-
Gene signatures predictive of response to therapy in NSCLC
Gene expression signatures predictive of bevacizumab/erlotinib
therapeutic benefit in advanced non-squamous non-small cell lung
cancer patients (SAKK 19/05 trial)
Anca Franzini1, Florent Baty1, Ina I. Macovei2, Oliver Dürr3,
Cornelia Droege4, Daniel Betticher5, Bogdan D. Griogriu2, Dirk
Klingbiel6, Francesco Zappa7, and Martin H. Brutsche1
1 Department of Pulmonary Medicine, Cantonal Hospital St.
Gallen, St. Gallen, Switzerland
2 Department of Pulmonary Diseases, University of Medicine and
Pharmacy, Iasi, Romania
3 Institute of Data Analysis and Process Design, Zürich
University of Applied Sciences, Winterthur, Switzerland
4 Männedorf Hospital, Männedorf, Switzerland
5 Cantonal Hospital Fribourg, Fribourg, Switzerland
6 Swiss Group for Clinical Cancer Research (SAKK) Coordinating
Center, Bern, Switzerland
7 Department of Medical Oncology, Clinica Luganese, Lugano,
Switzerland
Corresponding author: Martin H. Brustche, Cantonal Hospital St.
Gallen, 95 Rorschacher Strasse, 9007 St. Gallen, Switzerland.
Phone: 004171-494-1004; Fax: 004171-494-6118; E-mail:
[email protected]
Grant Support
Swiss Cancer League & Swiss Cancer Center
(KLS-2880-02-2012): Martin H Brutsche,
KSSG Medical Research Center (MFZF_2014_001): Anca Franzini,
Romanian–Swiss Research Program (IZERZO_142235/1): Ina I.
Macovei.
Abstract
Purpose: We aimed to identify gene expression signatures
associated with angiogenesis and hypoxia pathways with predictive
value for treatment response to bevacizumab/erlotinib (BE) of
non-squamous advanced NSCLC patients.
Experimental design: Whole genome gene expression profiling was
performed on 42 biopsy samples (from SAKK 19/05 trial) using
Affymetrix exon arrays, and associations with the following
endpoints: time-to-progression (TTP) under therapy, tumor-shrinkage
(TS), and overall survival (OS) were investigated. Next, we
performed gene set enrichment analyses using genes associated with
the angiogenic process and hypoxia response to evaluate their
predictive value for patients’ outcome.
Results: Our analysis revealed that both the angiogenic and
hypoxia response signatures were enriched within the genes
predictive of BE response, TS and OS. Higher gene expression levels
(GELs) of the 10-gene angiogenesis-associated signature and lower
levels of the 10-gene hypoxia response signature predicted improved
TTP under BE, 7.1 months vs. 2.1 months for low vs. high-risk
patients (P = 0.005), and median TTP 6.9 months vs. 2.9 months (P =
0.016), respectively. The hypoxia
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
2
response signature associated with higher TS at 12 weeks and
improved OS (17.8 months vs. 9.9 months for low vs. high risk
patients, P = 0.001). Conclusions: We were able to identify gene
expression signatures derived from the angiogenesis and hypoxia
response pathways with predictive value for clinical outcome in
advanced non-squamous NSCLC patients. This could lead to the
identification of clinically relevant biomarkers, which will allow
for selecting the subset of patients who benefit from the treatment
and predict drug response.
STATEMENT OF TRANSLATIONAL RELEVANCE
Clinical outcome of non-small cell lung cancer (NSCLC) could be
improved by molecular stratification of patients. Antiangiogenic
therapy is approved for treatment of advanced cancers, however
because only a subset of patients treated with angiogenesis
inhibitors show objective clinical response, there is an increased
need for predictive biomarkers. We identified 10-gene signatures
associated with angiogenesis and hypoxia-response pathways, which
have predictive value for response to combined anti-VEGF/anti-EGFR
in non-squamous advanced NSCLC. Subclassification of the patients
using these signatures indicated that patients most likely to
respond (low-risk) showed higher levels of angiogenesis associated
genes mainly involved in maintaining the vascular barrier integrity
and lower levels of hypoxia-response genes. Moreover, they showed
increased percentile tumor shrinkage and improved overall survival.
The inverse trend was observed for the high-risk patients. These
findings open the possibility for clinical use of these signatures
as predictive biomarkers for identifying patients who would benefit
from antiangiogenic therapies.
