European Journal of Cancer (2013) xxx, xxx xxx

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 Koberle, Bernd Kronenberger, Thomas Pleli, Jorg 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

KEYWORDS Abstract Background: The identication of new biomarkers to predict the aggressiveness of
HCC hepatocellular carcinoma (HCC) and supplement the current set of prognosis and treatment
microRNA algorithms is an important clinical need. Extracellular microRNAs (miRNAs) circulating in
miR-1 the blood are a new class of highly promising disease markers.
miR-122 Aim: Here we investigated the prognostic potential of miR-1 and miR-122 in sera from
Prognostic marker patients with HCC.
Methods: RNA was extracted from 195 sera of HCC patients and 54 patients with liver cir-
rhosis, 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 specic treatment.
Results: Patients with higher miR-1 and miR-122 serum levels showed longer OS than individ-
uals with lower miR-1 and miR-122 serum concentrations (hazard ratio [HR] 0.440, 95% con-
dence interval [CI] 0.2330.831, P = 0.011 for miR-1 and HR 0.493, 95% CI 0.2540.956,
P = 0.036 for miR-122, respectively). Serum miR-1 and miR-122 concentrations did not differ
signicantly 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 0.2280.856, 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 param-
eters of hepatic necroinammation, 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.
2013 Elsevier Ltd. All rights reserved.

Corresponding author: Tel.: +49 69 6301 87667; fax: +49 69 6301 87689.
E-mail address: piiper@med.uni-frankfurt.de (A. Piiper).

