with low-grade ccRCC, which could be the result of lower FBP1 expression in patients with high-grade ccRCC.


Introduction
Renal cell carcinoma (RCC) is one of the most prevalent tumors worldwide, with clear cell RCC (ccRCC) as the most common histologic type [1,2]. Surgical resection remains the most common treatment strategy for RCC [3,4]; however, approximately 25-30% of RCC patients are diagnosed with metastases, and many cases develop metastases after radical nephrectomy [5][6][7]. As a result, the overall 5year survival rate of RCC patients is still poor [4]. Clinicians must make an early diagnosis to improve the survival rate of RCC patients. As a result, the identification of new tumor markers that better reflects the biological characteristics of RCC is of great necessity.
Fluorine 18 ( 18 F) fluorodeoxyglucose-(FDG-) combined positron emission tomography (PET) and computed tomography (CT) has been widely used for evaluating tumor activity, which is based on the high rate of glucose metabolism in cancer cells [8][9][10][11]. However, previous studies have found that the sensitivity of 18 F-FDG PET/CT is not very high in the diagnosis of ccRCC because of low 18 F-FDG uptake in a considerable part of the ccRCC [12][13][14][15]. Because 18 F-FDG PET is increasingly being used as a diagnostic tool in RCC, a further characterization of this phenomenon is essential.
Previous studies have found that the maximum standardized uptake (SUVmax) is higher in high-grade ccRCC than in low-grade ccRCC [15]; however, the molecular mechanisms underlying these findings remain unclear. Glucose homeostasis is reciprocally controlled by anabolic gluconeogenesis and catabolic glycolysis. e kidney is one of the primary sites of anabolic gluconeogenesis, which is next only to the liver. Recently, attention has been mainly focused on the increased glycolysis in tumors, which is called the Warburg effect [16]. However, the possibility that this effect is also being facilitated by altered gluconeogenesis has not been studied.
Fructose 1,6-bisphosphatase 1 (FBP1) is a key enzyme in the gluconeogenesis pathway which catalyzes fructose 1,6bisphosphate into fructose 6-phosphate [17]. We and others have demonstrated that FBP1 plays an important role in the glucose metabolism of malignant tumors [17][18][19]. However, the relationship between FBP1 expression and 18 F-FDG uptake in ccRCC, along with the underlying molecular mechanisms, has not been examined so far.
In the present study, we investigated whether FBP1 expression is associated with 18 F-FDG uptake in ccRCC and whether 18 F-FDG uptake can be used to predict FBP1 status. In addition, we examined whether GLUT1 and HK2 expression are associated with 18 F-FDG uptake in ccRCC by immunohistochemical analysis.

Study Population.
is retrospective study included 54 patients with ccRCC (mean age, 58.9 years; age range, 31-82 years); 37 were men (mean age, 57.2 years; age range, 31-77 years) and 17 were women (mean age, 62.6 years; age range, 49-82 years). Patients underwent 18 F-FDG PET/CT before tumor resection at Ren Ji Hospital between 2009 and 2016. e inclusion criteria were as follows: diagnosis of ccRCC was confirmed by pathologic examination; complete clinical data, including age, sex, tumor size, lymph node metastasis, and tumor grade, were available; tissue specimens for immunohistochemical analysis were available; and follow-up information was available. ccRCCs were classified into 2 categories; tumors containing a nuclear G3 or G4 component and tumors consisting of G1 and G2 components (high-and low-grade ccRCC, respectively). No distant metastasis occurred. Our retrospective study was approved by the Institutional Review Board of Shanghai Jiao Tong University that is affiliated with Ren Ji Hospital, and the requirement to obtain informed consent was waived.

PET/CT Imaging and Analysis.
A combined PET/CT device (Biograph mCT; Siemens) was used for all PET/CT scans. PET imaging was carried out with an acquisition time of 3 minutes per bed position after CT scanning. All patients received an intravenous injection of 18 F-FDG (3.7 MBq/kg) after having fasted for at least 6 h. e mean uptake time was 50 ± 6 min. Blood glucose levels were measured and found to be less than 140 mg/dL at the time the 18 F-FDG was administered. PET images were iteratively reconstructed, and CT data were used for attenuation correction. For quantitative analysis, two experienced nuclear medicine physicians evaluated 18 F-FDG uptake on a workstation (Medex) by calculating the SUVmax of the regions of interest. Regions of interest were placed over the suspected lesions that may have exhibited increased 18 F-FDG uptake. Regions of interest were drawn according to previous contrast-enhanced CT scans in the lesions that exhibited no substantially increased 18 F-FDG uptake.
e SUVmax was calculated as follows: maximum pixel value in the decay-corrected ROI activity (MBq/kg)/(radioactivity of the injected dose (MBq)/body weight (kg)).

