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EGFR, FLT1 and Heparanase as Markers Identifying Patients at Risk of Short Survival in Cholangiocarcinoma

Authors:
  • KMG Klinik Silbermühle GmbH
  • Hämatologisch-Onmkologische Praxis Eppendorf

Abstract and Figures

Background Cholangiocarcinoma remains to be a tumor with very few treatment choices and limited prognosis. In this study, we sought to determine the prognostic role of fms-related tyrosine kinase 1/vascular endothelial growth factor receptor 1 (FLT1/VEGFR1), heparanase (HPSE) and epidermal growth factor receptor (EGFR) gene expression in patients with resected CCC. Methods 47 formalin-fixed paraffin embedded FFPE tumor samples from patients with resected CCC were analyzed. FFPE tissues were dissected using laser-captured microdissection and analyzed for FLT1, FLT4, HPSE, Hif1a, VEGFA/C, HB-EGF, PDGFA, PDGF-RA and EGFR mRNA expression using a quantitative real-time RT-PCR method. Gene expression values (relative mRNA levels) are expressed as ratios between the target gene and internal reference genes (beta-actin, b2mg, rplp2, sdha). Results EGFR, FLT1 and HPSE expression levels were significantly associated with overall survival (OS). FLT1 showed the strongest significant independent association with overall survival in a multivariate cox regression analysis when compared to the other genes and clinicopathological factors with a nearly 5 times higher relative risk (4.74) of dying earlier when expressed in low levels (p = 0.04). ROC Curve Analysis revealed that measuring EGFR potentially identifies patients at risk of a worsened outcome with a sensitivity of 80% and a specificity of 75% (p = 0.01). Conclusions EGFR and FLT1 seem to be potential markers to identify those patients at high risk of dying from cholangiocarcinoma. Therefore these markers may help to identify patient subgroups in need for a more aggressive approach in a disease that is in desperate need for new approaches.
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EGFR, FLT1 and Heparanase as Markers Identifying
Patients at Risk of Short Survival in Cholangiocarcinoma
Andreas-Claudius Hoffmann
1
*
.
, Eray Goekkurt
2.
, Peter V. Danenberg
3
, Sylvia Lehmann
4
,
Gerhard Ehninger
4
, Daniela E. Aust
5
, Jan Stoehlmacher-Williams
4
1Department of Medical Oncology, West German Cancer Center, University Duisburg-Essen, Essen, Germany, 2Department of Oncology, Hematology and Stem Cell
Transplantation, University Hospital Aachen, RWTH University Aachen, Aachen, Germany, 3Department of Biochemistry and Molecular Biology, and Norris Comprehensive
Cancer Center, University of Southern California, Los Angeles, California, United States of America, 4Internal Medicine Clinic I, Carl Gustav Carus University Hospital,
Dresden, Germany, 5Institute of Pathology, Carl Gustav Carus University Hospital, Dresden, Germany
Abstract
Background:
Cholangiocarcinoma remains to be a tumor with very few treatment choices and limited prognosis. In this
study, we sought to determine the prognostic role of fms-related tyrosine kinase 1/vascular endothelial growth factor
receptor 1 (FLT1/VEGFR1), heparanase (HPSE) and epidermal growth factor receptor (EGFR) gene expression in patients with
resected CCC.
Methods:
47 formalin-fixed paraffin embedded FFPE tumor samples from patients with resected CCC were analyzed. FFPE
tissues were dissected using laser-captured microdissection and analyzed for FLT1,FLT4,HPSE,Hif1a,VEGFA/C,HB-EGF,
PDGFA,PDGF-RA and EGFR mRNA expression using a quantitative real-time RT-PCR method. Gene expression values (relative
mRNA levels) are expressed as ratios between the target gene and internal reference genes (beta-actin, b2mg, rplp2, sdha).
Results:
EGFR,FLT1 and HPSE expression levels were significantly associated with overall survival (OS). FLT1 showed the
strongest significant independent association with overall survival in a multivariate cox regression analysis when compared
to the other genes and clinicopathological factors with a nearly 5 times higher relative risk (4.74) of dying earlier when
expressed in low levels (p = 0.04). ROC Curve Analysis revealed that measuring EGFR potentially identifies patients at risk of a
worsened outcome with a sensitivity of 80% and a specificity of 75% (p = 0.01).
Conclusions:
EGFR and FLT1 seem to be potential markers to identify those patients at high risk of dying from
cholangiocarcinoma. Therefore these markers may help to identify patient subgroups in need for a more aggressive
approach in a disease that is in desperate need for new approaches.
Citation: Hoffmann A-C, Goekkurt E, Danenberg PV, Lehmann S, Ehninger G, et al. (2013) EGFR, FLT1 and Heparanase as Markers Identifying Patients at Risk of
Short Survival in Cholangiocarcinoma. PLoS ONE 8(5): e64186. doi:10.1371/journal.pone.0064186
Editor: Soheil S. Dadras, University of Connecticut Health Center, United States of America
Received August 24, 2012; Accepted April 13, 2013; Published May 21, 2013
Copyright: ß2013 Hoffmann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Response Genetics Inc. (http://www.responsegenetics.com), and ‘‘Kampf dem Krebs’’ eV of the German Cancer Society
(Deutsche Krebsgesellschaft). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: This study was partly supported by Response Genetics Inc. RNA was isolated from microdissected tumor samples following a proprietary
procedure at Response Genetics Inc (Los Angeles, CA; US patent No. 6248,535). There are no further patents, products in development or marketed products to
declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
* E-mail: ach@o117.com
.These authors contributed equally to this work.
Introduction
The treatment of solid tumors has seen a lot of progress over the
last few years with significant survival benefits in diseases like
breast and colorectal cancer especially through the development of
several new molecular entities. Nonetheless in cholangiocarcinoma
respectively cancers of the biliary tract the treatment choices
remain very limited [1,2]. Cholangiocarcinoma seems to be a
cancer with an inhomogeneous genetic design influenced by
multiple molecular aberrations limiting the successful application
of conservative approaches to find new treatments by simply
adding new molecular entities (NME, i.e. small molecules) to
classical cytotoxic regimes [3]. Identifying patient subgroups with
more aggressive subtypes of CCC at risk for a shortened survival
may lead to improved trial designs and hence to a more effective
strategy in treating this disease.
By previous work we already identified several candidate
biomarkers that are associated with the overall survival of patients
in various cancer types. These genes have a strong correlation with
angiogenesis (Hif1a,FLT1) [4,5] and lead to alterations of the
extracellular matrix and remodeling of subepithelial and suben-
dothelial basal membranes (heparanase, HPSE) [6,7] and therefore
seem to be directly involved in the aggressiveness of cancers.
