Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocellular carcinoma

European Journal of Cancer (2013) 49, 3442–3449

Available at www.sciencedirect.com

journal homepage: www.ejcancer.com

Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocellular carcinoma Verena Ko¨berle, Bernd Kronenberger, Thomas Pleli, Jo¨rg Trojan, Esther Imelmann, Jan Peveling-Oberhag, Martin-Walter Welker, Mohammed Elhendawy, Stefan Zeuzem, Albrecht Piiper ⇑, Oliver Waidmann Department of Medicine I, Division of Gastroenterology and Hepatology, Johann Wolfgang Goethe University Hospital Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany

Available online 26 June 2013

KEYWORDS HCC microRNA miR-1 miR-122 Prognostic marker

Abstract Background: The identification of new biomarkers to predict the aggressiveness of hepatocellular carcinoma (HCC) and supplement the current set of prognosis and treatment algorithms is an important clinical need. Extracellular microRNAs (miRNAs) circulating in the blood are a new class of highly promising disease markers. Aim: Here we investigated the prognostic potential of miR-1 and miR-122 in sera from patients with HCC. Methods: RNA was extracted from 195 sera of HCC patients and 54 patients with liver cirrhosis, obtained at the time of study enrolment. miR-1 and miR-122 levels were correlated with overall survival (OS), Cancer of the Liver Italian Program (CLIP) score, Barcelona Clinic Liver Cancer stage, clinical chemistry parameters and tumor specific treatment. Results: Patients with higher miR-1 and miR-122 serum levels showed longer OS than individuals with lower miR-1 and miR-122 serum concentrations (hazard ratio [HR] 0.440, 95% confidence interval [CI] 0.233–0.831, P = 0.011 for miR-1 and HR 0.493, 95% CI 0.254–0.956, P = 0.036 for miR-122, respectively). Serum miR-1 and miR-122 concentrations did not differ significantly between patients with HCC and liver cirrhosis. An age-, sex-, tumor stage and treatment-adjusted multivariate Cox regression analysis revealed that miR-1 serum levels (HR 0.451, 95% CI 0.228–0.856, P = 0.015) were independently associated with OS, whereas serum miR-122 was not. miR-1 serum levels showed no relevant correlation with clinical chemistry liver parameters, whereas serum miR-122 correlated with clinical chemistry parameters of hepatic necroinflammation, liver function and synthetic capacity. Conclusion: Our data indicate that serum miR-1 is a new independent parameter of OS in HCC patients and may therefore improve the predictive value of classical HCC staging scores. Ó 2013 Elsevier Ltd. All rights reserved.

⇑ Corresponding author: Tel.: +49 69 6301 87667; fax: +49 69 6301 87689.

E-mail address: [email protected] (A. Piiper). 0959-8049/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejca.2013.06.002

V. Ko¨berle et al. / European Journal of Cancer 49 (2013) 3442–3449

1. Introduction

2. Materials and methods

Hepatocellular carcinoma (HCC) is an increasing burden in the world and is the second leading cause of cancer-related mortality.1 More than 90% of HCC cases develop in chronically inflamed liver as a result of viral hepatitis, alcohol abuse and in increasing incidence in patients with non-alcoholic fatty liver disease.2 Potentially curative treatments, mostly surgical in nature (i.e. resection and liver transplantation), are feasible only at very early or early tumor stages.3 At intermediate or advanced stages curative options are missing.4 HCC is pathologically and clinically heterogeneous. The prognosis depends on the aggressiveness of the HCC and residual liver function. Thus, the prediction of the prognosis and accurate patient stratification are crucial to optimise personalised treatment. This is currently performed by several staging scores, including the Barcelona Clinic Liver Cancer (BCLC) stage and the Cancer of the Liver Italian Program (CLIP) score.5,6 Modifications of these staging systems by the addition of new biomarkers, in particular those better reflecting tumor aggressiveness, are likely to improve the prognostic assessment of HCC patients and could therefore fulfill a clinical need. The discovery of microRNAs (miRNAs), small (18– 25 ribonucleotides) non-coding RNAs involved in the regulation of virtually all cell functions has opened new avenues for cancer diagnosis, prognosis and prediction of treatment response.7 miRNA signatures in HCC tissue have been shown to be associated with patient survival.8–10 In particular, miR-122, a miRNA showing highly abundant and almost exclusive expression in the liver, and miR-1 have been shown to act as tumor suppressors in HCC.8,10–15 miRNAs also circulate in the blood in a cell-free form,16,17 stabilised by incorporation into microvesicles or RNA-binding proteins such as Ago2.18–20 Blood serum- or plasma-derived miRNA biomarkers would be advantageous compared with the examination of tumor tissue due to the minimally invasive nature of the sampling, ease of standardisation of sample analysis, and the possibility of repeated sampling. miRNAs obtained from serum have been reported to provide prognostic information in patients with non-small-cell lung cancer and pancreatic cancer, indicating that serum-derived miRNAs can indeed serve as prognostic markers in cancer patients.21,22 As miR-1 and miR-122 act as tumor suppressors and can reach the circulation, we investigated if serum miR-1 and miR-122 levels can provide prognostic information in HCC patients. To this end we performed a prospective clinical study in which the levels of miR-1 and miR-122 were assessed in sera from HCC patients (mainly hepatitis C- or alcohol-related) and were correlated with patients’ survival and clinical variables.

