The Liver Maximum Capacity Test (LiMAx) Is Associated with Short-Term Survival in Patients with Early Stage HCC Undergoing Transarterial Treatment

Simple Summary The liver maximum capacity test (LiMAx) represents a useful tool to estimate liver function in patients with chronic liver disease. LiMAx results correlate with short-term survival in patients with early stage HCC after transarterial chemo- or radioembolization. Low LiMAx levels might enable the identification of patients with poor hepatic function and decreased short-term survival after treatment. Abstract Transarterial chemoembolization (TACE) and transarterial radioembolization (TARE) are recommended to treat patients with early or intermediate hepatocellular carcinoma (HCC). The liver maximum capacity test (LiMAx) has been supposed to predict the risk of post-interventional liver failure. We investigated the correlation of LiMAx with short-term survival as primary endpoint and the occurrence of adverse events after therapy as secondary endpoint. Our study cohort prospectively included 69 patients receiving TACE (n = 57) or TARE (n = 12). LiMAx test and serological analyses were performed on the day before and 4 weeks after treatment. Hepatic and extrahepatic complications were monitored for 4 weeks. The LiMAx results were not associated with altered liver function and the occurrence of adverse events. The survival rates of patients with BCLC A with LiMAx ≤ 150 μg/kg/h were lower after 30 days (75.0 ± 15.3% vs. 100%, p = 0.011), 90 days (62.5 ± 17.7% vs. 95.8 ± 4.1%, p = 0.011) and 180 days (50.0 ± 17.7% vs. 95.8 ± 4.1%, p = 0.001) compared to those with higher LiMAx levels. The LiMAx test is not suitable to predict liver function abnormalities or the occurrence of complications 4 weeks after therapy but enables the identification of patients with early stage HCC and reduced short-term survival after treatment.


Introduction
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death worldwide [1]. Transarterial chemoembolization (TACE) is a widely used first-line therapy for treatment of unresectable HCC in patients with early or intermediate-stage disease according to the Barcelona Clinic Liver Cancer Classification (BCLC) [2,3]. Transarterial radioembolization (TARE) has been proposed as an effective alternative to TACE, and it is the most common treatment option for patients with locally advanced HCC [2][3][4][5][6]. Both transarterial treatment strategies can help control local tumor growth, reduce palliate symptoms, prolong survival, or bridge the time to liver transplantation [7,8].

Patients and Study Design
Our study was conducted on patients who underwent TACE or TARE between November 2017 and April 2020 the University Medical Center. Patients were successively included into the study by availability. A multidisciplinary HCC tumor board made the decision to perform TACE or TARE in the enrolled patients. Liver function was assessed on the day before as well as at 4 weeks after TACE or TARE procedures using the LiMAx test and the well-established serological analyses. Our study was approved by the Ethics Committees of Medical Research of the University of Leipzig (vote no. 213/17-ek) in accordance with the Declaration of Helsinki from 1975 (revision 2013) and the International Conference on Harmonization/Committee for Proprietary Medicinal Products' "Good Clinical Practice" guidelines. All patients provided written informed consent. The following data were prospectively collected: patient demographic and laboratory data, cancer characteristics, CTP score, MELD score, ALBI score, and LiMAx results, as well as the occurrence of adverse effects of treatment at 4 weeks after TACE/TARE, and survival until 34 month after treatment. Adverse events of treatment were categorized according to the Society of Interventional Radiology (SIR) Adverse Event Classification in (a) mild: no or nominal therapy; (b) moderate: modest escalation of care, requiring intervention, extremely prolonged outpatient observation or overnight admission after outpatient procedure; (c) severe: marked escalation of care or complex intervention; (d) life-threatening or disabling event, e.g., cardiopulmonary arrest, shock, organ failure, unanticipated dialysis, paralysis, loss of limb or organ; and (e) patients death [13]. Furthermore, the occurrence of REILD was assessed and considered as classic with symptoms of fatigue, abdominal pain, increased abdominal girth, hepatomegaly, anicteric ascites 1-3 months after TARE, and a twofold increase of the alkaline phosphatase; or non-classic with dysregulated hepatic functions with jaundice and/or remarkably elevated serum transaminases [35].

Transarterial Chemoembolization (TACE)
All TACE procedures were routinely performed in the clinic using a standard protocol consisting of doxorubicin, mitomycin C, and lipiodol. According to the guideline, a coaxial 2.7 French microcatheter was placed into the hepatic artery to selectively visualize the tumor vessels. Then, doxorubicin, mitomycin C and lipiodol were selectively applied to the tumor vessels. Several weeks after TACE, all patients received a local computer tomography scan to evaluate the embolized liver volume. TACE was performed based on interdisciplinary tumor board decision as palliative treatment or as bridging treatment before liver transplantation.

