CART is an established treatment for refractory ascites, but no detailed information on biological parameters before and after CART has been published to date. Our study revealed three major points. First, the levels of four of the 27 examined cytokines, i.e., IL-5, IL-6, IL-10, and MCP-1, were elevated in the concentrated AF, and their levels were also increased in blood immediately after reinfusion of concentrated ascites reduction, but decreased 24 h after reinfusion. Furthermore, among the cytokines in the concentrated AF, FGF-basic, G-CSF, IFN-γ, and TNF-α were present at lower levels in the concentrated AF than in the original AF. Second, the BT and WBC count increased immediately after reinfusion, but decreased after 24 h, unlike the CRP level, which increased 24 h after reinfusion. Moreover, a positive correlation was found between CRP and IL-6 at 24 h after reinfusion. Third, in the QOL assessment, we found that the CFS score was significantly lower the day after reinfusion than before AF collection, indicating that QOL improved after CART. These three points are further discussed below.

First, regarding cytokines in AF, the prognostic impact of inflammatory cytokines in patients with malignant tumors has been investigated in a study that reported the presence of IL-1β, IL-6, IL-8, IL-12, TNF-α, and IL-10 in AF. The presence of IL-10 was associated with a prolonged life expectancy after CART. In contrast, there was no significant correlation between the other cytokines and adverse events, such as fever, and the WBC count and CRP level, which are indicators of an inflammatory response [11]. In the present study, we similarly detected the presence of IL-1β, IL-6, IL-8, TNF-α, and IL-10 in AF; the IL-6 values were lower, the TNF-α and IL-10 values were slightly higher, and the IL-1β and IL-8 values were comparable to the values reported in the previous studies. In addition, we found that the levels of FGF-basic, G-CSF, IFN-γ, and TNF-α were lower in the concentrated AF than in the original AF. G-CSF, IFN-γ, and TNF-α are cytokines that play a role in enhancing inflammation. They are filtered out and removed by the AF filtration unit (AHF-MO). Rather than suppressing inflammation, the removal of these cytokines in CART does not cause inflammation. In particular, TNF-α is a cytokine that is secreted in the early stages of inflammation, and it has a half-life of less than 20 min; despite its short half-life, it is believed to induce the secretion of various inflammatory and anti-inflammatory cytokines, such as IL-6, IL-8, IFN-γ, and IL-10 [16]. The IL-5, IL-6, IL-10, and MCP-1 levels in blood were increased immediately after reinfusion, but were decreased 24 h later, likely because these cytokines have short half-lives ranging from a few minutes to around 20 h [17]. These results indicate that the levels of the cytokines induced by TNF-α that were found in the concentrated AF would have also decreased naturally in the body, suggesting that even though the reinfusion of concentrated AF returns the inflammatory cytokines into the body, the effect of the cytokines is transient. Thus, suggesting that CART is safe.

Regarding the second point, the BT and WBC count increased immediately after reinfusion, but decreased 24 h later, and that the CRP level increased 24 h after reinfusion. In addition, we observed a positive correlation between the CRP and IL-6 levels at 24 h after reinfusion. Previous studies found no correlation between cytokines and adverse events, such as an elevated BT, WBC count, and CRP level, which are associated with inflammatory responses [11]. In our study, the results of the single regression analysis showed that the CRP level at 24 h after reinfusion was positively correlated with the IL-6 level. Moreover, the BT, WBC count, and CRP level immediately after reinfusion, and the BT and CRP level on the day after reinfusion tended to be increased with increasing IL-6 levels. IL-6 is a very important cytokine in the early host response to infection, and an increase in IL-6 precedes an increase in CRP. IL-6 has a very short half-life, becoming undetectable within 24 h in most infected patients. CRP is synthesized in the liver within 6–8 h in response to inflammation, peaking at 24–48 h, and then decreasing with time as the inflammation subsides [18]. Previous studies found that the CRP level was increased 3 h after IL-6 administration, and was further increased at 16 h. It has also been reported that the number of neutrophils was increased by IL-6 administration, peaking at 2 h after administration, and returning to the pre-administration level by the next day [19]. In the present study, the CRP level did not change immediately after reinfusion, but was increased 24 h later; in contrast, the WBC count increased immediately after reinfusion, and was decreased after 24 h. This suggested that the WBC count increased immediately after reinfusion and the CRP level increased 24 h later due to the immediate increase in IL-6 that occurred after reinfusion. Thus, the inflammation caused by CART is considered to be temporary, as the WBC count and IL-6 level peaked out at 24 h after reinfusion. In addition, fever was observed immediately after reinfusion, but it resolved 24 h later. We consider that the fever was induced by inflammatory cytokines, since the blood levels of inflammatory cytokines, such as IL-6, increased immediately after reinfusion. The fever encountered in the present study was a Grade 1 (mild) adverse event according to the Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0. Thus, our results indicate that CART can be safely performed with no adverse events other than fever, which can be managed with non-steroidal anti-inflammatory drugs and acetaminophen.

