cancer discovery-2014-horwitz-730-43
TRANSCRIPT
730 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org
RESEARCH ARTICLE
Human and Mouse VEGFA -Amplifi ed Hepatocellular Carcinomas Are Highly Sensitive to Sorafenib Treatment Elad Horwitz 1 , Ilan Stein 1 , 3 , Mariacarla Andreozzi 5 , Julia Nemeth 6 , Avivit Shoham 1 , Orit Pappo 3 , Nora Schweitzer 12 , Luigi Tornillo 5 , Naama Kanarek 1 , Luca Quagliata 5 , Farid Zreik 3 , Rinnat M. Porat 3 , Rutie Finkelstein 3 , Hendrik Reuter 7 , Ronald Koschny 11 , Tom Ganten 11 , Carolin Mogler 9 , Oren Shibolet 4 , Jochen Hess 6 , 8 , 10 , Kai Breuhahn 9 , Myriam Grunewald 2 , Peter Schirmacher 9 , Arndt Vogel 12 , Luigi Terracciano 5 , Peter Angel 6 , Yinon Ben-Neriah 1 , and Eli Pikarsky 1 , 3
ABSTRACT Death rates from hepatocellular carcinoma (HCC) are steadily increasing, yet thera-
peutic options for advanced HCC are limited. We identify a subset of mouse and
human HCCs harboring VEGFA genomic amplifi cation, displaying distinct biologic characteristics.
Unlike common tumor amplifi cations, this one seems to work via heterotypic paracrine interactions;
stromal VEGF receptors (VEGFR), responding to tumor VEGF-A, produce hepatocyte growth factor
(HGF) that reciprocally affects tumor cells. VEGF-A inhibition results in HGF downregulation and
reduced proliferation, specifi cally in amplicon-positive mouse HCCs. Sorafenib—the fi rst-line drug
in advanced HCC—targets multiple kinases, including VEGFRs, but has only an overall mild benefi cial
effect. We found that VEGFA amplifi cation specifi es mouse and human HCCs that are distinctly sensi-
tive to sorafenib. FISH analysis of a retrospective patient cohort showed markedly improved survival
of sorafenib-treated patients with VEGFA -amplifi ed HCCs, suggesting that VEGFA amplifi cation is a
potential biomarker for HCC response to VEGF-A–blocking drugs.
SIGNIFICANCE: Using a mouse model of infl ammation-driven cancer, we identifi ed a subclass of HCC
carrying VEGFA amplifi cation, which is particularly sensitive to VEGF-A inhibition. We found that a simi-
lar amplifi cation in human HCC identifi es patients who favorably responded to sorafenib—the fi rst-line
treatment of advanced HCC—which has an overall moderate therapeutic effi cacy. Cancer Discov; 4(6);
730–43. ©2014 AACR.
See related commentary by Luo and Feng, p. 640.
Authors’ Affi liations: 1 The Lautenberg Center for Immunology; 2 Depart-ment of Developmental Biology and Cancer Research, IMRIC, Hadassah Medical School, Hebrew University; 3 Department of Pathology, Hadassah-Hebrew University Medical Center, Jerusalem; 4 Liver Unit, Tel Aviv Sour-asky Medical Center, Tel Aviv, Israel; 5 Institute of Pathology, University Hospital Basel, Basel, Switzerland; 6 Division of Signal Transduction and Growth Control (A100), 7 Division of Molecular Genetics (B060), and 8 Jun-ior Group Molecular Mechanisms of Head and Neck Tumors (A102), Ger-man Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance; 9 Institute of Pathology, University Hospital Heidelberg; Departments of 10 Otolaryngol-ogy, Head and Neck Surgery and 11 Internal Medicine, University Hospital Heidelberg, Heidelberg; and 12 Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).
Corresponding Authors: Eli Pikarsky, The Lautenberg Center for Immunol-ogy, IMRIC, Hadassah Medical School, Hebrew University, Kiryat Hadas-sah, P.O.B. 12272, Jerusalem 91120, Israel. Phone: 972-2-675-8202; Fax: 972-2-642-6268; E-mail: [email protected] ; and Yinon Ben-Neriah, The Lautenberg Center for Immunology, IMRIC, Hadassah Medical School, Hebrew University, Kiryat Hadassah, P.O.B. 12272, Jerusalem 91120, Israel. Phone: 972-2-675-8718; E-mail: [email protected]
doi: 10.1158/2159-8290.CD-13-0782
©2014 American Association for Cancer Research.
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
JUNE 2014�CANCER DISCOVERY | 731
INTRODUCTION Hepatocellular carcinoma (HCC) is the third leading cause of
cancer-related mortality worldwide, with the highest increase
rate in North America ( 1, 2 ). Sorafenib is the mainstay of
therapy for advanced HCC and the only systemic drug that has
shown any survival advantage in HCC so far ( 3, 4 ). However,
patients’ response is modest, and sorafenib treatment is asso-
ciated with side effects ( 3–8 ). Thus, several studies looked for
predictive markers for sorafenib response ( 9–11 ), yet no such
biomarkers have entered the clinical setting. Predictive biomar-
kers, identifying patient subsets to guide treatment choices, are
usually based on distinct pathogenetic mechanisms, and are
the cornerstone of personalized medicine ( 12, 13 ). Prominent
examples include ERBB2 amplifi cation and KRAS mutations
that serve as key determinants of treatment with trastuzumab
or cetuximab, respectively ( 14, 15 ).
Sorafenib is a multikinase inhibitor, the targets of which
include BRAF, CRAF, PDGFR2, c-KIT, and the VEGFA recep-
tors (VEGFR; refs. 10 , 16 ). Although VEGFRs were postulated
as mediators of sorafenib response in HCC, testing for VEGF-A
serum levels was not found to be predictive of sorafenib
treatment success ( 10 ). Moreover, bevacizumab, an antibody
against VEGF-A, only shows minimal responses in HCC
( 17–20 ). A possible explanation for this is that the mecha-
nism of action of sorafenib is predominantly independent of
VEGF-A inhibition. Alternatively , a small subset of patients
who can respond to VEGF-A blockers may exist that was
underrepresented in bevacizumab studies.
VEGF-A is a master regulator of angiogenesis whose role
in tumor vessel recruitment is well established ( 21 ). However,
additional roles for VEGF-A in tumorigenesis are emerging.
VEGF-A was shown to promote tumor cell growth in an
autocrine manner, in skin and lung cancer cells expressing
VEGFRs ( 22, 23 ). Moreover, VEGF-A also has nonangiogenic
functions in normal physiology. In liver tissue, VEGF-A elicits
hepatocyte proliferation by elevating expression of hepato-
cyte mitogens in liver sinusoidal endothelial cells ( 24, 25 ).
HCC is most commonly the outcome of chronic injury and
infl ammation, resulting in hepatocyte regeneration and dys-
regulated growth factor signaling ( 1 , 26 ). It has become clear
that infl ammatory signaling pathways can support survival,
growth, and progression of cancer. Accordingly, secreted
cytokines and effector molecules, which are abundant in the
tumor microenvironment, could constitute suitable targets
for treatment and primary prevention of HCC ( 26 ). Here, we
used Mdr2 -defi cient mice ( Mdr2 −/− ), which develop chronic
liver infl ammation, eventually leading to infl ammation-
induced liver tumors similar to human HCC ( 27, 28 ), to look
for candidate treatment targets modulating the tumor micro-
environment that are relevant to human HCC.
RESULTS Array CGH Reveals Recurrent Gains in the VEGFA Locus Identifying a Molecularly Distinct Tumor Subpopulation
In search of microenvironment-affecting factors whose
amplifi cation or deletion plays a role in infl ammation-induced
HCC development, we applied array-based comparative
genomic hybridization (aCGH) to 10 HCCs obtained from
16-month-old Mdr2 −/− mice (Supplementary Fig. S1A). We
detected several amplifi cations and deletions, including an
amplifi cation in the qB3 band of murine chromosome 17
(Chr17qB3; Supplementary Table S1), encoding among oth-
ers the gene for VEGF-A. Genomic amplifi cation of Vegfa was
of interest as it is a cytokine gene that can modulate several
components of the tumor microenvironment and induce liver
cell growth ( 24, 25 ). To determine the frequency of this ampli-
fi cation, we tested a larger cohort of Mdr2 −/− tumors by quan-
titative PCR (qPCR) of tumor DNA (Supplementary Fig. S1B)
and chromogenic in situ hybridization (CISH; Fig. 1A ); 13 of
93 (∼14%) HCCs harbored this amplicon. To map the minimal
amplifi ed region of this amplicon, we used DNA qPCR directed
at several loci along murine chromosome 17 in a cohort of
tumors bearing this amplifi cation ( Fig. 1B ). We found that
the common proximal border lies between 43.3 and 48.5 mega
base pairs (Mbp) from the chromosome 17 start. This minimal
amplifi ed region spanned 53 genes (Supplementary Table S2).
Amplifi cation of human Chr6p21 (the region syntenic for
murine Chr17qB3) and whole chromosome gains were previ-
ously reported in several whole-genome analyses of human
HCC with a frequency ranging between 7% and 40% ( 29–33 ).
Accordingly, through FISH, we found VEGFA amplifi cations
and chromosome 6 polysomies in 11% of human HCCs we
tested (21 of 187; Fig. 1C ; Supplementary Table S3).
To elucidate the tumor relevance of these chromosomal
gains, we analyzed the expression of mouse Chr17qB3 ampli-
con genes in amplicon-positive (herein Amp pos ) and amplicon-
negative (Amp neg ) tumors. Matched increases in mRNA and
DNA levels were found for Vegfa , Tjap1 , and Xpo5 ( Fig. 1D
and Supplementary Fig. S2A). We further found a correlation
between Chr17qB3 amplifi cation and VEGF-A protein levels
in both tumor extracts and serum ( Fig. 1E and Supplemen-
tary Fig. S2B). Furthermore, double immunostaining for
VEGF-A and E-cadherin in Amp pos tumors showed that the
tumor cells are indeed the main origin of VEGF-A in these
tumors ( Fig. 1F ). Thus, amplifi cation of murine Chr17qB3 is
a recurrent event in HCC associated with an elevated expres-
sion of several resident genes, including Vegfa .