0959-8049/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ejca.2013.06.002

Please cite this article in press as: Koberle V. et al., Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocel-
lular carcinoma, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.002
2 V. Koberle et al. / European Journal of Cancer xxx (2013) xxxxxx
1. Introduction
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 inamed liver as a result of viral
hepatitis, alcohol abuse and in increasing incidence in
patients with non-alcoholic fatty liver disease.2 Poten-
tially curative treatments, mostly surgical in nature
(i.e. resection and liver transplantation), are feasible
only at very early or early tumor stages.3 At intermedi-
ate 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 stratication are
crucial to optimise personalised treatment. This is cur-
rently 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
Modications of these staging systems by the addition
of new biomarkers, in particular those better reecting
tumor aggressiveness, are likely to improve the prognos-
tic assessment of HCC patients and could therefore ful-
ll 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 predic-
tion of treatment response.7 miRNA signatures in HCC
tissue have been shown to be associated with patient sur-
vival.810 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 sup-
pressors in HCC.8,1015
miRNAs also circulate in the blood in a cell-free
form,16,17 stabilised by incorporation into microvesicles
or RNA-binding proteins such as Ago2.1820 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 prospec-
tive 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 corre-
lated with patients survival and clinical variables.
Please cite this article in press as: Koberle V. et al., Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocel-
lular carcinoma, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.002
2. Materials and methods
2.1. Study subjects
HCC patients who were treated between February
2009 and July 2012 in our department as in- or outpa-
tients were prospectively enrolled into the present mono
center case control study. Inclusion criteria were histo-
logically conrmed HCC or pathognomonic results in
dynamic imaging and an underlying chronic liver dis-
ease.23 Exclusion criteria were an age of below 18 years,
a history of another malignant disease within the last
ve 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 examina-
tion, imaging (dynamic computer tomography, mag-
netic 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 per-
formed 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 brosis with com-
parable 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 bio-
marker 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 fol-
lowed 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.
 V. Koberle et al. / European Journal of Cancer xxx (2013) xxxxxx
2.4. Detection of miRNAs by quantitative real-time
reverse-transcription (RT)-PCR
The isolation of total RNA from sera and quantica-
tion of serum miRNA levels were performed as
described previously.2729 Total RNA was extracted
from 500 ll serum with the miRVana RNA isolation
kit and TriReagent LS (SigmaAldrich, St. Louis,
MO, United States of America (USA)) and chloroform
according to the mirVanae miRNA isolation kit proto-
col. 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 specic 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.
2.5. Statistical methods
Data were analysed using the BiAS software for Win-
dows (version 9.11, Epsilon-Verlag, Darmstadt, Ger-
many) and SPSS version 20 (IBM, Chicago, IL).
Predictors of survival were determined using a univari-
ate Cox regression hazard model. OS of patients was
shown by survival curves which were calculated with
the Cox regression model. For assessment of indepen-
dent predictors of survival a multivariate Cox regression
hazard model with forward stepwise (likelihood ratio)
entry was applied. Dierences between dierent groups
were determined using the nonparametric Wilcoxon
MannWhitney test. P values < 0.05 were considered
to be signicant. The correlation coecients (r) were cal-
culated by using the Spearman correlation.
3. Results
3.1. Clinicopathological data
One hundred and ninety ve patients with HCC were
prospectively enrolled in the present study. Fifty-four
patients with liver cirrhosis or severe liver brosis served
as control cohort. Patients characteristics are summa-
rised in Table 1. The mean duration of follow-up of
HCC patients was 211 271 days. In 132 patients the
diagnosis of HCC was conrmed 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-
Please cite this article in press as: Koberle V. et al., Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocel-
lular carcinoma, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.002
3
ter not exceeding 5 cm. Forty-two patients died within
the observation time.
3.2. MicroRNA-1 is a prognostic marker in HCC patients
miR-122 and miR-1 are involved in hepatocyte dif-
ferentiation and tumor suppression,8,1015 and low
miR-122 serum levels are associated with a poor prog-
nosis 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 miR-
1 concentrations using the 25% percentile of the serum
miRNA concentration as cut-o 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 signicant associa-
tion between the miR-1 serum concentration and OS
(P = 0.011, hazard ratio [HR] 0.440, 95% condence
interval [CI] 0.2330.831), as well as a signicant associ-
ation between miR-122 serum concentration and OS
(P = 0.036, HR 0.493, 95% CI 0.2540.956) (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 0.1440.497) (Table 2). Likewise, a
lower BCLC stage (A and B versus C and D) was asso-
ciated with a longer OS (P = 0.004, HR 0.403, 95% CI
0.2180.745) (Table 2).
In addition, the relations between gender and age and
OS were assessed. Both parameters are potential prog-
nostic attributes associated with mortality. Whereas
gender was not associated with OS (P = 0.965, HR
1.017, 95% CI 0.4862.126 for male gender), age tended
to be associated with OS (P = 0.066, HR 1.827, 95% CI
0.9613.474 for an age 664 years) (Table 2).
To exclude the kind of treatment for HCC as con-
founding 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 multivar-
iate 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).
4 V. Koberle et al. / European Journal of Cancer xxx (2013) xxxxxx

Table 1
Patients characteristics.
Parameter HCC cohort Liver cirrhosis P value
Epidemiology
Patients 195 54
Gender, m/f (%) 153/42 (78.5/21.5) 33/21 (61.1/38.9) <0.001
Age, years, mean, SD 62.6 10.4 53.8 11.2 <0.001
Etiology of liver disease
Alcohol abuse, n (%) 65 (33.3) 16 (29.6)
Hepatitis C, n (%) 87 (44.6) 41 (75.9)
Hepatitis B, n (%) 33 (16.9) 2 (3.7)
Cryptogenic, n (%) 9 (4.6) 0 (0.0)
NASH1, n (%) 14 (7.2) 0 (0.0)
Haemochromatosis, n (%) 8 (4.1) 0 (0.0)
BCLC stage
A, n (%) 47 (24.1)
B, n (%) 77 (39.5)
C, n (%) 58 (29.7)
D, n (%) 13 (6.7)
CLIP score
0, n (%) 23 (11.8)
1, n (%) 68 (34.9)
2, n (%) 25 (12.8)
3, n (%) 38 (19.5)
4, n (%) 22 (11.3)
5, n (%) 16 (8.2)
6, n (%) 3 (1.5)
Child-Pugh stage 0.010
A, n (%) 124 (63.6) 29 (53.7)
B, n (%) 50 (25.6) 17 (31.5)
C, n (%) 21 (10.8) 8 (14.8)
MELD, mean, SD 11.4 5.0 13.1 6.1 0.030
Treatment
Resection 18 (9.2)
Local ablation2 104 (53.3)
Sorafenib 46 (23.6)
Liver transplantation 21 (10.8)
Laboratory results
Sodium (mmol/l), mean, SD 138 5 139 4 0.148
ALT3 (U/l), mean, SD 98 196 58 52 0.010
AST4 (U/l), mean, SD 119 125 84 54 0.115
GGT5 (U/l), mean, SD 278 309 137 182 <0.001
ALP6 (U/l), mean, SD 174 136 109 44 0.001
Albumin (mg/dl), mean, SD 3.6 0.7 3.4 0.7 0.079
Bilirubin (mg/dl), mean, SD 2.1 3.0 2.4 3.0 0.180
INR7, mean, SD 1.24 0.28 1.45 0.44 <0.001
Creatinine (mg/dl), mean, SD 1.02 0.55 0.93 0.65 0.081
CRP8 (mg/dl), mean, SD 2.21 3.83 0.87 1.70 0.002
AFP9 (ng/ml), mean, SD 3709 12815 12 12 <0.001
1
Non-alcoholic steatohepatitis.
2
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.