In this study, we investigated the prognostic values of fms-
related tyrosine kinase 1/vascular endothelial growth factor
receptor 1 (FLT1/VEGFR1), heparanase (HPSE), hypoxia induc-
ible factor 1, alpha subunit (Hif1a), fms-related tyrosine kinase 4/
vascular endothelial growth factor receptor 3 (FLT4/VEGFR3),
vascular endothelial growth factor A & C (VEGFA/C), platelet-
PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e64186
derived growth factor alpha polypeptide A (PDGFA), PDGF
receptor, alpha polypeptide (PDGF-RA) heparin-binding EGF-like
growth factor (HB-EGF), and epidermal growth factor receptor
(EGFR) gene expressions as well as their interrelationships in
cholangiocarcinoma. We measured the mRNA expression levels of
these genes with quantitative real-time reverse transcriptase-PCR
(RT-PCR) in laser-microdissected formalin-fixed paraffin-embed-
ded (FFPE) tissue samples of cholangiocarcinoma. This approach
was taken to get a more precise result of gene expression than
previously available since stromal tissue might significantly
influence results and identification of significant genes [8]. We
then further analyzed the abovementioned genes and their
correlation with clinical and histopathological variables such as
primary tumor stage (pT, based on the International Union
Against Cancer, UICC, 1997), regional lymph node metastasis,
grading and overall survival.
Materials and Methods
Study Population and Tumor Samples
FFPE samples were obtained from patients with cholangiocar-
cinoma with a median age of 73 years (range 48–93 years) at time
of diagnosis. All patients received gemcitabine-based chemother-
apy and were treated at the University hospital of Dresden,
Germany between 2001 and 2007. Patient demographics are listed
in Table 1. TNM staging was performed according to the criteria
of the International Union Against Cancer [9]. All samples where
reviewed by a board certified clinical pathologist. Clinical
diagnosis and staging was done according to local guidelines
using among others endoscopy and CT-Scans. This study and the
herein used retrospective genetic analysis was approved by the
local ethics committee in Dresden, Germany. The Name of the
ethics committee is ‘‘Ethikkommission der Medizinischen Fakulta¨t,
TU Dresden’’ (Fetscherstrasse 74, 01307 Dresden) and the
Reference Number for the ethic statement is EK137072006.
The requirement for patient consent was specifically waived by the
approving IRB.
The clinico-pathological characteristics of all patients were
reviewed by a surgical pathologist. Representative hematoxillin
and eosin-stained slides of Formalin-fixed, paraffin-embedded
(FFPE) tissue blocks obtained at cholecystectomy or from biopsies
were reviewed in order to estimate the tumor load per sample. For
laser-captured microdissection (P.A.L.M. Microlaser Technologies
AG, Munich, Germany) slides of 10 mm thickness were obtained.
All tumor slides were prepared as described previously [4].
Quantitative Real-time Polymerase Chain Reaction
RNA was isolated from microdissected tumor samples following
a proprietary procedure at Response Genetics Inc (Los Angeles,
CA; US patent No. 6248,535). The resulting tumor RNA was
reverse transcribed into cDNA as described previously [4].
Expression of FLT1,FLT4,HPSE,Hif1a,VEGFA/C,HB-EGF,
PDGFA,PDGF-RA,EGFR and internal reference genes (beta-actin, b2mg,
rplp2, sdha) was quantified by real-time fluorescence detection of
amplified cDNA (ABI PRISM 7900 Sequence Detection System
[TaqMan], Perkin-Elmer Applied Biosystems, Foster City, CA).
The reverse transcription and polymerase chain reaction (RT-
PCR) assay was implemented as described previously [4]. All
primers were selected using the Gene Express software (Applied
Biosystems, Foster City, CA), but were adapted to the require-
ments of cDNA generated from RNA, which was extracted from
FFPE tissue. All primers were validated following a previously
described protocol [5]. All genes were run on all samples in
triplicates, i.e. one sample was run with each gene three times on
the same plate to identify potential outliers. The detection of
amplified cDNA results in a cycle threshold (Ct) value, which is
reciprocal to the amount of cDNA contained in the sample.
Normal colon, liver, and St. Universal Mix RNA (Stratagene, La
Jolla, CA) were used as control calibrators on each assay plate.
Gene expression levels were described as ratio between two
absolute measurements (gene of interest/endogenous reference
genes) to control for inter-sample variation. Before statistical
analysis, all ratios were logarithmically transformed including a
multiplier, which accounted the average Ct values obtained for
each gene during the validation process. This procedure facilitated
the comparison samples, which were run on different assay plates.
Depending on the used genes and mutlipliers the inter-plate
variation is around 5%.
Statistical Analyses
Associations of gene expression levels and progression-free or
overall survival were tested for each gene by the Kaplan-Meier
method. Survival differences between the high and low expression
group were analyzed by the log-rank test. To detect independent
prognostic factors associated with overall and progression-free
survival, multivariate Cox proportional hazards regression analysis
with stepwise selection was applied. After adjustment for potential
confounders, the following parameters were accounted for:
pathological tumor stage (pT), lymph node involvement (pN),
tumor grade (G) and the gene set. In addition, Receiver Operating
Characteristic (ROC) curve analysis was performed to test the
ability of the chosen cut-points to discriminate short survivors from
long survivors [10,11]. Recursive descent partition analysis was
used to identify the strongest divisor of all factors and the most
significant split determined by the largest likelihood-ratio chi-
square statistic in relation to clinical response as described
previously [12,13]. The split was chosen to maximize the
difference in the responses between the two branches of the split.
The level of significance was set to P,0.05. All P values were
based on two-sided tests. All statistical analyses were performed
Table 1. Patient Characteristics.
Age
median (range), years 74 (48–93)
Gender
female 37 (78.7%)
male 10 (21.3%)
Primary tumor expansion (pT)
pT1 6 (12.8%)
pT2 17 (36.2%)
pT3 20 (42.6%)
pT4 3 (6.4%)
Lymph node involvement (pN)
pN0 37 (78.7%)
pN1 10 (21.3%)
Grade of Dedifferentiation (G)
G2 17 (36.2%)
G3 28 (59.6%)
Overall Survival
median (range), months 13 (0–69)
doi:10.1371/journal.pone.0064186.t001
EGFR, FLT1, HPSE & Survival in Cholangiocarcinoma
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using the Software Packages Medcalc, Version 12.4.0 (Mariakerke,
Belgium) and JMP 10.0 (SAS Institute, Cary, NC, USA).
The level of significance was set to p,0.05. All p values
reported were based on two-sided tests. All statistical analyses were
performed using the Software Packages SPSSHfor Windows
(Version 16.0, Chicago, Il, USA) and JMP 7.0 Software (SAS,
Cary, NC, USA).
Results
Study group and tumor samples
Tissue blocks suitable for RNA extraction were retrieved from
47 patients and subjected to further analysis.
Comparison of gene expression levels throughout
subgroups
The gene expression levels of EGFR and HPSE showed a
significant inverse correlation (p = 0.03). FLT1 expression was
correlated to HPSE expression (p,0.0001; Correlation Coeffi-
cient, CC = 0.74). Furthermore FLT1 expression showed an
inverse correlation to PDGFA expression (p = 0.006; CC = 20.55)
and a positive correlation to the PDGFR expression (PDGF-RA;
p = 0.0008; CC = 0.59). HPSE expression was also significantly
associated with the expression of PDGFR-A (p,0.0001;
CC = 0.78) but not to its substrate PDGF. The correlation of the
other gene expression values are listed in Table 2.
Gene Expression and Survival
We used recursive decent partition tree analysis to find the
factors showing the strongest association to survival and define the
optimal cut-point of these factors. We used all available
clinicopathological data and the measured genes for this model.