Introduction
Solid tumor cells in their primary goal to avoid senescence and
achieve uncontrolled
proliferation co-opt neighboring stromal cells to assist them
with the expansion of the
malignant tissue.1, 2 These tumor-associated stromal components
have been demonstrated
to have an important role in tumor growth, disease progression3
as well as in response and
resistance to therapeutic agents.4, 5 Moreover, tumors are
strongly dependent on
angiogenesis, the formation of neovessels from the preexisting
vasculature, to obtain the
nutrients and oxygen essential for their rapid growth.6
Consequently, antiangiogenic
therapeutic strategies were extensively exploited in the last
decade in search for more
efficient cancer treatments.7
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
3
Though multiple therapeutic strategies have been developed
against vascular endothelial
growth factor (VEGF),8 bevacizumab,9 a monoclonal antibody
targeting VEGFA, is the first
approved antiangiogenic drug for clinical use. Bevacizumab is
mostly used in combination
with standard chemotherapy for treatment of metastatic
cancers,10-12 and as single agent in
recurrent glioblastoma.13 However, survival benefits are
observed only in a subset of
patients, as a significant number of patients show only modest
response presumably
because of intrinsic or rapidly acquired resistance to
antiangiogenic therapy.14 One proposed
mechanism of resistance to antiangiogenic agents is the onset of
hypoxia within the tumor
as a result of vessel regression during the course of
antiangiogenic therapy.15, 16 Moreover,
effective inhibition of neovascularization using antiangiogenic
therapy was shown in some
cases to change the phenotype of tumors by increasing their
invasion and metastatic
potential.17 Additionally, significant rates of adverse effects
were reported for patients
receiving anti-VEGF therapy, as well as a mortality rate of 1%,
which was a direct
consequence of bevacizumab administration.18, 19 Thus,
understanding all cascades of
vascular signaling involved in the response to antiangiogenic
therapy and subsequent
resistance is critical to achieve full potential of this
therapeutic approach.
Despite these obvious limitations of antiangiogenic therapy,
because a subset of cancer
patients treated with angiogenesis inhibitors show objective
clinical response, there is an
increased need to identify robust predictive biomarkers,20 which
could allow for selecting
the subgroup of patients who would benefit from the
treatment.
In a recent study, aimed at identifying novel biomarkers for
response to combined anti-
VEGFA/anti-EGFR (epidermal growth factor receptor) therapy in
non-squamous NSCLC by
exploring gene expression at exon-level, we identified EGFR exon
18 as a predictive marker
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
4
for patients with metastatic non-squamous NSCLC who have
received no previous therapy.21
The gene expression profiles obtained from this set of
microarray data are derived, however,
from highly heterogeneous clinical biopsies consisting of both
tumor and activated stromal
cells. In a previous study, Baty et al. revealed that prediction
of survival was independent of
tumor cell content present in each NSCLC biopsy.22 This suggests
a strong predictive
contribution from the tumor microenvironment compartments in
NSCLC. Additionally,
recent studies have demonstrated that tumor microenvironment can
provide independent
and reliable predictors of clinical outcome.23 A key constituent
of the tumor
microenvironment is the blood vasculature, which undergoes
angiogenesis to sustain the
high proliferative rate of the tumor, and is the direct target
of antiangiogenic therapies.
Because there is a high degree of cross-talk between epidermal
growth factor receptor
EGF(R) and VEGF(R) pathways, they have been identified as
potentially synergistic for dual
targeting.24 EGFR pathway is involved in growth factor–induced
angiogenesis,
transcriptionally up-regulating VEGF expression.25 Additionally,
multiple studies
demonstrated that hypoxia can trigger the angiogenic switch in
solid tumors.26 Therefore,
we aimed to investigate whether angiogenesis and
hypoxia-associated gene expression
signatures could predict the combined anti-VEGF/anti-EGFR
treatment response in advanced
non-squamous therapy naїve NSCLC patients unselected for EGFR
mutation status. Our
analysis identified 10-gene angiogenesis-associated and
hypoxia-response signatures
predictive of therapeutic response to bevacizumab/erlotinib (BE)
having time-to-progression
(TTP) and tumor shrinkage (TS) as endpoints. We also identified
for 10-gene signatures with
prognostic value. These signatures hold great potential for
clinical application allowing for
identification of biomarkers, which can identify the patients
most likely/ less likely to
respond to targeted therapy.
