Original Paper Received: September 16, 2014 Accepted after revision: November 26, 2014 Published online: January 30, 2015
Digestion 2015;91:117–127 DOI: 10.1159/000370212
Glutamate Dehydrogenase and Alkaline Phosphatase as Very Early Predictors of Hepatocellular Carcinoma Recurrence after Liver Transplantation Katja Piras-Straub a, b Khaleda Khairzada a, b Guido Gerken b Fuat Saner a Jürgen Treckmann a Andreas Paul a Ali Canbay b Kerstin Herzer a, b
Departments of a General, Visceral and Transplantation Surgery and b Gastroenterology and Hepatology, University Hospital Essen, Essen, Germany
Key Words HCC · NASH · GLDH · Tumor recurrence · Liver transplantation
Abstract Background: Although long-term survival rates for patients undergoing liver transplant (LT) for hepatocellular carcinoma (HCC) are good, the relatively high rate of tumor recurrence after LT necessitates the identification of biological parameters that supplement morphological predictors of recurrence. Method: From chart review we identified 175 patients who received liver transplantation due to HCC at our center between January 2000 and December 2013. We documented demographic and clinical data, as well as clinicopathological characteristics of the tumors, with a focus on liver values at the time of LT. Results: HCC recurred in 23% of LT patients. Most recurrences (59%) occurred within 12 months after LT; hardly any recurrence was detected later than 3 years after LT. Recurrence was positively correlated with tumor size, tumor stage and alpha-fetoprotein level (AFP), and it was most likely with certain causes of liver disease. Interestingly, tumor recurrence was independently predicted by serum levels of glutamate dehydrogenase (GLDH) and alkaline phosphatase (AP) at the time of LT. Conclusions: Because all HCC recurrence occurs within 36 months after LT, HCC detected more than 3 years after LT
© 2015 S. Karger AG, Basel 0012–2823/15/0912–0117$39.50/0 E-Mail [email protected] www.karger.com/dig
may be considered de novo. Liver values, with GLDH and AP being the most preponderant, serve as easy-to-assess biomarkers which contribute to predict the risk of tumor recurrence. © 2015 S. Karger AG, Basel
Introduction
Hepatocellular carcinoma (HCC) is one of the most common tumors and the third leading cause of cancer mortality worldwide [1]. Although therapeutic options are scarce, liver transplant (LT) has become the treatment of choice for early-stage HCC because it offers complete tumor excision and removal of the carcinogenic liver [2]. However, tumor recurrence after LT is still the main cause of death for HCC patients [3]. In 1996, Mazzaferro et al. introduced the Milan criteria, which were aimed at optimizing oncologic outcomes after LT by defining the size and the number of tumors as assessed by pathomorphological findings. Since then, the Milan criteria have been used to select patients who could expect 5-year recurrence-free survival and overall survival rates as high as 90% after LT and as high as 70% for HCC [4]. Although the Milan criteria have demonstrated excellent results for HCC, the risk of recurrence is still 10–20%, even for patients who meet the selection criteria. Kerstin Herzer, MD Liver Transplantation Unit, University Hospital Essen Hufelandstrasse 55 DE–45122 Essen (Germany) E-Mail kerstin.herzer @ uk-essen.de
In contrast, HCC does not recur in diverse patients who undergo transplantation on the basis of expanded criteria beyond the Milan criteria, such as preoperative radiologic findings other than the two main determinants of cancer recurrence (poor tumor grade and microvascular invasion) [5]. However, morphological data based mainly on tumor size and number of nodules are imperfect in predicting the behavior of HCC after LT. In recent years, new strategies have been advocated with the intent of not only increasing the number of potential transplant candidates but also selecting recipients with the lowest biological risk of recurrence. Accomplishing these goals will require a comprehensive search for surrogate tumor markers that reflect the aggressiveness of HCC. Attempts to identify clinical variables that influence tumor recurrence have resulted in improved LT selection criteria for patients with favorable HCC. Some potential markers of recurrence are tumor size, tumor number, differentiation, vascular invasion, and the serum concentration of alpha-fetoprotein (AFP) [6]. Various biological and dynamic variables have been proposed in both Western and Eastern scenarios, such as AFP dynamics, radiological response to locoregional treatments, and several inflammatory markers, the most promising of which is the neutrophil-to-lymphocyte ratio [7]. The immunosuppressive regimen selected has also been found to influence HCC recurrence: calcineurin inhibitors (CNIs) and steroids have been shown to increase the risk of recurrence, whereas mammalian target of rapamycin (mTor) inhibitors seem to have the potential to reduce the risk of recurrence [8, 9]. Which immunosuppressive regimen can decrease tumor recurrence is still a matter of debate. However, basic liver values have not yet been studied with regard to their ability to predict HCC recurrence. The objective of this study was to retrospectively investigate the correlation of various clinicopathological features with HCC recurrence after LT in a large cohort of patients. We not only evaluated established predictors but also determined whether liver values at time of LT could predict posttransplant oncologic outcomes. Our results may supplement established selection criteria for patients with HCC who are most likely to benefit from LT. Material and Methods Patients Between January 2000 and December 2013, 193 patients underwent LT because of HCC at the University Hospital Essen. Of these patients, 18 were excluded from this study because of peri-
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Table 1. Patient characteristics
Age MELD BMI, kg/m2 AFP, U/ml ALT, U/l AST, U/l AP, U/l GGT, U/l CHE, U/ml GLDH, U/l Bilirubin, mg/dl
Median
Range
57.0 13.5 26.2 13.2 49.0 52.5 137.0 97.5 4.0 5.7 1.2
27–69 6–40 18–44 1.1–27,055 3–297 0.5–312 40–540 4.4–506 0.65–9.2 1.1–44.7 0.1–18.3
MELD = Model of end-stage liver disease; BMI = body mass index; AFP = alpha-fetoprotein; ALT = alanine-amino transferase; AST = aspartate-amino transferase; AP = alkaline phosphatase; GGT = gamma-glutamyl transpeptidase; CHE = cholesterol esterase; GLDH = glutamate dehydrogenase.