2.1. Study subjects

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HCC patients who were treated between February 2009 and July 2012 in our department as in- or outpatients were prospectively enrolled into the present mono center case control study. Inclusion criteria were histologically confirmed HCC or pathognomonic results in dynamic imaging and an underlying chronic liver disease.23 Exclusion criteria were an age of below 18 years, a history of another malignant disease within the last five years and a history of organ transplantation. At the day of inclusion into the study blood samples were obtained and Child-Pugh-score,24 BCLC stage5 and CLIP score6 were assessed by results of clinical examination, imaging (dynamic computer tomography, magnetic resonance imaging or abdominal ultrasound examination) and laboratory parameters. The model of end stage liver disease (MELD) score was calculated as described before.25 Treatment of patients was performed according to BCLC stage.4 Patients who were eligible for liver transplantation according to the Milan criteria were listed for liver transplantation.26 Organ allocation was performed by Eurotransplant according to the German and Eurotransplant guidelines. From the day of transplantation the patients were excluded from further analysis. Patients were followed up until death, liver transplantation or last contact. The primary end point was overall survival (OS). Additionally, we investigated the diagnostic accuracy of miRNAs assessed at the day of study inclusion to diagnose HCC in a cross sectional approach by using matched patients with liver cirrhosis or severe fibrosis with comparable liver function as a control cohort. All patients gave their written informed consent. The study was approved by the local Ethics Committee. The study was performed in accordance with the Declaration of 1975 Helsinki and the REMARK guidelines for biomarker studies. 2.2. Blood sampling Blood samples were taken from every patient at the day of enrolment into the clinical trial. Serum tubes were centrifuged at 1500g for 10 min at 4 °C, which was followed by an additional centrifugation at 2000g at 4 °C to completely remove any remaining cells. The serum samples were portioned in aliquots and stored at 80 °C until further use. 2.3. Clinical chemistry Standard parameters of liver and kidney function and alpha-fetoprotein (AFP) levels were measured at the central laboratory of the Frankfurt University Hospital.

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2.4. Detection of miRNAs by quantitative real-time reverse-transcription (RT)-PCR

ter not exceeding 5 cm. Forty-two patients died within the observation time.

The isolation of total RNA from sera and quantification of serum miRNA levels were performed as described previously.27–29 Total RNA was extracted from 500 ll serum with the miRVana RNA isolation kit and TriReagent LS (Sigma–Aldrich, St. Louis, MO, United States of America (USA)) and chloroform according to the mirVanae miRNA isolation kit protocol. cDNA was reverse transcribed from 5 ll of RNA with the TaqMan miRNA reverse transcription kit. miR-122, miR-1 and miR-16 levels were assessed by the TaqMan miRNA assay specific for the indicated miRNAs using StepOnee Plus Real-Time PCR System (ABI). Quantitative real-time PCRs were performed in duplicates and the mean was calculated.