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
5
Methods
Study design. Our study is based on clinical bronchoscopic
biopsies available from 42
patients for which genome-wide gene expression was studied in a
microarray platform.
These patients (88% adenocarcinoma, 57% female, 31% never
smoker) were enrolled in the
Swiss Group for Clinical Cancer Research (SAKK) 19/05 phase II
trial.27 Identification of
predictive gene-expression signatures from RNA gene expression
analysis was a predefined
goal of this trial. The detailed clinical information of these
patients was published
previously.21 For these patients with stage IIIB or IV (93%)
non-squamous NSCLC, BE was
used as first-line therapy (independent of the EGFR mutation
status) followed by standard
platinum-based/gemcitabine chemotherapy (CT) after disease
progression, (Figure 1). Time
to disease progression and percentage tumor shrinkage at 12
weeks (assessed by CT scans)
were defined according to RECIST criteria. To confirm that there
was no sample selection
bias for the 42 patients included in our study, we performed
statistical analysis and found no
significant differences between the study group and the patients
with no available biopsies.
The results are summarized in Supplementary Table 1.
Gene expression analysis
Total RNA from 42 bronchoscopic biopsy samples were extracted
using miRNeasy Mini Kit
(Qiagen) according to the manufacturer's recommendations.
Affymetrix Human Exon 1.0 ST
arrays (Affymetrix, Santa Clara, CA, USA) were used for mRNA
hybridization. The gene level
probesets were preprocessed, quality-checked and normalized
using the Robust Multi-array
Average (RMA) procedure.28 Data were expressed as log2 ratio of
fluorescence intensities of
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
6
the sample and the reference, for each element of the array.
Additional experimental details
for this section were previously reported by us.21
Statistical analysis of gene expression.
Survival and time-to-event analysis were performed by applying
univariate Cox proportional
hazards regression and principal component analysis (metagene
approach) according to a
previously described method.29 We built a binary score (low/high
risk) using the median of
the metagene scores. The classification accuracy of our
algorithm was assessed by leave-
one-out cross-validation (LOOCV). Time-to-event and survival
results were displayed using
Kaplan-Meier curves, and log-rank tests are reported.
Hierarchical cluster analysis was
carried out using the Euclidean distance together with the
complete linkage agglomerative
method. The median follow up time was estimated using the
reverse Kaplan-Meier method.
All statistical tests were performed using the R statistical
software version 3.1.0
(http://www.R-project.org). A P value of 0.05 was set as
threshold for significance for all
study outcomes.
Gene set enrichment analysis (GSEA). We performed enrichment
analysis using GSEA v2.1.0
software (http://www.broad.mit.edu/gsea) and GSEA-preranked
function.30 The gene lists
were ranked by using as metric –log10(P) resulted from the
Cox-regression analysis for all
endpoints.
Quantitative real-time PCR analysis. Forty samples of RNA
isolated from non-squamous
NSCLC biopsies and previously analyzed by microarrays were
available for reverse
transcription (RT). RT was performed using QuanTiect® Reverse
Transcription Kit from
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
7
Qiagen starting from 10 ng RNA for each qPCR reaction. The
quantity and integrity of RNAs
were assessed using an UVS-99 micro-volume spectrophotometer
(ACTGene). For this study
we investigated the expression levels of nine
angiogenesis-associated predictive genes. For
RT-qPCR experiments we used predesigned optimized primer sets
(annealing temperature
55 °C) for GPR116, EMCN, ITGA9, GNG11, KDR, PECAM1, S1PR1, JAM2
and RHOJ (QuantiTect
Primer Assay, Qiagen). For sequences please refer to the
Supplementary Table 2.
All samples were processed in duplicate for the qPCR with the
LightCycler®480 SYBR Green I
Master in a 20 µL reaction volume containing 4 µL water, 1 µL
PCR primer, 10 µL Master mix
and 5 µL cDNA (10 ng). Quantitative real-time PCR experiments
were performed on a
LightCycler® 480 II instrument using the initial denaturation at
95 °C for 10 minutes followed
by 45 cycles: 95 °C for 10 sec, 55 °C for 20 sec, 72 °C for 20
sec. Controls containing no
reverse transcriptase were included for each sample. The mRNA
expression levels were
calculated relative to HPRT1 house-keeping gene and the relative
quantification of the gene
expression was performed using 2─ΔCp method.31 The correlation
between the RT-qPCR gene
expression levels (GELs) and microarray GELs was measured by
means of Spearman's
correlation coefficient.
