Diffusely increased splenic 18F-fluorodeoxyglucose (18F-FDG) uptake is sometimes detected by chance while interpreting PET/computed tomography (CT) images using 18F-FDG. As hepatic 18F-FDG uptake is useful as an internal reference in clinical settings, splenic 18F-FDG uptake exceeding hepatic 18F-FDG uptake is considered abnormal 1. It has been demonstrated that diffuse splenic 18F-FDG uptake can be found in cases of lymphoma, HIV infection, sarcoidosis, malaria, congestive splenomegaly, toxoplasmosis, varicella infection, and granulomatous angitis 2–5. In addition, a previous study from our institution has shown that concurrent inflammation or anemia may be associated with splenic 18F-FDG uptake and that the serum C-reactive protein (CRP) level is positively correlated with the ratio of the maximum standardized uptake value (SUVmax) of the spleen and liver 6.
Inflammation, the response of tissue to injury, is characterized in the acute phase by increased blood flow and vascular permeability and in the chronic phase by the development of humoral and cellular immune responses to the pathogen present at the site of tissue injury 7. The spleen, the largest lymphoid organ of the human body, is an important organ that is capable of efficiently mounting both innate and adaptive immune responses 8. Immune response, the complex interactions among lymphoid cells, inflammatory cells, and hematopoietic cells, is mediated by cytokines that are secreted by white blood cells (WBC) and various other cells in the body in response to tissue injury, infection, and inflammation 9. Cytokines regulate the intensity and duration of the immune response by stimulating or inhibiting the activation, proliferation, or differentiation of various cells and by regulating the secretion of antibodies or other cytokines 9.
Currently, splenic 18F-FDG uptake during PET/CT has not been considered as being the result of differences in the patterns of production of specific cytokines. In this study, we focused on cytokines to investigate interactions during this phenomenon and clarify the significance of splenic 18F-FDG uptake in PET/CT.
A total of 40 patients suffering from cholangiocarcinoma (n=28), pancreatic cancer (n=9), or gallbladder cancer (n=3) were enrolled in this study. Nineteen patients who had undergone PET/CT showing splenic 18F-FDG uptake exceeding hepatic uptake from May 2010 to September 2011 were included in group A. Twenty-one patients showing hepatic 18F-FDG uptake exceeding splenic uptake between May 2011 and September 2011 were included in group B. The representative cases of both groups are illustrated in Fig. 1.
Patients with metastasis to the spleen, lymphoma involvement of the spleen, or with known causes of splenic uptake were excluded. After informed consent was obtained from each patient, blood sampling was carried out on the same day as PET/CT. Hematological indices including serum CRP level, WBC counts, red blood cell (RBC) counts, hemoglobin (Hb) concentration, hematocrit (Hct) levels, and platelet counts were recorded from laboratory studies conducted within 3 days of PET/CT. The study was approved by the clinical research ethics committee of our institution.
For quantitative analysis of cytokines, serum samples were analyzed using the Milliplex assay (Human Cytokine/Chemokine Kit; Millipore Corp., St Charles, Missouri, USA), which allows the simultaneous quantification of the following 22 human cytokines: interleukin-1α (IL-1α), IL-1β, IL-1 receptor antagonist (IL-1RA), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-13, IL-15, IL-16, IL-17α, IL-23, interferon-inducible protein-10 (IP-10), interferon-γ (IFN-γ), growth-regulated oncogene (GRO), granulocyte macrophage-colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), and soluble CD40 ligand (sCD40L). Procedures were followed as described by the manufacturer.
Standard patient preparation included a fast of at least 8 h and a serum glucose level of less than 120 mg/dl before 18F-FDG administration. 18F-FDG PET/CT imaging was performed 60 min after administration of 370 MBq of 18F-FDG. Patients were hydrated with 500 ml of water peroral before the PET/CT imaging. At 60 min after administration of 18F-FDG, a low-dose area from the base of the skull to the proximal thighs was imaged for the purpose of attenuation correction and precise anatomical localization.
All patients were examined using a dedicated PET/CT scanner (Biograph40; Siemens, Knoxville, Tennessee, USA). The emission scan time per bed position was 3 min; six bed positions were acquired. PET data were obtained using a high-resolution whole-body scanner with an axial field of view of 21.6 cm. The average axial resolution varied between 2.0 mm full-width at half-maximum in the center and 2.4 mm at 28 cm. The average total PET/CT examination time was 20 min. Attenuation correction was performed for all patients with iterative reconstruction.
