Association of PET Scan Parameters of Pulmonary Masses and Reticuloendothelial System with Hematologic Parameters

Objectives: The most active organs of the reticuloendothelial system (RES) are the liver, spleen and bone marrow, having immune mechanisms against malignancy including neutrophils and platelets. RES may be imaged by different modalities, like PET scan. Neutrophil lymphocyte ratio (NLR) and platelet lymphocyte ratio (PLR) have gained importance as proinflammatory markers in cancer, e.g. lung cancer. The aim of this study was to investigate the relationship between PET parameters of pulmonary mass or RES, and hematological parameters, and to evaluate the role of these factors in differentiating the pathological character of the mass. Methods: A retrospective analysis of the data of 131 patients, retrieved from the department archives, with pulmonary mass limited to mediastinum was made. Patients were grouped according to pathological results: benign mass (n=46), squamous cell carcinoma (n=38), and non-squamous cancer of lung (n=47). All patients underwent PET/CT scanning and images were analyzed retrospectively. Maximum and mean SUV were calculated from primary lesions and RES. NLR and PLR were calculated from CBC. Results: SUVmax and SUVmean of RES organs were similar for both groups with benign and malignant pulmonary masses, and among the subgroups. SUVmax ratios of pulmonary mass were significantly different between the groups (the highest value in the squamous cell carcinoma and the lowest in the benign groups). No significant difference was determined between the subgroups for NLR and PLR. NLR was significantly correlated with SUVmax ratios of spleen and the mass, and SUVmean ratios of spleen and bone marrow. PLR was significantly correlated with SUVmax ratios of spleen, bone marrow, the mass and SUVmean ratios of spleen, bone marrow. Conclusion: SUV of RES and primary mass were correlated with NLR and PLR, indicators of systemic inflammation. The associations between NLR and PLR, and SUV should be clearly defined by further investigations.


Introduction
The reticuloendothelial system (RES) was first described by Aschoff in 1924, and is also known as the monocyte macrophage system. The organs containing the most active reticuloendothelial cells are the liver, spleen and bone marrow. Monocyte-macrophage groups of cells residing in these organs play a role in inflammation and immunity, such as defense against pathogens, removal of dead cells, debris and malignant cells. The cells of this system are motile and phagocytic, so that they can ingest and destroy unwanted foreign material. They play important roles both in cellular and adaptive immunity, and in defense against tumors.
Fluorine-18-2-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET) can be used to diagnose, to stage and monitor patients with different kinds of malignant tumors, and to detect recurrence in such patients. This modality has also been adopted as an effective method to diagnose and monitor several benign conditions, such as infectious and inflammatory processes. Therefore, as RES is one of the effectors of systemic immunity, 18F-FDG PET could have an important place in determining the existence and amount of immune response in RES organs against malignancy. Combined PET/CT screening provides metabolic information from PET and anatomical information from CT (computed tomography). Maximum standardized uptake value (SUV max ) is one of the most frequently used parameters of PET/CT, reflecting metabolic activity of the tumor. Some hematological parameters in addition to platelets, leukocytes, neutrophils and lymphocytes have gained importance in recent years, such as neutrophil lymphocyte ratio (NLR), and platelet lymphocyte ratio (PLR). NLR and PLR have been revealed as proinflammatory markers in some studies [1][2][3] and in cancer patients. Previous studies have shown that neutrophils and platelets have different actions in tumor immunology. A significant correlation has been determined between increased neutrophil counts and tumor aggressiveness. These parameters have been investigated in cardiovascular studies many times, but few studies have investigated the correlations between these parameters and PET SUV max of tumoral masses and RES organs in cancer patients.
Lung cancer is known to be one of the major causes of cancerrelated death and 3 million new cases per year are reported worldwide. The overall 5 year survival rates of lung cancer increased to 15% [4,5]. It is also known that lung cancer is the second most common cancer in both males and females. The most common type of lung cancer encountered in patients is adenocancer, followed by squamous cell carcinoma of the lung. As in other types of cancer, the immune system is activated in lung cancer against malignant cells. This activation may be reflected both in the blood as alterations in hematological parameters and in RES organs such as the liver, spleen and bone marrow. Although hematological alterations may be measured by counting, changes in RES can be shown by imaging modalities, such as 18F-FDG PET/CT determining metabolic activity.