As for the third point, the CFS score was significantly lower on the day after reinfusion than before AF collection, indicating that QOL was improved by CART. There is no standardized protocol for evaluating QOL improvement with CART. Previous retrospective studies have reported that CART for cancer patients improved their QOL, with significant reductions in the circumferences of the abdomen and both thighs, and improved appetite [20]. In the previous systematic reviews, a numerical rating scale system (NRS) was used to evaluate symptom relief after CART. Abdominal distention, dyspnea, and fatigue were reduced by 6.0 (95% CI 5.59–6.51), 2.66 (95% CI 2.05–3.28), and 2.64 (95% CI 1.86–3.42) points, respectively, using a 0–10 numerical rating scale system. Overall, 17% (95% CI 0.03–0.31%) of patients reported improved performance status after CART [7]. In the previous studies, the NRS was used to separately assess abdominal distension, dyspnea, and fatigue. As a result, it was difficult to comprehensively and multidimensionally assess the QOL of individual patients. The present study, we used CFS as a scale to comprehensively and multidimensionally assess changes in QOL. The CFS assesses physical fatigue, mental fatigue, and cognitive fatigue, and can be used to quantify and objectively evaluate a patient’s sense of fatigue. In the present study, CART significantly reduced the CFS score, indicating that CART is effective in improving the cancer patients’ QOL. In the future, in evaluating the improvement in QOL provided by CART, it will be necessary to create a protocol for administering the CFS before treatment, the day after treatment, and 1 week after treatment.

There are two limitations to this study. The first is the small number of eligible patients that were analyzed. There were 13 patients who had ovarian cancer with refractory malignant ascites and underwent CART, and only 11 patients were analyzed as data were insufficient for two cases; this small number may have increased the susceptibility to random errors. The second is that the assessment after CART was only performed at 24 h. As a prospective study, it was not possible to predict changes in cytokines and CRP after 24 h before the study. In addition, the evaluation was only conducted at 24 h in this study, as the increased number of blood samples would have increased patient invasiveness. Therefore, only short-term effects are apparent. Blood tests after 24 h would allow further investigation of the impact of CART on inflammation. Future evaluation at 48 and 72 h is an issue. Continuous QOL assessment on a weekly basis, taking into account the time until reaccumulation of ascites, would further validate the effectiveness of CART. A prospective randomized-controlled phase II trial comparing ascites puncture cases with CART cases is currently underway (the JGOG9006 trial). Although the cytokine levels in AF are not being monitored, a QOL study will be conducted. We are looking forward to the results of this study.

In conclusion, the reinfusion of concentrated AF in CART decreased the levels of inflammatory cytokines after 24 h, even though the cytokines in the AF were returned back into the body. Furthermore, the CFS score was significantly lower the day after reinfusion when compared to before AF collection, and QOL improved after CART. As this study demonstrated the safety and benefits of CART, we hope that CART will be used for treating patients with refractory cancerous ascites in the future.