Using CT images of the 18F-FDG PET/CT, circular regions of interest (ROIs) were placed on the lumbar vertebrae (L1–L3 vertebral bodies), which were averaged in the same manner as that for the bone marrow (BM). We manually placed one a round the ROI at the center of the spleen. In addition, an elliptical ROI was placed on the right lobe of the liver, in the middle part, to avoid a mismatch between the CT and PET images and the artifact by respiratory motion. The ROIs were used to measure SUVmax of the BM, liver, and spleen in each patient. Spleen SUVmax/liver SUVmax (S/L ratio), liver SUVmax/BM SUVmax (L/B ratio), and spleen SUVmax/BM SUVmax (S/B ratio) were calculated.
Data were analyzed using the MedCalc v.9.3 software (MedCalc, Mariakerke, Belgium). The Mann–Whitney test and Fisher’s exact test were used to determine statistical differences between the two groups. The correlation between splenic 18F-FDG uptake and hematological indices was determined using Pearson’s correlation coefficient. Logistic regression was used to analyze the relationship between splenic 18F-FDG uptake and variables. Results were considered statistically significant when the P-value was less than 0.05.
Patient characteristics of the two groups are shown in Table 1. Group A (11 women and eight men) showing splenic 18F-FDG uptake exceeding hepatic 18F-FDG uptake included 19 patients, whereas group B (six women and 15 men) showing hepatic 18F-FDG uptake exceeding splenic 18F-FDG uptake included 21 patients. Spleen SUVmax (P<0.0001), BM SUVmax (P=0.0209), S/L ratio (P<0.0001), and S/B ratio (P<0.0001) were higher in group A, whereas the L/B ratio (P=0.0172) of group A was lower than that of group B. Among the hematological indices, group A showed higher WBC counts (P=0.0383) and CRP levels (P=0.0018). The RBC count (P=0.1670), Hb level (P=0.0704), Hct level (P=0.1037), and platelet count (P=0.6041) did not show significant differences between the groups.
Comparison of cytokines
Table 2 shows the results of comparison among the 22 cytokines. Levels of IL-1β (P=0.0478), IL-1RA (P=0.0044), IL-4 (P=0.0118), IL-6 (P=0.0375), IL-7 (P=0.0478), and IL-13 (P=0.0081) were statistically higher in group A compared with group B. Levels of IL-1α (P=0.4439), IL-2 (P=0.1099), IL-3 (P=0.3097), IL-5 (P=0.5273), IL-8 (P=0.1631), IL-9 (P=0.8022), IL-15 (P=0.0580), IL-16 (P=0.3201), IL-17α (P=0.8811), IL-23 (P=0.1194), IP-10 (P=0.1714), IFN-γ (P=0.5308), GRO (P=0.2847), GM-CSF (P=0.0913), G-CSF (P=0.5244), and sCD40L (P=0.1714) were not statistically significantly different between the groups. Figure 2 shows the data comparison graphs of cytokines versus mean values.
Correlations between 18F-FDG uptake and hematological indices
Correlations between spleen SUVmax, BM SUVmax, S/L ratio, and hematological indices are shown in Table 3. SUVmax of the spleen was positively correlated with WBC counts (r=0.3894, P=0.0130) and CRP levels (r=0.4750, P=0.0020). BM SUVmax was positively correlated with WBC counts (r=0.4479, P=0.0037) and CRP levels (r=0.5987, P<0.0001). The S/L ratio was positively correlated with WBC counts (r=0.3688, P=0.0192) and CRP levels (r=0.3835, P=0.0146). The S/L ratio was negatively correlated with Hb (r=−0.3529, P=0.0255) and Hct levels (r=−0.3414, P=0.0311). Scatter diagrams and regression lines of each variable are presented in Fig. 3.
Prediction of splenic 18F-FDG uptake exceeding hepatic 18F-FDG uptake
Using the logistic regression analysis, IL-13 among cytokines and CRP among hematologic indices were significant predictors of splenic 18F-FDG uptake exceeding hepatic 18F-FDG uptake (Table 4).
The current study investigated the relationship between cytokines and splenic 18F-FDG uptake. Our previous study reported that diffuse splenic 18F-FDG uptake may be related to inflammation 6 but could not present definite proof for that phenomenon.
In this study, we subdivided the patients into two groups according to the S/L ratio. Spleen SUVmax, BM SUVmax, WBC counts, and CRP levels were higher in group A, which was in accordance with the previous study 6. CRP generated by the liver in response to IL-6, tumor necrosis factor-α, and IL-1β is a representative marker for inflammation and plays an important role in the immune system 10. The elevation in the CRP level indicates that patients may also encounter inflammation.