Amp pos Tumors Display Distinct Histologic Features
Tumor cell proliferation is a strong and consistent marker
of poor prognosis in human HCC ( 34 ). Bromodeoxyuridine
(BrdUrd ) immunostaining revealed that Amp pos mouse HCCs
displayed a 2-fold higher proliferation index compared with
Amp neg tumors ( Fig. 2A and B , top). No differences were
noted in apoptosis (Supplementary Fig. S2C) or in the pres-
ence of neutrophils or fi broblasts (data not shown). Other
histologic features, differing between Amp pos and Amp neg
mouse HCCs, include a 6-fold higher vessel density ( Fig.
2A and B , middle) and 4-fold higher macrophage content
( Fig. 2A and B , bottom). Moreover, several signature genes
of tumor-associated macrophages (TAM), including those
encoding Arginase 1, TGFβ, and YM1 ( 35, 36 ), were elevated
in the Amp pos group ( Fig. 2C ), whereas markers of classically
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
732 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org
Horwitz et al.RESEARCH ARTICLE
A B
n.s.
WT
liver
Ampneg
tumor
Amppos
tumor
***
** ***
*** ***
Cul9
D E
0
1
2
3
4
5
6
7
1 2 1 2 3 4 1 2 3 4
Vegfa mRNA (qPCR)
VEGF-A protein (ELISA)
Fo
ld in
du
ctio
n (
qP
CR
) p
g/µ
g p
rote
in (
EL
ISA
)
Mdr2 –/– tumors
C
Normal Polysomic Amplified
Murine HCCs
Tumor #1 Tumor #2
Tumor #3
Human HCCs
Tumor #4
Am
pneg
Am
ppos
Mbp fro
m c
hro
mosom
e 1
7 s
tart
Murine Amppos HCCs
43.3
43.8
44.4
45.0
45.745.9
46.16
46.4
46.9
48.5
46.15–46.17Vegfa
45.82–45.84Mrpl14
45.78–45.82Tmem63b
45.77–45.79Capn11
46.39–46.41Tjap1
46.33–46.37Xpo5
45.71–45.71Hsp90ab1
46.24–46.26Mrps18a
45.69–45.70Nfkbie
46.43–46.44Egfl9
45.64–45.66Aars2
44.36–44.41Cdc5L
46.63–46.68Parc
46.86–46.87Gnmt47.4
47.9
Amplified
Nonamplified
Minimal amplified region
F Hoechst E-cadherin VEGF-A Merge
Am
pp
os t
um
or
10
8
6
4
2
0
5
4
3
2
1
0
Cdc5L
Tjap1 Xpo5
Hsp90ab1 Vegfa
6
4
2
0
Rela
tive e
xpre
ssio
n
40
30
20
10
0
8
6
4
2
0
6
4
2
0
Rela
tive e
xpre
ssio
n
Rela
tive e
xpre
ssio
n
Rela
tive e
xpre
ssio
n
Rela
tive e
xpre
ssio
n
Rela
tive e
xpre
ssio
n
WT
liver
Ampneg
tumor
Amppos
tumor
WT
liver
Ampneg
tumor
Amppos
tumor
WT
liver
Ampneg
tumor
Amppos
tumor
WT
liver
Ampneg
tumor
Amppos
tumor
WT
liver
Ampneg
tumor
Amppos
tumor
WT
liverAmpneg Amppos
Figure 1. A recurrent gain in the VEGFA locus identifying a molecularly distinct tumor subpopulation. A, representative photomicrographs of CISH using probes specifi c for the murine Chr17qB3. B, DNA qPCR analysis using primers specifi c for different loci on the qB3 arm of chromosome 17. Each vertical line represents a single Amp pos tumor. The thin line represents nonamplifi ed regions, and the thick line represents amplifi ed regions. The list includes several of the residing genes (a full list is available as Supplementary Data). C, representative photomicrographs of FISH of human HCC using Chr6p12 probe (red) and chromosome 6 centromere probe (green). D, qPCR analysis of the mRNA levels of murine genes encoded on the amplifi ed region. Each dot represents a different tumor. The cross line signifi es geometric mean (n.s., not signifi cant; **, P < 0.01; ***, P < 0.0001). E, qPCR and ELISA analyses of VEGF-A performed on extracts of wild-type (WT) livers, Amp neg , and Amp pos Mdr2 −/− tumors in matching pairs show a correlation between the increase in mRNA and protein levels. F, confocal microscopy images of an Mdr2 −/− Amp pos tumor immunostained for E-cadherin and VEGF-A. Hoechst 33342 marks nuclei. Scale bar, 20 μm.
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
JUNE 2014�CANCER DISCOVERY | 733
VEGFA Amplifi cation in HCC RESEARCH ARTICLE
Figure 2. Amp pos tumors are a distinct tumor subpopulation. A, representative IHC photomicrographs for BrdUrd, vWF, and F4/80. Scale bar, 50 μm (BrdUrd) and 100 μm (vWF and F4/80). B, IHCs were quantifi ed using automated image analysis ( n ≥ 7; *, P < 0.01; **, P < 0.05). C, qPCR analysis of tumor-associated (protumorigenic) and classically activated (antitumorigenic) macrophage markers. Each dot represents a different tumor. The cross line signifi es geometric mean (n.s., not signifi cant; **, P < 0.01; ***, P < 0.001). D, immunofl uorescent stain for MRC1 on Amp neg and Amp pos tumors. Scale bar, 40 μm. E, quantifi cation of MRC1 immunofl uorescence. Bars represent geometric mean; **, P < 0.01.
B
C
n.s. n.s. n.s.
Antitu
morigenic
mark
ers
***** **
Pro
tum
origenic
mark
ers
Ampneg tumor Amppos tumor
Amppos tumor
Ampneg tumor
WT liver
Ampneg tumor Amppos tumor
Brd
Urd
0
5
10
15
20
vWF
Perc
enta
ge p
ositiv
e n
ucle
i
** BrdUrd
vW
F
0
2
4
6
8
10
12
14
Perc
enta
ge p
ositiv
e a
rea
vWF
*
F4/8
0
0
2
4
6
8
10
12
14
Perc
enta
ge p
ositiv
e a
rea
*
F4/80
A
D
MR
C1
E MRC1
**
1.5
Arg1
Nos2 Cxcl10
Tgfb Ym1
Tnfa
8 40
30
20
10
0
6
4
2
0
1.0
0.5
Rela
tive
expre
ssio
nR
ela
tive
expre
ssio
n
Rela
tive
expre
ssio
n
Rela
tive
expre
ssio
n
Perc
enta
ge p
ositiv
e a
rea
Rela
tive
expre
ssio
n
Rela
tive
expre
ssio
n
0.0
600 50
40
30
20
10
0
15
10
5
0
60
40
20
0
40020015
10
5
0
WT
liver
Ampneg
tumor
Amppos
tumor WT
liver
Ampneg
tumor
Amppos
tumor
WT
liver
Ampneg
tumor
Amppos
tumor
WT
liver
Ampneg
tumor
Amppos
tumor
WT
liver
Ampneg
tumor
Amppos
tumor
Ampneg
Amppos
WT
liver
Ampneg
tumor
Amppos
tumor
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
734 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org
Horwitz et al.RESEARCH ARTICLE
activated macrophages, including TNFα, inducible nitric
oxide synthase (iNOS ), CXCL10, and IL12p40, were similar
in both tumor groups ( Fig. 2C and Supplementary Fig. S3A).
Along with these, immunofl uorescent staining for the protu-
morigenic macrophage marker MRC1 revealed a higher pres-
ence of MRC1-expressing cells in Amp pos tumors ( Fig. 2D and
E ). Of note, VEGF-A was shown to act as a chemoattractant
for naïve myeloid cells, which facilitate the generation of new
blood vessels ( 37 ). Together, these data signify Amp pos tumors
as a distinct subgroup of HCCs characterized by enhanced
presence of specifi c microenvironmental components and
increased proliferation rate.
Histologic analysis of human HCCs revealed several char-
acteristic traits of HCCs harboring VEGFA gains. A higher
incidence of vascular invasion was found in the Amp pos
group (9 of 20 Amp pos tumors vs. 1 of 20 Amp neg tumors;
P < 0.01; Supplementary Table S4). A lower incidence of fi brosis
within tumor tissue was found in the Amp pos group as well. Few
additional traits nearly reaching statistical signifi cance were
identifi ed (Supplementary Table S4). We found no differences
in several clinical characteristics of HCC including underlying
disease, gender, or tumor size (Supplementary Fig. S4A–S4C).
Altogether, these results indicate that in both murine Mdr2− /−
and human HCCs, tumors that harbor genomic gains in the
VEGFA locus are distinct from those that do not.
Macrophage–Tumor Cell Cross-talk in Amp pos Tumors
Previous studies have delineated hepatocyte–endothelial
cross-talk taking place in non-neoplastic liver, wherein VEGF-A
stimulates endothelial cells to secrete several mitogens includ-
ing hepatocyte growth factor ( HGF; refs. 24, 25 ). We hypothe-
sized that Amp pos HCCs exploit this interaction for promoting
tumor cell proliferation. Following this notion, we detected a
3-fold elevation of Hgf mRNA levels in Amp pos versus Amp neg
mouse tumors ( Fig. 3A ). We did not fi nd signifi cant changes
in other angiocrine-produced molecules ( 24, 25 )—Wnt2, IL6,
and heparin binding EGF-like growth factor (HB-EGF; data
not shown). Immunostaining detected HGF expression only
in Amp pos tumors, exclusively in the non-neoplastic stromal
cells ( Fig. 3B ). Immunofl uorescent staining for von Willebrand
Factor (vWF), F4/80, and HGF suggested that macrophages are
the major cell type expressing HGF ( Fig. 3C ).
