The aim of this study was to determine whether coffee consumption decreases HCC recurrence and promotes survival following orthotopic OLT and to examine putative mechanisms of action.

The health benefits of coffee and its relationship to human diseases inclusive of cancer has been suggested, yet the underlying mechanisms remain largely elusive. 10 - 12 Extracellular adenosine (ADO) is a well‐known molecular signal that could drive cancer cell growth through various purinergic receptors, namely A1, A2A, A2B and A3. Caffeine, a natural A2A receptor antagonist displaying tumouricidal activity in experimental settings, has been postulated to be, at least in part, responsible for such action. 13

Recent studies identified several risk factors for HCC recurrence, such as biological and radiological progression on the waiting list, number of tumour nodules and poor differentiation. 5 , 6 However, underlying mechanisms are still poorly understood and potential strategies after OLT to prevent HCC recurrence are still lacking. There is an emerging clinical evidence that coffee consumption might be associated with a more favourable outcome in chronic liver disease and chronic hepatitis. 7 , 8 Subgroup analysis emphasised that the benefit is also dose dependent and strong coffee drinkers have a decreased risk of HCC development in comparison to low or noncoffee drinkers. 9

Hepatocellular carcinoma (HCC) is the most common primary malignant tumour of the liver and its incidence has increased continuously in Western countries over the last decades. 1 Chronic liver disease represents the major risk factor for the development of HCC, with multiple known etiological factors, eg viral hepatitis, alcohol abuse or non‐alcoholic steatohepatitis. 2 , 3 Orthotopic liver transplantation (OLT) represents a therapeutic option with the most favourable outcome, as it offers both radical removal of the tumour and eliminates the underlying chronic liver disease in selected patients or those with early stage HCC. However, HCC recurrence following OLT remains a serious issue with up to 20% experiencing this lethal complication. 4

For comparing numerical data we used a two‐sample t test unless denoted otherwise. Fisher's exact test was used in cases with cell sizes smaller than five observations and imbalanced margin sums. Survival analysis was based around Kaplan‐Meier estimations with log‐rank tests for univariate or Cox' proportional hazards models for multivariate analysis. All analyses were done using r software environment for statistical computing with the “survival” package. 19

For mechanistic studies in vitro, human HepG2 cells were analysed for expression patterns of adenosine receptors and ectoenzymes by qPCR and Western blot, as previously described. 14 - 16 Extracellular adenosine metabolism was detected by thin layer chromatography (TLC) as previously established in the laboratory. 17 , 18 Cells were also treated with adenosine (50 µmol/L) for various times, in the presence or absence of 8‐(3‐Chlorostyryl)‐caffeine (CSC, 60 nmol/L; a xanthine and an A2A antagonist), or alloxazine (50 µmol/L; an A2B antagonist). Cell growth was determined using Cell Counting Kit‐8 and metastatic potential was examined by soft agar 3D colony formation assay as reported recently. 16 Alterations in key adenosine‐mediated cancer pathways inclusive of MAPK (ERK and JNK) and NF‐kappa B were evaluated by Western blot.

Patients and first‐degree relatives were interviewed on the precise number of daily consumed cups of coffee before and after OLT. Between the type of caffeinated coffee was not differentiated. If the consumption ranged between two cups, eg 1‐2 or 2‐3, the lower number was used in all patients for statistical analysis.

Patients or first‐degree relatives (in deceased patients) were retrospectively asked about their routine daily intake of coffee before and after OLT during telephone interviews between April 2014 and May 2014 by two surgical residents (GW and UL). The interviewers were blinded to patients´ data, except for the fact whether or not the patient was still alive.

Patients were seen routinely in our outpatient clinic, after being discharged from the hospital following OLT. AFP levels, abdominal ultrasound and radiological imaging were performed in a routine manner. A minimum follow‐up of 1 year was mandatory to include the patients in this analysis. Every patient alive at the time of interview had either abdominal ultrasound and/or CT/MRI scans as well as AFP levels detected in the last 12 months (time of study). The routine immunosuppressive regimen was based on tacrolimus, mycophenolate mofetil and corticosteroids. Four weeks later, it was normally changed over to an mTOR inhibitor (Everolimus or Sirolimus) if there were no contraindications.