It is clear that granulocytosis, anemia, and thromobocytosis are associated with the acute-phase response mediated by inflammation-associated cytokines and hormones 11. Among the hematological indices, only the WBC count of group A was statistically higher than that of group B in this study. In group A, all 19 patients had lower levels of hemoglobin compared with the normal range (women 12.5–15.0 g/dl; men 14.0–17.0 g/dl), whereas 75% of patients in group B (15/20) had anemia. Production of cytokines by activated macrophages and fibroblasts in the BM is believed to play a major role in the response of BM to inflammation 11. In this study, group A showed a higher BM SUVmax compared with group B. Generally, 18F-FDG uptake of the BM moves in the same direction as 18F-FDG uptake of the spleen. In addition, diffuse BM 18F-FDG uptake was assumed to be related to an inflammatory change in the BM 12.
Studies on cytokines have demonstrated that proinflammatory mediators such as IL-1β and IL-6 induce a systemic inflammatory response 13. IL-6 induces the growth and differentiation of immune cells, the production and expression of other cytokines, and acute-phase protein synthesis 14. In addition, IL-6 acts as a growth factor for mature B cells and induces their final maturation into antibody-producing plasma cells 7. IL-6 enhances the effect of IL-1β on the induction of the expression of IL-1RA 15. The proinflammatory function of IL-1β also can be inhibited by IL-1RA by a negative feedback 7. IL-4 is produced by CD4+ helper T cells, mast cells, and basophils 7. IL-4 stimulates CD4+ helper T cells to differentiate into Th2 cells and to produce IgE antibodies 16. IL-13 was originally described as an IL-4-like molecule, but studies have reported that IL-4 contributes to Th2 cell differentiation, whereas IL-13 causes Th2 inflammation and tissue remodeling 17,18. IL-7 stimulates the development of pre-B and pre-T cells 19. In the current study, levels of IL-1β, IL-1RA, IL-4, IL-6, IL-7, and IL-13 in group A were higher than those in group B among the 22 cytokines evaluated.
The S/L ratio, spleen SUVmax, and BM SUVmax were strongly correlated with CRP, the acute-phase protein, which reflects the concurrent inflammation seen in group A patients. The BM SUVmax was positively correlated with WBC counts but was not significantly correlated with RBC and platelet counts, which corresponds to the results of the study by Murata et al. 20. They hypothesized that 18F-FDG uptake of the BM reflects the hematopoietic activity of the BM and that the life span of granulocytes is shorter than that of other blood cells, which can explain the correlation between BM SUVmax and WBC counts. Anemia can be induced by inflammation-associated cytokines, resulting in adequate erythropoietin production and decreased sensitivity of erythroid precursors to the actions of erythropoietin 21. In our study, the S/L ratio was negatively correlated with Hb and Hct levels.
We hypothesized that the increase in splenic 18F-FDG uptake in group A indicates the enhancement of glucose metabolism as a consequence of activation of the immune system in the spleen. In the spleen, the white pulp consisting of T-cell-rich periarteriolar lymphoid sheaths and B-cell-rich follicles is involved in adaptive immunity 22, whereas the marginal zone is involved in both innate and adaptive immunity through macrophages and marginal-zone B cells. Iyengar et al. 23 documented that increased splenic 18F-FDG uptake can reflect massive stimulation of B cells in the spleen during HIV infection. In addition, in human malarial infection both the high density and functional activity of splenic macrophages may contribute to an increase in splenic 18F-FDG uptake 24. Macrophages play a major role as antigen-presenting cells and are essential for T-cell-clone formation 25. According to the results of our study, the increase in the levels of IL-4 and IL-13 in group A may be connected to the humoral immunity of the spleen. However, other than humoral immunity, inflammatory responses may also be involved in the phenomenon of splenic 18F-FDG uptake.
The present study has several limitations. The number of patients in our study was small. Splenic 18F-FDG uptake without an abnormal CT finding corresponding anatomically was rarely visualized in the clinical setting. In addition, we included patients with hepatobiliary and pancreatic cancers in order to avoid heterogeneity in the sample populations. In a preliminary study, we collected PET/CT images showing splenic 18F-FDG uptake exceeding hepatic 18F-FDG uptake, and we found that patients with those cancers had a tendency to show splenic 18F-FDG uptake compared with patients with other cancers. The exact reason for this tendency has not been found yet.
In summary, our data show that splenic 18F-FDG uptake is associated with inflammation, and patients showing that phenomenon had higher levels of IL-1β, IL-1RA, IL-4, IL-6, IL-7, and IL-13. This may be connected to humoral immune response. Further studies with a larger sample size and animal research are needed.
This study was supported by Biomedical Research Grant (2012-17) of Pusan National University Hospital.
Conflicts of interest
There are no conflicts of interest.
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