Of the tested genes only EGFR, FLT1 and HPSE showed a
correlation to survival in Spearman’s test and the recursive decent
partition tree analysis. Patients expressing EGFR above the 35
th
percentile had a significantly worsened outcome (P = 0.04, hazard
ratio [HR] = 2.84, 95% CI = 1.0959 to 7.3692; Figure 1) with a
median overall survival of 8.5 months, whereas patients with lower
EGFR expression had a median survival time of more than 3 years
(38.5 months) as tested with Kaplan-Meier analysis. The same
statistical test revealed that to the contrary patients with a higher
FLT1 expression had a higher chance for prolonged survival with
Table 2. Spearman rank correlation between genes of interest.
EGFR FLT1 FLT4 HB-EGF HIF1a HPSE PDGFA PDGFRA VEGF VEGFC
CC 20.242 20.243 20.295 20.015 20.491 20.307 20.242 20.307 0.189
EGFR P 0,2915 0,2417 0,1821 0,9418 0,0327 0,1537 0,2657 0,1537 0,365
n 212522 25192323 2325
CC 20.242 0.044 0.014 20.268 0.735 20.551 0.587 20.024 20.143
FLT1 P 0,2915 0,837 0,9503 0,1944
,
0.0001 0,0064 0,0008 0,9101 0,5036
n21 2422 25272329 2424
CC 20.243 0.044 0.709 0.270 0.156 0.263 0.128 0.626 0.322
FLT4 P 0,2417 0,837
,
0.0001 0,1109 0,487 0,1269 0,5079
,
0.0001 0,0555
n2524 38 36223529 3936
CC 20.295 0.014 0.709 0.526 0.437 0.549 0.244 0.708 0.539
HB-EGF P 0,1821 0,9503
,
0.0001 0,0014 0,0615 0,0008 0,2288
,
0.0001 0,001
n222238 34193426 3934
CC 20.015 20.268 0.270 0.526 0.241 0.708 0.296 0.597 0.855
HIF1a P 0,9418 0,1944 0,1109 0,0014 0,2797
,
0.0001 0,1195 0,0001
,
0.0001
n25253634 224029 3640
CC 20.491 0.735 0.156 0.437 0.241 20.020 0.780 0.256 20.100
HPSE P 0,0327
,
0.0001 0,487 0,0615 0,2797 0,9348
,
0.0001 0,263 0,6581
n19272219 22 2027 2122
CC 20.307 20.551 0.263 0.549 0.708 20.020 0.182 0.466 0.678
PDGFA P 0,1537 0,0064 0,1269 0,0008
,
0.0001 0,9348 0,3638 0,0048
,
0.0001
n23233534 4020 27 3539
CC 20.242 0.587 0.128 0.244 0.296 0.780 0.182 0.354 0.332
PDGF-RA P 0,2657 0,0008 0,5079 0,2288 0,1195
,
0.0001 0,3638 0,0645 0,0789
n23292926 292727 2829
CC 20.307 20.024 0.626 0.708 0.597 0.256 0.466 0.354 0.580
VEGF P 0,1537 0,9101
,
0.0001
,
0.0001 0,0001 0,263 0,0048 0,0645 0,0003
n23243939 36213528 35
CC 0.189 20.143 0.322 0.539 0.855 20.100 0.678 0.332 0.580
VEGFC P 0,365 0,5036 0,0555 0,001
,
0.0001 0,6581
,
0.0001 0,0789 0,0003
n25243634 40223929 35
doi:10.1371/journal.pone.0064186.t002
EGFR, FLT1, HPSE & Survival in Cholangiocarcinoma
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a median overall survival time of 23.6 months and 40% of patients
surviving longer than 3 years (P = 0.006, [HR] = 0.28, 95%
CI = 0.07–1.14; Figure 2), whereas patients with a low expression
of FLT1 survived 5.3 months in median and none of the patients in
this group survived longer than 2 years. Similar to FLT1 patients
with a high HPSE expression had a longer median survival time
(P = 0.02, [HR] = 0.34, 95% CI = 0.11–1.04; Figure 3) with nearly
one third of the patients living after 3 years, whereas none of the
patients with a HPSE expression lower than the 35
th
percentile
reached this time point (median OS = 10.2 months).
Only the genes with a significant (p,0.05) correlation to
survival in univariate analysis (see above) were then put into a
stepwise multivariate Cox proportional hazards regression model.
In addition to FLT1,HPSE,EGFR mRNA expression clinical
factors such as Age at Diagnosis, primary tumor expansion (pT),
lymph node involvement (pN) and dedifferentiation grade were
included in the model. The overall model fit had a significance
level of p = 0.047. The factor that had the strongest significant
independent association with survival-time in this patient cohort
was low FLT1 mRNA expression (20
th
percentile cut-off) with a
significance level of p = 0.04. Patients with a low FLT1 expression
had a nearly five times higher relative risk (4.74) of dying earlier.
Receiver Operating Characteristic (ROC)
All three factors (expression of FLT1,HPSE and EGFR) were
tested with ROC Analysis for their potential predictive capabilities
in identifying patients with cholangiocarcinoma with a prolonged
overall survival time (.3 years). The 35
th
percentile cut-off of the
EGFR mRNA expression showed 80% sensitivity (true positive
rate) of and 75% specificity (true negative rate) for the diagnosis
prolonged (.3 years) overall survival. The area under the curve
was 0.775 (CI 0.583 to 0.908) with a significance level of p = 0.012.
The positive likelihood ratio (true positive rate/false positive rate)
was 3.2 and the negative likelihood ratio (false negative rate/true
negative rate) 0.27. Both, FLT1 and HPSE, were also significant
when tested with the same criteria on ROC Curve Analysis, but
with a specificity less than 40% (Figure 4).
Discussion
We determined the gene expressions of FLT1,FLT4,HPSE,
Hif1a,HB-EGF,PDGFA,PDGF-RA and EGFR in FFPE samples of
patients with cholangiocarcinoma. By using laser capture micro-
dissection to isolate tumor tissue from the clinical specimens along
with quantitative RT-PCR, we hoped to achieve a more precise
characterization of the associations of these gene expressions with
each other and with patients’ prognosis than was previously
available. Of the chosen candidate genes three expression profiles
seemed promising to be used in further studies, the fms-related
tyrosine kinase 1 respectively vascular endothelial growth factor
receptor 1 (FLT1/VEGFR1), heparanase (HPSE) and epidermal
growth factor receptor (EGFR).