3.2. MicroRNA-1 is a prognostic marker in HCC patients

2.5. Statistical methods Data were analysed using the BiAS software for Windows (version 9.11, Epsilon-Verlag, Darmstadt, Germany) and SPSS version 20 (IBM, Chicago, IL). Predictors of survival were determined using a univariate Cox regression hazard model. OS of patients was shown by survival curves which were calculated with the Cox regression model. For assessment of independent predictors of survival a multivariate Cox regression hazard model with forward stepwise (likelihood ratio) entry was applied. Differences between different groups were determined using the nonparametric Wilcoxon– Mann–Whitney test. P values < 0.05 were considered to be significant. The correlation coefficients (r) were calculated by using the Spearman correlation. 3. Results 3.1. Clinicopathological data One hundred and ninety five patients with HCC were prospectively enrolled in the present study. Fifty-four patients with liver cirrhosis or severe liver fibrosis served as control cohort. Patients’ characteristics are summarised in Table 1. The mean duration of follow-up of HCC patients was 211 ± 271 days. In 132 patients the diagnosis of HCC was confirmed by histopathological examination of biopsy material, whereas in the other 63 patients HCC was diagnosed by pathognomonic results in dynamic imaging. Twenty-one patients underwent liver transplantation and were excluded from further analysis from the day of transplantation. Forty-seven HCC patients had limited disease within the Milan criteria with up to three tumors with a maximum diameter of 3 cm or one tumor with a diame-

miR-122 and miR-1 are involved in hepatocyte differentiation and tumor suppression,8,10–15 and low miR-122 serum levels are associated with a poor prognosis in patients with liver cirrhosis.30 Therefore, the levels of miR-122 and miR-1 were determined in the sera of the HCC patients. The patients were grouped in subjects with low or high serum miR-122 or miR1 concentrations using the 25% percentile of the serum miRNA concentration as cut-off value and OS analyses with univariate Cox regression models were performed. The univariate Cox regression analyses for miR-1 and miR-122 serum levels revealed a significant association between the miR-1 serum concentration and OS (P = 0.011, hazard ratio [HR] 0.440, 95% confidence interval [CI] 0.233–0.831), as well as a significant association between miR-122 serum concentration and OS (P = 0.036, HR 0.493, 95% CI 0.254–0.956) (Table 2). Survival curves for miR-1 and miR-122 are shown in Fig. 1A and B. CLIP score and BCLC stage, i.e. the most widely used HCC staging systems, have been reported to be of prognostic relevance for the prediction of OS in HCC patients.31,32 Univariate Cox regression analyses for HCC patients in the present cohort revealed that a low CLIP score (CLIP score 6 2 versus CLIP score > 2) was strongly associated with a longer OS (P < 0.001, HR 0.268, 95% CI 0.144–0.497) (Table 2). Likewise, a lower BCLC stage (A and B versus C and D) was associated with a longer OS (P = 0.004, HR 0.403, 95% CI 0.218–0.745) (Table 2). In addition, the relations between gender and age and OS were assessed. Both parameters are potential prognostic attributes associated with mortality. Whereas gender was not associated with OS (P = 0.965, HR 1.017, 95% CI 0.486–2.126 for male gender), age tended to be associated with OS (P = 0.066, HR 1.827, 95% CI 0.961–3.474 for an age 664 years) (Table 2). To exclude the kind of treatment for HCC as confounding factor for miR-1 and miR-122 as biomarker for OS the dichotomised qualitative variables such as resection, local therapy including TACE, RFA or LITT and sorafenib treatment were introduced in a multivariate Cox regression model. Additionally the model was corrected by age, gender and tumor stages (BCLC stage and CLIP score). In the multivariate analysis only local therapy, an age >64 years, a CLIP score 62 and a high serum miR-1 level were associated with a longer OS (Table 2).

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Table 1 Patients’ characteristics. Parameter

HCC cohort

Liver cirrhosis

P value

Epidemiology Patients Gender, m/f (%) Age, years, mean, SD

195 153/42 (78.5/21.5) 62.6 ± 10.4

54 33/21 (61.1/38.9) 53.8 ± 11.2

<0.001 <0.001

Etiology of liver disease Alcohol abuse, n (%) Hepatitis C, n (%) Hepatitis B, n (%) Cryptogenic, n (%) NASH1, n (%) Haemochromatosis, n (%)

65 (33.3) 87 (44.6) 33 (16.9) 9 (4.6) 14 (7.2) 8 (4.1)

16 (29.6) 41 (75.9) 2 (3.7) 0 (0.0) 0 (0.0) 0 (0.0)

BCLC stage A, n (%) B, n (%) C, n (%) D, n (%)