Please cite this article in press as: Koberle V. et al., Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocel-
lular carcinoma, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.002
 V. Koberle et al. / European Journal of Cancer xxx (2013) xxxxxx 5

Table 2
Univariate and multivariate analyses of parameters associated with overall survival of all HCC patients.
Parameter Univariate analysis Multivariate analysis
HR 95% CI P value HR 95% CI P value
High miR-1 0.440 0.2330.831 0.011 0.451 0.2380.856 0.015
High miR-122 0.493 0.2540.956 0.036
Low CLIP score 0.268 0.1440.497 <0.001 0.287 0.1480.555 <0.001
Low BCLC stage 0.403 0.2180.745 0.004
Age 6 64 years 1.827 0.9613.474 0.066 2.235 1.1614.300 0.016
Male gender 1.017 0.4862.126 0.965
Resection 0.870 0.3412.217 0.770
Local therapy 0.308 0.1630.579 <0.001 0.417 0.2140.813 0.010
Sorafenib 0.711 0.3551.424 0.336
Abbreviations: HR, hazard ratio, CI, condence interval.

A 1.0
3.4. Serum levels of miR-122, but not of miR-1, correlate
high miR-1 with liver function and inammatory disease activity in the
Cumulative survival

0.8 liver
0.6
low miR-1 To investigate if the prognostic signicance of serum
0.4 miR-1 is related to parameters of liver damage and func-
0.2 P = 0.011 tion, we assessed parameters of hepatic necroinamma-
tion (alanine aminotransferase [ALT] and aspartate
0.0
aminotransferase [AST]), liver function or synthetic
Time in the study (d) 0 200 400 600 800 capacity (bilirubin, international normalised ratio
Patients at risk (high miR) 148 76 41 19 8
Patients at risk (low miR) 47 20 7 4 1
[INR], total protein and serum albumin) in the HCC
patients (Table 3). The only signicant correlation
between miR-1 serum levels and clinical chemistry
B 1.0 parameters was a weak correlation with serum creati-
high miR-122 nine levels. In contrast, the serum miR-122 levels
Cumulative survival

0.8
strongly correlated with the serum ALT, AST and
0.6 GGT levels and negatively with the MELD score
low miR-122 (Table 3). Serum levels of the ubiquitously expressed
0.4
miR-16 showed no signicant relation with parameters
0.2 of liver damage or liver function (data not shown).
P = 0.036
0.0
Time in the study (d) 0 200 400 600 800
Patients at risk (high miR) 147 78 42 21 8 4. Discussion
Patients at risk (low miR) 48 19 6 2 1

