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Diffuse reduction of spleen density is a novel prognostic marker for intrahepatic cholangiocarcinoma after curative resection

BACKGROUND Diffuse reduction of spleen density (DROSD) is related to cancer prognosis; however, its role in intrahepatic cholangiocarcinoma (ICC) remains unclear. AIM To assess the predictive value of DROSD in the prognosis of ICC after curative resection. METHODS In this multicenter retrospective cohort study, we enrolled patients with ICC who underwent curative hepatectomy between 2012 and 2019. Preoperative spleen density was measured using computed tomography. Overall survival (OS) and recurrence-free survival (RFS) rates were calculated and compared utilizing the Kaplan–Meier method. Univariable and multivariable Cox regression analyses were applied to identify independent factors for OS and RFS. A nomogram was created with independent risk factors to predict prognosis of patients with ICC. RESULTS One hundred and sixty-seven ICC patients were enrolled. Based on the diagnostic cut-off values (spleen density ≤ 45.5 Hounsfield units), 55 (32.9%) patients had DROSD. Kaplan–Meier analysis indicated that patients with DROSD had worse OS and RFS than those without DROSD (P < 0.05). Cox regression analysis revealed that DROSD, carcinoembryonic antigen level, carbohydrate antigen 19-9 level, length of hospital stay, lymph node metastasis, and postoperative complications were independent predictors for OS (P < 0.05). The nomogram created with these factors was able to predict the prognosis of patients with ICC with good reliability (OS C-index = 0.733). The area under the curve for OS was 0.79. CONCLUSION ICC patients with DROSD have worse OS and RFS. The nomogram is a simple and practical method to identify high-risk ICC patients with poor prognosis.

Author contributions: Deng LM, Wang Y, and Yang JH contributed equally to this work; Deng LM, Wang Y, Yang JH, Jin B, Yu ZP, and Chen G conceptualized and designed the study; Chen KY, Yao XF, Bao WM, Li JL, Chen ZY, Zheng CM, and Zheng JY collected the data and performed the analyses; Deng LM and Wang Y drafted the initial version of the manuscript; Jin B and Chen G had full access to all data in the study and had responsibility for the integrity of the data, accuracy of the analyses, and final decision to submit the manuscript for publication; All authors contributed to the interpretation of the results, critically reviewed revisions of the manuscript, and contributed important intellectual content.

INTRODUCTION
Intrahepatic cholangiocarcinoma (ICC) is a subtype of cholangiocarcinoma associated with poor prognosis and limited treatment options. The incidence of ICC in the United States currently is nearly 1.65 times higher than it was 30 years ago, and a similar trend has been observed worldwide, highlighting the need for immediate attention [1][2][3]. Surgery is the most effective treatment for ICC patients [4,5]. Previous studies have demonstrated that the 5-year overall survival (OS) rate in ICC patients with negative surgical margins is 39%-41% [6][7][8][9]. However, factors that affect long-term survival after surgical resection remain equivocal. Therefore, it is imperative to explore useful prognostic predictors for ICC patients to implement suitable therapies and follow-up strategies.
The spleen is crucial in regulating immune homeostasis [10], which is vital for a favorable prognostic outcome [11]. Studies have reported the occurrence of diffuse reduction of spleen density (DROSD) in patients with gastric cancer and acute pancreatitis, and speculated that DROSD might be associated with poor prognosis [12,13]. We have observed this phenomenon in some ICC patients; however, the predictive value of DROSD in the prognosis of ICC still needs exploration.
In this multicenter retrospective cohort study, we focused on the prognostic value of DROSD in ICC patients who underwent surgical resection. We also developed a useful predictive model for the prognosis of ICC based on DROSD to inform surgeons' clinical decisions. To the best of our knowledge, this is the first study to explore the impact of DROSD on the long-term outcome of patients with ICC who underwent curative partial hepatectomy.

Patient selection
The study was based on data from two cohorts of ICC patients who underwent curative resection between August 2012 and October 2019. The two clinical cohorts included were: The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) and Qilu Hospital of Shandong University (Jinan, China). This study was performed according to the Declaration of Helsinki, and ethical approval was obtained from the Institutional Ethics Committees of The First Affiliated Hospital of Wenzhou Medical University and Qilu Hospital Shandong University. The study participants' clinicopathological information was reviewed with their written informed consent.
All patients enrolled were histologically confirmed to have ICC. Patient inclusion criteria were partial hepatectomy for ICC. The exclusion criteria were as follows: history of previous anticancer therapy or history of other malignancies, a history of splenic disease, presence of hematological disease, perioperative mortality, lack of preoperative abdominal computed tomography (CT) to evaluate spleen density, palliative resection, and loss to follow-up after discharge. The study flow diagram is illustrated in Figure 1.