The latter gene has already been described and known to play a
major role in tumorigenesis and aggressiveness of cancer, amongst
others in lung cancer. Recent publications evaluated the role of
anti-EGFR therapies in biliary tracts carcinomas. Chiorean and
colleagues tested Erlotinib and Docetaxel in Advanced and
Refractory Hepatocellular and Biliary Cancers in a Phase II Trial
of the Hoosier Oncology Group GI06-101 [14]. They came to the
conclusion that anti-EGFR therapy remains to be an important
possibility in these tumors but only with a molecular ‘‘targeted’’
approach. We were able to show that a high EGFR mRNA
expression level (.35th percentile) is associated to patients’
survival and confers a significantly worsened chance to survive
longer than one year (P = 0.04, hazard ratio [HR] = 2.84), whereas
patients with lower EGFR expression had a median survival time
Figure 1. Kaplan-Meier plot, estimating overall survival. Differences in survival between the high (interrupted line) and the low (continuous
black line) EGFR expression group were analyzed with the log-rank test.
doi:10.1371/journal.pone.0064186.g001
EGFR, FLT1, HPSE & Survival in Cholangiocarcinoma
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Figure 3. Kaplan-Meier plot, estimating overall survival. Differences in survival between the high (interrupted line) and the low (continuous
black line) HPSE expression group were analyzed with the log-rank test.
doi:10.1371/journal.pone.0064186.g003
Figure 2. Kaplan-Meier plot, estimating overall survival. Differences in survival between the high (interrupted line) and the low (continuous
black line) FLT1 expression group were analyzed with the log-rank test.
doi:10.1371/journal.pone.0064186.g002
EGFR, FLT1, HPSE & Survival in Cholangiocarcinoma
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of more than 3 years (38.5 months). These results are in agreement
with conclusions from other groups [15] that also indicated a
higher chance for better outcome in low expression groups. With
receiver operating characteristic (ROC) curve analysis we were
able to show that the 35th percentile cut-off of the EGFR mRNA
expression could be useful in identifying those patients at risk for
shortened survival with a sensitivity (true positive rate) of 80% and
a specificity (true negative rate) of 75%. As Andersen and
colleagues recently published the selection of patients from high
risk groups may indicate the necessity of modified treatment and
seems to be useful also in cholangiocarcinoma [16].
It has already been discussed by other groups that measuring
genes from the angiogenesis pathway seems to be a promising
approach in tumors of the biliary tract and pancreas, especially
due to their hypoxic nature [17]. In preceding works we were able
to shed light on a strong association of Hif1a expression with
survival in pancreatic cancer and soft tissue sarcomas [4,5].
However, this association was not significant in the examined
study group of patients with cholangiocarcinoma which is in
concordance with discoveries from other groups examining Hif1a
in CCC [18] who were also not able to show a correlation of Hif1a
expression to survival.
VEGFR 2/3 expression was tested in several studies so far [19].
There is however only limited data available for the expression of
FLT1/VEGFR1 in cholangiocarcinoma though Rogler and others
suggested a potential association with a more aggressive phenotype
[20]. We were able to show for the first time that FLT1 seems to be
independently associated with overall survival of patients with a
biliary tract tumor. Interestingly Kaplan-Meier Analysis revealed
that patients with a higher FLT1 expression potentially have a
better outcome, though one would anticipate high expression to
indicate a more aggressive tumor. Patients with a high expression
showed a median overall survival time of 23.6 months and 40% of
patients surviving longer than 3 years (P = 0.006, [HR] = 0.28).
The independent association of high FLT1 expression with better
outcome was supported by a stepwise multivariate Cox propor-
tional hazards regression model. In this study group FLT1 was the
strongest independent factor associated with overall survival.
Especially due to the destructive locally invasive behavior and a
high rate of distant metastasis we already tested HPSE in
pancreatic cancer [21]. Although we were not able to affirm a
significant correlation to overall survival we revealed the link of
HPSE expression to a higher rate of lymph node invasion, hence a
more aggressive tumor type. In this study we were able to show
that similar to FLT1 none of the patients with a HPSE expression
lower than the 35
th
percentile reached the 2-year mark (median
OS = 10.2 months). Although FLT1 and HPSE proved to be
significantly linked with overall survival they were - in contrast to
EGFR - not usable to distinguish patients at high risk for worsened
overall survival with sufficient sensitivity and specificity – at least
not in the examined study group. This may be explainable by the
size of the study group or may indicate that although FLT1 and
HPSE are potential markers to predict outcome they are not usable
as such as stand-alone markers, which may explain the discordant
Figure 4. Receiver Operating Characteristic (ROC) Curve Analysis for the potential of distinct EGFR expression to identify patients
with risk of short vs. long (3–5 years) Survival.
doi:10.1371/journal.pone.0064186.g004
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findings in relation to other studies. Interestingly the dichotomized
(cut-point) FLT1 mRNA expression showed a significant but
inverse correlation to the pathological tumor stage (p = 0.001;
Table 3).
It should be pointed out that our present study has been
retrospectively conducted in samples collected from patients that
were treated consecutively in our clinic. Accordingly, the results
may have been influenced by confounders that have occurred
during the follow-up period but were not reported, and by
additional bias [22]. Studies like the one we conducted may
therefore not be used to directly be translated into clinical practice
but may help understand a tumor that until now defies classical
unselected treatment approaches. The results have to be validated
in prospectively collected study groups. Nonetheless - as discussed
before - identifying genes that are associated with an aggravated
outcome is an important method to form a candidate oncogene
pool that is available for further work, such as in vitro studies or
biomarker guided therapy trials [4]. Further studies are warranted
and currently employed by our group to validate these genes
including tactics to identify these genes in circulating tumor cells
[23,24].
Conclusions
The significant association of high EGFR expression on survival
probability and the high sensitivity and specificity of measuring
EGFR expression to identify patients at risk for a shorter survival
suggests that EGFR may be a useful candidate for treatment
selection in cholangiocarcinomas. Furthermore FLT1 was inde-
pendently associated with survival in our study group and stronger
associated with outcome than other clinicopathologic parameters.
The significant associations of EGFR,FLT1 and HPSE gene
expression with survival warrant prospective evaluation of their
usability in selecting more efficient treatment strategies for patients
with cholangiocarcinoma.
Author Contributions
Conceived and designed the experiments: ACH JSW EG. Performed the
experiments: ACH SL. Analyzed the data: ACH JSW. Contributed
reagents/materials/analysis tools: DEA GE JSW ACH PD. Wrote the
paper: ACH EG JSW. Proofread the manuscript: EG JSW GE DEA.
Collection and assembly of data: ACH JSW EG. Approved the final
manuscript: ACH EG JSW GE.
References
1. Valle J, Wasan H, Palmer DH, Cunningham D, Anthoney A, et al. (2010)
Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. The New
England journal of medicine 362: 1273–1281.
2. Zhu AX, Meyerhardt JA, Blaszkowsky LS, Kambadakone AR, Muzikansky A, et
al. (2010) Efficacy and safety of gemcitabine, oxaliplatin, and bevacizumab in
advanced biliary-tract cancers and correlation of changes in 18-fluorodeox-
yglucose PET with clinical outcome: a phase 2 study. Lancet Oncol 11: 48–54.
3. Zhu AX, Hezel AF (2011) Development of molecularly targeted therapies in
biliary tract cancers: reassessing the challenges and opportunities. Hepatology
53: 695–704.
Table 3. Spearman rank correlation between clinicopathological characteristics and dichotomized expression values.