operative mortality (up to 6 months after LT) or incomplete documentation of data. The remaining 175 patients were included in the study. Demographic data, patient characteristics, and clinicopathological findings were retrieved from the database of the Liver Transplantation Unit of the University Hospital Essen. All analyses were performed in accordance with the Declaration of Helsinki, and the study was approved by our local Institutional Review Board. Pathological Variables Each explant was examined by a pathologist and categorized on the basis of tumor number, tumor size and tumor differentiation. The Milan criteria (tumor size and number) were applied. Tumors that were not recognized before transplant but were detected in the explanted liver were regarded as incidental. When multiple lesions were detected in the liver explants, the histological grade of the largest tumor was recorded. Tumor differentiation was separated into four grades, according to the Edmondson-Steiner criteria. The tumor stage was determined according to the pathological TNM (pTNM) classification [AJCC, 2009] and was summarized as follows: one single tumor without vascular invasion, one single tumor with vascular invasion, or multiple tumors, none of which was larger than 5 cm (pT 1–2); or a single tumor or multiple tumors with vascular invasion, with direct invasion of adjacent organs other than the gallbladder, or with perforation of the visceral peritoneum (pT 3–4). Postoperative Follow-Up and Treatment No adjuvant chemotherapy was administered to any patient after transplant. Sorafenib was selectively given to patients with multiple pulmonary metastases. Immunosuppressive therapy after LT consisted of tacrolimus (TAC) alone or combined with mycophenolate mofetil (MMF) or everolimus (EVR). Steroid dosages were tapered to discontinuation by 3 months after LT. Patients were followed up approximately every 3 months during the first
Piras-Straub/Khairzada/Gerken/Saner/ Treckmann/Paul/Canbay/Herzer
Table 2. Clinicopathological parameters in relation to HCC recurrence rate
Patients Recurrence p** ratio, % rate, % Gender Male Female Age 60 Tumor stage pT I–II pT III–IV Grading Highly differentation [G = 1–2] Moderate differentation [G = 3] AFP, U/l 400 ALT, U/l 35 AST, U/l 35 AP, U/l 140 GGT, U/l 35 GLDH, U/l 10 CHE, U/l 10
74 26
24 24
0.911
79 31
28 15
0.073
75 25
19 49
0.0004
87 13
22 38
0.102
44 31 12.5 12.5
17 18 40 40
0.036
38 62
23 24
0.956
31 69
17 26
0.204
52 48
19 25
0.391
13 87
29 23
0.583
75 25
15 35
0.038
67 27 6
20 20 14
0.930
2 years and every 6 months thereafter. During the routine followup visits, imaging studies, including computed tomography of the abdomen and chest, were performed 6, 12, 24, and 36 months after LT, and the AFP level was also assessed for AFP-positive tumors. Statistical Analysis Numeric data (serum activity of liver enzymes) are presented as means ± SEM, unless stated otherwise. Categorical variables are given as counts or percentages. The differences in numeric liver values of large groups (n ≥40) were analyzed with two-tailed Student’s t tests with Welch’s correction. Numeric liver values of small groups (n ≤30) were compared with the Mann-Whitney U test. Categorical variables were analyzed with χ2-tests with Pearson approximation. Independent prognostic markers were determined by multivariate analysis. The level of statistical significance was set at p ≤ 0.05. Statistical analyses were performed with SPSS 19 statistical software (IBM SPSS).
HCC Recurrence after Liver Transplant
Patients Recurrence p** ratio, % rate, % Etiology ASH HCV HBV NASH Other Milan-criteria Within Outside BMI, kg/m2 36 months post LT
3
p 0.175 50 HCC recurrence rate (%)
HCC patients (%)
100
d
44% 4
40
9
30
24% 19
20
17%
13% 10 0
39%
5
4
ASH
HCV
HBV
NASH
Other
Fig. 1. For retrospective analysis, 175 patients were included. a Ratio of patients who did or did not experience recurrence of hepatocellular carcinoma (HCC) after liver transplant (LT). b Time in-
terval to the incidence of HCC recurrence. c Causes of underlying liver disease for the enrolled HCC patients. d Risk of HCC recurrence according to underlying liver disease.
Overall Outcome Although 23% of the patients who underwent LT because of HCC experienced tumor recurrence (fig. 1a); more than half (59%) of the tumors reoccurred 6 to 12 months after LT. Almost all patients who completed follow-up experienced HCC recurrence within 24 months after LT (fig. 1b). For 3 patients, tumor recurrence was documented more than 36 months after LT, 2 at month 36 after LT and one at month 41 after LT. The main indication for LT because of HCC was viral hepatitis (63%); the predominant indication was HCV (46%), followed by nutritive toxic liver diseases (NASH, 5%; ASH, 19%) and other diseases that rather rarely cause HCC (hemochromatosis, autoimmune disorders, Wilson’s disease, primary biliary cirrhosis, and cryptogenic
cirrhosis), which in this report are categorized as ‘others’ (fig. 1c). To clarify the influence of the underlying liver disease on the probability of HCC recurrence, we separately analyzed the relationship between recurrence and the cause of the liver disease (fig. 1d). We found the highest risk of tumor recurrence among patients with NASH-associated HCC (44%), followed by those with HCV (24%) and HBV (17%). Only 13% of the patients with ASH experienced HCC recurrence. With regard to the high probability of HCC recurrence in NASH patients, we analyzed the impact of insulin sensitivity on the HCC recurrence rate. In our cohort, patients with diabetes mellitus do not show higher incidences of HCC relapse than patients without (data not shown).