Collection of baseline characteristics
Patients' demographic characteristics, preoperative laboratory indicators, clinicopathological information, and operation-related variables were retrieved from the hospital database and retrospectively reviewed. Data collected included age, sex, history of abdominal surgery, length of hospital stay, comorbidities, hepatitis B surface antigen, albumin (ALB), carcinoembryonic antigen (CEA), alpha-fetoprotein, carbohydrate antigen 19-9 (CA19-9), total cholesterol, triglycerides, platelet/lymphocyte ratio (PLR), neutrophil/lymphocyte ratio (NLR), and lymphocyte/monocyte ratio (LMR). According to the Fudan score, CA19-9 cut-off values were set at 37 U/mL [14]. We also retrieved data regarding portal hypertension, tumor node metastasis (TNM) stage (8 th staging system for ICC), tumor number, maximum tumor diameter, degree of tumor differentiation, lymph node metastasis, perineural invasion, vascular invasion, type of surgical procedure, extent of hepatectomy, intraoperative blood loss, postoperative complications (PCs), and operating time. Resection of three or more Couinaud liver segments was defined as major hepatectomy, while resection of fewer than three segments constituted minor hepatectomy. PCs were graded using the Clavien-Dindo classification system, which were recorded from the day of surgery until discharge [15]. Clavien-Dindo grade II or higher were considered as relevant complications.

Measurement of spleen density
The spleen density of each patient was retrospectively reviewed and measured on cross-sectional plain CT images without contrast enhancement. The CT positioning conditions were as follows: Tube voltage 120 kV, tube rotation time 750 ms, tube current 50 mA, layer spacing 5 mm, and layer thickness 5 mm. A special processing system (version 3.0.11.3 BN1732 bit; INFINITT Healthcare Co. Ltd., Seoul, South Korea) was used to measure the CT values at the levels of the upper pole, hilum, and lower pole of the spleen (Figure 2A-C) [12,16]. On each plane, we took two points as density values for calculations. The spleen density was taken as the average value of six CT measurements. Quality control and analysis of all images were performed by two trained physicians, separately. We used the R survminer package to calculate the cut-off value of spleen density and define DROSD ( Figure 2D). Based on this cut-off value, patients were then divided into DROSD and non-DROSD subgroups.

Outcomes
Patients were followed up once every 3 mo for the first year after surgery, once every 6 mo for the next 3 years after surgery, and once every year thereafter. OS and August 15, 2021 Volume 13 Issue 8  recurrence-free survival (RFS) were the primary endpoints. OS was calculated from the date of surgery to the date of patient death or last follow-up. RFS was defined from the date of surgery to the date of first ICC recurrence, death, or last follow-up visit. The last follow-up of the study took place on May 20, 2020.

Statistical analyses
Statistical analyses were performed using the R program (version: 3.6.1). Continuous variables are expressed as the mean ± SD or median (interquartile range). Categorical variables are expressed in terms of frequency (percentage). Missing data were imputed using multiple imputation by logistic regression. The imputation was repeated five times; the results were checked by comparing multivariable distribution of the observed and imputed data. Rubin's rule was applied when the results were pooled. Student's t-test was used to compare groups when a continuous variable showed normal distribution; otherwise, the Mann-Whitney U test was used. The χ 2 test was applied to compare differences between independent groups of categorical variables. August  The optimal sex-specific cut-off value of DROSD was selected by the survminer package in R program. The Kaplan-Meier method and log-rank test were used to estimate OS and RFS among the different subgroups. Median follow-up was measured using the R survival package. Univariable and multivariable Cox regression analyses were applied to assess the prognostic factors related to OS and RFS. The R forestplot package was used to visualize the Cox regression analysis results. The multivariable analysis results were used to create a nomogram using the rms package in R software. The C-index was applied to evaluate the performance of the nomogram. The area under the receiver operating characteristic (ROC) curve (AUC) was used to evaluate prediction accuracy by using the R survival ROC package. P < 0.05 was considered statistically significant.

Patients' characteristics
In total, 251 primary ICC patients who underwent partial hepatectomy were shortlisted from the multi-institutional database between August 2012 and October 2019. Of these, 167 patients who satisfied the inclusion criteria constituted the study cohort.  Table 1. There were no significant differences in demographic and clinicopathological features between the DROSD and non-DROSD subgroups.

Creation of a nomogram to predict OS in patients with ICC
We constructed a prognostic nomogram based on the independent factors for OS identified by multivariable Cox regression analysis. The nomogram demonstrated that DROSD and CEA were the main weighting factors in the scoring system ( Figure 6A). The C-index for OS prediction was 0.733 (95%CI: 0.68-0.79). The calibration chart for 3year postoperative survival rates demonstrated satisfying coherence between August   nomogram predictions and actual observations ( Figure 6B). The AUC of OS was 0.79 ( Figure 6C).