Results
10-gene angiogenesis-associated and hypoxia-response signatures
predict response to BE
therapy. We hypothesized that the genes which could associate
with the response to BE
therapy are genes involved in angiogenic signaling pathways. To
investigate this, we
performed GSEA30 using a core gene signature (43 genes) specific
for angiogenesis
transcriptional program predefined by integrative meta-analysis
of the expression profiles of
over 1,000 primary human cancers.32
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
8
GSEA revealed a significant enrichment of the
angiogenesis-associated genes within the
genes that associate with TTP under BE therapy endpoint (mean
rank = 4246, P=0.004,
Figure 2A). As VEGF expression is directly activated under
hypoxic conditions by the
transcription factor hypoxia inducible factor 1 alpha (HIF1α),
we decided to additionally
investigate hypoxia-response genes by GSEA. For this analysis we
used a previously derived
common hypoxia metagene (51 genes) across cancer types.33 Within
the genes which
associate with TTP under BE therapy, we identified by GSEA a
significant enrichment of the
hypoxia-response genes (mean rank = 4798, P = 0.001, Figure 2B).
The top 12-ranked
angiogenesis-associated and hypoxia-response genes, which
significantly correlate with TTP
under BE therapy are given in Figure 2C and 2D.
We then applied unsupervised hierarchical clustering to the top
10-ranked
angiogenesis-associated genes to investigate whether there are
gene expression patterns
correlating with the BE treatment response. The clustering
output for the angiogenesis-
associated signature is displayed in Figure 3A. We identified
three distinct gene clusters:
Cluster A1 (low risk, 9 patients, 21 %) was characterized by an
increased expression of the
angiogenesis-associated signature and associated with improved
TTP under BE 7.1 months,
95% confidence interval (95% CI): 4.0 ─ ∞; Cluster A2 (high
risk, 12 patients, 29 %) was
characterized by a decreased expression of the
angiogenesis-associated gene signature and
associated with reduced response to BE treatment with a mean TTP
under BE of 2.1 months
(95% CI: 1.4 ─ ∞). The patients within the third cluster A3
(medium risk, 21 patients, 50 %)
showed intermediate angiogenesis-associated GELs and a median
TTP under BE of 4.1
months (95%CI: 3.1 ─ 7). The Kaplan-Meier TTP curves are shown
in Figure 3C. GELs for the
top 10-ranked angiogenesis-associated genes for patients
included in each cluster are given
in Supplementary Figure 1.
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
9
Hierarchical clustering showing the variability of gene
expression of hypoxia-response top
10-ranked genes is given in Figure 3B. For this signature, we
identified two significant
clusters: Cluster H1 (18 patients, 43 %) with lower
hypoxia-response GELs and Cluster H2 (24
patients, 57 %) with hypoxia-response higher GELs (Supplementary
Figure 2).
Dichotomization of the patients into low-risk (Cluster H1) and
high-risk (Cluster H2),
subgroups based on the gene expression levels of the
hypoxia-response signature revealed a
marked difference in TTP under BE between the two groups (Figure
3D). We obtained a
median TTP for the high-risk patients of 2.9 months (95%CI: 1.8
─ 4.1) and of 6.9 months
(95% CI: 4.0 ─ 9.7) for the low-risk patients.
Hypoxia-response gene signature predictive of tumor shrinkage
after BE treatment in non-squamous NSCLC patients.
An additional secondary endpoint of interest was tumor shrinkage
(TS) measured at 12
weeks after BE treatment, which indicates clinically relevant
direct anti-tumor activity.
Because of lack of measurements at 12 weeks, 14 patients had to
be excluded from this
analysis. We analyzed the genes correlating with TS in our
patients using GSEA using both
the angiogenesis-associated and hypoxia-response gene signatures
(Figure 4A and B). Both
gene signatures where significantly enriched in the gene set
correlating with TS (mean rank =
5104, P = 0.002 and mean rank = 7795, P = 0.038, respectively).