Age Gender Grading pN pT EGFR Split FLT1 Split HPSE Split
CC 20,248 20,205 20,292 0,078 0,009 20,315 20,089
Age P 0,0933 0,1766 0,2402 0,6074 0,9643 0,09 0,6592
n 4745 184629 30 27
CC 20,248 0,086 0,158 20,047 20,099 0,017 20,246
Gender P 0,0933 0,5754 0,5309 0,7567 0,608 0,9281 0,2155
n47 45 184629 30 27
CC 20,205 0,086 0,553 0,082 0,345 20,022 20,098
Grading P 0,1766 0,5754 0,0172 0,5942 0,0722 0,9084 0,6353
n4545 184528 29 26
CC 20,292 0,158 0,553 0,012 0,501 0,051 20,267
pN P 0,2402 0,5309 0,0172 0,9631 0,0814 0,8675 0,428
n181818 1813 13 11
CC 0,078 20,047 0,082 0,012 0,107 20,572 20,258
pT P 0,6074 0,7567 0,5942 0,9631 0,5896 0,0012 0,2024
n n 46 46 45 18 28 29 26
EGFR CC 0,009 20,099 0,345 0,501 0,107 20,208 20,519
Split P 0,9643 0,608 0,0722 0,0814 0,5896 0,3649 0,0228
n292928 1328 21 19
FLT1 CC 20,315 0,017 20,022 0,051 20,572 20,208 0,596
Split P 0,09 0,9281 0,9084 0,8675 0,0012 0,3649 0,001
n303029 132921 27
HPSE CC 20,089 20,246 20,098 20,267 20,258 20,519 0,596
Split P 0,6592 0,2155 0,6353 0,428 0,2024 0,0228 0,001
n272726 112619 27
doi:10.1371/journal.pone.0064186.t003
EGFR, FLT1, HPSE & Survival in Cholangiocarcinoma
PLOS ONE | www.plosone.org 7 May 2013 | Volume 8 | Issue 5 | e64186
4. Hoffmann AC, Danenberg KD, Taubert H, Danenberg PV, Wuerl P (2009) A
three-gene signature for outcome in soft tissue sarcoma. Clin Cancer Res 15:
5191–5198.
5. Hoffmann AC, Mori R, Vallbohmer D, Brabender J, Klein E, et al. (2008) High
expression of HIF1a is a predictor of clinical outcome in patients with pancreatic
ductal adenocarcinomas and correlated to PDGFA, VEGF, and bFGF.
Neoplasia 10: 674–679.
6. Hoffmann A-C, Mori R, Vallboehmer D, Brabender J, Drebber U, et al. (2008)
High expression of heparanase is significantly associated with dedifferentiation
and lymph node metastasis in patients with pancreatic ductal adenocarcinomas
and correlated to PDGFA and via HIF1a to HB-EGF and bFGF. Journal of
Gastrointestinal Surgery 12: 1674–1681.
7. Cohen-Kaplan V, Naroditsky I, Zetser A, Ilan N, Vlodavsky I, et al. (2008)
Heparanase induces VEGF C and facilitates tumor lymphangiogenesis.
Int J Cancer 123: 2566–2573.
8. Collisson EA, Sadanandam A, Olson P, Gibb WJ, Truitt M, et al. (2011)
Subtypes of pancreatic ductal adenocarcinoma and their differing responses to
therapy. Nature medicine 17: 500–503.
9. Sobin LH, Fleming ID (1997) TNM Classification of Malignant Tumors, fifth
edition (1997). Union Internationale Contre le Cancer and the American Joint
Committee on Cancer. Cancer 80: 1803–1804.
10. Metz CE (1978) Basic principles of ROC analysis. Semin Nucl Med 8: 283–298.
11. Zweig MH, Campbell G (1993) Receiver-operating characteristic (ROC) plots: a
fundamental evaluation tool in clinical medicine. Clin Chem 39: 561–577.
12. Hoffmann A-C (2009) A Three-Gene Signature for Outcome in Soft Tissue
Sarcoma (vol 15, pg 5191, 2009). Clinical Cancer Research 15: 6472–6472.
13. Hoffmann A-C, Mori R, Vallbohmer D, Brabender J, Klein E, et al. (2008) High
expression of HIF1a is a predictor of clinical outcome in patients with pancreatic
ductal adenocarcinomas and correlated to PDGFA, VEGF, and bFGF.
Neoplasia 10: 674–679.
14. Chiorean EG, Ramasubbaiah R, Yu M, Picus J, Bufill JA, et al. (2012) Phase II
Trial of Erlotinib and Docetaxel in Advanced and Refractory Hepatocellular
and Biliary Cancers: Hoosier Oncology Group GI06-101. Oncologist 17: 13–
e26.
15. Sergeant G, Lerut E, Ectors N, Hendrickx T, Aerts R, et al. (2011) The
prognostic relevance of tumor hypoxia markers in resected carcinoma of the
gallbladder. Eur J Surg Oncol 37: 80–86.
16. Andersen JB, Spee B, Blechacz BR, Avital I, Komuta M, et al. (2012) Genomic
and genetic characterization of cholangiocarcinoma identifies therapeutic targets
for tyrosine kinase inhibitors. Gastroenterology 142: 1021–1031 e1015.
17. Tempero MA, Berlin J, Ducreux M, Haller D, Harper P, et al. (2011) Pancreatic
cancer treatment and research: an international expert panel discussion. Ann
Oncol.
18. Chetboul V, Lichtenberger J, Mellin M, Mercera B, Hoffmann AC, et al. (2012)
Within-day and between-day variability of transthoracic anatomic M-mode
echocardiography in the awake bottlenose dolphin (Tursiops truncatus). J Vet
Cardiol 14: 511–518.
19. Bengala C, Bertolini F, Malavasi N, Boni C, Aitini E, et al. (2010) Sorafenib in
patients with advanced biliary tract carcinoma: a phase II trial. Br J Cancer 102:
68–72.
20. Xu L, Hausmann M, Dietmaier W, Kellermei er S, Pesch T, et al. (2010)
Expression of growth factor receptors and targeting of EGFR in cholangio-
carcinoma cell lines. BMC Cancer 10: 302.
21. Hoffmann AC, Mori R, Vallbohmer D, Brabender J, Drebber U, et al. (2008)
High expression of heparanase is significantly associated with dedifferentiation
and lymph node metastasis in patients with pancreatic ductal adenocarcinomas
and correlated to PDGFA and via HIF1a to HB-EGF and bFGF. J Gastrointest
Surg 12: 1674–1681; discussion 1681–1672.
22. Hoffmann A-C, Wild P, Leicht C, Bertz S, Danenberg KD, et al. (2010) MDR1
and ERCC1 Expression Predict Outcome of Patients with Locally Advanced
Bladder Cancer Receiving Adjuvant Chemotherapy. Neoplasia 12: 628–636.
23. Gauler TC, Theegarten D, Parr A, Schuhr I, Schmid KW, et al. (2011) Decrease
of Circulating Tumor Cells Associates with Response to Platinum-Based
Chemotherapy in Patients with Non-Small Cell Lung Cancer, but Not with
Small Cell Lung Cancer. Journal of Thoracic Oncology 6: S1114–S1114.
24. Christoph DC, Hoffmann A-C, Gauler TC, Asuncion BR, Loewendick H, et al.
(2012) Detection of Circulating Lung Cancer Cells with Strong Thymidylate
Synthase Reactivity in the Peripheral Blood of a Patient with Pulmonary
Adenocarcinoma Treated with Pemetrexed. Journal of Thoracic Oncology 7:
766–767 710.1097/JTO.1090b1013e3182460fa3182469.