To understand the VEGF-A–HGF relationship, we isolated
hepatocytes and macrophages from Mdr2 −/− livers at the age
of 8 months ( Fig. 3D and Supplementary Fig. S3B), a time
point signifi ed by marked dysplasia, yet no overt HCC for-
mation ( 27 ). mRNA profi ling of these fractions showed that
the genes encoding VEGFRs (FLT1 and KDR) and corecep-
tors [Neuropilin (Nrp)1 and 2] were higher in macrophages
whereas the HGF receptor (c-MET) was more abundant in
hepatocytes ( Fig. 3E ). This aligns with previous work showing
that hepatocytes are inert to direct activation with VEGF-A
( 24 ). Immunostaining for KDR in Amp pos tumors demon-
strated that its expression in these tumors was restricted to
non-neoplastic cells ( Fig. 3F ). This correlated with a modest
increase in mRNA levels of both Kdr and Flt1 in total tumor
lysates, which was comparable with the increase in mRNA
levels of recruited macrophage and endothelial markers Msr1
and Cd105 , respectively ( Fig. 3G ). Recapitulating this interac-
tion in vitro , we treated peritoneal macrophages with recom-
binant VEGF-A and detected an increase in Hgf mRNA levels
( Fig. 3H ). This raises the possibility that VEGF-A in Amp pos
tumors does not provide autocrine signals to hepatocytes,
but rather acts through manipulation of the microenviron-
ment to induce HGF secretion.
VEGF-A Increases Cellular Proliferation in HCC To prove the functional role of VEGFA in this genomic ampli-
fi cation, we set out to inhibit VEGF-A in Mdr2 −/− tumors. To
this end, we injected intravenously adenoviral vectors encoding
GFP alone or GFP and the soluble VEGFR1 (sFLT), a potent
inhibitor of VEGF-A ( 38 ), into 56 tumor-harboring Mdr2− /−
mice of ages 14 to 18 months and sacrifi ced them 10 days fol-
lowing injection (Supplementary Fig. S5A). Vegfa amplifi cation
status was determined after sacrifi ce by both DNA qPCR and
Vegfa mRNA expression ( Fig. 4C and data not shown). Liver
damage, measured through plasma aspartate aminotransferase
(AST) activity, was similar in all groups (Supplementary Fig.
S5B). Immunostaining for BrdUrd, Ki67, and phosphorylated
histone H3 (pHH3) revealed that blocking VEGF-A markedly
inhibited tumor cell proliferation in Amp pos tumors, but not
in Amp neg ones ( Fig. 4A and B and Supplementary Fig. S5C
and S5D). This decrease in proliferation was accompanied by
reduced Hgf mRNA levels ( Fig. 4C ). Treatment with adenovi-
rus encoding GFP alone did not induce any change in either
group. Macrophage infi ltration and protumorigenic macro-
phage expression profi le did not decrease following the sFLT
treatment (Supplementary Fig. S5C and S5D and data not
shown). Macroscopic and histologic analyses revealed multiple
foci of coagulative necrosis only in sFLT-treated Amp pos tumors
(3 of 6 vs. 0 of 10 in Amp neg ; P < 0.05; Fig. 4D ). In these specifi c
tumors, we also found an elevation of the hypoxia-inducible
factor-1α (HIF1α) target genes Glut1 and Pgk1 ( Fig. 4C ), indica-
tive of tissue hypoxia. Immunostaining for vWF revealed a
trend of decrease in vasculature only in VEGF-A–blocked
Amp pos tumors, particularly in the hypoxic tumors (Supple-
mentary Fig. S5C and S5D). These data denote Amp pos tumors
as hypersensitive to direct inhibition of VEGF-A.
Overexpression of VEGF-A in HCC Cells Increases Proliferation Only In Vivo
To further substantiate the tumor–stroma relationship with
respect to VEGF amplifi cation, in particular hepatocyte VEGF-
A eliciting a macrophage HGF–heterotypic circuit in tumor
growth, we injected human Hep3B HCC cells transduced with
a lentivector overexpressing human VEGF-A into immuno-
defi cient mice (Supplementary Fig. S6A). The in vivo growth
rate of VEGF-A–overexpressing cells was higher than control
vector–transduced cells ( Fig. 5A and Supplementary Fig. S6B).
This correlated with higher proliferation rate and increased
HGF expression ( Fig. 5B–D and Supplementary Fig. S6C and
S6D), phenocopying the Amp pos Mdr2 −/− HCCs. Supporting
the non–cell-autonomous role, VEGF-A overexpression did
not affect the in vitro growth rate of Hep3B cells ( Fig. 5E ). We
also found no difference in expression of the hypoxia markers
prolyl hydroxylase domain 3 (PHD3) and lactate dehydroge-
nase A (LDHA) in these xenografts, lending further support to
a nonangiogenic role for VEGF-A in HCC (Supplementary Fig.
S6E). Immunofl uorescence confi rmed VEGF-A expression in
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
JUNE 2014�CANCER DISCOVERY | 735
VEGFA Amplifi cation in HCC RESEARCH ARTICLE
Figure 3. A macrophage-tumor cell cross-talk within Amp pos tumors. A, qPCR analysis of Hgf mRNA in WT livers, Amp neg , and Amp pos tumors. The cross line signi-fi es geometric mean (**, P < 0.001). B, representative photomicrographs of IHC for HGF on Amp neg and Amp pos tumors. Scale bar, 100 μm. C, representative confocal microscopy images of Amp pos tumor immunostained for vWF, F4/80, and HGF. Hoechst 33342 marks nuclei. Scale bar, 40 μm. D, representative cytospin preparations of macrophage and hepatocyte fractions from Mdr2 −/− liv-ers. E, qPCR analysis of hepatocyte and macrophage fractions ( n = 11 different mice), isolated from livers of Mdr2 −/− mice. Bars represent geometric mean (**, P < 0.001; ***, P < 0.0001). F, representa-tive photomicrograph of IHC for the VEGFR KDR in Amp neg and Amp pos tumors. Note that the expression of KDR is confi ned to endothelial and stromal cells. Scale bar, 100 μm. G, mRNA qPCR analysisfor the VEGFRs Kdr and Flt1 and the macrophage and endothelium markers Msr1 and Cd105 on tissue lysates from the indicated groups. Each dot represents a different tumor. The cross line represents geo-metric mean (***, P < 0.0001). H, peritoneal macrophages were cultured under serum-free conditions followed by 8 hours of exposure to recombinant murine VEGF-A (100 ng/mL). Hgf expression was measured by qPCR. Bars represent geometric mean; n ≥ 3; *, P < 0.05.
C
B
HG
F
**
D
FE
KD
R
**
***
0
2
4
6
8
10
12
14
16
18
Flt1 Kdr Nrp1 Nrp2 Met
Rela
tive e
xpre
ssio
n
Hepatocytes
Macrophages **
**
**
***
Kdr
***
Flt1
***
Msr1
***
Cd105
A
G
H&
E
H
*
Hgf
Peritoneal
macrophages
vWF F4/80 HGF
Macrophages Hepatocytes
Merge
Ampneg tumor
Ampneg
Amppos tumor
Amppos
Am
pp
os tu
mo
r
WT
Hgf
6
4
2
Rela
tive e
xpre
ssio
n
0
liver
Ampneg
tumor
Amppos
tumor
3 52.5
2.0
1.5
1.0
0.5
0.0NT 8h rVEGF-A
4
3
2
1
0
2
1
Rela
tive e
xpre
ssio
nR
ela
tive e
xpre
ssio
n
Rela
tive e
xpre
ssio
nR
ela
tive e
xpre
ssio
n
Rela
tive e
xpre
ssio
n
0
10 3
2
1
0
8
6
4
2
0
WT
liver
Ampneg
tumor
Amppos
tumor
WT
liver
Ampneg
tumor
Amppos
tumor
WT
liver
Ampneg
tumor
Amppos
tumor
WT
liver
Ampneg
tumor
Amppos
tumor
transduced tumor cells and revealed HGF expression in mac-
rophages (Supplementary Fig. S7A).
Next, we generated single-cell suspensions from VEGF-A–
overexpressing or control Hep3B ZsGreen-labeled xenografts
and isolated macrophages (ZsGreen − CD45 + F4/80 + ) and endo-
thelial cells (ZsGreen − CD45 − Meca32 + ) using FACS sorting.
qPCR analysis of the macrophage-specifi c and endothelium-
specifi c genes, Msr1 and Cd105 , respectively, confi rmed
successful separation of cell populations (Supplementary
Fig. S7B). Although VEGF-A and control tumor groups
did not show signifi cant differences in the TAM genes Tgfb ,
Ym1 , Tnfa , and Nos2 (Supplementary Fig. S7C), we found
a 2-fold increase in Hgf expression in macrophages, but
not endothelial cells, isolated from VEGF-A–overexpress-
ing tumors ( Fig. 5F ). Thus, VEGF-A overexpression by HCC
cells is suffi cient to induce upregulation of HGF, mostly in
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
736 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org
Horwitz et al.RESEARCH ARTICLE
Figure 4. VEGF-A inhibition impedes proliferation in Amp pos tumors. Mdr2 −/− mice were treated with adenovectors expressing either GFP alone or GFP and sFLT for 10 days. A, representative photomicrographs of IHC for BrdUrd. Tumor-infi ltrating cells remain proliferative. Scale bar, 100 μm. B, BrdUrd immunostaining was quantifi ed using automated image analysis ( n ≥ 6; *, P < 0.05). C, mRNA qPCR analysis of the indicated genes in Amp neg and Amp pos tumors treated with the indicated adenovectors. Each dot represents a different tumor. The cross line signifi es geometric mean (***, P < 0.0001). D, left, histologic section stained with H&E depicting necrosis, representing three out of the six sFLT-treated Amp pos tumors. Scale bar, 500 μm. Right, a macro-scopic picture of a tumor with hemorrhagic necrosis. Scale bar, 0.5 cm.