DISCUSSION
ICC is a highly aggressive malignancy with a poor clinical prognosis. Radical hepatectomy is by far the most effective therapeutic strategy for patients with ICC. The prognosis of patients with ICC who have undergone curative resection is a common concern for both surgeons and patients. Therefore, it is essential to explore the indicators and establish models that can accurately predict ICC patients' prognosis. In our study, DROSD was investigated as a new predictor of prognosis in ICC patients who underwent curative hepatectomy. We found that the OS and RFS in ICC patients with DROSD were worse than in those without DROSD. Our study revealed that lymph node metastasis, length of hospital stay, DROSD, PCs, CEA and CA19-9 were independent prognostic factors for OS and RFS. Spleen density is a novel indicator of patient prognosis. It has been reported that DROSD is an excellent prognostic predictor in patients with gastric cancer and pancreatitis [12,13]. In our study, the incidence of DROSD was 32.9% in patients with ICC. For OS and RFS of patients with ICC, DROSD was an independent prognostic factor. Further studied in other patients cohorts are required to validate these findings.
The mechanism for DROSD in patients with ICC is unclear. One study revealed that the decrease of spleen density is related to lipid metabolism [17]; however, a previous study demonstrated that lipid deposition is not the cause of DROSD [13]. In our study, we found no difference in total cholesterol or triglyceride levels between DROSD and non-DROSD subgroups. Other studies have demonstrated that hemoperfusion of the spleen could impact its physical density, which is reflected as its density on CT [13,18]. The indicators of increased splenic blood perfusion include hypertension, portal hypertension, and increased hemoglobin, etc. However, in our study, no differences were found between the above-mentioned indicators in the DROSD group and the non-DROSD group, especially portal hypertension, there were 11 patients with portal hypertension, 4 patients were in the DROSD group, and 7 patients in the non-DROSD group, our data did not reflect that the spleen density of patients with external August 15, 2021 Volume 13 Issue 8 hypertension was higher. The reason may be due to less data of patients with portal hypertension, so it was not statistically significant. However, whether portal hypertension in cirrhosis can affect the change of spleen density needs to be studied. Many studies have suggested that cancer prognosis is closely associated with immune function [19][20][21]. NLR is an indicator of immune system activity. Some studies have discussed that since NLR is higher in DROSD gastric patients, it could be an indicator of prognosis in gastric cancer patients [12,22]. Thus, NLR may be one of the factors affecting DROSD. Our research revealed that PLR, NLR and LMR were not significantly different between DROSD and non-DROSD subgroups. Nevertheless, NLR and LMR affected the OS of patients in our study, consistent with other reports [20]. There is, therefore, a need to further explore the underlying mechanisms of DROSD in ICC patients extensively.
Several studies have shown that a nomogram has better predictive accuracy for survival than conventional staging systems [23][24][25]. Other studies have reported that CA19-9 and CEA are independent factors affecting ICC prognosis [26,27]. Our nomogram included DROSD, a new independent prognostic factor for ICC, and included CEA and CA19-9. Since this model combined conventional and new indicators, it is likely to predict ICC prognosis more accurately than its predecessors.
Our study had several limitations. First, as a retrospective study, selection bias was inevitable. Second, DROSD was observed on CT with no corresponding disease of the spleen. Since we were unable to explain the pathogenesis of DROSD, more research is needed to confirm the cause of this phenomenon. Third, due to the small number of patients with portal hypertension, no correlation between spleen density and portal hypertension was found in this study, which requires further study. Finally, the nomogram was created based on data collected from two institutions, and the accuracy of the model needs to be verified in more hospital settings.

CONCLUSION
DROSD is a novel prognostic marker for OS in ICC patients who underwent curative resection. The nomogram is a practical predictor and can accurately predict the prognosis of ICC patients who underwent curative resection.

Research background
Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor with poor prognosis and limited treatment options. Radical surgery is the only effective method, so it is necessary to explore prognostic predictors for patients with ICC after radical surgery.

Research motivation
The diffuse reduction of spleen density (DROSD) is related to the prognosis of cancers; however, its role in ICC remains unclear.

Research objectives
This study assessed the predictive value of DROSD on the prognosis of ICC patients after curative resection.

Research methods
Patients with ICC who underwent curative hepatectomy from 2012 to 2019 were enrolled. Preoperative spleen density was measured using computed tomography scans. Overall survival (OS) and recurrence-free survival (RFS) rates were calculated and compared utilizing the Kaplan-Meier method. Univariable and multivariable Cox regression analyses were applied to identify independent factors for OS and RFS. A nomogram was created with independent risk factors to predict prognosis of patients with ICC.

Research results
A total of 167 ICC patients were enrolled, and 55 (32.9%) had DROSD. Kaplan-Meier analysis indicated that patients with DROSD had worse OS and RFS than those without DROSD. Cox regression analysis revealed that DROSD, carcinoembryonic antigen level, carbohydrate antigen 19-9 level, length of hospital stay, lymph node metastasis, and postoperative complications were independent predictors for OS. The nomogram created with these factors proved to be able to predict the prognosis of ICC patients with good reliability (OS C-index = 0.733).

Research conclusions
ICC patients with DROSD have worse OS and RFS. The nomogram is a simple and practical method to identify high-risk ICC patients with poor prognosis.

Research perspectives
Our study found that DROSD is a novel predictor of prognosis in ICC patients after curative resection. It could best stratify patients with ICC to the appropriate screening.