Diffuse splenic FDG uptake is predictive of clinical outcomes in patients with rectal cancer

This study aimed to investigate the correlations between diffuse splenic Fluorine-18-fluorodeoxyglucose (18F-FDG) uptake on positron emission tomography/computed tomography (PET/CT) and inflammatory markers and to evaluate the prognostic significance of splenic FDG uptake in rectal cancer patients who underwent curative surgery. We retrospectively analyzed the data from 161 patients who underwent splenic FDG PET/CT staging and subsequent curative surgical resection of rectal cancer between July 2006 and September 2014. The spleen-to-liver uptake ratio (S/L ratio) was calculated by dividing the spleen SUVmean by liver SUVmean. We found significant positive correlations between the S/L ratio and neutrophil-to-lymphocyte ratio (P = 0.013) and platelet-to-lymphocyte ratio (P = 0.007). In a Kaplan–Meier analysis, patients with S/L ratio ≤0.815 had a significantly higher recurrence-free survival rate than those with S/L ratio >0.815 (P = 0.028). Also, patients with S/L ratio ≤0.731 had a significantly higher overall survival rate than those with S/L ratio >0.731 (P = 0.036). In multivariate analysis, higher S/L ratio, as well as male, poor differentiation, higher TNM stage, perineural invasion, and larger tumor size, was independently predictive of cancer recurrence (>0.815 vs ≤0.815, hazard ratio [HR]: 2.04, P = 0.046). With regard to OS, S/L ratio was also an independent prognostic factor for death during follow-up (>0.731 vs ≤0.731, HR: 3.81, P = 0.017). Our results show significant correlations between S/L ratio on PET/CT and systemic inflammatory markers. Further, S/L ratio was an independent prognostic factor for predicting recurrence and death in patient with rectal cancer after curative surgery.

Laboratory studies and cancer staging. Laboratory data, including white blood cell (WBC) (/mm 3 , normal range 4,000-10,000), lymphocyte (%, normal range 20.5-51.1) platelet (/mm 3 , normal range 150,000-450,000), hemoglobin (g/dL, normal range 12.0-16.0), albumin (g/dL, normal range 3.5-5.1), carcinoembryonic antigen (CEA) (U/ml, normal range -5.0), and carbohydrate antigen 19-9 (CA19-9) (U/ml, normal range -27.0) were collected from laboratory studies performed within 10 days before or after 18 F-FDG PET/CT study. The median period from FDG PET/CT to surgery was 36 days (range 1-237 days). Tumor-node-metastasis (TNM) stages of the study subjects were assessed according to the American Joint Committee on Cancer (AJCC) staging guidelines 24 . 18 F-FDG PET images were obtained using an Allegro PET scanner (Philips-ADAC Medical Systems, Cleveland, OH, USA) or a Biograph mCT (Siemens Medical Solutions, Erlangen, Germany). Patients were instructed to fast for at least 6 hours before visiting our institutional PET center. About 5.18 MBq/kg of FDG was intravenously injected after confirming the level of fasting blood glucose was less than 140 mg/dL in all patients. Patients were asked to rest for 60 minutes prior to image acquisition. The covering range of PET or PET/CT was from the skull base to the thigh with the patient in the supine position. For PET, a transmission scan using the point source of 137 Cs was performed for attenuation correction, which was then followed by an emission scan. For PET/CT, a low-dose CT scan was obtained without contrast enhancement for attenuation correction and, subsequently, an emission scan of 2 minutes per bed positions using a three-dimensional mode was acquired and reconstructed using a three-dimensional OSEM iterative algorithm. The PET images of all patients were interpreted by two experienced nuclear medicine physicians who were unaware of the clinical outcomes. Regions of interest (ROIs) confined to the spleen were drawn, and mean standardized uptake values (SUV) were measured and defined as spleen SUV mean . The liver SUV mean was measured in ROIs placed on the right lobe of the liver. To prevent respiratory motion-induced artifacts and uptake by adjacent organs (e.g., the bowel), the ROI was placed in the middle part of the organ in the cranio-caudal direction 21 . In patients with a single hepatic metastasis, the liver SUV mean was measured in a ROI that did not encompass metastatic lesions. The spleen-to-liver uptake ratio (S/L ratio) was calculated by dividing the spleen SUV mean by liver SUV mean for each patient. For the precise measurement of metabolic activity, the placement of ROI was carefully determined and adjusted to avoid possible spillover uptake from neighboring tissue (e.g., the bowel). Correlations between diffuse splenic FDG uptake and inflammatory markers. To evaluate the association between diffuse splenic FDG uptake and systemic inflammatory markers, we assessed S/L ratio, WBC, NLR, and PLR. In addition, we also assessed albumin and hemoglobin ( Table 2). We found that S/L ratio was positively correlated with NLR (r s = 0.195, P = 0.013, Fig. 2A) and PLR (r s = 0.213, P = 0.007, Fig. 2B). In addition, a negative correlation was observed between the S/L ratio and hemoglobin (r s = −0.292, P < 0.001, Fig. 2C) and albumin (r s = −0.318, P < 0.001, Fig. 2D).