C D
B
***
0
1
2
3
4
5
Brd
Urd
Ampneg
GFP
Ampneg
sFLT
Amppos
GFP
Amppos
sFLT
BrdUrd
Pe
rce
nta
ge
po
sitiv
e n
ucle
i
*
A
8
30
20
10
0
5
4
3
2
1
0
6
4
2
0
Rela
tive e
xpre
ssio
n
8
6
4
2
0
Re
lative e
xpre
ssio
n
Re
lative e
xpre
ssio
n
Rela
tive e
xpre
ssio
n
Ampneg
sFLT
Ampneg
GFP
Amppos
GFPAmp
pos
sFLTAmp
neg
sFLT
Ampneg
GFP
Amppos
GFP
Amppos
sFLT
Ampneg
sFLT
Ampneg
GFP
Amppos
GFP
Amppos
sFLT
Ampneg
sFLT
Ampneg
GFP
Amppos
GFP
Amppos
sFLT
Vegfa
Glut1 Pgk1
Hgf
Ampneg GFP Ampneg sFLT Amppos GFP Amppos sFLT
H&
E
Amppos sFLT
Figure 5. VEGF-A overexpression enhances tumor cell proliferation. Immunodefi cient mice were subcutaneously injected with Hep3B cells transduced with control vector or with a human VEGF-A vector. A, growth curve of xenografts transduced with control vector (black line) or with VEGF-A lentivec-tor (gray line). Tumor volumes were measured topically with a caliper (*, P < 0.05; **, P < 0.01). B, representative photomicrographs of IHC for BrdUrd in control or VEGF-A lentivector-transduced xenografts. Scale bar, 100 μm. C, BrdUrd immunostaining was quantifi ed using automated image analysis ( n ≥ 3; *, P < 0.05; a representative experiment of two performed is shown). D, mRNA qPCR analysis in control or VEGF-A lentivector-transduced xenografts. Bars represent geometric mean ( n ≥ 4; *, P < 0.05; **, P < 0.01). E, XTT in vitro proliferation assay of the indicated lentivector-transduced cultured cells. F, expression of Hgf in macrophage and endothelial fractions from tumors overexpressing VEGF-A and controls determined by qPCR. Bars represent geometric mean; n ≥ 3; *, P < 0.05; ND, not detected—amplifi cation did not occur in any of the sample’s wells.
D
B
E
A C
F*
ND ND
Mac. End. Mac. End.
Control
tumor
VEGF-A
tumor
Control VEGF-A
Brd
Urd
*Control
VEGF-A
Control
VEGF-A
2,000
1,800
1,600
1,400
1,200
1,000
800
600
400
200
0
6 1.9
Control
VEGF-A
BrdUrd
35
30
25
20
15
10
5
0
Pe
rce
nta
ge
po
sitiv
e n
ucle
i
Hgf
2.5
2.0
1.5
1.0
0.5
0.0
1.7
1.5
1.3
1.1
0.9
0.7
0.50 1 2 3
Days in culture
4
Control
VEGF-A
5 6
Tum
or
volu
me
(m
m3)
Re
lative
exp
ressio
n
Ab
so
rba
nce
(4
50
nm
)
Re
lative
exp
ressio
n
5
4
3
2
1
0
0 10
***
**
*
*
**
Days
hVEGFA mHgf
20 30
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
JUNE 2014�CANCER DISCOVERY | 737
VEGFA Amplifi cation in HCC RESEARCH ARTICLE
macrophages, and leads to increased proliferation of tumor
cells.
Profi ling Proangiogenic-Factor Expression in Mdr2 −/− HCC
As Amp neg tumors did not respond to sFLT treatment, we
profi led other proangiogenic factors that can support tumor
vascularization. mRNA qPCR analysis of the genes encod-
ing angiopoietin 1 and 2, angiopoietin like 2, FGF 1 and 2,
platelet-derived growth factor (PDGF)-A, -B, and -C, placental
growth factor (PLGF) , and VEGF-B revealed that several of
these factors were overexpressed in Mdr2 −/− HCCs compared
with normal livers, irrespective of the amplicon status. Nota-
bly, Pdgfc levels were signifi cantly higher (2.4-fold) in Amp neg
compared with Amp pos tumors ( Fig. 6 ). This is in line with
a previous report showing that PDGF-C can promote ang-
iogenesis in a VEGF-A–independent manner ( 39 ) and could
provide a plausible explanation for the lack of effect on vessel
density in Amp neg tumors in response to sFLT.
Murine Amp pos Tumors Are Uniquely Sensitive to Sorafenib
Sorafenib is the only systemic drug showing a clinical
advantage in patients with advanced HCC who are not eligi-
ble for local therapies and is a fi rst-line treatment for these
patients ( 3 ). As sorafenib inhibits VEGFRs and BRAF—a
downstream effector of both VEGFRs and the HGF receptor
c-MET ( 21 , 40 , 41 )—we tested whether sorafenib may have a
selective advantage in Amp pos tumors. We treated 58 Mdr2 −/−
mice ages 14 to 18 months with sorafenib or vehicle alone
for 3 days, after which they were sacrifi ced and tumor tissue
was analyzed (experimental design depicted in Supplemen-
tary Fig. S5A). Amplifi cation status was assessed after sac-
rifi ce by DNA qPCR and verifi ed by Vegfa mRNA expression
( Fig. 7C and data not shown). Immunostaining for BrdUrd
and pHH3 demonstrated decreased proliferation in soraf-
enib-treated mice in Amp pos but not Amp neg HCCs ( Fig. 7A
and B and Supplementary Fig. S8A and S8B). Similar to
VEGF-A inhibition, Hgf levels were decreased only in soraf-
enib-treated Amp pos tumors ( Fig. 7C ). Although no effects
were observed on tumor macrophage density (Supplemen-
tary Fig. S8A and S8B), expression of the TAM marker TGFβ
was decreased in sorafenib-treated Amp pos tumors (Sup-
plementary Fig. S8C). Blood vessel density changes or signs
of hypoxia were absent (Supplementary Fig. S8A–S8C),
possibly due to the short treatment duration, implying
that the early inhibitory effect of sorafenib in VEGF-A–
amplifi ed tumors may be independent of angiogenesis.
Figure 6. Angiogenic factors elevated in murine Mdr2 −/− HCC. mRNA qPCR analysis of Mdr2 −/− tumors for the indicated angiogenesis regulators. Values shown are fold over normal livers’ average. Each dot represents a different tumor. The cross line signifi es geometric mean, signifi cance marks immedi-ately above each group refer to comparison with normal livers. Topmost signifi cance marks represent the comparison between Amp neg and Amp pos tumors ( n = 4 normal liver; n ≥ 6 in tumor groups; n.s., not signifi cant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). PDGF, platelet-derived growth factor.
Angpt1 Angpt2 Angptl2 Fgf1 Fgf2
n.s.
50
40
30
20
10
5
Rela
tive to n
orm
al liv
er
Rela
tive to n
orm
al liv
er
4
3
2
1
0
n.s.n.s.
*
*n.s.
n.s.
n.s.n.s.n.s.
n.s.n.s.
n.s.
*****
Pdgfa Pdgfb Pdgfc Pgf Vegfb
n.s.
****
n.s.
n.s.* * n.s.*
n.s.
n.s.n.s.
n.s.
n.s.*
Ampneg Amppos Ampneg Amppos Ampneg Amppos Ampneg Amppos Ampneg Amppos
Ampneg Amppos Ampneg Amppos Ampneg Amppos Ampneg Amppos Ampneg Amppos
8070605040302010
5
4
3
2
1
0
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
738 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org
Horwitz et al.RESEARCH ARTICLE
Figure 7. Amp pos tumors are uniquely sensitive to sorafenib. A, representative BrdUrd immunostains. Scale bar, 100 μm. B, BrdUrd immunostaining was quantifi ed using automated image analysis ( n ≥ 6; *, P < 0.05). C, qPCR analysis of Amp neg and Amp pos tumors treated as indicated. Each dot represents a different tumor. The cross line signifi es geometric mean (n.s., not signifi cant; *, P < 0.01). D, growth curves of xenografts transduced with control (dashed lines) or VEGF-A (solid lines) lentivectors, treated daily with sorafenib (gray lines) or nontreated (NT, black lines). Tumor volumes were measured with a caliper ( n ≥ 6; *, P < 0.05). E, Kaplan–Meier curves showing survival of resected HCC patients negative ( n = 96) or positive ( n = 14) for VEGFA gain (p log-rank > 0.05). F, Kaplan–Meier curves showing survival of patients with HCC treated with sorafenib, negative ( n = 47, 10 months median) or positive for VEGFA gain ( n = 7; median undefi ned; plog-rank = 0.029). G and H, genomic gains in VEGFA promote tumorigenesis through the microenvironment. G, an increase in VEGFA gene copy number in liver tumor cells leads to elevated VEGF-A secretion. VEGF-A modulates the tumor microenvironment in favor of tumor cell growth through several modes: (i) recruitment of TAMs expressing the mitogen HGF and (ii) activating the liver endothelium to secrete angiocrine factors and enhance the tumor blood supply. H, inhibition of VEGF-A through soluble receptor or sorafenib results in decreased HGF signaling and blood supply, impeding tumor growth.
Resected patients treated with sorafenib
B
00.20.40.60.8
11.21.41.61.8
2 *
Brd
Urd
Ampneg
vehicle
Ampneg
sorafenib
Amppos
vehicle
Amppos
sorafenib
Pe
rce
nta
ge
po
sitiv
e n
ucle
i
BrdUrd
Ampneg
Veh
Ampneg
Sor
Amppos
Veh
Amppos
Sor
C
*
Hgf
0
1
2
3
4
Rela
tive e
xpre
ssio
n
Vegfa
0
2
4
6
8
10
Rela
tive e
xpre
ssio
n
n.s.
A
Resected HCC patients
Perc
enta
ge s
urv
ival
VEGFA gains
Normal VEGFA
VEGFA gains
Normal VEGFA
F E
H G
Daily treatment period
Tum
or
volu
me (
mm
2)
D 1,500Control - NT
Control - Sorafenib
VEGFA - NT
VEGFA - Sorafenib1,000
500
00
100
50
0
0 1,000 2,000
Days
3,000 4,000 5,000 00
Perc
enta
ge s
urv
ival
100
50
Plogrank = 0.029
500 1,000
Days
1,500 2,000
5 10
Days
15 20
*
*
Ampneg
vehicle
Ampneg
sorafenib
Amppos
vehicle
Amppos
sorafenib
Ampneg
vehicle
Ampneg
sorafenib
Amppos
vehicle
Amppos
sorafenib
VEGFA–amplified
malignant hepatocyte
Protumorigenic
macrophages
Angiocrine
factorsHGF
VEGF-A
Activated
endothelial
cells
VEGF-A
VEGF receptors
VEGF genomic region
HGF
C-Met
VEGFA–amplified
malignant hepatocyte
Decrease in mitogensAngiocrine
factorsHGF
VEGF-A
Decreased
vascularity
Soluble VEGF receptor
Sorafenib
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
JUNE 2014�CANCER DISCOVERY | 739
VEGFA Amplifi cation in HCC RESEARCH ARTICLE
Amp neg tumors did not show any measurable response to
sorafenib.