Immunohistochemistry.
Immunohistochemical staining was performed on paraffin embedded ccRCC tissues. After microtome sectioning (5 μm), the slides were stained with anti-GLUT1 (Proteintech), anti-HK2 (Proteintech), and anti-FBP1 (Sigma) antibodies. Immunohistochemical analyses were conducted by two experienced pathologists. e slides were scored according to staining intensity (0-3). Slides with a score of 2 or 3 were considered high expression, and slices with a score of 0 or 1 were considered low expression.

Statistical
Analyses. e data were presented as mean ± SD. e Mann-Whitney U test was used to assess the association between SUVmax and FBP1 expression. e relationship between FBP1 expression and clinicopathologic characteristics of RCC patients was assessed by using the Fisher exact or x 2 test. e Mann-Whitney U test was also used to assess the association between the expression of GLUT1 and HK2 and SUVmax. Pearson's rank correlation was applied to determine the association between FBP1 expression and the expression of GLUT1 or HK2. e receiveroperating characteristic curve was used to assess the optimal value of SUVmax for predicting FBP1 expression. P < 0.05 was considered significantly different. All statistical analyses were conducted with SPSS software (SPSS, version 13.0). Table 1. Among the 54 patients, 37 had low-grade ccRCC and 17 had high-grade ccRCC. Although the majority (82.8%, 39/54) of primary tumors showed positive 18 F-FDG uptake, 17.8% (15/54) of primary tumors showed negative 18 F-FDG uptake compared with normal liver tissues. e SUVmax for the primary tumors ranged from 1.7 to 22.1, with an average of 6.21. A total of 46.3% (25/54) of the tumors showed high FBP1 expression, and 53.7% (29/54) of the tumors showed low FBP1 expression.
Next, we sought to determine the SUVmax threshold for optimal differentiation between high and low FBP1 expression groups. Receiver-operating characteristic curve analysis revealed that the highest accuracy (66.7%) was obtained with an SUVmax cutoff of 3.55 and that the area under the curve was 0.67 ± 0.07. Sensitivity and specificity for the prediction of FBP1 expression were 88.9% (22/29) and 58.3% (14/25), respectively (Figure 1(b)).

Association of 18 F-FDG Uptake and FBP1
Expression with GLUT1 and HK2 Expression. We observed a positive association between SUVmax and GLUT1 expression in ccRCC (Figure 3(a)) but not between SUVmax and HK2 expression (Figure 3(b)). e SUVmax was significantly higher in the e expression of FBP1 in ccRCC tissues (0.46 ± 0.07) was significantly lower than that in corresponding peritumor tissues (0.91 ± 0.04) (P < 0.0001). (b) Receiver-operating characteristic curve analysis of SUVmax in primary tumor to predict FBP1 expression in ccRCC. With an SUVmax of 3.35 as the optimal value, sensitivity and specificity for prediction FBP1 expression were 88.9% and 58.3%, respectively. e area under the receiver-operating characteristic curve was 0.67 (95% confidence interval: 0.53-0.81; P � 0.032).