Hepatocellular carcinoma was verified histopathologically in all analysed patients either before and/or after OLT. Routine histological examination of pre‐operative tumour biopsies and the explanted liver, respectively, was conducted by well‐experienced pathologists. HCC in the explanted liver were classified according to the 6th UICC TNM classification between 2002 and 2009 and according to the 7th UICC TNM classification from 2009 on. Following histopathological data were obtained: Number of tumour nodules were defined as 1 = 1 nodule; 2 = 2 nodules; 3 = 3 nodules or 4 = >3 nodules, maximum tumour size (cm), free resection margins (R0 vs R1), microvascular invasion (yes vs no), lymphatic vessel invasion (yes vs no), perineural invasion (yes vs no), MILAN‐criteria (yes vs no), UCSF‐criteria (yes vs no) and Up‐to‐7‐criteria (yes vs no).

Pre‐transplant staging included computed tomography (CT), magnetic resonance tomography (MRI), transarterial chemoembolisation (TACE), abdominal ultrasound, AFP levels and liver biopsies, as indicated. Different therapy approaches and interventional bridging procedures were performed before patients were scheduled for OLT and included TACE, radio frequency ablation (RFA), percutaneous ethanol injection (PEI), liver resection and selective internal radiation therapy (SIRT) (bridging procedures yes vs no).

A retrospective analysis of patients who underwent OLT for HCC at the Department of Surgery, University Hospital Leipzig, Germany from January 2002 to December 2012 was conducted. The following demographic variables were assessed: sex (male vs female), age (years), BMI (kg/m 2 ) etiology of primary liver disease (viral, alcohol or others), death (yes vs no), 90‐day mortality (yes vs no), overall survival (days), disease‐free survival (days) and pre‐transplant serum levels of AFP (ng/dL). Patients were excluded from analysis for the following reasons: 90‐day in hospital mortality after initial OLT, microscopically positive margins, not reachable for interview, no vital tumour cells detectable in the explanted liver because of pre‐transplant interventional therapies.

ADO‐elicited activation of intracellular inflammatory elements is inhibited by both A2A and A2B antagonism. HepG2 cells were stimulated with exogenous ADO (50 µmol/L) for various time points, or in the absence or presence of CSC (60 nmol/L) or alloxazine (5 µmol/L). Cell lysates were analysed by Western blot using antibodies as indicated (n = 3)

As both A2A and A2B receptor signalling have been linked to MAPK and NFκB pathways 22 - 24 , we next examined these pathways transduction in response to ADO and receptor blockade. As demonstrated in Figure 5 , ADO markedly induced activation of MAPK (ERK and JNK) and NFκB pathways, which could be blocked by A2A or A2B antagonism.

Similar phenomena were noted with regard to colony formation capacity (Figure 4 B,C). ADO increased both number and size of colonies, when compared to vehicle controls. A2A receptor antagonist CSC drastically blocked such effects. In contrast, blockade of A2B receptors had no effect on the number of colonies, but decreased the size of colonies. These results imply that ADO promotes HCC cell growth and metastasis, at least in part, in an A2A receptor‐dependent manner.

Growth‐ and metastasis‐promoting effects of ADO are counteracted by A2A receptor blockade. HepG2 cells were exposed to exogenous ADO (50 µmol/L), with or without CSC (8‐(3‐Chlorostyryl)caffeine, 60 nmol/L) or alloxazine (5 µmol/L) for 48 h. (A) Cell viability was evaluated by CCK‐8 that measures the activity of cellular dehydrogenases (correlating with cell proliferation) (n = 4‐8). (B, C) Metastatic potential was assessed by soft agar 3D colony formation assay (n = 3). Colonies were quantified (B) and representative images of colonies were shown (40 × magnification) (C). CSC: a selective A2A antagonist; alloxazine, a selective A2B antagonist. Error bars, M ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.0001, ns, nonsignificant

Next, cells were treated with exogenous ADO in the presence or absence of antagonist to A2A receptor (8‐(3‐Chlorostyryl)caffeine, CSC), or a more specific antagonist to A2B receptor (alloxazine), followed by assessments of growth capacity (by CCK‐8) and metastatic potential (by soft agar 3D colony formation). Figure 4 A shows that ADO significantly stimulated HCC cell growth, which could be blocked by A2A antagonist CSC, but not by the A2B antagonist alloxazine. Indeed, Alloxazine alone also promoted cell growth.