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Piras-Straub/Khairzada/Gerken/Saner/ Treckmann/Paul/Canbay/Herzer
a
49%
50 40 30 20
19%
10 0 pT I–II
pT III–IV
HCC recurrence rate (%)
HCC recurrence rate (%)
p S
50
40%
40 30
18%
17%
20
40%
10 0
b
AFP 400 IU/ml IU/ml IU/ml
S
HCC recurrence rate (%)
50
Fig. 2. Risk of recurrence of hepatocellular carcinoma (HCC) according to tumor size (a), serum concentration of alpha-fetoprotein (AFP) (b), and serum activity of glutamate dehydrogenase (GLDH) (c). Statistical significance was set at the level of p ≤ 0.05
c
40
35%
30 20
15%
10 0 GLDH >10 U/l
GLDH 40) and p+: Mann-Whitney U test for small groups (n 100 IU/ml AP >100 U/l
c
Table 4. Patient characteristics in dependence on the etiology of the underlying liver disease HBV
Age MELD BMI, kg/m2 AFP, U/ml ALT, U/l AST, U/l AP, U/l GGT, U/l CHE, U/ml GLDH, U/l Bilirubin, mg/dl
NASH
ASH
HCV
median
range
median
range
median
range
median
range
55.0 12.5 27.0 9.5 44.0 52.0 101.0 81.0 5.8 5.3 1.0
29.0±69.0 6.0±29.0 17.8±35.3 1.7±10,411.0 4.0±98.0 13.0±999.0 68.0±322.0 5.0±336.0 2.1±357.0 1.9±33.7 0.1±15.1
59.5 14.5 32.1 16.0 32.0 57.0 171.0 128.0 4.3 6.6 2.6
50.0±65.0 6.0±34.0 24.0±44.0 4.5±27,055.0 18.0±89.0 25.0±73.0 80.0±278.0 53.0±291.0 1.1±7.9 1.3±9.6 0.5±5.8
60.0 13.5 26.8 6.7 33.5 41.0 130.0 103.5 4.3 4.8 1.3
35.0±69.0 6.0±40.0 21.6±37.7 1.3±7,436.0 4.0±188.0 18.0±282.0 40.0±376.0 4.4±424.0 1.0±9.2 1.1±44.7 0.2±13.1
56.0 13.0 26.0 15.3 64.0 73.0 141.0 96.0 3.3 6.0 1.4
43.0±69.0 7.0±34.0 18.0±41.8 1.8±5,770.0 3.0±297.0 1.1±312.0 68.0±362.0 9.0±506.0 0.7±10.3 1.9±42.9 0.3±18.3
p value 0.016 0.438 0.005 0.329 0.536 0.618 0.223 0.671 0.008 0.832 0.847
MELD = Model of end-stage liver disease; BMI = body mass index; AFP = alpha-fetoprotein; ALT = alanine-amino transderase; AST = aspartate-amino transferase; AP = alkaline phosphatase; GGT = gamma-glutamyl transpeptidase; CHE = cholesterin esterase; GLDH = glutamate dehydrogenase.
25%. The influence of GLDH levels becomes even more apparent in patients with an advanced tumor stage. Because of this, the probability of HCC recurrence increases from 43% in patients with low GLDH levels to 56% in patients with elevated GLDH levels (p ≤ 0.003).
Combination of AFP and GLDH levels does apparently further improve HCC recurrence prediction (fig. 4b). Patients with none or one of both elevated parameters show a maximum rate of HCC relapse of 25%, whereas patients with elevated AFP and elevated GLDH
HCC Recurrence after Liver Transplant
Digestion 2015;91:117–127 DOI: 10.1159/000370212
123
Patients ratio*
Recurrence rate
Immuno supressive therapy after LTx 25% 21% 29%
Fig. 5. Rate of recurrence of hepatocellular carcinoma (HCC) in enrolled patients according to the immunosuppressive therapy received after liver transplant (LT). Statistical significance was set at the level of p ≤ 0.05 and was determined with the χ2-test with Pearson approximation.
8% 40
30
Influence of Immunosuppressive Treatment To evaluate the effect of immunosuppressants on HCC recurrence after LT, we analyzed HCC recurrence according to the diverse immunosuppressive regimens used. Most patients received a tacrolimus (TAC)-based immunosuppressive regimen followed by cyclosporine A (CyA) (table 2; fig. 5). Interestingly, the HCC recurrence rate was lower for both calcineurin inhibitors (CNIs) when they were combined with MMF: TAC, 25% recurrence without MMF and 17% with MMF; CyA, 29% recurrence without MMF and no recurrence with MMF (fig. 5). An mTOR inhibitor-based immunosuppressive regimen achieved the lowest rate of HCC recurrence (8%). However, the differences in HCC recurrence according to immunosuppressive therapy did not achieve statistical significance.
Discussion
It is widely accepted that the pathologic status of HCC is closely related to the risk of HCC recurrence after LT. Patient survival has essentially improved with the estabDigestion 2015;91:117–127 DOI: 10.1159/000370212
10
0.339
Tacrolimus
55%
26%
+MMF
21%
21%
Cyclosporin A
10%
29%
+MMF
5%
0%
mTor inhibitor
9%
8%
0
Recurrence rate (%)
levels show an HCC reoccurrence in 83% of cases (p ≤ 0.001). The most accurate prediction of HCC recurrence can be observed by the combined analysis of GLDH, AP and AFP levels (fig. 4c). Patients with elevated levels of all three lab values show an HCC reoccurrence in 100% of cases, whereas the probability of all other groups (none, one or two elevated lab values) together does not achieve a probability of more than 22% (p ≤ 0.012).