LiMAx
The LiMAx test (Humedics GmbH, Berlin, Germany) was performed after a minimum of 3 h fasting. As previously described, the test procedure is based on intravenous administration of 2 mg/kg body weight 13 C-methacetin, which is selective substrate of the hepatic cytochrome P450 1A2 enzyme [30]. The liver specific enzyme demethylates 13 C-methacetin into acetaminophen and 13 CO 2 , which is subsequently exhaled. The ratio of 13 CO 2 / 12 CO 2 concentration was constantly monitored online in the exhaled breath over a period of 60 min maximum using an infrared absorption spectroscopy method. The baseline ratio of 13 CO 2 / 12 CO 2 concentration was recorded in the native exhaled air before substrate injection. LiMAx value was calculated according to the previously described formula [30]. Results are given in µg/kg/h and available directly after test termination. The LiMAx values > 315 µg/kg/h were considered normal [36].

Statistical Analysis
Statistical analyses of epidemiological associations were performed using SPSS software (SPSS Inc., version 25.0, Chicago, IL, USA). Values are presented in median and interquartile range if not otherwise specified. Categorical variables are shown as frequencies and percentage. The Chi-squared test was applied for categorical variables and the Mann-Whitney U-test and Wilcoxon signed-rank test to compare quantitative variables. Correlations were calculated using the Spearman correlation coefficient. Univariate and multivariate logistic regression analyses (inclusion model) were used to determine the association between different parameters. The regression coefficient (RC), standard error (SE), odds ratio (OR) and the 95% confidence interval (CI) were calculated. Survival analyses were performed with Kaplan-Meier estimator and Cox regression analysis for 30-, 60-, 90-, and 180-day survival as well as for overall survival. Multivariate regression analysis was performed by using p < 0.05 for inclusion and p > 0.1 for exclusion of parameters in the final model. All tests were two-sided and p values of <0.05 were considered significant.
The CTP score is based on total serum bilirubin and albumin and the international normalized ratio for prothrombin time (INR) as wells on the quantification of the severity of ascites and hepatic encephalopathy from none to mild to severe [37,38]. Patients were classified in Child A with CTP points 5-6 and in Child B with CTP points 7-9. The MELD sore included the serum levels of bilirubin and creatinine and INR, and is calculated according to the formula: MELD = 3.78 × ln (serum bilirubin [md/dL]) + 11.2 × ln (INR) + 9.57 × ln (serum creatinine [mg/dL]) + 6.43 [39].

Study Population
LiMAx was performed in 91 patients between November 2017 and April 2020. In total, 22 patients were excluded because of concomitant cancer diseases such as cholangiocarcinoma (n = 3), colon carcinoma (n = 2), and bile duct carcinoma (n = 2), other carcinoma (n = 1), as well as missing data sets (n = 4). TACE or TACE was cancelled in 10 patients after evaluation because of contraindications such as arteriovenous shunts, metastases, coronary diseases, or consent withdrawal ( Figure 1).
were performed with Kaplan-Meier estimator and Cox regression analysis for 30-, 60-, 90-, and 180-day survival as well as for overall survival. Multivariate regression analysis was performed by using p < 0.05 for inclusion and p > 0.1 for exclusion of parameters in the final model. All tests were two-sided and p values of <0.05 were considered significant.
The CTP score is based on total serum bilirubin and albumin and the international normalized ratio for prothrombin time (INR) as wells on the quantification of the severity of ascites and hepatic encephalopathy from none to mild to severe [37,38]. Patients were classified in Child A with CTP points 5-6 and in Child B with CTP points 7-9. The MELD sore included the serum levels of bilirubin and creatinine and INR, and is calculated according to the formula: MELD = 3.78 × ln (serum bilirubin [md/dL]) + 11.2 × ln (INR) + 9.57 × ln (serum creatinine [mg/dL]) + 6.43 [39].

Study Population
LiMAx was performed in 91 patients between November 2017 and April 2020. In total, 22 patients were excluded because of concomitant cancer diseases such as cholangiocarcinoma (n = 3), colon carcinoma (n = 2), and bile duct carcinoma (n = 2), other carcinoma (n = 1), as well as missing data sets (n = 4). TACE or TACE was cancelled in 10 patients after evaluation because of contraindications such as arteriovenous shunts, metastases, coronary diseases, or consent withdrawal ( Figure 1).