18
F-FDG PET/CT has been widely used for diagnosis, staging, and monitoring of therapeutic response in many malignant tumors [8][9][10][20][21][22]. However, 18 F-FDG PET/ CT has not been recommended for routine evaluation of renal tumors because of false-negative cases. However, the underlying mechanism for relatively low 18 F-FDG uptake in some ccRCC remains unclear. Our study showed that SUVmax was significantly higher in ccRCCs with low FBP1 expression compared with high FBP1 expression, indicating that 18 F-FDG uptake might reflect FBP1 expression levels in patients with ccRCC. e kidney is one of the major organs of gluconeogenesis which is only next to the liver. Our previous study showed that the key enzyme of gluconeogenesis, FBP1, was downregulated and played an important role in regulating 18 F-FDG uptake in hepatocellular carcinoma [18]. Similar to the expression of FBP1 in hepatocellular carcinoma [18], our current study showed that FBP1 was also decreased in   [17]. ese results indicate that FBP1 is suppressed in some patients with ccRCC and that FBP1 might play a critical role during tumor development of ccRCC and possibly can serve as a novel molecular biomarker for ccRCC. Our study also showed that FBP1 expression was associated with tumor size, tumor grade, and SUVmax; thus, tumor size, tumor grade, and SUVmax may be good predictors for FBP1 in ccRCC.
We also assessed the association between the expression of FBP1, 18 F-FDG uptake tumor, and tumor differentiation grade in patients with ccRCC. In ccRCC specimens, we found that SUVmax was significantly higher in high-grade ccRCC than in low-grade ccRCC; this finding is consistent with previous studies [15]. Furthermore, we showed that FBP1 expression was significantly lower in high-grade ccRCC tumors than in low-grade tumors. Consistent with these results, an inverse association was observed between FBP1 expression and SUVmax. ese results suggest that the low uptake of 18 F-FDG in some ccRCCs was due to the high FBP1 expression. 18 F-FDG uptake in malignant tumors depends mainly on hexokinases and glucose transporters, both of which are overexpressed in many malignant tumors [10]. Several previous studies have found that 18 F-FDG uptake in RCC was associated with the expression of GLUT1 [23][24][25][26]. Likewise, our results showed that there was a positive association between SUVmax and GLUT1 expression in ccRCC. Our results also showed that there was an inverse association between GLUT1 expression and FBP1 expression, whereas HK2 expression was not associated with FBP1 expression. In addition, our findings showed that the higher the FBP1 expression in ccRCC patients, the lower the SUVmax. Consistent with our clinical data, a previous study reported that in vitro overexpression of FBP1 in RCC cell lines led to a significant decrease in 18F-FDG uptake and GLUT1 expression [17]. us, we suggest the hypothesis that FBP1 might decrease 18 F-FDG uptake via the downregulation of GLUT1 expression in ccRCC. Of course, the specific mechanism on how FBP1 affects 18 F-FDG uptake in ccRCC should be further confirmed.
FBP1 expression was lower in ccRCC patients than in patients with normal kidneys. In addition, the expression levels of FBP1 varied widely in patients with different grades of ccRCC. FBP1 expression in patients with high-grade ccRCC was lower compared with low-grade ccRCC patients, whereas SUVmax was significantly higher in patients with high-grade ccRCC; thus, 18 F-FDG PET/CT detected poorly differentiated RCCs with high sensitivity. In contrast, FBP1 expression was relatively high in patients with lowgrade ccRCC, which corresponded to low 18 F-FDG uptake. Consequently, 18 F-FDG PET exhibited relatively low sensitivity for the detection of low-grade ccRCC, which has been noted in previous studies [15].
Our study had some limitations. First, the sample size of this study was small because our study was conducted at only one center. Second, there was unavoidable selection bias because our study was a retrospective study. ird, the kidneys are sites of physiological radioactive urine collection, and it is difficult to distinguish pathological uptake from the physiological radioactive urine collection in some cases. Furthermore, though the correlation between FBP1 and SUVmax is present, it is not very high. Large prospective studies that include more sample sizes and academic centers are needed to confirm our results in the further.
In conclusion, we found that in patients with ccRCC, 18 F-FDG uptake and tumor grade (from low-grade to highgrade) were both inversely correlated with FBP1 expression. Our study also showed that FBP1 expression was correlated with tumor size, tumor grade, and SUVmax. FBP1 appears to inhibit 18 F-FDG uptake via regulating GLUT1. ese results suggest the underlying mechanisms of low 18 F-FDG uptake in partial ccRCC. ese results also lay the foundation for the development of a new imaging agent in the diagnosis of ccRCC with high sensitivity.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
All procedures performed in studies involving human participants were in accordance with the principles of the Institutional Review Board of Shanghai Jiao Tong Universityaffiliated Ren Ji Hospital and the 1975 Declaration of Helsinki, as revised in 2013. is article does not contain any studies with animals performed by any of the authors.

Consent
For this type of retrospective study, formal consent is not required.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Ruohua Chen and Xiang Zhou are contributed equally to this work.