Collectively, these data suggest that the dominant ectoenzymes responsible for extracellular purine metabolism of HCC cells are most likely ENTPD3 and ADA1. As there is no specific inhibitor for ENTPD8, the involvement of this enzyme, that we show to be highly expressed by HepG2 cells, cannot be ruled out.

Next, co‐incubation of each radio‐labelled substrate with different combinations of ectoenzyme inhibitors was employed to understand the enzymes participated in each degradation step 17 , 20 , 21 (Figure 3 E; inhibitor specificity was listed at the bottom). POM6 markedly inhibited ADO production from ADP, suggesting the involvement of ENTPD3; whereas POM1 also worked with decreased potency. When using AMP as substrate, CD73 inhibition by APCP failed to block ADO generation, suggesting a CD73 independent mechanism of AMP dephosphorylation. Inhibition of ADA1 using EHNA completely blocked degradation of radio‐labelled ADO. In contrast, ADA2 blockade by pentostatin exhibited no effects on extracellular ADO degradation.

Thin layer chromatography analysis of ectoenzyme activity and extracellular ADO turnover by live cells was performed using radio‐labelled substrates (ADP, AMP or ADO) in the presence of the adenosine uptake inhibitor dipyridamole. As shown in Figure 3 D, HepG2 cells can not only generate ADO from exogenously added ADP or AMP, but also metabolise ADO further to inosine and hypoxanthine in a dynamic manner.

Purinergic profiles of HepG2 cells. (A) qPCR analysis of mRNA expression of four ADO receptors. (B) Western blot of protein levels of ectoenzymes. (C) Adenosine A2A receptor expression in HCC and adjacent cirrhotic liver. Immunohistochemistry performed on two different biopsies using antibody from Abcam to A2A receptor on paraffin fixed tissues, developed with DAB and counterstained with Gill's hematoxylin II. The expression of A2A receptor is noted in tumor cells and infiltrating myeloid type cells. Arrows indicate area of section magnified further and depicted in side panel. The major image is at ×200 magnification and insert (with borders) to show cellular localization is at ×400. (D) Thin layer chromatography (TLC) analyses of enzymatic activity and extracellular ADO turnover in the presence of adenosine uptake blocker dipyridamole, using various numbers of cells. (E) Impacts of combination of ectoenzyme inhibitors on extracellular purine metabolism assessed by TLC. Inhibitor specificity is listed as following: POM1: ENTPD1, ENTPD3; POM6: ENTPD2, ENTPD3; EHNA: ADA1; Pentostatin: ADA2; APCP: CD73 (n = 3)

HepG2 cells express abundant levels of A2A and A2B receptors as well as several membrane located ectoenzymes that are involved in extracellular adenosine metabolism, namely ecto‐nucleoside triphosphate diphosphohydrolase (ENTPD) 3, 5 and 8, CD73, and adenosine deaminase 1 (ADA1) (Figure 3 A,B). In accordance, human HCC samples appear to express more A2AR when compared to cirrhotic livers. The desmoplastic response also has cellular elements that express A2AR (Figure 3 C).

The influence of several recorded factors on survival was tested in a Cox proportional hazards model (see Table 2 ). Only post‐operative coffee consumption and meeting the MILAN criteria were independent factors with a Hazard ratio 0.2936 (95% CI 0.1222‐0.7053; P = 0.0061) and 0.1425 (95% CI 0.0229‐0.8877; P = 0.0368) for improved long‐term survival (see Figure 1 and 2 ). When aggregating the cohorts those with less than three cups coffee intake daily showed a significantly shorter survival (7.48 ± 0.76 a) than those with higher intake (11.00 ± 0.52 a) ( χ 2 (1) = 4.7, P = 0.029). Donor criteria did not show any significant influence.