Fig. 1. Survival curves for patients with high and low miR-1 (A) and In patients with malignant diseases an accurate
miR-122 serum levels (B). The P values were calculated with the Cox assessment of the prognosis is essential to enable indi-
regression model. vidualised therapeutic decisions. This is particularly
challenging in HCC patients because it requires staging
and grading of both tumor and the chronic liver disease.
3.3. No signicant dierences in miR-1 and miR-122 Therefore, novel markers that reect the aggressiveness
serum levels between patients with HCC and liver of the disease would complement the current set of prog-
cirrhosis nostic and treatment algorithms. In the present study,
we identied the miR-1 serum level as a new prognostic
To investigate if the serum levels of miR-1 or miR- parameter in HCC patients that is independent from
122 might be useful for the diagnosis of HCC, their lev- BCLC stage, CLIP score and tumor-specic treatment.
els were compared between the cohort of HCC patients Thus, serum miR-1 levels may supplement the predictive
and a cohort of patients with liver cirrhosis without value of classical HCC staging scores. In addition,
HCC. The patients characteristics of the cirrhotic serum miR-122 was also associated with OS in our
patients are shown in Table 1. However, serum miR-1 cohort of HCC patients. However, dierent from
and miR-122 levels did not signicantly dier between patients with liver cirrhosis without concomitant
patients with and without HCC (P = 0.569 and 0.060, HCC,30 it was not independently associated with OS,
for miR-1 and miR-122, respectively). indicating that miR-122 serum levels mostly reect liver

Please cite this article in press as: Koberle V. et al., Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocel-
lular carcinoma, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.002
6 V. Koberle et al. / European Journal of Cancer xxx (2013) xxxxxx