124
20
p*
lishment of the Milan criteria for selecting transplant candidates. Although the Milan criteria have contributed to reducing the incidence of tumor recurrence to a remarkably low 10%, increasing evidence indicates that large numbers of patients were wrongfully excluded from curative liver transplant when their test results exceeded these criteria [10]. Recently, a multicenter study retrospectively explored the likelihood of survival after LT for HCC by applying additional variables beyond those included in the Milan criteria. This study found that if the rule of ‘up to seven’ was met (HCC with a score of 7, calculated as the sum of the size of the largest tumor in cm and the number of tumors), the 5-year survival rate could reach 70% [11]. The Metroticket calculator was then developed as a statistical tool that could predict the likelihood of 5-year survival for any given patient on the basis of several morphologic and pathologic characteristics: total size of the nodules, size of the largest nodule, and presence or absence of vascular invasion [12, 13]. Although predictions of survival have recently become more accurate and precise, physicians have not yet reached any agreement on the question of which survival rate is considered acceptable in balancing the highest likelihood of cure with the availability of organs for transplant. In the current study we analyzed the effect of pathological findings, basal liver values, and the patient’s exposure to immunosuppressive agents on early recurrence of HCC. Consistent with previous reports, our study showed that tumor stage and AFP levels were independently and positively correlated with HCC recurrence [14, 15]. However, although AFP concentrations higher than 400 U/l have been reported to be sensitive not only in diagnosing HCC but also in predicting recurrence after LT, we found Piras-Straub/Khairzada/Gerken/Saner/ Treckmann/Paul/Canbay/Herzer
that the risk of tumor recurrence was significantly higher when AFP levels were above a threshold concentration of only 100 U/l. Apparently, AFP-negative tumors are significantly less likely to recur. Of all the predictors that have been discussed previously, basic liver values have not yet been investigated in relation to tumor recurrence. In our cohort we saw a significant association (Pearson-R: 0.19; p ≤ 0.046) between elevated GLDH levels at the time of LT and HCC recurrence after LT. Moreover, when patients in whom HCC recurred were compared with those in whom it did not recur, the recurrence-free group exhibited significantly lower AP and GLDH activity at the time of LT. In particular, the combination of GLDH and AFP serum levels shows significant implications on the development of a HCC relapse. Patients with elevated serum levels of both show an HCC recurrence rate of 83% compared to patients with none or only one elevated parameter (p ≤ 0.001). This combination is more accurate than the combination of tumor stage and AFP levels, which results in a prediction of 69% probability for HCC relapse in patients with advanced tumor size and AFP levels >100 U/ml (pT I–II, AFP 100 IU/ml: 25%; pT III–IV, AFP 5 years after LT) is rarely observed. Thus, prolonged active surveillance for HCC recurrence beyond 3 years after orthotopic liver transplant may not be useful if the intention is to provide a survival benefit for these patients [24]. A high risk of recurrence early after LT may be associated with the rather high trough levels of immunosuppressive agents during this period. Tumor recurrence may be associated not only with the size and aggressiveness of the tumor but also with the immunosuppressive regimen used. Although postoperative immunosuppression can accelerate tumor growth, only a few studies have investigated the possible influence of various immunosuppressive agents on HCC recurrence after LT [25, 26]. In this regard, we examined the relationship between the immunosuppressive regimen used and early HCC recurrence (within one year). Patients who were given everolimus for immunosuppression were least likely to experience a recurrence. However, because everolimus was not approved for immunosuppression after LT until November 2012, the number of patients in our study who were given this immunosuppressive regimen is small, and conclusions should be drawn with caution. Surprisingly, in our cohort, recurrence occurred less frequently when a CNI (TAC or CyA) was combined with MMF. This
HCC Recurrence after Liver Transplant
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finding contradicts previous findings of a correlation between MMF and higher tumor recurrence rates after LT [27]. Thus, additional prospective trials are needed to thoroughly elucidate the influence of immunosuppressive agents on HCC recurrence. In general, careful and well-considered patient selection is the key to success of LT for HCC. Although tumor size and tumor number are used to predict whether LT will be successful, the weighting of these two variables has not been determined. In addition to the size and stage of HCC, its morphology also predicts its behavior. Well-circumscribed lesions, in general, are less aggressive than those with poorly defined borders [28]. However, the paradigm suggesting that HCC patients should be selected according to morphological aspects (tumor stage and diameter) alone, a paradigm based on the nearly 20-yearold success story of the Milan criteria, should be modified by combining these variables with newer biological tumor markers so that patient selection for LT can be further refined. Such a therapeutic algorithm will allow additional improvements in patient selection and in the outcome of HCC patients after LT. Certainly, it is essential that the criteria used for selecting HCC patients for LT should be easily applicable and
fair to all transplant candidates. However, each of the described biomarkers is imperfect when used alone, and each is limited in the sensitivity and specificity with which it detects HCC. Presumably, a combination of these biomarkers would be a practical way to improve their performance. In this respect, in our cohort, the combination of AFP, AP and GLDH levels results in an even higher predictive value. Patients with elevated levels of all three parameters experience an HCC relapse in 100% of cases; thus, combining these parameters provides a prediction of recurrence with a high significance. Therefore, novel and easy-to-assess biomarkers of HCC are being sought for use in diagnosing the disease and optimizing treatment modalities, in predicting prognosis or recurrence, and in discovering novel targets for therapeutic intervention.
Acknowledgment The study was supported by a grant from the Deutsche Krebshilfe to K.H. We thank Florence M. Witte (Bluegrass Editorial Services Team, LLC, Winchester, Ky., USA) for editorial assistance, which was supported by the deanery of the faculty of medicine, university of Duisburg-Essen. The work forms part of the thesis of K.P.-S.