EGFR, FLT1, HPSE & Survival in Cholangiocarcinoma
PLOS ONE | www.plosone.org 8 May 2013 | Volume 8 | Issue 5 | e64186
... Another report revealed that EGFR is highly expressed in 44.7% (17/38) of ICC patients, and its upregulation is closely correlated with poor differentiation, lymph node metastasis, and aberrant p53 expression [10]. Expression of EGFR and vascular endothelial growth factor (VEGF) were highly elevated in 27.4% (29/109) and 53.8 (57/106) of ICC patients, respectively, and 19.2% (25/130) and 59.2% (77/130) of ECC patients, respectively [11,12]. EGFR is significantly associated with stage, lymph node metastasis, lymphatic vessel invasion, and perineural invasion in ECC. ...
... EGFR is significantly associated with stage, lymph node metastasis, lymphatic vessel invasion, and perineural invasion in ECC. High EGFR expression also leads to reduced overall survival (OS) and increased tumor recurrence [11,12]. Moreover, HB-EGF and amphiregulin as EGFR ligands are highly elevated in various types of cancer cells, including CC cells, and significantly enhances the survival of cancer cells by activation of EGFR and the extracellular-signal-regulated kinase (ERK) pathway [13]. ...
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The erythroblastic leukemia viral oncogene homolog (ErBb) family consists of the receptor tyrosine kinases (RTK) epidermal growth factor receptor (EGFR; also called ERBB1), ERBB2, ERBB3, and ERBB4. This family is closely associated with the progression of cholangiocarcinoma (CC) through the regulation of cellular networks, which are enhanced during tumorigenesis, metastasis, and chemoresistance. Additionally, the constitutive activation of cellular signaling by the overexpression and somatic mutation-mediated alterations conferred by the ErBb family on cholangiocarcinoma and other cancers enhances tumor aggressiveness and chemoresistance by contributing to the tumor microenvironment. This review summarizes the recent findings on the molecular functions of the ErBb family and their mutations during the progression of cholangiocarcinoma. It also discusses the developments and applications of various devising strategies for targeting the ErBb family through different inhibitors in various stages of clinical trials, which are essential for improving targeted clinical therapies.
... Epidermal growth factor receptor expression, determined by IHC, has been observed in 19.2% of 130 eCCA cases, and confers significantly higher rates of lymph node metastasis, lymphatic vessel invasion and perineural invasion [68]. The overexpression of EGFR and HER2 has been identified as an independent marker of poor prognosis within BTC [69][70][71]. Somatic activating EGFR mutations occur in eCCA, but their frequency within pCCA is difficult to determine, because data on mutational frequency are rarely stratified by anatomical subtype [72]-somatic EGFR mutations were found in 9.5% of 137 resected BTCs in one study [73], and in 13.6% of 22 consecutive resected CCAs in another [74]. A next-generation sequencing study featuring 248 eCCAs revealed that 9.7% of patients harboured aberrations within the EGFR family, of which 53.6% had mutated HER2 [30]. ...
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Perihilar cholangiocarcinoma (pCCA) is the anatomical sub-group of biliary tract cancer (BTC) arising between the second-order intrahepatic bile ducts and the cystic duct. Together with distal and intrahepatic cholangiocarcinoma (dCCA and iCCA; originating distal to, and proximal to this, respectively), gallbladder cancer (GBC) and ampulla of Vater carcinoma (AVC), these clinicopathologically and molecularly distinct entities comprise biliary tract cancer (BTC). Most pCCAs are unresectable at diagnosis, and for those with resectable disease, surgery is extensive, and recurrence is common. Therefore, the majority of patients with pCCA will require systemic treatment for advanced disease. The prognosis with cytotoxic chemotherapy remains poor, driving interest in therapies targeted to the molecular nature of a given patient’s cancer. In recent years, the search for efficacious targeted therapies has been fuelled both by whole-genome and epigenomic studies, looking to uncover the molecular landscape of CCA, and by specifically testing for aberrations where established therapies exist in other indications. This review aims to provide a focus on the current molecular characterisation of pCCA, targeted therapies applicable to pCCA, and future directions in applying personalised medicine to this difficult-to-treat malignancy.
... FLT-1, also known as vascular endothelial growth factor receptor 1 (VEGFR-1), is a high-affinity tyrosine kinase receptor for VEGF. Previous studies have demonstrated that FLT1 expression is significantly correlated with OS in several cancers, including CRC, cholangiocarcinoma, and esophageal cancer (35)(36)(37). Additionally, Han et al. found that FLT1-rs9513111 was associated with a decreased risk of cervical (I) Identified immune molecules and factors verified by differential analysis between low CIFI group and high CIFI group. ...
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Cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) are among the most common malignancies of the female genital tract. Ferroptosis and immunity regulate each other and play important roles in the progression of CESC. The present study aimed to screen ferroptosis- and immune-related differentially expressed genes (FI-DEGs) to identify suitable prognostic signatures for patients with CESC. We downloaded the RNAseq count data and corresponding clinical information of CESC patients from The Cancer Genome Atlas database; obtained recognized ferroptosis- and immune-related genes from the FerrDb and ImmPort databases, respectively; and screened for suitable prognostic signatures using a series of bioinformatics analyses. We identified eight FI-DEGs ( CALCRL , CHIT1 , DES , DUOX1 , FLT1 , HELLS , SCD , and SDC1 ) that were independently correlated with the overall survival of patients with CESC. The prediction model constructed using these eight FI-DEGs was also independently correlated with overall survival. Both the sensitivity and specificity of the prediction model constructed using these eight signatures were over 60%. The comprehensive index of ferroptosis and immune status was significantly correlated with the immunity of patients with CESC. In conclusion, the risk assessment model constructed with these eight FI-DEGs predicted the CESC outcomes. Therefore, these eight FI-DEGs could serve as prognostic signatures for CESC.
... It has been reported that in epithelial cells of breast and non-small cell lung cancers, the increased expression of VEGFR-1 is associated with short survival time [38][39][40]. In contrast, reduced expression of VEGFR-1 in bile duct cancer and diffuse large B-cell lymphoma (DLBCL) is considered a negative prognostic factor [41,42]. Immunohistochemical studies of other research centers also showed that VEGFR-1 was stained in tumor cells [43,44]. ...
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Background VEGF-A, VEGF-B, VEGFR-1 and VEFR-2 are important proteins involved in the induction and development of a new blood vessel network through which the tumor is properly nourished and oxygenated. Objectives The aim of the study was to evaluate changes in the expression of VEGF-A, VEGF-B, VEGFR-1 and VEGFR-2 in endometrial cancer depending on its grade and to determine the VEGFR-1 to VEGFR-2 concentration ratio. Methods The study group consisted of 45 patients diagnosed with endometrial cancer (G1, 17; G2, 15; G3, 13). The control group included 15 patients. VEGF-A, VEGF-B, VEGF-R1, VEGFR-2 expression was assessed using the immunohistochemical method. Statistical analysis was carried out using the Statistica 12 PL program (StatSoft, USA). It included the one-way ANOVA and Tukey's post-hoc test (p<0.05). Results Statistically significant differences in the level of VEGF-A, VEGF-B, VEGF-R1, VEGFR-2 were observed between the majority of analyzed groups (except for VEGF-B; G3 vs. G1, p=0.997700). The expression pattern of VEGF-A, VEGF-R1, VEGFR-2 was as follows: G3>G2>G1>C; VEGF-B: G2> G3> G1>C. A lower concentration of VEGFR-1 than VEGFR-2 was found regardless of the cancer grade. Conclusion VEGF-A, VEGF-B, VEGF-R1, VEGFR-2 are key proteins involved in tumor angiogenesis. The analysis of the entire panel of proteins involved in a given process is an important element of modern diagnostics. The concentration ratio of VEGFR-1 to VEGFR-2 appears to be a determining factor in the patients' survival prognosis.