Clinical factors predicting recurrence during follow-up.
To evaluate the association of clinical features, systemic inflammatory markers and diffuse splenic FDG uptake with recurrence, Cox proportional hazards regression analyses were performed ( Table 3). The optimal cutoff values determined by the ROC curve analysis were 5.0 cm for tumor size, 2.05 for NLR, 177.6 for PLR, 0.815 for S/L ratio, 12.5 g/dL for hemoglobin, and 3.9 g/ dL for albumin for the RFS.
Clinical factors predicting mortality during follow-up. The optimal cutoff values determined by the ROC curve analysis were 3.7 cm for tumor size, 2.17 for NLR, 145.4 for PLR, 0.731 for S/L ratio, 11.5 g/dL for hemoglobin, and 3.8 g/dL for albumin for the OS.

Discussion
In the present study, we found a notable association between S/L ratio and systemic inflammatory markers existed. S/L ratio, NLR, and PLR were significantly associated with recurrence and death in the univariate analysis, while only S/L ratio was an independent prognostic factor for recurrence and death in patients with rectal cancer after curative surgical resection. Several studies have reported that the degree of splenic FDG uptake is related to inflammation and hematological parameters 19,[21][22][23]25 . Meanwhile, we considered that the spleen SUV itself would not be appropriate parameter because it can be influenced by various biological and technical factors 26 . Instead, we calculated the S/L ratio by dividing the spleen SUV mean by the liver SUV mean , which is widely used as an internal reference. The hepatic FDG uptake is known as an useful internal reference in clinical diagnostic settings due to its homogeneous values 8,9,27 . In our study, S/L ratio was significantly correlated with NLR, PLR, and hemoglobin as well as albumin. Previous studies have revealed that a significant positive associations of S/L ratio with systemic inflammatory markers such as WBC, neutrophil and CRP were present in patients diagnosed with lung cancer and cholangiocarcinoma 21,28,29 . According prior investigations, presumably, diffusely increased splenic uptake FDG is considered to be a systemic inflammatory response-related phenomenon [28][29][30][31] . The spleen consists of red pulp and white pulp, which are separated by the marginal zone containing macrophages and marginal zone B cells 32,33 . The white pulp is involved in adaptive immunity and the marginal zone is involved in both innate and adaptive immunity 12 . An increased splenic FDG uptake can indicate the activation of immune systems in the spleen and can reflect the presence of active systemic inflammation [34][35][36][37] . Inflammation has powerful effect on cancer promoters, facilitating genomic instability, and stimulating angiogenesis 14,38,39 . Among inflammatory cells, neutrophils can contribute to cancer promoters and cancer-activated platelets can drive cancer progression and invasion in rectal cancer, whereas lymphocytes can act on cancer-killing responses [40][41][42] . Albumin is also related to the cancer cell-induced inflammatory reaction 43 Table 2. Correlation between Fluorodeoxyglucose uptake of spleen-to-liver and systemic inflammatory and hematologic markers. Abbreviations: r s , Spearman correlation coefficients; NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio; WBC, White Blood Cells; S/L ratio, spleen-to-liver mean standardized uptake ratio. inflammatory markers 39,44,45 . Similarly to 18 F-FDG uptake of the spleen, 18 F-FDG uptake of bone marrow (BM) in patients with cancer has been reported to reflect the degree of systemic inflammation 19 . In that study, BM SUV was positively associated with systemic inflammatory markers including NLR, PLR, and CRP 19,31,35 . Further, BM was found to be an independent predictor for recurrence in non-small cell lung cancer, lymphoma, and colorectal cancer 10,23,31,46 .