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Copyright: S. Karger AG, Basel 2015. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Digestion 2015;91:117–127 DOI: 10.1159/000370212
Glutamate Dehydrogenase and Alkaline Phosphatase as Very Early Predictors of Hepatocellular Carcinoma Recurrence after Liver Transplantation Katja Piras-Straub a, b Khaleda Khairzada a, b Guido Gerken b Fuat Saner a Jürgen Treckmann a Andreas Paul a Ali Canbay b Kerstin Herzer a, b
Departments of a General, Visceral and Transplantation Surgery and b Gastroenterology and Hepatology, University Hospital Essen, Essen, Germany
Key Words HCC · NASH · GLDH · Tumor recurrence · Liver transplantation
Abstract Background: Although long-term survival rates for patients undergoing liver transplant (LT) for hepatocellular carcinoma (HCC) are good, the relatively high rate of tumor recurrence after LT necessitates the identification of biological parameters that supplement morphological predictors of recurrence. Method: From chart review we identified 175 patients who received liver transplantation due to HCC at our center between January 2000 and December 2013. We documented demographic and clinical data, as well as clinicopathological characteristics of the tumors, with a focus on liver values at the time of LT. Results: HCC recurred in 23% of LT patients. Most recurrences (59%) occurred within 12 months after LT; hardly any recurrence was detected later than 3 years after LT. Recurrence was positively correlated with tumor size, tumor stage and alpha-fetoprotein level (AFP), and it was most likely with certain causes of liver disease. Interestingly, tumor recurrence was independently predicted by serum levels of glutamate dehydrogenase (GLDH) and alkaline phosphatase (AP) at the time of LT. Conclusions: Because all HCC recurrence occurs within 36 months after LT, HCC detected more than 3 years after LT
© 2015 S. Karger AG, Basel 0012–2823/15/0912–0117$39.50/0 E-Mail [email protected] www.karger.com/dig
may be considered de novo. Liver values, with GLDH and AP being the most preponderant, serve as easy-to-assess biomarkers which contribute to predict the risk of tumor recurrence. © 2015 S. Karger AG, Basel
Introduction
Hepatocellular carcinoma (HCC) is one of the most common tumors and the third leading cause of cancer mortality worldwide [1]. Although therapeutic options are scarce, liver transplant (LT) has become the treatment of choice for early-stage HCC because it offers complete tumor excision and removal of the carcinogenic liver [2]. However, tumor recurrence after LT is still the main cause of death for HCC patients [3]. In 1996, Mazzaferro et al. introduced the Milan criteria, which were aimed at optimizing oncologic outcomes after LT by defining the size and the number of tumors as assessed by pathomorphological findings. Since then, the Milan criteria have been used to select patients who could expect 5-year recurrence-free survival and overall survival rates as high as 90% after LT and as high as 70% for HCC [4]. Although the Milan criteria have demonstrated excellent results for HCC, the risk of recurrence is still 10–20%, even for patients who meet the selection criteria. Kerstin Herzer, MD Liver Transplantation Unit, University Hospital Essen Hufelandstrasse 55 DE–45122 Essen (Germany) E-Mail kerstin.herzer @ uk-essen.de
In contrast, HCC does not recur in diverse patients who undergo transplantation on the basis of expanded criteria beyond the Milan criteria, such as preoperative radiologic findings other than the two main determinants of cancer recurrence (poor tumor grade and microvascular invasion) [5]. However, morphological data based mainly on tumor size and number of nodules are imperfect in predicting the behavior of HCC after LT. In recent years, new strategies have been advocated with the intent of not only increasing the number of potential transplant candidates but also selecting recipients with the lowest biological risk of recurrence. Accomplishing these goals will require a comprehensive search for surrogate tumor markers that reflect the aggressiveness of HCC. Attempts to identify clinical variables that influence tumor recurrence have resulted in improved LT selection criteria for patients with favorable HCC. Some potential markers of recurrence are tumor size, tumor number, differentiation, vascular invasion, and the serum concentration of alpha-fetoprotein (AFP) [6]. Various biological and dynamic variables have been proposed in both Western and Eastern scenarios, such as AFP dynamics, radiological response to locoregional treatments, and several inflammatory markers, the most promising of which is the neutrophil-to-lymphocyte ratio [7]. The immunosuppressive regimen selected has also been found to influence HCC recurrence: calcineurin inhibitors (CNIs) and steroids have been shown to increase the risk of recurrence, whereas mammalian target of rapamycin (mTor) inhibitors seem to have the potential to reduce the risk of recurrence [8, 9]. Which immunosuppressive regimen can decrease tumor recurrence is still a matter of debate. However, basic liver values have not yet been studied with regard to their ability to predict HCC recurrence. The objective of this study was to retrospectively investigate the correlation of various clinicopathological features with HCC recurrence after LT in a large cohort of patients. We not only evaluated established predictors but also determined whether liver values at time of LT could predict posttransplant oncologic outcomes. Our results may supplement established selection criteria for patients with HCC who are most likely to benefit from LT. Material and Methods Patients Between January 2000 and December 2013, 193 patients underwent LT because of HCC at the University Hospital Essen. Of these patients, 18 were excluded from this study because of peri-
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Table 1. Patient characteristics
Age MELD BMI, kg/m2 AFP, U/ml ALT, U/l AST, U/l AP, U/l GGT, U/l CHE, U/ml GLDH, U/l Bilirubin, mg/dl
Median
Range
57.0 13.5 26.2 13.2 49.0 52.5 137.0 97.5 4.0 5.7 1.2
27–69 6–40 18–44 1.1–27,055 3–297 0.5–312 40–540 4.4–506 0.65–9.2 1.1–44.7 0.1–18.3
MELD = Model of end-stage liver disease; BMI = body mass index; AFP = alpha-fetoprotein; ALT = alanine-amino transferase; AST = aspartate-amino transferase; AP = alkaline phosphatase; GGT = gamma-glutamyl transpeptidase; CHE = cholesterol esterase; GLDH = glutamate dehydrogenase.