... Here, we investigated prognostic value of EGFR mRNA expression by mining the data deposited in the GEO and Oncomine databases. Although there are studies revealing that high EGFR mRNA [13][14][15][16][17][18] or even gene copy number [19] was correlated with poor clinical outcomes or pathological characteristics, a more systematic evaluation of published studies did not validate the proposed impact of EGFR mRNA expression. The inconsistency partially may be attributed to the choice of the EGFR probe. ...
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Epidermal growth factor receptor (EGFR) as a prevalent oncogene regulates proliferation, apoptosis and differentiation and thereby contributes to carcinogenesis. Even though, the documentation on its clinical relevance is surprisingly heterogeneous in the scientific literature. Here, we systematically investigated the correlation of mRNA to survival time and pathological parameters by analyzing 30 datasets in silico. Furthermore, the prognostic value of membrane-bound, cytoplasmic (mcEGFR) and nuclear expression (nEGFR) of EGFR was experimentally analyzed by immunohistochemical staining of 502 biopsies from 27 tumor types. We found that protein expression of EGFR showed better prognostic efficiency compared to mRNA, and that mcEGFR expression was positively correlated with nEGFR expression (p < 0.001). Unexpectedly, both mcEGFR and nEGFR expression were associated with low T stage (p < 0.001 and p = 0.004; respectively). Moreover, positive mcEGFR was significantly related to high differentiation (p = 0.027). No significant correlation was found with any other pathological parameters. Collectively, our results imply that the oncogenic function of EGFR may be more related to nascent stages of carcinogenesis than to advanced and progressive tumors, which may as well explain at least partially the occurrence of secondary resistance against EGFR-directed therapy. Copyright: Yan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... This divergence has also been observed for other cancer types. High VEGFR-1 expression in epithelial tumor cells of breast cancers and non-small cell lung carcinomas is associated with short survival [19][20][21], but low VEGFR-1 expression in tumor cells of cholangiocarcinomas and diffuse large B cell lymphomas (DLBCL) is also associated with short survival [22,23]. ...
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... This chromosomal area also contains Transcription Factor Dp-1 (TFDP1), which plays a crucial role in cell cycle progression 32 and the oncogene Fms Related Tyrosine Kinase 1 (FLT1), known to induce metastasis in breast cancer models 33 . Overexpression of the latter was also significantly associated with poor survival in cholangiocarcinoma 34 . ...
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Cholangiocarcinoma (CCA) is a malignant cancer that is associated with high mortality rates. The relationship between laminin γ 2 chain gene (LAMC2) and epidermal growth factor receptor (EGFR) has been previously documented in gastric cancer and oral squamous cell carcinoma. This study investigates the role of LAMC2 in epithelial-mesenchymal transition (EMT) and angiogenesis in CCA and explores the underlying mechanism(s). Differentially expressed genes related to CCA were initially screened using a microarray analysis, and the interaction between LAMC2 and the EGFR signaling pathway was identified. To determine the regulatory effects of LAMC2 on CCA progression, LAMC2 was silenced or overexpressed and the EGFR signaling pathway was activated or blocked. Subsequently, the regulation effects of LAMC2 were evaluated on the expression of EMT markers, invasion and migration of CCA cells, as well as microvessel density in nude mice. Microarray analysis demonstrated that highly expressed LAMC2 is linked to CCA development, which involves the EGFR signaling pathway. When LAMC2 expression was increased, the EGFR signaling pathway and EMT were activated in CCA tissues. Silencing of LAMC2 as well as EGFR signaling pathway inhibition led to suppression of EMT, cell invasion, and migration abilities in vitro, as well as angiogenesis in vivo. This study demonstrates that LAMC2 silencing suppresses the activity of the EGFR signaling pathway, thus functioning as a tumor suppressor in CCA.
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Objective: to evaluate the expression of the epithelial growth factor receptor (EGFR) by immunohistochemistry, and to verify its association with prognostic factors and survival of patients operated by cholangiocarcinoma. Methods: we verified the immunohistochemical expression of EGFR in 35 surgical specimens of cholangiocarcinoma (CCA). We obtained survival curves with the Kaplan-Meier method. Results: we found significant EGFR expression in ten (28.6%) of the 35 CCAs, eight with score 3 and two with score 2. Advanced stages (III and IV) presented higher EGFR expression (p=0.07). The clinical characteristics that were most associated with positive EGFR expression were female gender (p=0.06) and absence of comorbidities (p=0.06). Overall survival at 12, 24, 36 and 48 months was 100%, 82.5%, 59% and 44.2%, respectively. The survival of EGFR positive patients at 12, 24, 36 and 48 months was 100%, 75%, 50% and 0%, whereas for negative EGFR patients it was 100%, 87.5%, 65.6% and 65.6%, respectively. Conclusion: EGFR expression occurred in 28.6% of the cases studied and was associated with lower survival.