47 77 58 13

CLIP score 0, n (%) 1, n (%) 2, n (%) 3, n (%) 4, n (%) 5, n (%) 6, n (%)

23 (11.8) 68 (34.9) 25 (12.8) 38 (19.5) 22 (11.3) 16 (8.2) 3 (1.5)

Child-Pugh stage A, n (%) B, n (%) C, n (%) MELD, mean, SD

124 (63.6) 50 (25.6) 21 (10.8) 11.4 ± 5.0

Treatment Resection Local ablation2 Sorafenib Liver transplantation

18 (9.2) 104 (53.3) 46 (23.6) 21 (10.8)

Laboratory results Sodium (mmol/l), mean, SD ALT3 (U/l), mean, SD AST4 (U/l), mean, SD GGT5 (U/l), mean, SD ALP6 (U/l), mean, SD Albumin (mg/dl), mean, SD Bilirubin (mg/dl), mean, SD INR7, mean, SD Creatinine (mg/dl), mean, SD CRP8 (mg/dl), mean, SD AFP9 (ng/ml), mean, SD

138 ± 5 98 ± 196 119 ± 125 278 ± 309 174 ± 136 3.6 ± 0.7 2.1 ± 3.0 1.24 ± 0.28 1.02 ± 0.55 2.21 ± 3.83 3709 ± 12815

1

(24.1) (39.5) (29.7) (6.7)

0.010 29 (53.7) 17 (31.5) 8 (14.8) 13.1 ± 6.1

0.030

139 ± 4 58 ± 52 84 ± 54 137 ± 182 109 ± 44 3.4 ± 0.7 2.4 ± 3.0 1.45 ± 0.44 0.93 ± 0.65 0.87 ± 1.70 12 ± 12

0.148 0.010 0.115 <0.001 0.001 0.079 0.180 <0.001 0.081 0.002 <0.001

Non-alcoholic steatohepatitis. Including transarterial chemoembolisation (TACE), radiofrequency ablation (RFA) and Laser interstitial thermal therapy (LITT). 3 Alanine aminotransferase. 4 Aspartate aminotransferase. 5 c-Glutamyltransferase. 6 Alkaline phosphatase. 7 International normalised ratio. 8 C-reactive protein. 9 Alpha-Fetoprotein. 2

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Table 2 Univariate and multivariate analyses of parameters associated with overall survival of all HCC patients. Parameter

Univariate analysis

High miR-1 High miR-122 Low CLIP score Low BCLC stage Age 6 64 years Male gender Resection Local therapy Sorafenib

Multivariate analysis

HR

95% CI

P value

HR

95% CI

P value

0.440 0.493 0.268 0.403 1.827 1.017 0.870 0.308 0.711

0.233–0.831 0.254–0.956 0.144–0.497 0.218–0.745 0.961–3.474 0.486–2.126 0.341–2.217 0.163–0.579 0.355–1.424

0.011 0.036 <0.001 0.004 0.066 0.965 0.770 <0.001 0.336

0.451

0.238–0.856

0.015

0.287

0.148–0.555

<0.001

2.235

1.161–4.300

0.016

0.417

0.214–0.813

0.010

Abbreviations: HR, hazard ratio, CI, confidence interval.

A

3.4. Serum levels of miR-122, but not of miR-1, correlate with liver function and inflammatory disease activity in the liver

Cumulative survival

1.0 high miR-1

0.8 0.6 low miR-1

0.4

P = 0.011

0.2 0.0

Time in the study (d)

0

Patients at risk (high miR) 148 47 Patients at risk (low miR)

B

200

400

600

76

41

19

800 8

20

7

4

1

Cumulative survival

1.0 high miR-122

0.8 0.6 low miR-122

0.4 0.2

P = 0.036

0.0 Time in the study (d)

0

200

400

600

Patients at risk (high miR)

147

78

42

21

8

Patients at risk (low miR)

48

19

6

2

1

To investigate if the prognostic significance of serum miR-1 is related to parameters of liver damage and function, we assessed parameters of hepatic necroinflammation (alanine aminotransferase [ALT] and aspartate aminotransferase [AST]), liver function or synthetic capacity (bilirubin, international normalised ratio [INR], total protein and serum albumin) in the HCC patients (Table 3). The only significant correlation between miR-1 serum levels and clinical chemistry parameters was a weak correlation with serum creatinine levels. In contrast, the serum miR-122 levels strongly correlated with the serum ALT, AST and GGT levels and negatively with the MELD score (Table 3). Serum levels of the ubiquitously expressed miR-16 showed no significant relation with parameters of liver damage or liver function (data not shown).

