[ 18 F ] FDG PET imaging evaluation on non-alcoholic fatty liver disease and hepatocellular carcinoma model treated with sorafenib

Aim: Evaluate the effect of sorafenib in a rat model of non-alcoholic fatty liver disease (NAFLD) related to hepatocellular carcinoma (HCC) by quantifying the correlation between changes in glucose metabolism on PET imaging and degree of tumor differentiation. Methods: NAFLD related HCC was induced by the combination of high fat and choline deficient diet with diethylnitrosamine (100 mg/L) for 16 weeks. Then carcinogenic stimuli were suspended, liver nodules were identified by abdominal ultrasound and two groups were randomized: control (n = 10) and sorafenib (n = 20). Rats received daily gavage administration of 1 mL saline or sorafenib (5 mg/kg/day) for more 3 weeks. After treatment, [F]FDG PET scan was performed on animals. Results: [F]FDG uptake was lower in the sorafenib group than that in the control group (3.3 ± 0.48 vs . 5.5 ± 1.5, P = 0.01). Direct correlation was found between poorly-differentiated HCC and TumorSUVmax/MuscleSUVmax ratio (R = 0.54, P = 0.006). Treatment was associated with significantly more residual tumors that were well differentiated (Grades I/II) than in the untreated control group (39% vs . 5%, respectively, P = 0.01). Conclusion: Sorafenib shows promise as a treatment for reducing the aggressiveness of HCC as demonstrated by [F] FDG PET and immunohistochemistry.


INTRODUCTION
Non-alcoholic, fatty liver disease (NAFLD) is associated with obesity and known to progress to nonalcoholic, steatohepatitis (NASH), cirrhosis and then hepatocellular carcinoma (HCC), or directly progress from NASH to HCC [1][2][3] .Liver cancer is the 16th cause of global mortality and HCC accounts for up to 90% of all primary liver cancers [4,5] .Observational studies showed that diabetes, obesity, and iron overload are risk factors for development of HCC and NAFLD [2] .
Animal models are crucial to elucidate the physiopathology of HCC and to test potential therapeutic targets [6,7] .The ideal model of NAFLD-related HCC should replicate human HCC development, given a high caloric diet, obesity, insulin resistance, dyslipidemia, similar hepatic markers, natural evolution of HCC, genetic aspects and activation of the same signaling pathways [8,9] .There are many animal models for HCC that include genetically altered animals and orthotopic tumor implantation, however they are not ideal to replicate NAFLD as they do not exhibit liver and metabolic changes [7,10] .Previous work used a mixed experimental model of NAFLD-related HCC with fat and choline deficient diets together with diethylnitrosamine (DEN) in drinking water to achieve HCC development within 16 weeks; a shorter period than usual [7,11] .DEN has been used as a carcinogen and is capable of inducing HCC with intra-and intertumor variability as it occurs in humans [12][13][14] .
Earlier studies performed with HCC patients, treated with sorafenib, demonstrated that higher values of 2-deoxy-2-[ 18 F]-fluoro-D-glucose ([ 18 F]FDG) uptake (expressed as SUVmax) were correlated with lower overall survival and more advanced HCC [15][16][17][18][19][20][21] .In HCC cell lines, the absence of p53 expression is indicative of a worse prognosis, as is the increased [ 18 F]FDG uptake [22] .A recent metabolic study of HCC by positron emission tomography ([ 18 F]FDG PET) showed that this imaging technique could be a prognostic tool, as results correlate with long-term survival and early recurrence of HCC after liver transplantation [21,23] .This tool can be used to identify the most undifferentiated and aggressive tumors to select patients who should undergo liver transplantation [17] .
Sorafenib was the first drug approved by the FDA for treatment of advanced HCC (BCLC C) [24,25] .The usual dose for humans is 800 mg/day, providing an average of 127 μmol/L/h of plasmatic concentration (AUC 0-12h ), while the equivalent dosage in rats is 5 mg/kg/day [26][27][28] .It had been showed that sorafenib (2.5 mg/kg/day) has the capacity to prevent hepatic fibrosis, mitochondrial dysfunction, and reduce inflammation (interleukines 6 and 10) in NAFLD animal models [29] .Furthermore, Yang et al. [30] tested it for 8 weeks (5 mg/kg/day) and demonstrated that sorafenib improved hepatic venous dysregulation, inhibited recruitment and activation of leukocytes, and reduced splanchnic vasodilatation and ascites.
This study aimed to evaluate the effect of sorafenib in a rat model of NAFLD related to HCC by using [ 18 F] FDG PET imaging as a tool to quantitate the degree of HCC differentiation in vivo.