In addition, we treated mice bearing Hep3B xenografts,
with or without VEGF overexpression, with sorafenib for 10
days. Similar to the Mdr2− /− HCCs, this treatment markedly
reduced growth, proliferation, and HGF expression in VEGF-
A–overexpressing HCCs but did not affect control HCCs ( Fig.
7D and Supplementary Fig. S9A–S9D). Despite the longer
duration of treatment, we still could not detect changes in
macrophage or blood vessel densities (Supplementary Fig.
S10A–S10C). Of note, a differential response to sorafenib was
not evident in vitro , emphasizing the importance of microen-
vironmental factors (Supplementary Fig. S10D).
Benefi cial Effect of Sorafenib Treatment in Human Patients with HCCs Bearing VEGFA Gains
Noting the predictive potential of Vegfa gains in the mouse
model, we analyzed samples from patients with HCC who
underwent tumor resection. This retrospective cohort was col-
lected from three different centers. To assess the correlation
between VEGFA gain and survival, we analyzed human tumor
samples by FISH ( Fig. 1C ). Survival of patients with HCC who
did not receive sorafenib was independent of VEGFA status
( Fig. 7E ). However, markedly improved survival was seen in
the VEGFA -gain group compared with the non-gain group
in sorafenib-treated patients (indefi nable median survival
from sorafenib treatment start and 11 months, respectively;
p log-rank = 0.029; Fig. 7F ). Taken together, our mouse data
and retrospective analysis of a human cohort imply that
VEGFA gains correlate with a particularly benefi cial response
to sorafenib, and possibly other VEGF-A inhibitors, in HCC.
DISCUSSION Using a mouse model of infl ammation-induced HCC,
we identifi ed and characterized a unique group of HCC in
humans and mice. These tumors are defi ned by genomic gains
of a region encompassing VEGFA , and are distinct in histologic
appearance, rate of proliferation, and microenvironmental
content. We delineated cytokine-based heterotypic cross-talk
between malignant Amp pos hepatocytes and tumor stromal
cells. Importantly, we showed that mouse Amp pos tumors are
uniquely sensitive to VEGF-A inhibition and to sorafenib. A
retrospective analysis of human HCCs indicates that genomic
gains of VEGFA can predict response to sorafenib.
The Mdr2 −/− model of infl ammation-induced HCC yields
primary tumors each holding specifi c genetic changes. There-
fore, we denote it as a sound platform to test the pro-
tumorigenic effects of recurring changes in an unbiased
manner. Moreover, the infl ammatory background in this
model is particularly relevant for studying tumor–microenvi-
ronment interactions in clinically relevant settings. Previous
work showed that systemic elevation of VEGF-A induces
proliferation of normal hepatocytes through factors secreted
from endothelial cells ( 24 ). Although hepatocytes are inert
to VEGF-A, liver sinusoidal cells respond to VEGF-A with
counter-secretion of HGF ( 24 ). Furthermore, in regenerat-
ing livers, hepatocyte proliferation was shown to depend on
VEGF-A–induced expression of HGF from endothelial cells
( 25 ). Here, we show that recurring genomic gains in VEGFA ,
detected in a subset of HCCs, can promote tumor growth
through a similar cell–cell interaction module.
TAMs are key players in tumor progression and are known
to modulate invasion, angiogenesis, immune response, and
metastasis ( 36 ). TAMs are characterized by a specifi c phe-
notype, distinguished by a unique expression signature.
Previous studies have shown that VEGF-A recruits myeloid
cells that play an active part in vessel growth processes ( 37 ,
42 ). In agreement, we observed that Amp pos tumors harbor
higher numbers of TAMs compared with Amp neg tumors and
detected features of protumorigenic macrophages. Our fi nd-
ings therefore raise the interesting possibility that the VEGFA
amplicon contributes to the infl ammatory microenviron-
ment, which supports developing tumors.
Our data suggest that VEGFA genomic gains facilitate
tumor development by several different modes: (i) providing
a microenvironment rich in TAMs; (ii) promoting prolif-
eration via stroma-derived HGF secretion (and possibly other
cytokines as well); and (iii) enhancing tumor angiogenesis.
Interestingly, all these modes entail heterotypic cellular inter-
actions, distinguishing this particular genomic gain from
other studied amplicons ( 43 ). We maintain that the unique
sensitivity of tumors with VEGFA gains to VEGF-A block-
ade stems from these multiple protumorigenic functions of
VEGF-A ( Fig. 7G and H ).
An amplicon spanning VEGFA was noted in several differ-
ent human cancers ( 29–31 , 33 , 43–51 ). A linear correlation
was found between mRNA levels of VEGFA and extent of
amplifi cation in human HCC ( 29 ). Amplifi cations in the
VEGFA locus and juxtaposed regions were associated with
advanced-stage HCC ( 30 ). In colorectal carcinoma and breast
cancer, this amplifi cation was found in correlation with vas-
cular invasion and shorter survival ( 46 , 51 ). Cumulative anal-
ysis of these reported human studies shows that gains and
amplifi cations of VEGFA are found in 7% to 30% of human
HCCs ( 29–33 ). Our interventional studies in the Mdr2 −/−
model indicate that Vegfa is a major driver of this amplicon,
which minimally harbors 53 genes in Mdr2 −/− murine tumors
and 11 genes in humans ( 32 ). Xenograft experiments reveal
that overexpression of VEGF-A in a human HCC cell line is
suffi cient to upregulate HGF expression and increase tumor
cell proliferation in vivo and that the tumor growth advantage
gained through this overexpression can be negated by sora-
fenib. Nevertheless, we cannot completely exclude the contri-
bution of other amplicon genes to tumorigenesis.
The fi rst line of treatment for advanced HCC is the multi-
kinase inhibitor sorafenib, which prolongs median survival
by 10 to 12 weeks ( 3, 4 ). The response to sorafenib seems
to be variable, and treatment is associated with signifi cant
side effects ( 3–8 ). Importantly, there are no clinically applied
biomarkers for predicting sorafenib response in HCC ( 10 ).
Among the multiple targets of sorafenib ( 16 ) are VEGFRs
and BRAF—a downstream effector of both VEGFRs and the
HGF receptor c-MET ( 21 , 40 ). Indeed, in line with our mouse
results, sorafenib treatment in patients led to a decrease in
serum HGF ( 10 ). Unlike most tumors, advanced HCC is
usually diagnosed and treated without obtaining tumor tis-
sue, making it diffi cult to establish tissue-based predictive
biomarkers. On the basis of our small-scale retrospective
study, we show that VEGFA gains may predict response to
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
740 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org
Horwitz et al.RESEARCH ARTICLE
sorafenib in HCC, thus enabling the tailoring of treatment
to only those patients who may benefi t from this side effect–
prone therapy. Notably, the same amplifi cation was also
found in lung, colorectal, bone, and breast cancers ( 44–48 );
thus, it is plausible that similar considerations could be
applied to other tumors harboring VEGFA gains.
METHODS Human Tissue Samples and Tissue Microarray
Human HCC tissues were obtained from resected patients from
the institutes of Basel University Hospital (Basel, Switzerland), Han-
nover Medical School Hospital (Hannover, Germany), and Heidel-
berg University Hospital (Heidelberg, Germany). Clinical information
included age at diagnosis, tumor diameter, gender, and survival time
information. Examination of tumor hematoxylin and eosin (H&E)
sections was performed by an expert liver pathologist (O. Pappo).
Samples from Heidelberg were also reviewed by Heidelberg patholo-
gists (C. Mogler and P. Schirmacher). The study was approved by
each of the institutions’ ethics committees—numbered 206/05
(Heidelberg), 660-2010 (Hannover), and EKBB20 (Basel). Required
cohort size was computed by power analysis to yield a power of at
least 90% with an α value of 0.05. Construction of the tissue micro-
array (TMA) was performed as follows: tissue samples were fi xed
in buffered 10% formaldehyde and embedded in paraffi n. H&E-
stained sections were made from each selected primary block (named
donor blocks) to defi ne representative tissue regions. Tissue cylinders
(0.6 mm in diameter) were then punched from the region of the
donor block with the use of a custom-made precision instrument
(Beecher Instruments). Afterward, tissue cylinders were transferred to
a 25 mm × 35 mm paraffi n block to produce the TMAs. The resulting
TMA block was cut into 3-μm sections that were transferred to glass
slides by use of the Paraffi n Sectioning Aid System (Instrumedics).
Sections from the TMA blocks were used for FISH analysis.
Mice Male and female Mdr2 −/− mice on FVB background were held in
specifi c pathogen-free conditions. Experiments on Mdr2 −/− mice were
performed in cohorts of 10 to 20 mice each time; results show the
combined data from at least four different experiments. Wild-type
(WT) controls were age-matched FVB mice. Mouse body weights dur-
ing the experiments were 30 to 45 g. Sorafenib (Xingcheng Chemp-
harm Co., Ltd.) was administered daily (50 mg/kg) by oral gavage.
Cremophor EL (Sigma)/ethanol/water (1:1:6) was used as a vehicle.
Two hours before sacrifi ce, mice were injected with 10 μL BrdUrd
(Cell Proliferation labeling reagent; Amersham) per 1 g body weight.
Mice were anesthetized with ketamine and xylazine and the liver was
perfused via the heart with PBS–Heparin solution followed by 4%
formaldehyde. Following perfusion, livers were removed and sub-
jected to standard histologic procedures. Xenograft experiments were
performed by subcutaneous injection of transduced Hep3B cells (2.5
× 10 6 ) suspended in 100 μL PBS and 100 μL Matrigel (Becton Dickin-
son) into NOD/SCID mice. Tumor volumes were assessed by external
measurement with calipers. All animal experiments were performed
in accordance with the guidelines of the institutional committee for
the use of animals for research. In all mouse experiments, the different
groups were housed together in the same cages.