In this study, we also identified the prognostic value of S/L ratio for predicting recurrence and death in rectal cancer. Regarding this issue, 18 F-FDG uptake of the spleen has been already found to be significantly associated with death in patients with unresectable cholangiocarcinoma 29 . Furthermore, we reported previously that diffuse splenic 18 F-FDG uptake is significantly related to the RFS and OS of patients with gastric cancer 25 . In the current study, univariate analyses demonstrated that S/L ratio, NLR, and PLR were significantly associated with recurrence and death. Among them, only S/L ratio was independently predictive of recurrence and death in multivariate analysis. This suggests that S/L ratio can be more helpful in the prediction of clinical outcome as compared with other systemic inflammatory markers. Patients with a high S/L ratio showed worse RFS and OS than did those with a low S/L ratio. Cancer recurrence occurred in 22.3% of patients with a high S/L ratio within 2-years after surgery, whereas it developed in 15.7% of those with a low S/L ratio. Similarly, 8.0% of patients with a high S/L ratio died within 2-years of surgical resection, whereas 5.2% of patients with a low S/L ratio died within that period. We thus hypothesize that among patients with rectal cancer, a higher S/L ratio is predictive of poor  Table 3. Clinical factors associated with the recurrence during follow-up. Abbreviations: HR, hazard ratio; CI, confidence interval; TNM, tumor-node-metastasis; NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-tolymphocyte ratio; S/L ratio, spleen-to-liver mean standardized uptake ratio. prognosis after curative surgical resection. Followed by previous study for gastric cancer 25 , this suggests that diffuse splenic FDG uptake, using hepatic FDG uptake as internal reference, has prognostic utility in patients with cancer of the gastrointestinal tract. In addition, we found a significant negative correlation between the hemoglobin level and the S/L ratio. Therefore, anemia is predictive of death in patients with rectal cancer. Few studies have reported that anemia is associated with diffuse splenic FDG uptake on PET/CT in cancer patients 21,22,28 . We assume that concurrent anemia during PET/CT study may be one of the causes of diffuse splenic FDG uptake. This assumption can be explained by the fact that spleen is a secondary hematopoietic organ 12,32,33 . Thus, when diffuse splenic FDG uptake is observed on clinical PET/CT study, the degree of anemia in patients with rectal cancer who underwent curative surgical resection should be checked for correct interpretation.
The present study has several limitations. First, the number of the study subjects was relatively small, thus weakening the strength of our results. Further larger scaled studies are needed to clarify the implications of diffuse splenic FDG uptake. Second, to homogenize the patient population, we included 161 rectal cancer patients who underwent FDG PET/CT scan for staging and who had hematological data within 10 days before or after PET/CT scan. We excluded patients with colon cancer in order to avoid heterogeneity in the sample population, which might have led to selection bias. Third, diffuse splenic FDG uptake may be influenced by other factors, particularly hemoglobin level. Because anemia stimulates hematopoiesis, hemoglobin level can be associated with FDG uptake of the spleen 12,32 . Fourth, we did not consider pre-or post-operative chemotherapy or radiotherapy, which may affect the prognosis of patients with rectal cancer. Fifth, hepatic micrometastasis, which cannot be detected by PET or PET/CT, might bias the liver SUV mean data, despite exclusion of patients with multiple or huge hepatic metastases. In patients with a single hepatic metastasis, the liver SUV mean was measured in ROIs that did not encompass metastatic lesions. Sixth, the use of two different PET system, a stand-alone PET and PET/CT, is a major limitation. Though the SUV is the most common method in PET studies, using the parameter is not appropriate in our study because technologic factors from different scanners are known to affect SUV measurement. Thus, we used the SUV ratio (S/L ratio) rather than the SUV itself. Last, information on serum CRP results is needed to confirm the correlations between diffuse splenic FDG uptake and the systemic inflammatory markers. The level of CRP, an important acute-phase protein, provides a more accurate estimate of the inflammatory status than other systemic markers of inflammation 16,19,21,35,45 . In our hospital the CRP level is not evaluated during the preoperative work-up of patients with rectal cancer not suspected to have infection or inflammation. The patients in this study did not have infection or inflammation and, thus, did not undergo measurement of the CRP level.
In conclusion, S/L ratio on FDG PET/CT imaging was significantly correlated with systemic inflammatory markers. Moreover, S/L ratio was an independent prognostic factor for the prediction of recurrence or death in rectal cancer during follow-up after curative surgery. Accordingly, we suggest that physicians need to pay particular attention to patients with a high S/L ratio among those with rectal cancer who undergo curative surgical resection.  Table 4. Clinical factors associated with the death during follow-up. Abbreviations: HR, hazard ratio; CI, confidence interval; TNM, tumor-node-metastasis; NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-tolymphocyte ratio; S/L ratio, spleen-to-liver mean standardized uptake ratio.