METHODS
This study was approved by the ethical committee for animal use of the University of Sao Paulo Medical School (protocol 108/14), following the current standards of small animal care.
Thirty male Sprague-Dawley rats weighing 300-400 g at 8 weeks of age were used.HCC secondary to NAFLD was induced by high fat and choline deficient diets [35% of total fat, enriched with 54% of trans-fatty acids (Rhoster Ltd., BR)], with a DEN (Sigma-Aldrich Chemical, St. Louis, MO, USA) dose of 100 mg/L in the drinking water ad libitum for 16 weeks.After this period stimuli were suspended and the animals were randomly split into 2 groups.The control group (n = 10) received 1 mL of saline solution (0.9%) daily by gavage for 3 more weeks.The sorafenib group (n = 20) received 5 mg/kg/day of sorafenib (Bayer Healthcare Pharmaceuticals, Cologne, GY) by gavage for 3 more weeks.
At 16 weeks, rats were anesthetized with ketamine 80 mg/kg (Cristalia, BR) and xylazine 10 mg/kg (Bayer, BR) intraperitoneally and then submitted to abdominal ultrasound (US) to quantify, measure, and localize hepatic nodules [31] .Board-certificated radiologist (M.C.C) with 24 years of experience performed the procedure using a Philips Ultrasound IU 22 system (Bothell, WA, USA) with a VL13-5 transducer.Only nodules larger than 0.2 cm were considered and catalogued in the distribution of parenchyma: left lobe, right lobe, medium lobe, and caudate lobe.
PET images with [ 18 F]FDG were acquired at the end of treatment (3 weeks after initial sorafenib) in a small animal PET scanner (LabPET4 Gamma Medica-Ideas, Northridge, CA, USA), using methods similar to those outlined in Park et al. [15] .Animals were anesthetized with isoflurane 5% in oxygen 100% for induction and 2%-3% for maintenance.[ 18 F]FDG was injected in the penile vein (37.7 ± 6.29 MBq) and the animals were allowed to wake up after injection for better tracer distribution.After 45 min of tracer injection the animals were anesthetized again and the image acquired for 30 min.Computed tomography (CT) images were also obtained with 65 kVp, 165 μA in 512 projections and magnification of 1.3 for anatomic correlation.
PET images were reconstructed by the ordered subsets expectation-maximization 3D (OSEM-3D) method [32] with 20 interactions, 4 subsets, a transverse field of view of 100 mm, and a matrix of 240 × 240, for pixel resolution of 0.42 mm × 0.42 mm.The CT images were reconstructed with a filtered back projection method, a matrix of 512 × 512, and pixel resolution of 0.17 mm × 0.17 mm.CT images were used for attenuation correction of the PET images.
Images were analyzed by the PMOD™ software (PMOD Technologies Ltd., Zurich, CH) to obtain quantitative measures of [ 18 F]FDG uptake in defined regions of interest in the rats in each group.The visualization interface in the software allowed regions of interest (ROI) to be drawn specifically and entirely within tumor lesions, liver tissue and muscle.The uptake values were expressed as using the dose-normalized parameter standardized uptake value (SUV).SUV = radioactivity concentration (kBq/mL)/[injected dose (kBq)/animal weight (g)].
The maximum value of the SUV within a region of interest is expressed as SUVmax.
Three days after the PET scan (week 19), the rats were euthanized with dextroketamine (Cristalia, BR) 120 mg/kg and xylazine (Bayer, BR) 10 mg/kg intraperitoneally.Liver samples of the right and left lobes and the larger tumors evidenced in PET/CT were collected for histological analysis.The liver specimens were fixed in 4% formaldehyde and stained with hematoxylin-eosin (HE).These samples were blindly scored by a veterinary hepatopathologist (B.C) with 12 years of experience, using a modified classification standardized by Kleiner et al. [33] .The variables analyzed were steatosis (0-3), lobular inflammatory changes (0-3), hepatocyte ballooning (0-2), fibrosis (0-4), and ductular reaction (0-3) through the NALFD activity score (NAS) [33] .HCC was diagnosed with characteristics defined by Thoolen et al. [34] for rats and then classified by Schlageter et al. [35] .Histological classifications are considered the gold standard for assessment of HCC and are used in this study to evaluate the performance of the non-invasive characterization using 18 F-FDG PET.
Statistical analysis was done with Excel® and GraphPad Prism® 7.0.The Student t-test was used for Gaussian distribution variables and the Mann-Whitney test was used for non-Gaussian distribution variables.Calculation of descriptive statistics: mean, median, and standard deviation, was performed using the appropriate form of the statistic for the distribution pattern of variables.To compare histological findings with PET findings, chi-square, linear regression, Kruskal-Wallis and Tukey post-hoc tests were used.Only P values less than 0.05 were considered statistically relevant.

