Cisterna chyli as an optimal marker of tolvaptan response in severe cirrhotic ascites

For patients with cirrhosis, no definitive predictor of the efficacy and prognosis of tolvaptan treatment exists. We assessed the cisterna chyli’s utility as an optimal marker. We retrospectively enrolled 172 patients with cirrhosis. The effect of tolvaptan was evaluated using post-treatment survival time. The overall response to tolvaptan was 52.3%. The median cisterna chyli diameter was 4.1 mm. Of 172 patients, 100 were included in the pilot set and 72 in the validation set. According to the Youden index, the cisterna chyli diameter’s cutoff value was 4 mm, with a sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio of 92%, 83%, 86%, 91%, 5.43, and 0.09, respectively, in the pilot set. The area under the curve of the cisterna chyli diameter for evaluating tolvaptan’s effect was 0.911 and 0.988 in the pilot and validation sets, respectively. During multivariate analysis, cisterna chyli narrowing and furosemide treatment were significant predictive factors for tolvaptan’s insufficient effect. Cumulative liver transplantation-free survival rates were significantly higher in patients with cisterna chyli dilatation than in those without (p = 0.028). Our findings suggest a strong association of cisterna chyli with tolvaptan treatment response in patients with cirrhosis and hepatic edema.

of Disse predominantly flows through the space of Mall and subsequently into lymphatic capillaries. Some of the lymphatic fluid in the space of Disse flows into the interstitium surrounding the central vein or underneath the hepatic capsule 25 . Lymphatic capillaries in the portal tract coalesce into collecting vessels and subsequently drain into the cisterna chyli and enlarged origin of the thoracic duct. Lymphatic fluid, through the thoracic duct, drains into the left subclavicular vein and returns to the systemic blood circulation 22 . During excessive lymphatic fluid drainage, lymphatic drainage dysfunction is considered to occur at one of the sites in patients with decompensated cirrhosis. Between these sites, it is feasible to detect cisterna chyli using multi-detector row computed tomography (CT) 26 .
Therefore, this study aimed to determine the utility of the cisterna chyli as an optimal marker of tolvaptanadministration response in patients with severe cirrhotic ascites.

Results
Baseline characteristics of patients. Initially, the study included 218 patients. Patients with heart failure (n = 4), without body weight records after tolvaptan treatment (n = 10), in whom cisterna chyli could not be detected on CT (n = 4), on whom contrast-enhanced CT was not performed before tolvaptan initiation (n = 3), and with an estimated Glomerular Filtration Rate (eGFR) < 30 mL/min/1.73m 2 (n = 25) were excluded. Finally, 172 patients were included in this study (58 women; median age, 67.0 years; Fig. 1). To define the cut-off value, 100 patients were assigned to the pilot set in the order of entry. The remaining 72 patients were assigned to the validation set to confirm the efficacy of tolvaptan. Patient demographics and clinical characteristics are shown in Table 1. The number of patients who responded to tolvaptan was 9 (11.0%) and 85 (94.4%) for patients with cisterna chyli diameters < 4 mm and ≥ 4 mm, respectively. Patient demographics and baseline characteristics between tolvaptan-responders vs. non-responders. are shown in Table 2.  Adverse effect. Adverse effects are shown in Table 5. All events were infrequent. There was no significant difference in adverse events between the responder and non-responder groups. There was no patient with elevated alanine aminotransferase levels.