operative mortality (up to 6 months after LT) or incomplete documentation of data. The remaining 175 patients were included in the study. Demographic data, patient characteristics, and clinicopathological findings were retrieved from the database of the Liver Transplantation Unit of the University Hospital Essen. All analyses were performed in accordance with the Declaration of Helsinki, and the study was approved by our local Institutional Review Board. Pathological Variables Each explant was examined by a pathologist and categorized on the basis of tumor number, tumor size and tumor differentiation. The Milan criteria (tumor size and number) were applied. Tumors that were not recognized before transplant but were detected in the explanted liver were regarded as incidental. When multiple lesions were detected in the liver explants, the histological grade of the largest tumor was recorded. Tumor differentiation was separated into four grades, according to the Edmondson-Steiner criteria. The tumor stage was determined according to the pathological TNM (pTNM) classification [AJCC, 2009] and was summarized as follows: one single tumor without vascular invasion, one single tumor with vascular invasion, or multiple tumors, none of which was larger than 5 cm (pT 1–2); or a single tumor or multiple tumors with vascular invasion, with direct invasion of adjacent organs other than the gallbladder, or with perforation of the visceral peritoneum (pT 3–4). Postoperative Follow-Up and Treatment No adjuvant chemotherapy was administered to any patient after transplant. Sorafenib was selectively given to patients with multiple pulmonary metastases. Immunosuppressive therapy after LT consisted of tacrolimus (TAC) alone or combined with mycophenolate mofetil (MMF) or everolimus (EVR). Steroid dosages were tapered to discontinuation by 3 months after LT. Patients were followed up approximately every 3 months during the first
Piras-Straub/Khairzada/Gerken/Saner/ Treckmann/Paul/Canbay/Herzer
Table 2. Clinicopathological parameters in relation to HCC recurrence rate
Patients Recurrence p** ratio, % rate, % Gender Male Female Age 60 Tumor stage pT I–II pT III–IV Grading Highly differentation [G = 1–2] Moderate differentation [G = 3] AFP, U/l 400 ALT, U/l 35 AST, U/l 35 AP, U/l 140 GGT, U/l 35 GLDH, U/l 10 CHE, U/l 10
74 26
24 24
0.911
79 31
28 15
0.073
75 25
19 49
0.0004
87 13
22 38
0.102
44 31 12.5 12.5
17 18 40 40
0.036
38 62
23 24
0.956
31 69
17 26
0.204
52 48
19 25
0.391
13 87
29 23
0.583
75 25
15 35
0.038
67 27 6
20 20 14
0.930
2 years and every 6 months thereafter. During the routine followup visits, imaging studies, including computed tomography of the abdomen and chest, were performed 6, 12, 24, and 36 months after LT, and the AFP level was also assessed for AFP-positive tumors. Statistical Analysis Numeric data (serum activity of liver enzymes) are presented as means ± SEM, unless stated otherwise. Categorical variables are given as counts or percentages. The differences in numeric liver values of large groups (n ≥40) were analyzed with two-tailed Student’s t tests with Welch’s correction. Numeric liver values of small groups (n ≤30) were compared with the Mann-Whitney U test. Categorical variables were analyzed with χ2-tests with Pearson approximation. Independent prognostic markers were determined by multivariate analysis. The level of statistical significance was set at p ≤ 0.05. Statistical analyses were performed with SPSS 19 statistical software (IBM SPSS).
HCC Recurrence after Liver Transplant
Patients Recurrence p** ratio, % rate, % Etiology ASH HCV HBV NASH Other Milan-criteria Within Outside BMI, kg/m2 36 months post LT
3
p 0.175 50 HCC recurrence rate (%)
HCC patients (%)
100
d
44% 4
40
9
30
24% 19
20
17%
13% 10 0
39%
5
4
ASH
HCV
HBV
NASH
Other
Fig. 1. For retrospective analysis, 175 patients were included. a Ratio of patients who did or did not experience recurrence of hepatocellular carcinoma (HCC) after liver transplant (LT). b Time in-
terval to the incidence of HCC recurrence. c Causes of underlying liver disease for the enrolled HCC patients. d Risk of HCC recurrence according to underlying liver disease.
Overall Outcome Although 23% of the patients who underwent LT because of HCC experienced tumor recurrence (fig. 1a); more than half (59%) of the tumors reoccurred 6 to 12 months after LT. Almost all patients who completed follow-up experienced HCC recurrence within 24 months after LT (fig. 1b). For 3 patients, tumor recurrence was documented more than 36 months after LT, 2 at month 36 after LT and one at month 41 after LT. The main indication for LT because of HCC was viral hepatitis (63%); the predominant indication was HCV (46%), followed by nutritive toxic liver diseases (NASH, 5%; ASH, 19%) and other diseases that rather rarely cause HCC (hemochromatosis, autoimmune disorders, Wilson’s disease, primary biliary cirrhosis, and cryptogenic
cirrhosis), which in this report are categorized as ‘others’ (fig. 1c). To clarify the influence of the underlying liver disease on the probability of HCC recurrence, we separately analyzed the relationship between recurrence and the cause of the liver disease (fig. 1d). We found the highest risk of tumor recurrence among patients with NASH-associated HCC (44%), followed by those with HCV (24%) and HBV (17%). Only 13% of the patients with ASH experienced HCC recurrence. With regard to the high probability of HCC recurrence in NASH patients, we analyzed the impact of insulin sensitivity on the HCC recurrence rate. In our cohort, patients with diabetes mellitus do not show higher incidences of HCC relapse than patients without (data not shown).