RESULTS
All animals completed the first 16 weeks, but between the 16th and 19th week, when the treatment began, animals' mortality reached 60% in both groups.The main causes of death were pneumonia, hemorrhagic ascites due to tumor rupture, included during anesthesia induction for PET scan.Mean survival in the sorafenib group was 130 ± 4.9 days with a median of 133 days, and the control group was 126.3 ± 8.5 days with a median of 130 days (P = 0.07).The animals' body weight did not show a statistical difference between groups during the study, however it was different at time of euthanasia [Table 1].
US findings showed no difference between groups regarding the average liver nodules distribution per animal (4.88 ± 2.75 in the control group vs. 4.95 ± 3.11 in the sorafenib group, P = 0.48) or major nodule size [Table 2].The average number of nodules per animal detected by PET was 4.37 ± 1.59 in the sorafenib group and 8.5 ± 3.7 in the control group (P = 0.006) [Figure 2].4].Significant differences were seen between grade II vs. III (P = 0.023) and grade II vs. IV (P = 0.013), but between grade III and IV the differences were not significant (P = 0.449).Correlation between less differentiated/undifferentiated HCC (Grades III/IV) and the highest values of [ 18 F] FDG uptake, presented as tumor SUVmax (R 2 = 0.44, P = 0.01) or as a tumor ratio, either Tumor SUVmax/ Liver SUVmax ratio (R 2 = 0.42, P = 0.02) or Tumor SUVmax/SUVmax muscle ratio (R 2 = 0.54, P = 0.006) was found [Figure 5].