LiMAx Results and Other Parameters of Liver Function before and after Transarterial Treatment
When we compared patients with lower LiMAx levels (LiMAx ≤ 150 μg/kg/h) versus those with higher LiMAx levels (LiMAx > 150 μg/kg/h), significant differences in blood parameters and liver function scores were assessed. Patients with LiMAx ≤ 150 μg/kg/h showed significantly increased levels of liver enzymes and increased ALBI, MELD, and CTP scores, as well as decreased levels of albumin and platelets counts compared to patients with higher LiMAx levels before and after treatment. However, within both LiMAx groups no significant differences were observed in almost parameters but ALBI score (median  Table S3).

LiMAx Results and Adverse Events of Transarterial Treatment
Common adverse events were recorded for 4 weeks after treatment, which are summarized in Supplementary Table S4. Only 27 patients (39.1%) showed mild extrahepatic adverse events such as fatigue (11.6%) and epigastric pressure (7.2%) in both treatment groups. In the TACE group, 15.8% of patients suffered from severe complications related to liver dysfunction such as ascites and hepatic encephalopathy requiring intervention.   When the study cohort is stratified according to CTP and ABLI grade groups, significant differences in LiMAx were observed in the subgroups. The ALBI grade 1 group showed  Tables S1 and S2).
When we compared patients with lower LiMAx levels (LiMAx ≤ 150 µg/kg/h) versus those with higher LiMAx levels (LiMAx > 150 µg/kg/h), significant differences in blood parameters and liver function scores were assessed. Patients with LiMAx ≤ 150 µg/kg/h showed significantly increased levels of liver enzymes and increased ALBI, MELD, and CTP scores, as well as decreased levels of albumin and platelets counts compared to patients with higher LiMAx levels before and after treatment. However, within both LiMAx groups no significant differences were observed in almost parameters but ALBI score (median  Table S3).

LiMAx Results and Adverse Events of Transarterial Treatment
Common adverse events were recorded for 4 weeks after treatment, which are summarized in Supplementary Table S4. Only 27 patients (39.1%) showed mild extrahepatic adverse events such as fatigue (11.6%) and epigastric pressure (7.2%) in both treatment groups. In the TACE group, 15.8% of patients suffered from severe complications related to liver dysfunction such as ascites and hepatic encephalopathy requiring intervention. Two patients developed a kidney failure after treatment, and one patient showed a myocardial infarct. This patient had CTP Child A, MELD 17, ALBI grade 3 and BCLC A stage and a LiMAx level of 129 µg/kg/h before TACE and died 3 days after treatment because of acuteon-chronic liver failure (ACLF). Another patient also died due to ACLF within 4 weeks after treatment. In the TARE group, one patient with BCLC C stage developed symptoms of a non-classical REILD with jaundice and mild ascites as well as an increase in CPT score (from 5 to 9), MELD score (from 12 to 17) and in ALBI grade (from 1 to 3) 4 weeks after treatment, and died 71 days after therapy.

Association of LiMAx Results with Survival
The median survival time was 16 (range 0-34) month. Survival analyses of both treatment arms and LiMAx groups (≤150 µg/kg/h and >150 µg/kg/h) were performed after 30, 60, 90, and 180 days as well as after 34 month for overall survival.
Remarkably, when the patients were divided into groups according to the BCLC stage, significant differences in survival rates were detected between patients with LiMAx ≤ 150 µg/kg/h and with LiMAx > 150 µg/kg/h levels for BCLC A but not for BCLC B and C groups. In the BCLC B group, no patient died within 90 days, and in the BCLC C group, no patient died within 60 days. The 90-day survival rates of the BLCL C group were not significantly different according to the LiMAx ≤ 150 µg/kg/h and LiMAx >150 µg/kg/h groups (p = 0.392). In contrast, the survival rates of patients with BCLC A stage with LiMAx ≤ 150 µg/kg/h were significantly lower after 30 days (75.0 ± 15.3% vs. 100%, p = 0.011) and 90 days (62.5 ± 17.7% vs. 95.8 ± 4.1%, p = 0.011) compared to those patients with higher LiMAx levels. This was even more pronounced after 180 days for BCLC A with an estimate of 50.0 ± 17.7% vs. 95.8 ± 4.1%, p = 0.00; in contrast to BCLC B with an estimate of 100% vs. 75.0 ± 8.8%, p = 0.236; and BCLC C with an estimate of 100% vs. 66.7 ± 19.2%, p = 0.392, respectively (Figure 4).