Viral Vectors and Cultured Cells Adenoviral vectors encoding GFP or GFP and sFLT—a kind gift
from David Curiel (Washington University, St. Louis, MO) and Yosef
Haviv (Hadassah Hospital, Jerusalem, Israel)—were prepared in GH354
cells using standard procedures. A titer of 10 9 transducing units was
injected into mice tail veins. Mice whose livers did not yield minimal
60% adenovector transduction effi ciency (by tissue staining) were
excluded. Lentiviral-based vectors were prepared by subcloning the
PCR products of the human VEGFA 165 gene [from cDNA of decidual
natural killer (NK ) cells; a kind gift from Ofer Mandelboim, Hebrew
University, Jerusalem, Israel] into the pSC-B plasmid using the Strata-
Clone Kit (Stratagene), subsequently digested with Bam HI and NotI
and subcloned into the self-inactivating lentiviral vector pHAGE (gift
of Gustavo Mostoslavsky, Boston University School of Medicine, Bos-
ton, MA) digested with Bam HI and Not I. Lentivectors were produced
by cotransfection of the backbone vector plasmid with the gag-pol
and pMD.G plasmids and using standard procedures. Hep3B cells
(obtained from the ATCC) were grown in Dulbecco’s Modifi ed Eagle
Medium (DMEM; 10% FBS). Cells were tested free of Mycoplasma
before transduction and injection. Lentivector transduction effi ciency
was assessed by fl uorescent microscopy and was estimated as 80%.
In vitro proliferation was determined through XTT assay (Biological
Industries) using the manufacturer’s protocol. Murine recombinant
VEGF-A (R&D Systems) was used at the concentration of 100 ng/mL.
IHC, Immunofl uorescence, and ELISA Antibodies used for tissue immunostaining throughout the
work were—vWF (dilution at 1:300; Dako), pHH3 (1:800; Upstate),
cleaved caspase-3 (1:200; Cell Signaling Technology), F4/80 (1:300;
Seroteq), HGF (1:100; R&D Systems), BrdUrd (1:200; NeoMarkers),
KDR (1:400; Cell Signaling Technology), Ki67 (1:100; NeoMarkers),
E-cadherin (1:100; Cell Signaling Technology), and VEGF (1× as
supplied; Spring). IHC was performed on 5-μm paraffi n sections.
Antigen retrieval was performed in a decloaking chamber (Biocare
Medical) in citrate buffer for all antibodies except vWF and F4/80,
for which retrieval was performed with pronase (Sigma). Horserad-
ish peroxidase (HRP )–conjugated secondary antibodies for all IHC
antibodies used were Histofi ne (Nichirei Biosciences), except for anti-
mouse–derived antibodies that were detected with Envison (Dako).
3,3′-Diaminobenzidine (DAB; Lab Vision) was used as a chromogen.
Immunohistochemical stainings were quantitated when indicated
using an Ariol SL-50 system (Applied Imaging). For quantifi cation
of nuclear immunostaining, the ki-sight module of the Ariol-SL50
robotic image analysis system was applied. This system designates
classifi ers for positive (red-brown) and negative (azure) nuclei defi ned
by color intensity, size, and shape. Each tumor cell nucleus (distin-
guished by morphology) was designated as either positive or negative
by these parameters. The fraction of positive cells was calculated
from counting at least fi ve randomly selected fi elds in each tumor.
Immunofl uoresence was performed on snap-frozen tissue embed-
ded in OCT gel (Sakura Finetek) and sectioned to 8-μm slices.
Slides were incubated at 37°C and fi xed with both acetone and 4%
paraformaldehyde sequentially. Fluorophore-conjugated secondary
antibodies used were donkey anti-goat Alexa Fluor 647 (Invitro-
gen), donkey anti-rabbit Cy2/Cy5, donkey anti-mouse Cy3, and goat
anti-rat Cy3 (The Jackson Laboratory). Hoechst 33342 was used as
a nuclear marker (Invitrogen). Antibodies used for fl ow FACS sort-
ing were CD45-pacifi c blue, F4/80-PE (both 1:50; BioLegend), and
Meca32-biotin (1:50; BioLegend) used with streptavidin APC-Cy7
(BD Biosciences). Flow cytometry–based cell sorting was performed
in a FACSAria III cell sorter (BD Biosciences). VEGF-A ELISA was
performed using Quantikine mouse ELISA kit (R&D Systems).
DNA In Situ Hybridizations Probes for CISH analysis of mouse tumors were prepared from the
BAC clones RP24-215A3 for the murine Chromosome 17 (BACPAC
resources center). BAC clones were labeled with DIG using Nick-
Translation mix (Roche). Mouse Cot-1 DNA (Invitrogen) and soni-
cated murine genomic DNA were added to the probe for background
block. Tissues were prepared by boiling in pretreatment buffer and
digestion with pepsin (Zymed). Hybridization was performed at 37°C
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
JUNE 2014�CANCER DISCOVERY | 741
VEGFA Amplifi cation in HCC RESEARCH ARTICLE
overnight after denaturation at 95°C for 5 minutes. The SPoT-Light
Detection Kit (Invitrogen) was used for anti-DIG antibody and HRP-
conjugated secondary antibody.
FISH analysis for human HCCs was performed as follows. The
genomic BAC clone RPCIB753M0921Q (imaGENES), which covers the
human VEGFA gene region, was used for preparation of the FISH probe.
BAC-DNA was isolated using the Large-Construct Kit (Qiagen) accord-
ing to the instructions of the manufacturer. Isolated BAC-DNA (1 μg)
was digested with Alu I restriction enzyme (Invitrogen) and labeled with
Cy3-dUTP (GE Healthcare) using the BioPrime Array CGH Kit (Invit-
rogen). The labeling reaction was assessed by NanoDrop. Labeled DNA
was purifi ed with the FISH Tag DNA Kit (Invitrogen). TMAs and whole-
tissue sections were deparaffi nized in xylene for 20 minutes and subse-
quently washed with 100%, 96%, and 70% ethanol followed by a wash
with tap water (2 minutes each step). Slides were air dried at 75°C for 3
minutes. Slides were then boiled in pretreatment buffer [70% formamide,
2× saline-sodium citrate buffer (SSC)] at 100°C for 15 minutes followed
by a wash with tap water. Tissue was then subjected to Proteinase K
(Sigma) treatment at 37°C for 70 minutes followed by a wash in tap
water (2 minutes). Dehydration of slides was performed by serial immer-
sion of slides in 70%, 96%, and 100% ethanol (2 minutes each step). Slides
were then air dried at 75°C for 3 minutes. The FISH probe was applied
and slides were sealed with rubber cement. Following a denaturation
step (10 minutes at 75°C), slides were incubated overnight at 37°C.
Slides were washed in Wash Buffer (2× SSC, 0.3% NP40, pH 7–7.5) and
counterstained with 4′,6-diamidino-2-phenylindole (DAPI) I solution
(1,000 ng/mL; Vysis Abbott Molecular). As reference, a Spectrum Green-
labeled chromosome 6 centromeric probe (Vysis Abbott Molecular) was
used. Images were obtained with a Zeiss fl uorescence microscope using
a 63× objective (Zeiss) and the Axiovision software (Zeiss).
FISH results were evaluated according to (i) absolute VEGFA gene
copy number and chromosome 6 copy number and (ii) VEGFA gene/
chromosome 6 copy number ratio. The following classifi cation was
used: not amplifi ed— VEGFA /Chr6 ratio of less than 1.8; equivocal/
borderline— VEGFA /Chr6 ratio between 1.8 and 2.2; and amplifi ed—
VEGFA /Chr6 ratio higher than 2.2, as proposed by the ASCO/CAP
(American Society of Clinical Oncology/College of American Pathol-
ogists) guidelines for HER2 amplifi cation in breast cancer. High poly-
somy was defi ned as >3.75 copies of the CEP6 probe, low polysomy
was defi ned as cases displaying between 2.26 and 3.75 copies of the
CEP6 probe ( 52, 53 ). All cases displaying either amplifi cation or poly-
somy were collectively defi ned as VEGFA gain. FISH quantifi cation
and classifi cation were done by an expert molecular pathologist who
had no access to the clinical data (L. Tornillo).
aCGH and qPCR Genomic DNA was isolated using the QIAGEN DNAeasy Tissue
Kit. Samples were hybridized to mouse CGH 60-mer oligonucle-
otides microarrays (Agilent Technologies), washed, and scanned
according to the Agilent Technologies’ instructions. Data were ana-
lyzed using Feature Extraction software V8.1 (Agilent), GeneSpring
GX V7.3.1, and CGH Analytics V3.4.27 (Agilent) software. RNA
was extracted from tissues by mechanical grinding in TriReagent
(Sigma) with a Polytron tissue homogenizer (Kinematica) at maxi-
mum speed. cDNA was prepared with Moloney murine leukemia
virus (MMLV) reverse transcriptase (Invitrogen). qPCR analyses were
carried out with SYBR green (Invitrogen) in 7900HT Fast Real-Time
PCR System (Applied Biosystems). Results were analyzed using the
qBase v1.3.5 software. Primer sequences are available in Supplemen-
tary Table S5. In the xenografts experiment, murine Hgf mRNA levels
were assessed with TaqMan probe (Life Sciences). Human hypox-
anthine phosphoribosyltransferase ( HPRT ) and ubiquitin C ( UBC )
were used as reference genes in the xenograft experiment. Hprt and
peptidylprolyl isomerase A ( Ppia ) combined were used as reference
genes in all murine analyses except for the hepatocyte versus mac-
rophage comparison in which Ubc, B2m , and Tbp were additionally
applied. Primers detecting the murine chromosome 17 pericentro-
meric region were used as references in DNA qPCR analyses.
Cell Separation Hepatocytes and macrophages were isolated from Mdr2− /− mice
livers essentially as described by Kamimura and Tsukamoto ( 54 ).
Briefl y, livers were digested enzymatically with pronase and colla-
genase (Sigma) by in situ perfusion. Hepatocytes were isolated by cen-
trifugation at 50 × g for 2 minutes, and after three washes were frozen
immediately in liquid nitrogen for RNA preparation. Nonparenchy-
mal cells were pelleted by centrifugation at 150 × g for 8 minutes, laid
on top of a four-density gradient of Larcoll (Sigma) and centrifuged
at 20,000 rpm at 25°C for 30 minutes using a SW41Ti rotor (Beck-
man). Liver macrophages were recovered from the interface between
8% and 12% Larcoll, washed three times and immediately frozen in
liquid nitrogen. Purity of hepatocytes and macrophage fractions was
determined by H&E staining of cytospin preparations and always
exceeded 90%. Dissociation of cells from tumor xenografts was per-
formed using the gentleMACS dissociator (Miltenyi Biotec) accord-
ing to the manufacturer’s protocol.