Discussion
Certain factors have been reported to predict the efficacy of medical therapy for severe ascites; nonetheless, none has been conclusive. In this study, cisterna chyli dilatation emerged as an optimal marker of treatment response before the initiation of medical treatment. The relationship between the lymphatic system and hepatic ascites has recently attracted renewed attention. The liver produces vast quantities of lymph, estimated at 25-50% of the lymph, flowing through the thoracic duct 20,28 . In patients with cirrhosis, lymphatic fluid produced in the liver increases up to thirty-folds [29][30][31][32] . Lymphatic drainage impairment and interstitial fluid build-up are direct causes of ascites development [20][21][22] . However, the role of the lymphatic vascular system in the pathogenesis of ascites is yet to be fully elucidated. Therefore, we focused on lymphatic drainage dysfunction before lymph is returned to the systemic circulation 33,34 . Ghelfi et al. reported that patients with cirrhosis and portal hypertension exhibited a significant pressure gradient between the thoracic duct and left subclavian vein and that its correction after thoracic duct stenting might have induced ascites resolution. Thus, this suggests that lymphatic flow deactivation in the thoracic duct potentially aggravates hepatic lymphatic system dysfunction; however, this may not apply to all cases. Although Ghelfi et al. suggested lymphovenous-junction narrowing as a major cause of lymphatic drainage dysfunction, a figure in their study revealed narrowing of the entire thoracic duct. The thoracic duct and cisterna chyli are often reported to increase in diameter with increasing portal pressure 26,35 . However, the cisterna chyli diameter in patients with severe ascites 26 is noticeably narrower than that in healthy patients 36 . Thus, we hypothesized that the cisterna chyli and thoracic duct, which dilate as portal pressure increases, become narrower as liver disease progresses and fail to tolerate the increased internal pressure, and lymphatic vessel walls lose their ability to extend. Although not proven, similar hypotheses have been postulated in previous studies 26 . In patients with decompensated cirrhosis involving a narrow cisterna chyli (< 4 mm), lymphatic drainage into the systemic circulation is presumably unsuccessful. Additionally, albumin in plasma and water components potentially leaks into the interstitium, further contributing to hypoalbuminemia in patients with decompensated cirrhosis. Unexpectedly, there was only one case of chylous ascites in a patient with a narrowed cisterna chyli. According to Stewart et al. 37 , in patients with narrowed cisterna chyli, intestinal-derived lymph containing a high amount of triglycerides could not be merged with liver-derived lymph. Although MDCT was used in this study, MRI can be used to delineate the cisterna chyli. However, MRI takes more time than CT. Further, MRI may be inappropriate for patients with severe ascites because of signal attenuation. On comparing survival rate with cisterna chyli diameter, the prognosis worsened in patients with a narrower cisterna chyli diameter. This finding is consistent with that in previous  This study has several limitations. First, there was no validation cohort in other institutes. In the future, a multicenter validation trial should be conducted. The interactions between tolvaptan treatment response and lymphatic function and various other factors that affect ascites formation should also be analyzed with a large number of patients. Second, the pathological mechanism underlying cisterna chyli diameter narrowing in decompensated cirrhosis was not clarified. The irreversibility of this narrowing and its ability to cause lymphatic drainage dysfunction remain unclear. Animal experiments are required to elucidate these mechanisms. Third, the ascites developed in patients enrolled in this study is strictly different from refractory ascites. Because of the small stature of the Japanese population, treatment with tolvaptan is often initiated for patients with low-dose diuretics, as reported by Sakaida et al 39 . In the future, a group of patients with refractory ascites only should be analyzed. Finally, while AP2 and urine osmolality have been reported as predictors of tolvaptan's effectiveness, they could not be examined in this retrospective study 40 . A prospective study is required to analyze these factors.
In conclusion, cisterna chyli dilatation proved to be an effective optimal marker of tolvaptan's effectiveness in patients with severe cirrhosis. In cases where the cisterna chyli diameter is less than 4 mm, non-pharmacological treatments (e.g., TIPS) should be actively applied.