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a
49%
50 40 30 20
19%
10 0 pT I–II
pT III–IV
HCC recurrence rate (%)
HCC recurrence rate (%)
p S
50
40%
40 30
18%
17%
20
40%
10 0
b
AFP 400 IU/ml IU/ml IU/ml
S
HCC recurrence rate (%)
50
Fig. 2. Risk of recurrence of hepatocellular carcinoma (HCC) according to tumor size (a), serum concentration of alpha-fetoprotein (AFP) (b), and serum activity of glutamate dehydrogenase (GLDH) (c). Statistical significance was set at the level of p ≤ 0.05
c
40
35%
30 20
15%
10 0 GLDH >10 U/l
GLDH 40) and p+: Mann-Whitney U test for small groups (n 100 IU/ml AP >100 U/l
c
Table 4. Patient characteristics in dependence on the etiology of the underlying liver disease HBV
Age MELD BMI, kg/m2 AFP, U/ml ALT, U/l AST, U/l AP, U/l GGT, U/l CHE, U/ml GLDH, U/l Bilirubin, mg/dl
NASH
ASH
HCV
median
range
median
range
median
range
median
range
55.0 12.5 27.0 9.5 44.0 52.0 101.0 81.0 5.8 5.3 1.0
29.0±69.0 6.0±29.0 17.8±35.3 1.7±10,411.0 4.0±98.0 13.0±999.0 68.0±322.0 5.0±336.0 2.1±357.0 1.9±33.7 0.1±15.1
59.5 14.5 32.1 16.0 32.0 57.0 171.0 128.0 4.3 6.6 2.6
50.0±65.0 6.0±34.0 24.0±44.0 4.5±27,055.0 18.0±89.0 25.0±73.0 80.0±278.0 53.0±291.0 1.1±7.9 1.3±9.6 0.5±5.8
60.0 13.5 26.8 6.7 33.5 41.0 130.0 103.5 4.3 4.8 1.3
35.0±69.0 6.0±40.0 21.6±37.7 1.3±7,436.0 4.0±188.0 18.0±282.0 40.0±376.0 4.4±424.0 1.0±9.2 1.1±44.7 0.2±13.1
56.0 13.0 26.0 15.3 64.0 73.0 141.0 96.0 3.3 6.0 1.4
43.0±69.0 7.0±34.0 18.0±41.8 1.8±5,770.0 3.0±297.0 1.1±312.0 68.0±362.0 9.0±506.0 0.7±10.3 1.9±42.9 0.3±18.3
p value 0.016 0.438 0.005 0.329 0.536 0.618 0.223 0.671 0.008 0.832 0.847
MELD = Model of end-stage liver disease; BMI = body mass index; AFP = alpha-fetoprotein; ALT = alanine-amino transderase; AST = aspartate-amino transferase; AP = alkaline phosphatase; GGT = gamma-glutamyl transpeptidase; CHE = cholesterin esterase; GLDH = glutamate dehydrogenase.
25%. The influence of GLDH levels becomes even more apparent in patients with an advanced tumor stage. Because of this, the probability of HCC recurrence increases from 43% in patients with low GLDH levels to 56% in patients with elevated GLDH levels (p ≤ 0.003).
Combination of AFP and GLDH levels does apparently further improve HCC recurrence prediction (fig. 4b). Patients with none or one of both elevated parameters show a maximum rate of HCC relapse of 25%, whereas patients with elevated AFP and elevated GLDH
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Patients ratio*
Recurrence rate
Immuno supressive therapy after LTx 25% 21% 29%
Fig. 5. Rate of recurrence of hepatocellular carcinoma (HCC) in enrolled patients according to the immunosuppressive therapy received after liver transplant (LT). Statistical significance was set at the level of p ≤ 0.05 and was determined with the χ2-test with Pearson approximation.
8% 40
30
Influence of Immunosuppressive Treatment To evaluate the effect of immunosuppressants on HCC recurrence after LT, we analyzed HCC recurrence according to the diverse immunosuppressive regimens used. Most patients received a tacrolimus (TAC)-based immunosuppressive regimen followed by cyclosporine A (CyA) (table 2; fig. 5). Interestingly, the HCC recurrence rate was lower for both calcineurin inhibitors (CNIs) when they were combined with MMF: TAC, 25% recurrence without MMF and 17% with MMF; CyA, 29% recurrence without MMF and no recurrence with MMF (fig. 5). An mTOR inhibitor-based immunosuppressive regimen achieved the lowest rate of HCC recurrence (8%). However, the differences in HCC recurrence according to immunosuppressive therapy did not achieve statistical significance.
Discussion
It is widely accepted that the pathologic status of HCC is closely related to the risk of HCC recurrence after LT. Patient survival has essentially improved with the estabDigestion 2015;91:117–127 DOI: 10.1159/000370212
10
0.339
Tacrolimus
55%
26%
+MMF
21%
21%
Cyclosporin A
10%
29%
+MMF
5%
0%
mTor inhibitor
9%
8%
0
Recurrence rate (%)
levels show an HCC reoccurrence in 83% of cases (p ≤ 0.001). The most accurate prediction of HCC recurrence can be observed by the combined analysis of GLDH, AP and AFP levels (fig. 4c). Patients with elevated levels of all three lab values show an HCC reoccurrence in 100% of cases, whereas the probability of all other groups (none, one or two elevated lab values) together does not achieve a probability of more than 22% (p ≤ 0.012).