Histopathological factors associated with a significant increased risk of HCC‐recurrence were microvascular invasion ( P = 0.025), more than three tumour nodules ( P = 0.004), failure to meet MILAN criteria ( P = 0.017), UCSF ( P = 0.001) and up to seven criteria ( P = 0.003). Lymphatic vessel invasion ( P = 0.081) and tumour grading ( P = 0.422) were not significantly related to HCC‐recurrence. No significant relations between demographic data (age, sex and BMI) and recurrence were noted.

Sixteen (17.8%) patients, 14 male and two females with a median age of 60 (range 48‐73) and a median BMI of 26.8 (range 22.5‐34) experienced HCC recurrence after median time of 11.5 months (range 1‐40.5; M 16.3, SD 13.98) post‐transplant (see Table 1 ). Underlying disease in these patients was chronic alcohol abuse in nine patients, chronic viral hepatitis in four patients and cryptogenic or haemochromatosis induced liver cirrhosis in three patients. The mortality rate following HCC recurrence was 83.4% with a median survival after tumour recurrence of 5 months (range 1‐30, M 9.5, SD 9.52) and a median survival after OLT of 20 months (range 5‐61; M 23.6, SD 17.81), respectively. Three patients were still alive after a median time of 28.5 months (range 28‐29, M 28.5, SD 0.71) after recurrence and 43.5 months (range 42‐45, M 43.5, SD 212) after OLT. In one patient HCC‐recurrence was diagnosed during autopsy.

Eighty‐three patients (92.1%) drank caffeinated coffee on a regular basis before and/or after OLT. Seven patients (7.8%) did not drink any coffee before or after OLT. Before OLT, nine (10%) patients did not consume any coffee, 14 (15.6%) patients consumed less than one cup per day, 42 (46.7%) consumed one or two cup(s) per day and 25 (27.8%) consumed three or more cups per day. After OLT, 9 (10%) patients did not consume any coffee, nine (10%) patients consumed less than one cup per day, 51 (56.7%) consumed one or two cup(s) per day and 21 (23.2%) consumed three or cups per day.

According to the final histology, HCC were within MILAN in 58 patients (64.4%), according to the UCSF‐criteria in 68 patients (75.6%) and in 65 patients (72.1%) consisted with the Up‐to‐7‐criteria. Tumours were categorised as yT0 in two patients (2.1%), T1 in 37 patients (41%), T2 in 38 patients (42.1%), T3 in eight patients (8.9%) and T4 in three patients (3.2%). In six patients (6.7%) no histology was available because of pre‐transplant therapies, eg TACE or RFA; in all of these cases histology was confirmed pre‐operatively by fine needle biopsy. One tumour nodule was found in each of 46 patients (50%), two nodules in each of 11 patients (12%), three nodules in each of 12 patients (13.2%) and more than three nodules in each of 20 patients (22.1%). Median tumour size was 2.65 cm (range 0.3‐15). Regional lymph node metastases were not found in any patients, where no lymph nodes were available for analysis in 34 patients (37.7%). Vessel involvement (V1) was reported in 14 patients (15.5%) and lymphatic vessel invasion (L1) was seen in nine patients (10%). Perineural invasion (Pn1) was detected in none of the patients. Tumours were characterised as well differentiated (G1) in 28 patients (31%), moderately differentiated (G2) in 45 patients (50%), as poorly differentiated (G3) in three patients (3.2%), undifferentiated (G4) in one patient (1%) and in 13 patients (14.3%) grade could not be assessed (Gx).

OLT was performed with an end‐to‐end caval anastomosis, end‐to‐end anastomosis of the portal vein, splenic artery patch/gastroduodenal artery patch, side‐to‐side anastomosis of the bile duct with T‐drain insertion or an end‐to‐end anastomosis in patients with larger bile ducts. Re‐transplantation was indicated in 10 patients (8.4%) after a median post‐transplant time of 15 days (range 2‐296). Three of these patients died within 90 days after re‐transplantation.