Table 3 serum levels may improve clinical assessment of this

Correlation of serum miRNA levels and laboratory parameters. growing number of patients.
miR-1 Parameter Rank correlation coecient P The present study did not intend to evaluate the
(r) responses of dierent tumor specic treatment methods
ALT1 (U/l) 0.097 0.220 which have been extensively studied. Therefore, the kind
AST2 (U/l) 0.062 0.433 of treatment but not the response to therapy was
GGT3 (U/l) 0.110 0.164
Alkaline phosphatase (U/l) 0.033 0.679
included in the multivariate analysis. Our nding that
Total serum protein (mg/dl) 0.126 0.129 miR-1 serum levels correlated with OS of HCC patients
Serum albumin (mg/dl) 0.084 0.286 independently from the treatment mode indicates that
International normalised 0.065 0.409 the correlation between serum miR-1 and OS in HCC
ratio patients does not simply reect dierent treatment meth-
Bilirubin (mg/dl) 0.016 0.841
Creatinine (mg/dl) 0.165 0.036
ods of the patients.
MELD4 score 0.149 0.058 miR-1 has initially been described as a regulator of
myogenesis.35 Subsequently, it has been found that
miR-122 Parameter
miR-1 has a tumor suppressive function in cancer of
ALT1 (U/l) 0.383 <0.001
the urogenital tract.36 Dysregulation of miR-1 appears
AST2 (U/l) 0.234 0.003
GGT3 (U/l) 0.264 <0.001 to play an important role in HCC as miR-1 levels are
Alkaline phosphatase (U/l) 0.044 0.576 lower in HCC tissue in comparison to the adjacent
Total serum protein (mg/dl) 0.246 0.003 liver,14 and enforced expression of miR-1 reverses hepa-
Albumin (mg/dl) 0.232 0.003 tocyte dedierentiation.15 Thus, miR-1 might be a
International normalised 0.259 <0.001
tumor suppressor in HCC. The miR-1 serum level was
ratio
Bilirubin (mg/dl) 0.151 0.057 not associated with and independent from classical
Creatinine (mg/dl) 0.152 0.054 parameters of liver damage in our cohort of patients.
MELD4 score 0.348 <0.001 The only statistically signicant correlation between
1
Alanine aminotransferase. serum miR-1 and other clinical chemistry parameters
2
Aspartate aminotransferase. found here was a correlation with serum creatinine. As
3
c-Glutamyltransferase. miR-1 is an important regulator in muscle cells and is
4
Model of end stage liver disease.
highly expressed in these cells,35,37 low miR-1 levels
may be associated with cachexia which is accompanied
function, whereas miR-1 probably accounts for the can- by loss of muscle mass in advanced cancer disease and
cer disease. therefore correlate with OS.
Biomarkers related to the tumor biology are likely Intratumoral miR-122 levels correlate with the prog-
to provide more accurate assessment of the prognosis nosis of HCC patients.8 In the present study performed
of HCC patients than conventional pathology-based in patients with HCC formed in a liver showing
assessment. Previous studies identied prognostic advanced brosis or cirrhosis, we found a signicant
miRNA signatures in HCC that could be used as prog- correlation between miR-122 serum levels and OS.
nostic classiers.811,33 However, the use of non-invasive However, it was not an independent parameter in the
blood-based biomarkers is strongly preferred, in partic- multivariate analysis. Previous studies suggest that the
ular considering that HCC diagnosis is based on imag- serum level of miR-122 reects, depending on the con-
ing techniques without tumor biopsy in a substantial text and stage of liver disease, hepatic inammation
portion of cases. In patients with non-small cell lung and cell death in patients with HBV- or HCV-induced
cancer a particular set of serum miRNAs, including chronic hepatitis.27,28,30,3840 In agreement with those
miR-1, has been shown to distinguish patients with long studies, we found here that in HCC patients the miR-
and short OS.21,22 A recent pilot study in a small num- 122 serum level positively correlated with liver transam-
ber of patients has reported that the serum level of inases and negatively with the MELD score. In patients
miR-221 correlates with OS of patients suering from with liver cirrhosis the serum concentration of miR-122
HCC mainly due to chronic hepatitis B virus (HBV) correlates with hepatic necroinammation and the
infection.34 However, miR-221 serum levels also corre- MELD score, but also with OS.30 The latter correlation
lated with tumor stage and presence of liver cirrhosis could be explained by the suggestion that the miR-122
in that study and the number of patients was low. Thus, serum concentration also reects residual functional
the prognostic signicance of miR-221 in patients with liver tissue in patients with end stage liver disease. The
HBV-associated HCC requires validation. In the present weaker correlation between miR-122 serum levels and
study, we identied miR-1 as a new independent predic- OS in HCC patients found in the present study indicates
tor of OS in a cohort of patients, in which HCC was that the prognosis of HCC patients is also governed by
mainly a result of cirrhosis due to hepatitis C virus the aggressiveness of the HCC rather than by the resid-
(HCV) infection or former alcohol abuse. Thus, miR-1 ual functional liver capacity.

Please cite this article in press as: Koberle V. et al., Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocel-
lular carcinoma, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.002
 V. Koberle et al. / European Journal of Cancer xxx (2013) xxxxxx
Several studies indicate that circulating miRNAs,
including miR-122, have the potential to dierentiate
patients with HCC from those without, especially in
Asian patients suering from chronic HBV infec-
tion.4144 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 dier between
patients with and without HCC. However, our data
indicate that miR-1 and miR-122 are not particularly
useful to dierentiate patients with liver cirrhosis from
those with HCC.
A major part of extracellular miRNAs in the blood
appears to originate from blood cells.4547 Moreover,
miRNAs from serum or plasma proposed as diagnostic
miRNAs in cancer patients are strongly expressed in
blood cells and may reect changes in blood cells in can-
cer patients as compared to controls rather than cancer-
specic changes.46 miR-1 and miR-122 showing only
minor (miR-1) or virtually no (miR-122) expression in
blood cells46 are unlikely to reect changes of miRNA
release by blood cells.
In conclusion, we identied serum miR-1 as an inde-
pendent prognostic parameter for OS in patients suer-
ing 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 hepa-
tic necroinammation.
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 Universitat (to O.W.), the
Schauer foundation (to A.P.) and the Scolari
foundation (to B.K.).
Conict of interest statement
None declared.
Acknowledgement
We thank Ursula Karey and Yolanda Martinez for
excellent technical assistance.
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