124
20
p*
lishment of the Milan criteria for selecting transplant candidates. Although the Milan criteria have contributed to reducing the incidence of tumor recurrence to a remarkably low 10%, increasing evidence indicates that large numbers of patients were wrongfully excluded from curative liver transplant when their test results exceeded these criteria [10]. Recently, a multicenter study retrospectively explored the likelihood of survival after LT for HCC by applying additional variables beyond those included in the Milan criteria. This study found that if the rule of ‘up to seven’ was met (HCC with a score of 7, calculated as the sum of the size of the largest tumor in cm and the number of tumors), the 5-year survival rate could reach 70% [11]. The Metroticket calculator was then developed as a statistical tool that could predict the likelihood of 5-year survival for any given patient on the basis of several morphologic and pathologic characteristics: total size of the nodules, size of the largest nodule, and presence or absence of vascular invasion [12, 13]. Although predictions of survival have recently become more accurate and precise, physicians have not yet reached any agreement on the question of which survival rate is considered acceptable in balancing the highest likelihood of cure with the availability of organs for transplant. In the current study we analyzed the effect of pathological findings, basal liver values, and the patient’s exposure to immunosuppressive agents on early recurrence of HCC. Consistent with previous reports, our study showed that tumor stage and AFP levels were independently and positively correlated with HCC recurrence [14, 15]. However, although AFP concentrations higher than 400 U/l have been reported to be sensitive not only in diagnosing HCC but also in predicting recurrence after LT, we found Piras-Straub/Khairzada/Gerken/Saner/ Treckmann/Paul/Canbay/Herzer
that the risk of tumor recurrence was significantly higher when AFP levels were above a threshold concentration of only 100 U/l. Apparently, AFP-negative tumors are significantly less likely to recur. Of all the predictors that have been discussed previously, basic liver values have not yet been investigated in relation to tumor recurrence. In our cohort we saw a significant association (Pearson-R: 0.19; p ≤ 0.046) between elevated GLDH levels at the time of LT and HCC recurrence after LT. Moreover, when patients in whom HCC recurred were compared with those in whom it did not recur, the recurrence-free group exhibited significantly lower AP and GLDH activity at the time of LT. In particular, the combination of GLDH and AFP serum levels shows significant implications on the development of a HCC relapse. Patients with elevated serum levels of both show an HCC recurrence rate of 83% compared to patients with none or only one elevated parameter (p ≤ 0.001). This combination is more accurate than the combination of tumor stage and AFP levels, which results in a prediction of 69% probability for HCC relapse in patients with advanced tumor size and AFP levels >100 U/ml (pT I–II, AFP 100 IU/ml: 25%; pT III–IV, AFP 5 years after LT) is rarely observed. Thus, prolonged active surveillance for HCC recurrence beyond 3 years after orthotopic liver transplant may not be useful if the intention is to provide a survival benefit for these patients [24]. A high risk of recurrence early after LT may be associated with the rather high trough levels of immunosuppressive agents during this period. Tumor recurrence may be associated not only with the size and aggressiveness of the tumor but also with the immunosuppressive regimen used. Although postoperative immunosuppression can accelerate tumor growth, only a few studies have investigated the possible influence of various immunosuppressive agents on HCC recurrence after LT [25, 26]. In this regard, we examined the relationship between the immunosuppressive regimen used and early HCC recurrence (within one year). Patients who were given everolimus for immunosuppression were least likely to experience a recurrence. However, because everolimus was not approved for immunosuppression after LT until November 2012, the number of patients in our study who were given this immunosuppressive regimen is small, and conclusions should be drawn with caution. Surprisingly, in our cohort, recurrence occurred less frequently when a CNI (TAC or CyA) was combined with MMF. This
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finding contradicts previous findings of a correlation between MMF and higher tumor recurrence rates after LT [27]. Thus, additional prospective trials are needed to thoroughly elucidate the influence of immunosuppressive agents on HCC recurrence. In general, careful and well-considered patient selection is the key to success of LT for HCC. Although tumor size and tumor number are used to predict whether LT will be successful, the weighting of these two variables has not been determined. In addition to the size and stage of HCC, its morphology also predicts its behavior. Well-circumscribed lesions, in general, are less aggressive than those with poorly defined borders [28]. However, the paradigm suggesting that HCC patients should be selected according to morphological aspects (tumor stage and diameter) alone, a paradigm based on the nearly 20-yearold success story of the Milan criteria, should be modified by combining these variables with newer biological tumor markers so that patient selection for LT can be further refined. Such a therapeutic algorithm will allow additional improvements in patient selection and in the outcome of HCC patients after LT. Certainly, it is essential that the criteria used for selecting HCC patients for LT should be easily applicable and
fair to all transplant candidates. However, each of the described biomarkers is imperfect when used alone, and each is limited in the sensitivity and specificity with which it detects HCC. Presumably, a combination of these biomarkers would be a practical way to improve their performance. In this respect, in our cohort, the combination of AFP, AP and GLDH levels results in an even higher predictive value. Patients with elevated levels of all three parameters experience an HCC relapse in 100% of cases; thus, combining these parameters provides a prediction of recurrence with a high significance. Therefore, novel and easy-to-assess biomarkers of HCC are being sought for use in diagnosing the disease and optimizing treatment modalities, in predicting prognosis or recurrence, and in discovering novel targets for therapeutic intervention.
Acknowledgment The study was supported by a grant from the Deutsche Krebshilfe to K.H. We thank Florence M. Witte (Bluegrass Editorial Services Team, LLC, Winchester, Ky., USA) for editorial assistance, which was supported by the deanery of the faculty of medicine, university of Duisburg-Essen. The work forms part of the thesis of K.P.-S.
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