In two patients with HCC recurrence, three donor related risk factors were combined (age>60 years, BMI>35, Steatosis), but this constellation can be seen even in recipients without recurrence (12 patients).

In total, six donors had risk factors. The age over 60 years was the risk factor most frequently occurs, but to note is that three of six donors are less than 62 years.

Consequently, 90 patients were included for further analysis. The median age was 60 years (range 73‐37) and median BMI was 26.95 (range 18.3‐39.4). Median follow‐up time was 45.5 months (range 13‐143), and none of these patients was lost to follow‐up. Twenty‐one patients (23.2%) died after a median time of 16 months (4‐97) during follow‐up. 1‐, 3‐ and 5‐year overall survival was 90%, 80% and 72.6%, respectively. Underlying liver diseases included alcoholic cirrhosis in 59 patients (65.5%), virus‐related cirrhosis in 19 patients (hepatitis C, n = 12, 13.2%; hepatitis B, n = 7, 7.8%), cryptogenic cirrhosis in seven patients (7.8%), and others in five patients (5.6%).

One hundred and nineteen patients (female = 23; male = 96) underwent OLT for histologically confirmed HCC from January 2002 to December 2012. A total of 29 patients were excluded from further analysis due to 90‐day in hospital mortality (n = 18, 15.1%), microscopically positive margins (n = 1, 0.8%), not reachable for interview (n = 2, 1.6%), or no vital tumour cells detectable in the explanted liver because of pre‐transplant therapies, eg TACE or RFA (n = 8, 6.6%), respectively.

4 DISCUSSION

The recurrence of HCC contributes in a considerable manner to mortality after OLT. Even with improved patient selection and implementation of the Milan‐criteria, recurrence occurs in around 20%. Because of an ongoing organ donor shortage, it is essential to identify factors to better predict HCC recurrence after OLT.

Our results show that consumption of caffeinated coffee is associated with a decreased risk of HCC recurrence and beneficial survival following OLT. Experimental data suggest that such clinical benefits of coffee is associated, at least in part, with the antagonist activity of caffeine on adenosine‐receptor mediated growth‐promoting effects in HCC cells.

Several studies have demonstrated that specific factors are associated with an increased recurrence rate, mostly based on histological analysis of the explanted liver. In a meta‐analysis of 1198 patients, Sotiropoulos et al identified size of the tumour (>5 cm), poorly differentiation grade and macrovascular invasion as independent risk factors for HCC‐recurrence.25 Based on similar findings, Chan et al developed a predicting cancer recurrence score and suggested that the use of a predictive model could help to identify either low‐risk or high‐risk patients and lead them to an individual based cost‐effective approach to minimize expensive surveillance.26

Other studies evaluated the influence of pre‐operative parameters on perioperative outcome. Lai et al recently reviewed pre‐transplant tumour and AFP behaviour in patients inside or outside the Milan‐criteria, treated with locoregional therapy.27 AFP and radiological tumour progression were shown to be unique independent risk factors. They concluded that an integration of radiological and biological progression into the pre‐operative staging could represent an effective method of patients’ stratification before OLT.

Our analysis further confirms microvascular invasion, number of nodules, failure to meet Milan‐, UCSF‐ and Up‐to‐7‐criteria, respectively, as valuable predictive markers for HCC‐recurrence after OLT.

Most studies have aimed to identify perioperative risk factors to ensure better patient selection. The minority of studies has focused on potential therapeutic pathways, thus adjuvant treatment are not yet established.28 Thus, currently established strategies are limited to optimisation of immunosuppressive treatment by either decreasing the effective level of immunosuppression or switching immunosuppression to an antineoplastic agent like Inhibitor of the mammalian target of rapamycin (m‐TOR).29 There is a need for identifying modifiable risk factor for HCC recurrence and develop therapeutic strategies.