DISCUSSION
Our study is the first to evaluate the effect of sorafenib in a mixed experimental model of advanced HCC secondary to NAFLD using PET imaging with [ 18 F]FDG for quantitation of tumor growth.The decreased HCC nodules per animal in the treated group suggests the positive effect of sorafenib treatment, which is affirmed by the higher proportion of well-differentiated lesions (Edmondson-Steiner Grades I/II) in the treated group.The PET findings showing fewer lesions with high uptake per animal in the sorafenib group  Mattina et al. [36] published a meta-analysis summarizing the antitumor efficacy of sorafenib in preclinical studies.They found that 95% of the models used human xenotranplants to assess effectiveness of the drug.Although most cell lines show robust action of sorafenib in mice, others like McA-RH7777 did not respond [12] .In our study, we observed a decrease in the mean number of HCC lesions per animal, and a decrease in lesion aggressiveness; without a complete cure.
Groß et al. [12] compared differences in the response of sorafenib between the model with isolated use of DEN in water for 8 weeks and a model inoculated with cancerous cells in the liver by injecting them in the portal vein.The DEN model had intra-and inter-tumoral variability, as it does in humans, while the cancer cells  were more homogeneous.In addition, sorafenib only acted on the DEN model, decreasing tumor growth and perfusion [12] .Similar findings were demonstrated in another study that observed a low objective response (3%) by RECIST criteria [37] .This cytotoxic response pattern was also observed in another clinical study, in which approximately 42% of patients were stable or had a minor/partial response [38] .
In the current study, we found the presence of advanced fibrosis or cirrhosis in all animals, as well as advanced HCC with vascular invasion in 75% of control animals (n = 4) and 43% of animals in the treated group (n = 8).These findings highlight the clinical relevance of our model, as most preclinical studies used younger animals with less advanced disease, thus not reflecting the hepatic microenvironment observed in humans [36] .
Despite treatment with sorafenib, the mortality rate (60%) was similar in both groups, resulting from the severity of liver cirrhosis and advanced HCC; sometimes with decompensation in ascites (about 10% in both groups) and pulmonary metastasis.In humans, the prognosis of advanced HCC is bleak, with a median survival of 6 months or 25% in 1 year [24] .Park et al. [15] used PET/CT with [ 18 F]FDG to evaluate rats exposed only to intraperitoneal DEN, administered once a week for 16 weeks.They reported a mortality rate similar to our study, or about 65% in week 19.A more precise evaluation of the effect of sorafenib on survival was not possible because all animals were euthanized 3 days after the last PET scan to minimize risk of losing more animals prior to the endpoint of the study.
The development of a biochemical marker or diagnostic tool to identify the most undifferentiated and aggressive tumors has been studied in an attempt to better select patients for curative treatment [17,24] .
[ 18 F]FDG PET appears to be a potential tool, because it has been shown that higher values of [ 18 F]FDG uptake correlates with lower overall survival, advanced HCC, undifferentiated histology (loss of p53 expression), and increased liver transplantation recurrence [17,19,22,39] .Lee et al. [23] retrospectively evaluated patients who underwent liver transplantation for HCC and had [ 18 F]FDG PET prior to surgery.They found that HCC patients with lower [ 18 F]FDG uptake had more highly differentiated HCC (Grades I/II Edmondson-Steiner classification), with lower rates of microvascular invasion and no recurrence of HCC after a 3-year followup [23] : results that are similar to the results found in our work.
The absence of a pretreatment (16th week) [ 18 F]FDG PET scan is one limitation of our study, as we could not follow the evolution of the same node with one diagnostic tool throughout the experiment.This decision was based on two reasons: (1) high probability of increasing mortality rate on additional anesthesia during image acquisition (animals were already weak at that point) and (2) at pretreatment time point it was necessary to ensure the presence of lesions that could be detected by PET (> 1 mm) at a later point in time; therefore, a method with higher spatial resolution was chosen (US) to check homogeneity of lesion count and size between groups.US was able to show that the groups were homogeneous and that comparison between groups with PET at the end of the experiment was feasible.Although US is more sensitive in the detection of small lesions and offers good localization, it is not able to provide detailed information about tumor grade.Therefore we chose to use PET as a correlate for tumor aggression.
We showed that sorafenib could be responsible for reduced number of nodules and aggressiveness of HCC (more Grade I/II lesions than III/IV), as well as reduced tumor [ 18 F]FDG uptake.[ 18 F]FDG PET could be used as a diagnostic tool for in vivo assessment of the degree of histological differentiation of HCC, since higher values of tracer uptake were correlated with more poorly differentiated HCC.Our methods and animal model, NAFLD, which exhibited progression to advanced HCC, will be useful for further studies of hepatic carcinogenesis.

Figure 2 .
Figure 2. Illustrative images of the CT, PET with [ 18 F]FDG, and fusion PET/CT.Note that the sorafenib group shows 3 high uptake lesions in the liver, while the control group shows 5 high uptake lesions in the liver and 1 lesion in the right lung (indicated by white arrows in the fusion image)

Figure 3 .
Figure 3.Comparison of SUVmax values between nodules of the control and sorafenib groups

Figure 4 .
Figure 4. Comparison between SUVmax and HCC pathological classification

Figure 5 .
Figure 5. Dispersion graphs.(A) Plot of HCC Grade of differentiation and SUVmax taken by lesions; (B) plot of HCC Grade of differentiation and relation between tumor and liver tissue SUVmax; (C) plot of HCC Grade of differentiation and relation of tumor and muscle SUVmax.Correlation and P values are noted on the graphs