Discussion
In our study, we investigated the correlation between enzymatic liver function, based on the LiMAx test, and clinical outcome in patients with early or intermediate stage HCC who are eligible for transarterial treatments. We found that LiMAx results ≤ 150 µg/kg/h are strongly associated with decreased survival rates over 30 and 60 days, and with survival over 180 days in patients in BCLC stage A.
As liver cancer covers a wide range of stages-from very early to advanced disease, and with many treatment options from surgery to loco-regional treatments to immunotherapy-the treating physician is often faced with a great variety of therapy strategies.
In this context, measuring liver function by LiMAx might represent a useful tool in identifying patients with short survival, which was the primary aim of the study. This could be especially relevant for patients eligible to transarterial treatment with Li-MAx results ≤ 150 µg/kg/h which had higher 30-and 60-day mortalities as compared to patients with LiMAx results > 150 µg/kg/h. In addition, the long-term prognosis over 180 days was significantly lower for patients in BCLC stage A with LiMAx values ≤ 150 µg/kg/h. It needs to be investigated whether those patients might have had more benefit from different treatment approaches.
Furthermore, as a secondary aim, we found no significant association of LiMAx test results with adverse events associated with transarterial treatment. Indeed, the most common side effects to TACE, prevalent in 35-100% of patients, is the post-embolization syndrome, a constellation of fever, abdominal pain, nausea, and vomiting that is transient and rather mild, and therefore potentially underreported [8,11,12,41]. Potential severe complications of TACE include liver failure, biliary or hepatic artery injury, and infection, and mortality rates from TACE are less than 2% [42]. A reason for this low rate is that main risk factors for liver failure including decompensated cirrhosis, portal vein thrombosis, large bilobar tumors, a glomerular filtration rate (GFR) less than 30 mL/min, and extrahepatic spread are considered contraindications to TACE [43][44][45]. Similarly, severe side effects have also been rarely reported after TARE [12,41]. Therefore, our and other previous reports linking LiMAx results with side effects of transarterial treatment may have been underpowered to reliably detect associations with such rare events. Nevertheless, other studies suggested an association of LiMAx with tolerability of TACE. In fact, the only patient who had a liver failure after TACE in our cohort had a LiMAx result < 150 µg/kg/h before treatment, suggesting that a large scale study would be necessary to clarify the value of LiMAx for the prediction of adverse effects of transarterial treatment.
Several studies showed that the LiMAX test was appropriate to quantify the liver function capacity in different clinical settings of liver disease. Hence, the LiMAx test is comparable to conventional liver function tests (e.g., dynamic indocyanine green test or static tests such as bilirubin, INR, and lactate) for predicting liver function deterioration.
In some reports, LiMAx levels before TACE correlated with bilirubin and albumin levels and liver function scores, which are surrogate markers indicating liver function deteriorations after treatment [2,[14][15][16][17]. However, in our study protocol we assessed liver function 4 weeks after treatment to allow for liver regeneration.
Thus, in our study, there was no association of pre-treatment LiMAx levels with changes in liver function 4 weeks after TACE/TARE. This was also not detected when the patients were divided into groups with LiMAx ≤ 150 µg/kg/h and LiMAx > 150 µg/kg/h levels according to Stockman et al. [29]. The results are in agreement with the study of Barzakova et al. [36], where the patients fully recovered one month after treatment. It seems that the LiMAx test might only be successful to detect short-term changes in liver function. Thus, in the aforementioned studies of Barzakova et al. [33] and Reichert et al. [32], the individual LiMAx levels were significantly reduced by 10% and 7% one day after TACE.
Limitations of the study were the small size of the patient cohort, the diversity of liver tumors and the large number of pretreated patients with a wide range of treatment regimen. Despite the fact that this trial was noteworthy, most of the results were not significant and several issues need to be addressed in larger follow-up studies. Furthermore, the follow-up LiMAx test was only performed in approximately 50% of patients of the initial cohort. Future multicenter studies should aim at including a broad range of patients in different tumor stages and include sequential LiMAx measurements over time.

Conclusions
In conclusion, LiMAx measurement before therapy was no appropriate predictor of the occurrence and the severity of complications 4 weeks after TACE or TARE treatment and of tumor response. However, low LiMAx levels might enable the identification of patients with poor hepatic function and decreased short-term survival after treatment, especially in early stage HCC. In view of the rapidly developing field of systemic therapies for HCC, LiMAx could play a key role in the development of personalized therapy algorithms.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers14215323/s1, Table S1: Changes in LiMAx, laboratory parameters and liver function scores according Child A and Child B group. Table S2: Changes in LiMAx, laboratory parameters and liver function scores according ALBI grade 1 and ALBI grade 2/3 group. Table S3: Changes in LiMAx, laboratory parameters and liver function scores according LiMAx ≤ 150 µg/kg/h and LiMAx >150 µg/kg/h levels.   Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to legal issues.