The aim of this study was to investigate the relationship between SUV max in pulmonary mass limited to the mediastinum measured by PET scanning and hematological parameters, and to evaluate the importance of these values in the discrimination of pathological diagnosis of the mass as malignant or benign. It was also aimed to determine whether there was any association between PET/CT parameters reflecting RES activity and hematological parameters, and the value of these factors in differentiating the pathological character of the mass as benign or malignant.

Materials and Methods
Of the total 12,380 patients on whom PET/CT imaging was performed between June 2012 and June 2016, evaluation was made of 2256 patients who were referred with pre-diagnosis of mediastinal or pulmonary mass. Of these, 516 patients had no extra-thoracic involvement in PET/CT, so of these, 131 had pathological recordings and complete blood count in same week and were included in the study, while the others were excluded. The data of the patients admitted were analyzed retrospectively by retrieving the patient files from Department of Nuclear Medicine, Saglik Bilimleri University Diyarbakir Gazi Yasargil Training and Research Hospital, Diyarbakir, Turkey. The patients were grouped as benign mass (n=46), squamous cell carcinoma of the lung (n=38), and non-squamous cancer of the lung (n=47), according to the pathological results of the examined biopsy or excised material.
The PET/CT images of the patients were analyzed again retrospectively. All patients underwent routine PET/CT scanning with Biograph 6 PET/CT (Siemens Medical Systems, CTI, Knoxville, TN, USA). Images were taken after at least 6 hours fasting, and glucose levels in peripheral blood in all patients were confirmed to be 140 mg/dl or less before the FDG injection. Approximately 5.5 MBq/kg of FDG was administered intravenously 1 hour before image acquisition. After the initial low-dose CT (Discovery 600: Biograph 6: 40 mA, 120 kVp), standard PET imaging was performed from the skull base to the proximal thighs with an acquisition time of 3 min/bed in threedimensional mode. Images were then reconstructed using the ordered subset expectation maximization algorithm (2 iterations, 20 subsets). SUV max values were calculated from primary lesions in the mediastinum or lung in all patients. Volume of interest (VOI) of 2cm diameter was taken in the liver, spleen, abdominal aorta, and L2 vertebra, and thus SUV max and SUV mean values were obtained. The SUV values of the aorta and liver were accepted as reference values. SUV max ratios of liver/aorta, spleen/aorta, bone marrow/aorta, spleen/ liver, bone marrow/liver, and SUV mean ratios of liver/aorta, spleen/ aorta, bone marrow/aorta, spleen/liver, bone marrow/liver were calculated, and RES activity in PET/CT was calculated. SUV max ratios of mass/liver, mass/aorta were calculated and standardized in all patients.
In all patients, leukocyte, neutrophil, thrombocyte and lymphocyte counts were measured as the number of cells per microliter in complete blood count (CBC) analyzed by automatic hematology analyzer. NLR was calculated by dividing the neutrophil count by the lymphocyte count, and PLR by dividing the platelet count by the lymphocyte count. SPSS 22.0 (IBM Corporation, Armonk, New York, United States) program was used for analysis of variables. The conformity of data to normal distribution was evaluated with the Shapiro-Wilk test and homogeneity of variance with the Levene test. When comparing the quantitative data of 2 independent groups, the Independent Samples T test was used together with Bootstrap results, and Mann-Whitney U (Exact) test was used together with Monte Carlo results. When comparing the quantitative data of more than 2 groups, One-Way Anova and Kruskal-Wallis H Tests were used. Dunn's Test, Fisher's Least Significant Difference (LSD) and Games-Howell tests were applied for Post Hoc analyses. To examine the correlations of variables, the Pearson Chi-Square and Fisher Exact tests were applied with the Monte Carlo stimulation technique. Quantitative variables were shown as mean ± standard deviation (SD) and median range (maximumminimum), and categorical variables as number (n) and percentage (%) in tables. Variables were analyzed at 95% confidence level and a value of p <0.05 was accepted as statistically significant.