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Background: Patients with advanced hepatocellular (HCC) and biliary tract carcinomas (BTC) have poor prognosis. While the EGFR pathway is overactive in HCC and BTC, single agent anti-EGFR therapies confer modest activity. Preclinical data showed synergistic antiproliferative and proapoptotic effects between anti-EGFR therapies and taxanes. We conducted a phase I study of erlotinib and docetaxel in solid tumors, and noted good tolerability and sustained complete (5 years +) and partial responses in patients with HCC and BTC. This trial evaluated the efficacy of erlotinib with docetaxel in refractory hepatobiliary cancers. Methods: Eligible patients were allowed to have two prior systemic therapies. Docetaxel 30 mg/m2 i.v. was administered on days 1, 8, 15, and erlotinib 150 mg was dosed orally on days 2-7, 9-14, 16-28 of each 28-day cycle. The primary endpoint was 16 weeks progression-free survival (PFS), and secondary endpoints included response, stable disease, and overall survival. Tumor samples were analyzed for KRAS gene mutations and E-cadherin expression by immunohistochemistry (IHC). Patients with BTC and HCC were accrued and assessed in separate strata for the efficacy endpoints, but for the two-stage initial design of the study, combined PFS was considered. A Simon optimal two-stage design tested the hypothesis that the 16-week PFS is ≤ 15% (clinically inactive) versus the alternative of ≥ 30% (warranting further study). Results: Twenty-five patients, 14 with HCC and 11 with BTC, were enrolled. Common toxicities were rash (76%), diarrhea (56%), and fatigue (52%), mostly grade 1 or 2. No objective responses were seen. Seven BTC (64%) and 6 HCC patients (46%) had stable disease as best response, with a median duration of 16.1 weeks (95% CI 3.7-56.3) for BTC, and 17.6 weeks (95% CI 8.1-49.8) for HCC. The 16-week PFS was 64% for BTC (95% CI 29.7-84.5), and 38% for HCC (95% CI 14.1-62.8). Median overall survival was 5.7 and 6.7 months for BTC and HCC patients, respectively. BTC patients with grade ≥ 2 rash had higher median PFS (6.2 vs 2.2 months) and OS (14.2 vs. 4.2 months). HCC patients with negative/low E-cadherin expression had higher median PFS (6.7 vs. 2.1 months) and OS (14.5 vs. 4 months). Conclusion: Erlotinib with docetaxel met the 16-week PFS ≥ 30% endpoint, but overall survival was comparable to that seen with single-agent erlotinib. With the limitation of small numbers of patients, grade ≥ 2 rash (in BTC), and negative/low E-cadherin expression (HCC) were associated with higher PFS and OS. Discussion Refractory biliary tract and hepatocellular cancers are difficult to treat, and no chemotherapy or biologically targeted therapies have impacted survival. Based on preclinical synergism and prior phase I data, we conducted a multi-institutional study sequentially combining the EGFR-targeted agent erlotinib with docetaxel. Results from this study show that the primary endpoint, 16-week PFS of ≥ 30%, was met for the combined group of BTC and HCC patients (as originally planned in the study design), as well as in each disease category: 63.6% for BTC and 38.5% for HCC patients. Nevertheless, no patients attained an objective response and the median survival of 5.7 months for BTC, and 6.7 months for HCC patients (while heavily pretreated), is comparable to that seen with single-agent EGFR-targeted therapies. Safety analysis shows that this regimen was generally well tolerated, and most adverse events were grade 1 or 2. Few patients had reversible grade 3 transaminase elevation (8%), and severe anorexia, fatigue, and rash were uncommon. As expected, patients with grade ≥ 2 rash experienced higher PFS and OS, but this was noted only among the BTC group, likely because too few HCC patients had grade ≥ 2 rash. KRAS is an important predictive marker for anti-EGFR therapies for lung and colorectal cancers, but for HCC or the heterogeneous group of BTC (with 10-50% KRAS mutations) no significant correlations have been established. We were not able to identify a correlation between KRAS and benefit from erlotinib-based therapy, as all but one HCC patient had KRAS wild type gene status. Preclinical data in multiple tumor types showed that E-cadherin, a signature marker for an "epithelial" tumor phenotype when overexpressed, predicts EGFR pathway activation and determines sensitivity to EGFR-targeted agents. E-cadherin is often seen as a poor prognostic marker when downregulated, as noted during cancer progression. Not all studies demonstrate beneficial effects from E-cadherin overexpression, possibly due to histological expression variability or tumor type specificity for this biomarker. Six BTC and 8 HCC patients had evaluable tumor samples for E-cadherin analysis. While the numbers were small and conclusions should be viewed with caution, negative/low E-cadherin expression was associated with improved PFS and OS for hepatobiliary cancers (most significant in HCC) in this refractory patient population where we expected lower expression levels. In conclusion, the combination of erlotinib with docetaxel provided a 16-week PFS of ≥ 30% but showed no appreciable differences in overall survival from historical data with single-agent erlotinib. While EGFR represents an important target in this group of malignancies, it is clear that hepatobiliary cancers are heterogeneous, thus a meaningful improvement in survival most likely will require careful treatment selection based on patient tumor's molecular and genetic profiling.
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Pancreatic ductal adenocarcinoma (PDA) is a lethal disease. Overall survival is typically 6 months from diagnosis. Numerous phase 3 trials of agents effective in other malignancies have failed to benefit unselected PDA populations, although patients do occasionally respond. Studies in other solid tumors have shown that heterogeneity in response is determined, in part, by molecular differences between tumors. Furthermore, treatment outcomes are improved by targeting drugs to tumor subtypes in which they are selectively effective, with breast and lung cancers providing recent examples. Identification of PDA molecular subtypes has been frustrated by a paucity of tumor specimens available for study. We have overcome this problem by combined analysis of transcriptional profiles of primary PDA samples from several studies, along with human and mouse PDA cell lines. We define three PDA subtypes: classical, quasimesenchymal and exocrine-like, and we present evidence for clinical outcome and therapeutic response differences between them. We further define gene signatures for these subtypes that may have utility in stratifying patients for treatment and present preclinical model systems that may be used to identify new subtype specific therapies.
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The use of transthoracic echocardiography in dolphins has been limited so far owing to technical and anatomical specificities. Anatomic M-mode (AMM) is a postprocessing echocardiographic technique generating M-mode studies from two-dimensional (2D) cineloops independently of the ultrasound beam orientation. The aim of the present study was to determine the within-day (repeatability) and between-day (reproducibility) variability of AMM echocardiography in awake healthy bottlenose dolphins (BN, Tursiops truncatus). Four adult BN trained to lie in left recumbency at the water surface were involved in the protocol. A total of 96 echocardiographic examinations were performed on 4 different days by a trained observer examining each BN 6 times per day. Video clips of 2D left parasternal long-axis views showing the left ventricle (LV) ventrally and the aortic root dorsally were recorded at each examination and analyzed for AMM measurements in a random order. A general linear model was used to determine the within-day and between-day coefficients of variation (CV). All examinations were interpretable allowing calculation of 10 AMM variables (i.e., end-diastolic and end-systolic ventral and dorsal LV myocardial wall thicknesses as well as LV and aortic diameters, mean aortic diameter, and LV shortening fraction). Most within- and between-day CV values (18/20) were <15%, the lowest being observed for the end-diastolic LV diameter (1.6%). In conclusion, AMM provides a simple non-invasive evaluation of heart morphology and function in the awake BN with good repeatability and reproducibility of the measurements. Further studies are required to determine the corresponding reference intervals.
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Cholangiocarcinoma is a heterogeneous disease with a poor outcome that accounts for 5%-10% of primary liver cancers. We characterized its genomic and genetic features and associated these with patient responses to therapy. We profiled the transcriptomes from 104 surgically resected cholangiocarcinoma samples collected from patients in Australia, Europe, and the United States; epithelial and stromal compartments from 23 tumors were laser capture microdissected. We analyzed mutations in KRAS, epidermal growth factor receptor (EGFR), and BRAF in samples from 69 tumors. Changes in gene expression were validated by immunoblotting and immunohistochemistry; integrative genomics combined data from the patients with data from 7 human cholangiocarcinoma cell lines, which were then exposed to trastuzumab and lapatinib. Patients were classified into 2 subclasses, based on 5-year survival rate (72% vs 30%; χ(2) = 11.61; P < .0007), time to recurrence (13.7 vs 22.7 months; P < .001), and the absence or presence of KRAS mutations (24.6%), respectively. Class comparison identified 4 survival subgroups (SGI-IV; χ(2) = 8.34; P < .03); SGIII was characterized by genes associated with proteasomal activity and the worst prognosis. The tumor epithelium was defined by deregulation of the HER2 network and frequent overexpression of EGFR, the hepatocyte growth factor receptor (MET), pRPS6, and Ki67, whereas stroma was enriched in inflammatory cytokines. Lapatinib, an inhibitor of HER2 and EGFR, was more effective in inhibiting growth of cholangiocarcinoma cell lines than trastuzumab. We provide insight into the pathogenesis of cholangiocarcinoma and identify previously unrecognized subclasses of patients, based on KRAS mutations and increased levels of EGFR and HER2 signaling, who might benefit from dual-target tyrosine kinase inhibitors. The group of patients with the worst prognosis was characterized by transcriptional enrichment of genes that regulate proteasome activity, indicating new therapeutic targets.