Beneficial effects of coffee consumption in patients with liver ailments have been suggested by several studies. Modi et al recognised that the severity of hepatic fibrosis was improved in patients with daily coffee intake of two cups. In a large population‐based prospective cohort study with a median follow‐up of 18 years, coffee consumption was associated with a decreased risk of progression to cirrhosis, a lower mortality rate in cirrhosis patients, and a lower rate of HCC development. Patients with chronic hepatitis C showed a significant improvement in responses to antiviral therapy when three or more cups of coffee were consumed. Furthermore, rates of disease progression and fibrotic severity declined with increasing daily coffee intake.

Several studies reported an inverse association between coffee consumption and the risk of HCC. A recently published meta‐analysis concluded that the risk for HCC was reduced by 40% for any coffee drinking vs no coffee drinking.7 Furthermore, there is evidence that the amount of coffee intake plays an important role. In a Japan Public Health Center‐based Prospective Study Cohort II, which included 18 815 participants followed up for 13 years an RR of 0.54 was found for drinkers of three or more cups of coffee per day compared with never/almost never drinkers.

Our results support these findings and for the first time show an effect of coffee consumption on HCC recurrence after OLT. Patients with higher coffee intake, in particular post‐operative coffee intake, showed a significant decrease in HCC recurrence. We also confirmed the impact of the dose effect: Patients with only one to two cups per day could not profit of hepatoprotective effects in the same manner as individuals who consumed ≥3 cups of coffee. Of note, our results in terms of coffee consumption were in range with recent published data about the general coffee consumption in Germany. Only 4%‐10% of the German population mentioned to drink no coffee at all. With an average annual consumption of 7.2 kg coffee per capita, Germany ranks sixth in international comparison.30

Coffee is composed of more than 1000 substances of which any may have beneficial effects in chronic liver diseases. However, not all sources of coffee may have hepatoprotective or anti‐fibrotic responses and therefore, most studies have focused on caffeine, kahweol and cafestol.31, 32 There are a number of proposed mechanisms for the hepatoprotective effects of caffeine. One well‐known pharmacological effect of caffeine is the antagonistic effects on adenosine receptors, especially A2A receptor.13 In the setting of tissue injury or inflammation, as in liver fibrosis, there is an increase in extracellular ATP through the release from dead or dying cells and by active transport through the plasma membrane.33

ATP can then be degraded to adenosine, provided that specific cell surface located enzymes are present that catalyse the necessary dephosphorylation.

Our in vitro studies reveal that HepG2 cells express abundant levels of A2A and A2B receptors as well as several key ectoenzymes, which display intrinsic capacity to generate extracellular adenosine from ATP. In accordance, human HCC samples appear to express more A2AR when compared to cirrhotic livers. We show that exogenous adenosine promotes HCC cell growth and metastasis in vitro, mediated largely via A2A receptors. Adenosine also activates MAPK (ERK and JNK) and NF‐kappa B pathways, which can be abrogated by antagonism of A2A and A2B receptors.

The limitations of our study are the retrospective design, the interview of first grade relatives in some cases and that the interviewer was blind to patient's data but had to know which patient was deceased. Only 64% of all patients were transplanted within the Milan criteria. Consequently, further prospective trials should be focussing on patients within Milan criteria for reasons of better comparability. It has to be noted that no further differentiation between the specific coffee types was included, which should be included in upcoming prospective trials. Several studies have suggested that hepatoprotective effects are increased with the consumption of filtered coffee but not with unfiltered coffee.34, 35 The majority of US habitants consume filtered coffee whereas in Europe, espresso coffee is the more preferred beverage.36 Anty et al showed no beneficial effects of espresso on liver ailment, particularly in NAFLD and assumed that any added sucrose, was associated with increased severity of hepatic fibrosis.37

Lastly, our in vitro experiments focus on the effects of caffeine, while there are countless other ingredients in coffee that may well contribute to the observed effects. The aim of our mechanistic studies was to investigate whether caffeine might contribute to the proposed effect of coffee, rather than investigating all possible active ingredients in coffee. Consequently, we cannot rule out that other constituents in coffee might mediate or boost anti‐carcinogenic effects in HCC.