Results
Of the total patients, 73.9% with a benign pulmonary mass and 77.6% with a malignant pulmonary mass were male. The mean age of patients with a benign mass was 58.43 ± 13.10 years, in those with a malignant mass, it was 58.56 ± 12.03 years. No significant difference was determined between the patient groups with a benign or malignant pulmonary mass in respect of the SUV max and SUV mean ratios of the liver/aorta, spleen/aorta, and bone marrow/aorta (pSUV max 0.455, 0.894, 0.469, respectively; pSUV mean 0.203, 0.651, 0.553, respectively). No significant difference was determined between the 2 patient groups in respect of the SUV max and SUV mean ratios of the spleen/liver, and bone marrow/liver (pSUV max 0.821, 0.485, respectively; pSUV mean 0.107, 0.673, respectively). The SUV max ratios of the mass/aorta and mass/liver were significantly different between the groups (pSUV max 0.003 and 0.006, respectively). No significant difference was determined between the groups in respect of leukocyte, thrombocyte, lymphocyte, neutrophil counts, and NLR and PLR (    Male gender was determined in 73.9% of the patients with a benign mass, in 74.5% of non-squamous cancer cases, and in 81.6% of squamous cell tumor cases. The mean age of these groups was 58.43 ± 13.10 years, 54.77 ± 12.05 years, and 63.26 ± 10.36 years, respectively. The mean age was significantly higher in the squamous cell carcinoma group compared to the non-squamous carcinoma group (p=0.001). There were no significant differences between the groups in respect of SUV max and SUV mean ratios of liver/aorta, spleen/aorta, bone marrow/ aorta (pSUV max 0.621, 0.968, 0.596 respectively; pSUV mean 0.328, 0.595, 0.178, respectively). No significant differences were determined between the groups in respect of the SUV max and SUV mean ratios of spleen/liver and bone marrow/liver. A significant difference was determined between patients with a benign mass and squamous cell carcinoma in respect of the SUV max ratios of mass/aorta and mass/liver (p<0.001 and p=0.001, respectively), and between the non-squamous carcinoma and squamous cell carcinoma patients (p=0.049 and p=0.038, respectively).
These SUV max ratios were determined as highest in the squamous cell carcinoma group and lowest in the benign mass group. Although the number of platelets were highest in the squamous cell carcinoma group (p=0.034), no statistically significant differences were detected between pathological subgroups of patients for leukocyte, neutrophil, and lymphocyte numbers, and NLR and PLR (  Leukocyte numbers were not correlated with the SUV max and SUV mean ratios of RES organs and mass lesions. The SUV max ratios of mass/aorta and mass/liver were positively correlated with neutrophil counts, and no other significant correlations were found between neutrophil counts and SUV ratios. The SUV max bone marrow/aorta, SUV mean bone marrow/aorta, SUV mean spleen/liver, SUV mean bone marrow/liver, SUV max bone marrow/liver, SUV max mass/aorta, SUV max mass/liver ratios were positively correlated with thrombocyte counts. A significant negative correlation was determined between thrombocyte counts and the SUV mean ratio of liver/aorta. Significant negative correlations were determined between lymphocyte counts, and the SUV mean ratios of spleen/aorta and spleen/liver. NLR was significantly correlated with the SUV max ratios of spleen/aorta, spleen/liver, mass/ aorta, mass/liver, and the SUV mean ratios of spleen/aorta, bone marrow/aorta, spleen/liver, bone marrow/liver. PLR was significantly correlated with the SUV max ratios of spleen/liver, bone marrow/liver, mass/aorta, mass/liver, and the SUV mean ratios of spleen/aorta, bone marrow/aorta, spleen/liver, bone marrow/liver (  Table 3: Correlations between hematological parameters and SUV ratios.

Discussion
The immune system is composed of several mechanisms protecting the body against several kinds of pathogens [6][7][8]. As a part of the immune system, RES consists of cells located in lymph nodes, spleen, liver, and bone marrow, and has an important place in the fight against pathogens [9][10][11]. Each part of this system has a unique role in immunity; the liver has a high antigenic load from its blood flow and local immune coping mechanisms, the spleen contributes to both humoral and cellular immunity and bone marrow plays a role in producing immune cells [12][13][14].