Methods
Patients and study design. This study followed a retrospective design and was approved by the relevant institutional ethics committee (Ehime University of Medical Science). The study was conducted in accordance with the principles of the Declaration of Helsinki. All the subjects provided written informed consent. Two hundred and eighteen patients with decompensated liver cirrhosis and fluid retention (pleural effusion, ascites, or lower-limb edema) were included in this retrospective study at our hospital between December 2012 and August 2021. The inclusion criteria were as follows: (1) age between 20 and 90 years; (2) non-consideration of sex during the selection process; (3) diagnosis of liver cirrhosis by ultrasound, CT, or liver biopsy; (4) presence of ascites unresponsive to standard therapy, including diuretic therapy comprising furosemide, spironolactone, or both, in addition to the appropriate salt (< 5-7 g/day) restrictions; and (5) patients in whom pre-breakfast body weight was stable (within the range of ± 1 kg). The exclusion criteria, regarding which reference was made to a previous phase 3 trial 41 , were as follows: (1) hepatic encephalopathy stage 42,43 2 or higher; (2) vascular invasive hepatocellular carcinoma; (3) esophagogastric varices requiring treatment; (4) a history of cerebrovascular disorder; (5) type 1 hepatorenal syndrome; (6) body mass index > 35; (7) systolic blood pressure < 90 mmHg; (8) hemoglobin < 8.0 g/dL; (9) serum sodium < 120 mEq/L or > 147 mEq/L; (10) serum potassium > 5.5 mEq/L; (11) inability to take oral medication; (12) pregnancy; (13) inability to undergo contrast-enhanced CT; (14) inability to have body weight measured one week after tolvaptan initiation; and 15) eGFR < 30 mL/min/1.73m 2 . All authors had access to the study data and reviewed and approved the final manuscript. Treatment protocol. Patients were initially administered salt-restricted diet therapy (5-7 g/day) and conventional diuretics (furosemide, spironolactone, or both) for at least seven days. If body weight remained constant after the aforementioned treatments, tolvaptan (Samsca®, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) was orally administered at a dosage of 7.5 mg once a day for at least one week. Water intake was not restricted during the tolvaptan administration, and no albumin preparation was infused. Ascites and pleural effusion were not resolved by paracentesis during the first seven days of tolvaptan treatment. According to previous studies 14,39,44 , patients with a ≥ 1.5-kg decrease in body weight from baseline were defined as responders.
Laboratory test. Body weight and 24-h urine volume were measured and recorded daily from baseline to one week after treatment. Body weight was measured on wakening. The hematological test items shown in Table 1 were measured at tolvaptan initiation. Additionally, 24-h urine volume, urinary sodium, and creatinine secretion were measured at tolvaptan initiation.
Imaging study. Contrast-enhanced CT scans were acquired in the portal venous phase with patients in a supine position and arms above the head using a 128-slice spiral CT scanner. The patients fasted for at least 3 h before the contrast-enhanced CT examination. The CT images were taken with intermittent breath-holding after Table 5. Adverse effect. a More than grade 2 (Lethargy or moderate confusion was occurred). b Creatinine level increased by ≥ 0.3 mg/dL. c Alanine aminotransferase increased more than 1.5-fold. www.nature.com/scientificreports/ maximum inspiration. As in previous studies, the cisterna chyli was assessed 45 . The cisterna chyli was identified in the retrocrural space, right of the aorta, as an oblong structure that is isodense to water, typically extending cranially into the lower-caliber thoracic duct. Between Th12 and L2, the maximum axial diameter of the cisterna chyli was measured independently by two hepatologists, perpendicular to the long axis within four weeks before initiating tolvaptan treatment. Image analysis was performed on the basis of axial slices and additional coronal and sagittal reformation. Measurement of the largest cisterna chyli diameter (outer circumference to outer circumference) was manually performed by both readers (Fig. 3).

Statistical analysis.
After confirmation of data normality using the Shapiro-Wilk test, quantitative data were presented as the median (interquartile range). To compare clinical characteristics and effects between the two groups, the Mann-Whitney U-test was applied. Percentages were compared using the χ 2 or Fisher's exact test. Univariate and multivariate logistic regression analyses were performed to investigate factors predicting the achievement of a 1.5-kg body weight reduction from baseline. Sensitivity, specificity, PPV (positive predictive value), NPV (negative predictive value), LR + (positive likelihood ratio), and LR− (negative likelihood ratio) scores were calculated to evaluate tolvaptan's efficacy. The AUCs were determined by receiver-operating characteristic curve analysis. To assess survival and recurrence curves, the Kaplan-Meier method was used. All statistical analyses were performed using STATA (version 15; StataCorp, College Station, TX, USA). Statistical significance was set at p < 0.05.

Data availability
The data sets generated and/or analyzed in this study are not publicly available because consent for publication has not been obtained from all patients, but are available from the corresponding author upon reasonable request.