Statistical Analysis Data were analyzed using a paired two-tailed Student t test at P <
0.05. Histologic differences were analyzed using the Pearson χ 2 test
at P < 0.05. Data were processed using Microsoft Excel 2007. Graphs
were generated using either GraphPad Prism 5.0 or Excel software.
Kaplan–Meier calculations and graphs were performed in GraphPad
Prism 5.0. Log-rank (Mantel–Cox) was used to determine survival P
value. Throughout the work, error bars represent 1 SEM.
Disclosure of Potential Confl icts of Interest R. Koschny has received travel grants from Bayer Company.
E. Pikarsky has fi led for a provisional patent application based on
this article. No potential confl icts of interest were disclosed by the
other authors.
Authors’ Contributions Conception and design: E. Horwitz, I. Stein, J. Hess, P. Angel,
Y. Ben-Neriah, E. Pikarsky
Development of methodology: E. Horwitz, I. Stein, M. Andreozzi,
R.M. Porat, M. Grunewald
Acquisition of data (provided animals, acquired and managed
patients, provided facilities, etc.): E. Horwitz, I. Stein, J. Nemeth,
A. Shoham, O. Pappo, N. Schweitzer, L. Tornillo, N. Kanarek, L. Quagliata,
F. Zreik, R.M. Porat, R. Finkelstein, R. Koschny, T. Ganten, C. Mogler,
O. Shibolet, K. Breuhahn, M. Grunewald, P. Schirmacher, A. Vogel,
L. Terracciano
Analysis and interpretation of data (e.g., statistical analysis,
biostatistics, computational analysis): E. Horwitz, A. Shoham,
O. Pappo, N. Schweitzer, L. Quagliata, H. Reuter, C. Mogler, O. Shibolet,
J. Hess, A. Vogel, L. Terracciano, P. Angel, Y. Ben-Neriah, E. Pikarsky
Writing, review, and/or revision of the manuscript: E. Horwitz,
I. Stein, N. Schweitzer, R. Koschny, T. Ganten, C. Mogler, O. Shibolet,
J. Hess, P. Schirmacher, P. Angel, Y. Ben-Neriah, E. Pikarsky
Administrative, technical, or material support (i.e., reporting or
organizing data, constructing databases): E. Horwitz, A. Shoham,
N. Kanarek, L. Quagliata, T. Ganten, J. Hess, P. Schirmacher, A. Vogel,
L. Terracciano
Study supervision: Y. Ben-Neriah, E. Pikarsky
Acknowledgments The authors thank Rivka Ben-Sasson, Reba Condioti, Shaffi ka El-
Kawasmi, Etti Avraham, Mohamad Juma’, and Drs. Hila Giladi, Gus-
tavo Mostoslavsky, David Curiel, Yosef Haviv, Shemuel Ben-Sasson,
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
742 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org
Horwitz et al.RESEARCH ARTICLE
and Sharon Elizur for providing expertise and reagents. The authors
also thank Drs. Jacob Hannah, Hidekazu Tsukamoto, Ofer Man-
delboim, Noam Stern-Ginosar, Noa Stanietsky, Rachel Yamin, Seth
Salpeter, Temima Schnitzer-Perlman, Dan Lehmann, Rachel Horwitz,
and Chamutal Gur for expert advice and kind assistance. The authors
are indebted to Dr. Daniel Goldenberg for supplying aged Mdr2 −/−
mice and are grateful to Drs. Christoffer Gebhardt, Robert Goldstein,
Moshe Biton, Zvika Granot, and Tzachi Hagai for fruitful discussions.
Grant Support The research leading to these results has received funding from
the European Research Council under the European Union’s Seventh
Framework Programme (FP/2007-2013)/ERC Grant Agreements
281738 to E. Pikarsky and 294390 to Y. Ben-Neriah; the Dr. Miriam
and Sheldon G. Adelson Medical Research Foundation (AMRF), the
German Research Foundation (DFG, SFB-TR77), the Cooperation
Program in Cancer Research of the Deutsches Krebsforschungszen-
trum (DKFZ), and Israel’s Ministry of Science, Culture and Sport
(MOST) to E. Pikarsky, Y. Ben-Neriah, and P. Angel; the Israel Sci-
ence Foundation (ISF) Centers of Excellence to E. Pikarsky and
Y. Ben-Neriah; and an ISF Project Grant to I. Stein and O. Pappo.
Received October 21, 2013; revised March 26, 2014; accepted
March 26, 2014; published OnlineFirst March 31, 2014.
REFERENCES 1. Farazi PA , DePinho RA . Hepatocellular carcinoma pathogenesis:
from genes to environment . Nat Rev Cancer 2006 ; 6 : 674 – 87 .
2. El-Serag HB . Hepatocellular carcinoma . N Engl J Med 2011 ; 365 :
1118 – 27 .
3. Llovet JM , Ricci S , Mazzaferro V , Hilgard P , Gane E , Blanc JF , et al.
Sorafenib in advanced hepatocellular carcinoma . N Engl J Med
2008 ; 359 : 378 – 90 .
4. Cheng AL , Kang YK , Chen Z , Tsao CJ , Qin S , Kim JS , et al. Effi cacy
and safety of sorafenib in patients in the Asia-Pacifi c region with
advanced hepatocellular carcinoma: a phase III randomised, double-
blind, placebo-controlled trial . Lancet Oncol 2009 ; 10 : 25 – 34 .
5. Iavarone M , Cabibbo G , Piscaglia F , Zavaglia C , Grieco A , Villa
E , et al. Field-practice study of sorafenib therapy for hepatocellu-
lar carcinoma: a prospective multicenter study in Italy . Hepatology
2011 ; 54 : 2055 – 63 .
6. Jia N , Liou I , Halldorson J , Carithers R , Perkins J , Reyes J , et al. Phase I
adjuvant trial of sorafenib in patients with hepatocellular carcinoma
after orthotopic liver transplantation . Anticancer Res 2013 ; 33 : 2797 – 800 .
7. Balsom SM , Li X , Trolli E , Rose J , Bloomston M , Patel T , et al. A
single-institute experience with sorafenib in untreated and previously
treated patients with advanced hepatocellular carcinoma . Oncology
2010 ; 78 : 210 – 2 .
8. Brunocilla PR , Brunello F , Carucci P , Gaia S , Rolle E , Cantamessa
A , et al. Sorafenib in hepatocellular carcinoma: prospective study
on adverse events, quality of life, and related feasibility under daily
conditions . Med Oncol 2013 ; 30 : 345 .
9. Dai X , Schlemmer HP , Schmidt B , Hoh K , Xu K , Ganten TM , et al.
Quantitative therapy response assessment by volumetric iodine-
uptake measurement: initial experience in patients with advanced
hepatocellular carcinoma treated with sorafenib . Eur J Radiol
2013 ; 82 : 327 – 34 .
10. Llovet JM , Pena CE , Lathia CD , Shan M , Meinhardt G , Bruix J , et al.
Plasma biomarkers as predictors of outcome in patients with advanced
hepatocellular carcinoma . Clin Cancer Res 2012 ; 18 : 2290 – 300 .
11. Tsukui Y , Mochizuki H , Hoshino Y , Kawakami S , Kuno T , Fukasawa
Y , et al. Factors contributing to the overall survival in patients with
hepatocellular carcinoma treated by sorafenib . Hepatogastroenterol-
ogy 2012 ; 59 : 2536 – 9 .
12. Strebhardt K , Ullrich A . Paul Ehrlich’s magic bullet concept: 100
years of progress . Nat Rev Cancer 2008 ; 8 : 473 – 80 .
13. Hoshida Y , Toffanin S , Lachenmayer A , Villanueva A , Minguez B ,
Llovet JM . Molecular classifi cation and novel targets in hepatocellular
carcinoma: recent advancements . Semin Liver Dis 2010 ; 30 : 35 – 51 .
14. Piccart-Gebhart MJ , Procter M , Leyland-Jones B , Goldhirsch A , Untch
M , Smith I , et al. Trastuzumab after adjuvant chemotherapy in
HER2-positive breast cancer . N Engl J Med 2005 ; 353 : 1659 – 72 .
15. Karapetis CS , Khambata-Ford S , Jonker DJ , O’Callaghan CJ , Tu D ,
Tebbutt NC , et al. K-ras mutations and benefi t from cetuximab in
advanced colorectal cancer . N Engl J Med 2008 ; 359 : 1757 – 65 .
16. Wilhelm SM , Carter C , Tang L , Wilkie D , McNabola A , Rong H , et al.
BAY 43-9006 exhibits broad spectrum oral antitumor activity and tar-
gets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved
in tumor progression and angiogenesis . Cancer Res 2004 ; 64 : 7099 – 109 .
17. Govindarajan R , Siegel E , Makhoul I , Williamson S . Bevacizumab and
erlotinib in previously untreated inoperable and metastatic hepato-
cellular carcinoma . Am J Clin Oncol 2013 ; 36 : 254 – 7 .
18. Siegel AB , Cohen EI , Ocean A , Lehrer D , Goldenberg A , Knox JJ ,
et al. Phase II trial evaluating the clinical and biologic effects of
bevacizumab in unresectable hepatocellular carcinoma . J Clin Oncol
2008 ; 26 : 2992 – 8 .
19. Thomas MB , Morris JS , Chadha R , Iwasaki M , Kaur H , Lin E , et al.
Phase II trial of the combination of bevacizumab and erlotinib in
patients who have advanced hepatocellular carcinoma . J Clin Oncol
2009 ; 27 : 843 – 50 .
20. Yau T , Wong H , Chan P , Yao TJ , Pang R , Cheung TT , et al. Phase II
study of bevacizumab and erlotinib in the treatment of advanced
hepatocellular carcinoma patients with sorafenib-refractory disease .
Invest New Drugs 2012 ; 30 : 2384 – 90 .