In the current study, no significant difference was determined in respect of the maximum and mean SUV ratios of the liver, spleen, and bone marrow between the patient groups with a benign mass and squamous cell carcinoma and non-squamous cancers. In a study by Bural et al. [15] evaluation was made of 39 subjects evaluated for pulmonary nodules and who showed no evidence of activity on 18F-FDG PET imaging, and 30 subjects with lung cancer with and without distant metastases detected on 18F-FDG PET imaging. In contrast to the current study, the findings of that study revealed a statistically significant difference in respect of the SUV mean values of the RES organs (liver, spleen, bone marrow) in favor of patient group with malignant cancer(p<0.05). However, in that study, 20% of the cancer patients were diagnosed as squamous cell carcinoma, while in the current study the ratio was 44%. Thus it may be proposed that differences in distribution of pathological subclasses of lung cancer may lead to discrepancy when comparing SUV results. However, increased SUV mean in patients with lung cancer, as shown in this study, suggests that as a part of the immune system developing defense against malignancy, RES could present increased activity. The increased activity and phagocytic mechanisms causing increased glucose utilization and uptake lead to increased 18F-FDG uptake. Hence, the increased activity in the Several studies have investigated whether hematological parameters such as platelets, MPV, PLR, neutrophils, NLR, and leukocytes have any effect on the pathological results of tumoral masses [16]. Recent investigations have suggested that NLR could be a predictor for cardiovascular diseases and cancer [1][2][3]. NLR as a marker points to subclinical inflammation. In several studies, PLR has also been proposed as a new and simple marker that could be used as an indicator of inflammation accompanied by platelet mediators [17,18]. Some studies have also shown that NLR and PLR can be used as markers predicting survival in cancer patients including those with lung cancer [19][20][21][22]. Moreover, these markers may be easily obtained in daily practice [23,24]. Therefore, in the current study, NLR and PLR values were investigated in pathological subclasses of pulmonary masses to determine whether there was any correlation between NLR and PLR, and SUV ratios. No significant difference was determined between the groups according to NLR and PLR. Nikolić et al. [25] showed significantly higher NLR and PLR values in lung cancer patients (n=388) in comparison with a control group (n=47), but no difference was observed between subgroups of the lung cancer patients [25]. In the current study, NLR was significantly correlated with the SUV max ratios of spleen/aorta, spleen/liver, mass/aorta, mass/liver, and with the SUV mean ratios of spleen/aorta, bone marrow/aorta, spleen/ liver, and bone marrow/liver. PLR was significantly correlated with the SUV max ratios of spleen/liver, bone marrow/liver, mass/aorta, mass/ liver, and with the SUV mean ratios of spleen/aorta, bone marrow/aorta, spleen/liver, and bone marrow/liver. Sürücü et al. [26] investigated the association of NLR and PLR with SUV and metabolic tumor volume in a study of 52 esophageal squamous cell carcinoma patients and a control group (n=52). The NLR and PLR values were found to be higher in the patient group compared to the control group, which was consistent with literature. However, in contrast to the current study, it was demonstrated that the SUV max of esophageal tumor was not correlated with NLR or PLR. In that study, NLR was found to be correlated with MTV in the patient group, but as previously mentioned, pathological evaluation of metastases was not applied. Nam et al. showed significant correlations between the SUV max ratio of spleen/liver, and neutrophil and leukocyte counts [27]. Again contrasting with the current study results, in a study of 57 lung cancer patients, Sunnetcioglu et al. [28] reported no correlations of the SUV of the cancer mass with NLR or leukocyte counts. However, similar to the current study, it was shown that in squamous cell cancer patients the SUV max values of the tumor mass were higher than in patients with another histopathological diagnosis.