800

Fig. 1. Survival curves for patients with high and low miR-1 (A) and miR-122 serum levels (B). The P values were calculated with the Cox regression model.

3.3. No significant differences in miR-1 and miR-122 serum levels between patients with HCC and liver cirrhosis To investigate if the serum levels of miR-1 or miR122 might be useful for the diagnosis of HCC, their levels were compared between the cohort of HCC patients and a cohort of patients with liver cirrhosis without HCC. The patients’ characteristics of the cirrhotic patients are shown in Table 1. However, serum miR-1 and miR-122 levels did not significantly differ between patients with and without HCC (P = 0.569 and 0.060, for miR-1 and miR-122, respectively).

4. Discussion In patients with malignant diseases an accurate assessment of the prognosis is essential to enable individualised therapeutic decisions. This is particularly challenging in HCC patients because it requires staging and grading of both tumor and the chronic liver disease. Therefore, novel markers that reflect the aggressiveness of the disease would complement the current set of prognostic and treatment algorithms. In the present study, we identified the miR-1 serum level as a new prognostic parameter in HCC patients that is independent from BCLC stage, CLIP score and tumor-specific treatment. Thus, serum miR-1 levels may supplement the predictive value of classical HCC staging scores. In addition, serum miR-122 was also associated with OS in our cohort of HCC patients. However, different from patients with liver cirrhosis without concomitant HCC,30 it was not independently associated with OS, indicating that miR-122 serum levels mostly reflect liver

V. Ko¨berle et al. / European Journal of Cancer 49 (2013) 3442–3449 Table 3 Correlation of serum miRNA levels and laboratory parameters. miR-1 Parameter ALT1 (U/l) AST2 (U/l) GGT3 (U/l) Alkaline phosphatase (U/l) Total serum protein (mg/dl) Serum albumin (mg/dl) International normalised ratio Bilirubin (mg/dl) Creatinine (mg/dl) MELD4 score

Rank correlation coefficient (r)

P

0.097 0.062 0.110 0.033 0.126 0.084 0.065

0.220 0.433 0.164 0.679 0.129 0.286 0.409

0.016 0.165 0.149

0.841 0.036 0.058

0.383 0.234 0.264 0.044 0.246 0.232 0.259

<0.001 0.003 <0.001 0.576 0.003 0.003 <0.001

0.151 0.152 0.348

0.057 0.054 <0.001

miR-122 Parameter ALT1 (U/l) AST2 (U/l) GGT3 (U/l) Alkaline phosphatase (U/l) Total serum protein (mg/dl) Albumin (mg/dl) International normalised ratio Bilirubin (mg/dl) Creatinine (mg/dl) MELD4 score 1 2 3 4

Alanine aminotransferase. Aspartate aminotransferase. c-Glutamyltransferase. Model of end stage liver disease.

function, whereas miR-1 probably accounts for the cancer disease. Biomarkers related to the tumor biology are likely to provide more accurate assessment of the prognosis of HCC patients than conventional pathology-based assessment. Previous studies identified prognostic miRNA signatures in HCC that could be used as prognostic classifiers.8–11,33 However, the use of non-invasive blood-based biomarkers is strongly preferred, in particular considering that HCC diagnosis is based on imaging techniques without tumor biopsy in a substantial portion of cases. In patients with non-small cell lung cancer a particular set of serum miRNAs, including miR-1, has been shown to distinguish patients with long and short OS.21,22 A recent pilot study in a small number of patients has reported that the serum level of miR-221 correlates with OS of patients suffering from HCC mainly due to chronic hepatitis B virus (HBV) infection.34 However, miR-221 serum levels also correlated with tumor stage and presence of liver cirrhosis in that study and the number of patients was low. Thus, the prognostic significance of miR-221 in patients with HBV-associated HCC requires validation. In the present study, we identified miR-1 as a new independent predictor of OS in a cohort of patients, in which HCC was mainly a result of cirrhosis due to hepatitis C virus (HCV) infection or former alcohol abuse. Thus, miR-1