21. Ferrara N . Vascular endothelial growth factor . Arterioscler Thromb
Vasc Biol 2009 ; 29 : 789 – 91 .
22. Lichtenberger BM , Tan PK , Niederleithner H , Ferrara N , Petzelbauer P ,
Sibilia M . Autocrine VEGF signaling synergizes with EGFR in tumor
cells to promote epithelial cancer development . Cell 2010 ; 140 : 268 – 79 .
23. Chatterjee S , Heukamp LC , Siobal M , Schöttle J , Wieczorek C , Peifer
M , et al. Tumor VEGF: VEGFR2 autocrine feed-forward loop triggers
angiogenesis in lung cancer . J Clin Invest 2013 ; 123 : 1732 – 40 .
24. LeCouter J , Moritz DR , Li B , Phillips GL , Liang XH , Gerber HP ,
et al. Angiogenesis-independent endothelial protection of liver: role
of VEGFR-1 . Science 2003 ; 299 : 890 – 3 .
25. Ding BS , Nolan DJ , Butler JM , James D , Babazadeh AO , Rosenwaks
Z , et al. Inductive angiocrine signals from sinusoidal endothelium are
required for liver regeneration . Nature 2010 ; 468 : 310 – 5 .
26. Hernandez-Gea V , Toffanin S , Friedman SL , Llovet JM . Role of the
microenvironment in the pathogenesis and treatment of hepatocel-
lular carcinoma . Gastroenterology 2013 ; 144 : 512 – 27 .
27. Mauad TH , van Nieuwkerk CM , Dingemans KP , Smit JJ , Schinkel AH ,
Notenboom RG , et al. Mice with homozygous disruption of the mdr2
P-glycoprotein gene. A novel animal model for studies of nonsup-
purative infl ammatory cholangitis and hepatocarcinogenesis . Am J
Pathol 1994 ; 145 : 1237 – 45 .
28. Shukla R , Upton KR , Munoz-Lopez M , Gerhardt DJ , Fisher ME ,
Nguyen T , et al. Endogenous retrotransposition activates oncogenic
pathways in hepatocellular carcinoma . Cell 2013 ; 153 : 101 – 11 .
29. Chiang DY , Villanueva A , Hoshida Y , Peix J , Newell P , Minguez B , et al.
Focal gains of VEGFA and molecular classifi cation of hepatocellular
carcinoma . Cancer Res 2008 ; 68 : 6779 – 88 .
30. Chochi Y , Kawauchi S , Nakao M , Furuya T , Hashimoto K , Oga A , et al.
A copy number gain of the 6p arm is linked with advanced hepatocel-
lular carcinoma: an array-based comparative genomic hybridization
study . J Pathol 2009 ; 217 : 677 – 84 .
31. Patil MA , Gutgemann I , Zhang J , Ho C , Cheung ST , Ginzinger D , et al.
Array-based comparative genomic hybridization reveals recurrent
chromosomal aberrations and Jab1 as a potential target for 8q gain in
hepatocellular carcinoma . Carcinogenesis 2005 ; 26 : 2050 – 7 .
32. Beroukhim R , Mermel CH , Porter D , Wei G , Raychaudhuri S , Dono-
van J , et al. The landscape of somatic copy-number alteration across
human cancers . Nature 2010 ; 463 : 899 – 905 .
33. Moinzadeh P , Breuhahn K , Stutzer H , Schirmacher P . Chromosome
alterations in human hepatocellular carcinomas correlate with aetiology
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
JUNE 2014�CANCER DISCOVERY | 743
VEGFA Amplifi cation in HCC RESEARCH ARTICLE
and histological grade—results of an explorative CGH meta-analysis .
Br J Cancer 2005 ; 92 : 935 – 41 .
34. Ouchi K , Sugawara T , Ono H , Fujiya T , Kamiyama Y , Kakugawa Y , et al.
Mitotic index is the best predictive factor for survival of patients with
resected hepatocellular carcinoma . Dig Surg 2000 ; 17 : 42 – 8 .
35. Qian BZ , Pollard JW . Macrophage diversity enhances tumor progres-
sion and metastasis . Cell 2010 ; 141 : 39 – 51 .
36. Sica A , Larghi P , Mancino A , Rubino L , Porta C , Totaro MG , et al.
Macrophage polarization in tumour progression . Semin Cancer Biol
2008 ; 18 : 349 – 55 .
37. Grunewald M , Avraham I , Dor Y , Bachar-Lustig E , Itin A , Jung S , et al.
VEGF-induced adult neovascularization: recruitment, retention, and
role of accessory cells . Cell 2006 ; 124 : 175 – 89 .
38. Mahasreshti PJ , Navarro JG , Kataram M , Wang MH , Carey D , Siegal
GP , et al. Adenovirus-mediated soluble FLT-1 gene therapy for ovar-
ian carcinoma . Clin Cancer Res 2001 ; 7 : 2057 – 66 .
39. Crawford Y , Kasman I , Yu L , Zhong C , Wu X , Modrusan Z , et al.
PDGF-C mediates the angiogenic and tumorigenic properties of
fi broblasts associated with tumors refractory to anti-VEGF treat-
ment . Cancer Cell 2009 ; 15 : 21 – 34 .
40. Trusolino L , Bertotti A , Comoglio PM . MET signalling: principles
and functions in development, organ regeneration and cancer . Nat
Rev Mol Cell Biol 2010 ; 11 : 834 – 48 .
41. Whittaker S , Marais R , Zhu AX . The role of signaling pathways in the
development and treatment of hepatocellular carcinoma . Oncogene
2010 ; 29 : 4989 – 5005 .
42. Wyckoff JB , Wang Y , Lin EY , Li JF , Goswami S , Stanley ER , et al.
Direct visualization of macrophage-assisted tumor cell intravasation
in mammary tumors . Cancer Res 2007 ; 67 : 2649 – 56 .
43. Albertson DG . Gene amplifi cation in cancer . Trends Genet 2006 ; 22 :
447 – 55 .
44. Weir BA , Woo MS , Getz G , Perner S , Ding L , Beroukhim R , et al.
Characterizing the cancer genome in lung adenocarcinoma . Nature
2007 ; 450 : 893 – 8 .
45. Tsafrir D , Bacolod M , Selvanayagam Z , Tsafrir I , Shia J , Zeng Z , et al.
Relationship of gene expression and chromosomal abnormalities in
colorectal cancer . Cancer Res 2006 ; 66 : 2129 – 37 .
46. Vlajnic T , Andreozzi MC , Schneider S , Tornillo L , Karamitopoulou
E , Lugli A , et al. VEGFA gene locus (6p12) amplifi cation identifi es
a small but highly aggressive subgroup of colorectal patients . Mod
Pathol 2011 ; 24 : 1404 – 12 .
47. Horlings HM , Lai C , Nuyten DS , Halfwerk H , Kristel P , van Beers E ,
et al. Integration of DNA copy number alterations and prognostic
gene expression signatures in breast cancer patients . Clin Cancer Res
2010 ; 16 : 651 – 63 .
48. Lau CC , Harris CP , Lu XY , Perlaky L , Gogineni S , Chintagumpala
M , et al. Frequent amplifi cation and rearrangement of chromosomal
bands 6p12-p21 and 17p11.2 in osteosarcoma . Genes Chromosomes
Cancer 2004 ; 39 : 11 – 21 .
49. Andreozzi M , Quagliata L , Gsponer JR , Ruiz C , Vuaroqueaux V ,
Eppenberger-Castori S , et al. VEGFA gene locus analysis across 80
human tumour types reveals gene amplifi cation in several neoplastic
entities . Angiogenesis. 2013 Oct 11 . [Epub ahead of print] .
50. Yang J , Yang D , Sun Y , Sun B , Wang G , Trent JC , et al. Genetic ampli-
fi cation of the vascular endothelial growth factor (VEGF) pathway
genes, including VEGFA, in human osteosarcoma . Cancer 2011 ; 117 :
4925 – 38 .
51. Schneider BP , Gray RJ , Radovich M , Shen F , Vance G , Li L , et al.
Prognostic and predictive value of tumor vascular endothelial growth
factor gene amplifi cation in metastatic breast cancer treated with
paclitaxel with and without bevacizumab; results from ECOG 2100
trial . Clin Cancer Res 2013 ; 19 : 1281 – 9 .
52. Salido M , Tusquets I , Corominas JM , Suarez M , Espinet B , Corzo C ,
et al. Polysomy of chromosome 17 in breast cancer tumors showing
an overexpression of ERBB2: a study of 175 cases using fl uorescence
in situ hybridization and immunohistochemistry . Breast Cancer Res
2005 ; 7 : R267 – 73 .
53. Wolff AC , Hammond ME , Schwartz JN , Hagerty KL , Allred DC ,
Cote RJ , et al. American Society of Clinical Oncology/College of
American Pathologists guideline recommendations for human epi-
dermal growth factor receptor 2 testing in breast cancer . J Clin Oncol
2007 ; 25 : 118 – 45 .
54. Kamimura S , Tsukamoto H . Cytokine gene expression by Kupffer cells
in experimental alcoholic liver disease . Hepatology 1995 ; 22 : 1304 – 9 .
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782
2014;4:730-743. Published OnlineFirst March 31, 2014.Cancer Discovery Elad Horwitz, Ilan Stein, Mariacarla Andreozzi, et al. Are Highly Sensitive to Sorafenib Treatment
-Amplified Hepatocellular CarcinomasVEGFAHuman and Mouse
Updated version
10.1158/2159-8290.CD-13-0782doi:
Access the most recent version of this article at:
Material
Supplementary
http://cancerdiscovery.aacrjournals.org/content/suppl/2014/04/01/2159-8290.CD-13-0782.DC1.html
Access the most recent supplemental material at:
Cited articles
http://cancerdiscovery.aacrjournals.org/content/4/6/730.full.html#ref-list-1
This article cites 53 articles, 15 of which you can access for free at:
Citing articles
http://cancerdiscovery.aacrjournals.org/content/4/6/730.full.html#related-urls
This article has been cited by 5 HighWire-hosted articles. Access the articles at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at
Permissions
To request permission to re-use all or part of this article, contact the AACR Publications Department at
on March 27, 2016. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from
Published OnlineFirst March 31, 2014; DOI: 10.1158/2159-8290.CD-13-0782