Several kinds of modalities such as MRI, and bone marrow scintigraphy with technetium 99 m, have been used to image bone marrow [29]. However, PET imaging provides both structural and functional knowledge about bone marrow both in benign and malignant conditions [30]. Although there is generally moderate 18F-FDG uptake by bone marrow in normal individuals, some investigations have shown high uptake by normal individuals on PET imaging. Whether these changes determined on PET scans are important remains unknown as there has been insufficient research to determine which levels of 18F-FDG uptake of bone marrow can be considered abnormally increased. Some studies have suggested a range of 1.3-1.6, using SUV [31,32]. However, SUV changes with many factors different from the features of the relevant organ [31][32][33]. The uptake of FDG by bone marrow may also be changed according to whether the primary tumor is benign or not. For example, the bone marrow FDG uptake of patients with lung cancer has been found to be higher than that of patients with benign pulmonary nodules [15]. Moreover, bone marrow FDG uptake in patients with a malignancy has been determined to be associated with serum cytokine and CRP levels, and some blood parameters [34][35][36]. These findings suggest that FDG uptake by bone marrow could point to a systemic immune response. Therefore, the discrimination of the physiological uptake in bone marrow or uptake due to other causes is of great importance clinically. Logically, it could be assumed that FDG uptake by marrow could be correlated with hematopoietic activity of bone marrow. In furtherance of this idea, in studies where bone marrow was imaged after administration of G-CSF and GM-CSF, increased activity has been demonstrated, probably given by these factors [32,[37][38][39]. As mentioned previously, in the current study, positive significant correlations were determined between the SUV max ratios of bone marrow/aorta and thrombocyte count and there were also significant positive correlations between the SUV mean ratios of bone marrow/ aorta, and thrombocyte, NLR and PLR. There were positive significant correlations between the SUV max ratios of bone marrow/liver, and thrombocyte and PLR, and significant positive correlations between the SUV mean ratios of bone marrow/liver, and thrombocyte, NLR and PLR. Murata et al. [40] investigated the correlations of SUV ratios of bone marrow with hematological parameters in a study of 48 patients. In that study, the values of bone marrow SUV in the lower thoracic spine (Th 11-12) and the upper lumbar spine (L1-2) were calculated, and the uptake ratio (UR) was calculated by dividing the SUV of the bone marrow by the SUV of the longitudinal dorsal muscles.
In contrast to the current study, total leukocyte counts and neutrophil counts were correlated with the SUV and uptake ratios, and the uptake ratios of the bone marrow, respectively. Similar to the current study, there were no correlations between lymphocyte counts and SUV ratio. Lee et al. [41] reported similar results according to the association between the SUV mean ratio of bone marrow to liver (BLR) and hematological parameters, in a study of 110 non-small cell lung carcinoma (NSCLC) patients who had undergone curative resection. In that study, BLR was similar between the two groups, whether or not there was recurrence and there were significant positive correlations between BLR and some hematological parameters such as PLR, NLR and total leukocyte counts (p=0.001, p<0.001, p=0.03, respectively). In the same study, only cancer patients curable with resection had been enrolled to exclude the effect of tumor FDG uptake on bone marrow FDG uptake. Again, Lee et al. [41] showed significant positive correlations of BLR with leukocyte numbers but no correlations with NLR or PLR. These findings suggested that bone marrow could indicate that systemic immunity developed as a defense mechanism against primary tumor. As patients were analyzed retrospectively in the current study, predictors for mortality were not investigated. In contrast to the current study, Prévost et al. [42] showed that BLR was a prognostic factor in non-small cell lung carcinoma, in a study of 120 patients.
The results of the current study showed that SUV max ratios were determined as highest in the squamous cell carcinoma group and lowest in the benign mass group, but the data were not analyzed to obtain a threshold value of SUV. Several studies have investigated whether the SUV value of pulmonary mass has any role in predicting the mass as benign or malignant [43]. Bryant et al. [44] reported that the higher the SUVmax value, the higher the probability that the pulmonary mass would be malignant. However, in the same study, it was shown that malignancy was found in 24% of pulmonary nodules with SUV max between 0 and 2.5. Huang et al. [45] showed significantly increased SUV max values in malignant pulmonary lesions compared to benign lesions. Several SUV max values have been proposed as a threshold for this discrimination. Some studies have accepted 2.5 as the SUV max threshold [46][47][48][49], although Yi et al. [50] considered masses with a SUV max >3.5 as malignant. Nguyen stated that a common threshold value for different sites was not feasible, but also reported SUVmax threshold >3.6 as highly sensitive and specific for pulmonary nodules [51]. Besides these findings, in some studies, it has been shown that quantitative analysis by PET scan did not improve accuracy [52][53][54]. Lobrano et al. [55] investigated the effect of SUV of pulmonary mass in differentiating whether the masses were benign or malignant. A total of 73 patients with both PET images and reports and corresponding biopsy results were included in the study. Pulmonary malignancies were determined in 75%, and benign diagnoses in 25% according to the biopsy results. In that study, SUV was not determined to be a useful predictor in discriminating malignant lesions from benign masses. 18F-FDG uptake in mediastinal or hilar lymph nodes was an important predictor for the pulmonary lesion to be malignant (p=0.024).

Conclusion
The results of this study demonstrated that SUV ratios of RES organs in FDG-PET imaging were greater in patients with lung cancer in comparison to healthy subjects, and this effect may have resulted from the development of immune defense against cancer. Further studies are required on this subject.