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serum levels may improve clinical assessment of this growing number of patients. The present study did not intend to evaluate the responses of different tumor specific treatment methods which have been extensively studied. Therefore, the kind of treatment but not the response to therapy was included in the multivariate analysis. Our finding that miR-1 serum levels correlated with OS of HCC patients independently from the treatment mode indicates that the correlation between serum miR-1 and OS in HCC patients does not simply reflect different treatment methods of the patients. miR-1 has initially been described as a regulator of myogenesis.35 Subsequently, it has been found that miR-1 has a tumor suppressive function in cancer of the urogenital tract.36 Dysregulation of miR-1 appears to play an important role in HCC as miR-1 levels are lower in HCC tissue in comparison to the adjacent liver,14 and enforced expression of miR-1 reverses hepatocyte dedifferentiation.15 Thus, miR-1 might be a tumor suppressor in HCC. The miR-1 serum level was not associated with and independent from classical parameters of liver damage in our cohort of patients. The only statistically significant correlation between serum miR-1 and other clinical chemistry parameters found here was a correlation with serum creatinine. As miR-1 is an important regulator in muscle cells and is highly expressed in these cells,35,37 low miR-1 levels may be associated with cachexia which is accompanied by loss of muscle mass in advanced cancer disease and therefore correlate with OS. Intratumoral miR-122 levels correlate with the prognosis of HCC patients.8 In the present study performed in patients with HCC formed in a liver showing advanced fibrosis or cirrhosis, we found a significant correlation between miR-122 serum levels and OS. However, it was not an independent parameter in the multivariate analysis. Previous studies suggest that the serum level of miR-122 reflects, depending on the context and stage of liver disease, hepatic inflammation and cell death in patients with HBV- or HCV-induced chronic hepatitis.27,28,30,38–40 In agreement with those studies, we found here that in HCC patients the miR122 serum level positively correlated with liver transaminases and negatively with the MELD score. In patients with liver cirrhosis the serum concentration of miR-122 correlates with hepatic necroinflammation and the MELD score, but also with OS.30 The latter correlation could be explained by the suggestion that the miR-122 serum concentration also reflects residual functional liver tissue in patients with end stage liver disease. The weaker correlation between miR-122 serum levels and OS in HCC patients found in the present study indicates that the prognosis of HCC patients is also governed by the aggressiveness of the HCC rather than by the residual functional liver capacity.

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Several studies indicate that circulating miRNAs, including miR-122, have the potential to differentiate patients with HCC from those without, especially in Asian patients suffering from chronic HBV infection.41–44 In the present study, sera from patients mainly with HCV- or alcohol-induced chronic liver disease and HCC tended to show higher miR-122 levels than sera from patients with liver cirrhosis without HCC, whereas miR-1 serum concentration did not differ between patients with and without HCC. However, our data indicate that miR-1 and miR-122 are not particularly useful to differentiate patients with liver cirrhosis from those with HCC. A major part of extracellular miRNAs in the blood appears to originate from blood cells.45–47 Moreover, miRNAs from serum or plasma proposed as diagnostic miRNAs in cancer patients are strongly expressed in blood cells and may reflect changes in blood cells in cancer patients as compared to controls rather than cancerspecific changes.46 miR-1 and miR-122 showing only minor (miR-1) or virtually no (miR-122) expression in blood cells46 are unlikely to reflect changes of miRNA release by blood cells. In conclusion, we identified serum miR-1 as an independent prognostic parameter for OS in patients suffering from HCC, whereas miR-122 was also associated with OS. The miR-122 serum level, however, was not an independent factor for OS, but correlated with the MELD score and clinical chemistry parameters of hepatic necroinflammation. Financial supports This work was supported by grants from the foundation Dr. Paul und Ursula Klein, the foundation Marie Christine Held and Erika Hecker and in parts by the Nachwuchsforscherprogramm 2010 of the Medical Faculty, Goethe Universita¨t (to O.W.), the Schaufler foundation (to A.P.) and the Scolari foundation (to B.K.). Conflict of interest statement None declared. Acknowledgement We thank Ursula Karey and Yolanda Martinez for excellent technical assistance. References 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69–90. 2. Welzel TM, Graubard BI, Zeuzem S, El-Serag HB, Davila JA, McGlynn KA. Metabolic syndrome increases the risk of primary liver cancer in the United States: a study in the SEER-Medicare database. Hepatology 2011;54:463–71.

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