The resulting top 12-ranked
genes for both signatures are given in Figure 4C and D. Further,
we classified the patients
based on the metagene score calculated for the top 10-ranked
genes for each gene
signature. For the angiogenesis-associated signature, the
resulting two patients groups (low-
risk and high-risk) showed a non-significantly different median
TS, 13.7 % interquartile range
(IRQ): -0.8 ─ 26.2 vs. 0 %, IQR: -3.2 ─ 16.4, P = 0.755. In
contrast, when dichotomization was
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
10
performed using the 10-gene hypoxia-response signature, the
low-risk patients had a
significantly higher median TS than the high-risk patients (16.1
%, IRQ: 0 ─ 26.2 vs. -0.4 %,
IRQ: -2.5 ─ 2.6, P = 0.013), indicating a higher potential for
assessing treatment response
using this signature. A heat map showing the gene expression
levels of the top 10-ranked
hypoxia response genes predictive of tumor shrinkage is given in
Supplementary Figure 3.
Angiogenesis-associated and hypoxia-response gene signatures
have prognostic value for
non-squamous NSCLC patients. Lastly, we analyzed the prognostic
value of both
angiogenesis and hypoxia gene signatures by investigating the
genes correlating with the
overall survival (OS). The median follow-up time was 24.9 months
(95% CI, 23.9 - ∞). The
results of the GSEA for OS are given in Figure 5A and 5B, and
the derived top 12-ranked
genes are given in Figure 5C and 5D, respectively.
Both gene signatures where significantly enriched in the gene
set correlating with OS (mean
rank = 5064, P = 0.031 and mean rank = 4555, P = 0.001,
respectively). Using the top
10-ranked genes for both signatures to dichotomize the patients
in low-risk (longer OS) and
high-risk (shorter OS) revealed marked differences in OS.
Figures 5E and 5F show the Kaplan-
Meier OS curves for both gene signatures used. Using the
angiogenesis-associated gene
signature led to a median OS for the high-risk patients of 10.6
months [95%CI, 5.2 ─ 19.4]
and for the low-risk patients of 14.1 months [95% CI, 10.5 ─ ∞],
P = 0.035. The hypoxia-
response gene signature showed a higher prognostic value (P =
0.001) resulting in a median
OS for the high-risk patients of 9.9 months [95% CI 4.8 ─ 13.4]
and 17.8 months [95% CI 16.6
─ ∞] for the low-risk patients. A heat map displaying the gene
expression levels of the top
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
11
10-ranked hypoxia response genes significantly associated with
OS is given in Supplementary
Figure 4.
Validation of microarray gene expression levels (GELs) by
RT-qPCR. RT-qPCR was used to
assess the GELs that were established by microarray analysis.
RT-qPCR was performed on 40
out of 42 RNA samples and for nine genes, plus three control
genes. The Spearman’s rank
correlation coefficients (r) were between 0.51─0.71. The
correlation was significant for each
gene. We obtained P values for these associations of <
0.0008, demonstrating good
agreement between the two complementary methods in quantifying
the gene expression
levels (Figure 6). This outcome indicates a statistically
significant correlation between the
microarray GELs and GELs assessed by RT-qPCR, which is a more
convenient and less
expensive technique for routine application in a clinical
setting. These results are in
agreement with the expected level of correlation considering the
fact that there is no
designed sequence overlap between the qPCR primers and the
microarray probes.
Discussion
There are several gene expression signatures identified as
prognostic biomarkers for lung
cancer, mostly for lung adenocarcinoma.34 However, gene
expression signatures with
predictive value for treatment response to antiangiogenic
therapy are still lacking.
Therefore, the aim of this study was to evaluate the predictive
potential of angiogenesis-
associated and hypoxia-response gene signatures for the benefit
of BE treatment. We
performed GSEA for each endpoint, which led to the
identification of specific angiogenesis
and hypoxia–derived 10-gene signatures. These signatures were
then evaluated for their
predictive (TTP und TS endpoints) and prognostic value (OS) and
tested in an independent
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
12
data set. Our findings might shed light on the mechanism of
antiangiogenic treatment
response and resistance. Moreover, they may lead to the
identification of the causal
mechanism behind the high proportion of patients treated with
angiogenesis inhibitors
showing partial response, as demonstrated by increased
progression free survival (PFS),
however, with no improvement in their OS.
We took a closer look at the angiogenesis-associated genes with
predictive value for TTP
under BE therapy. The expression of the first ranked gene,
adhesion G-protein-coupled
receptor 116 (GPR116), was shown to be significantly correlated
with tumor progression,
recurrence, and poor prognosis in human breast cancer. However,
here we identify this gene
to play a protective role and its expression to be associated
with lower risk of disease
recurrence in NSCLC. The biological role of GPR116 in
angiogenesis and endothelial
proliferation remains to be assessed, however this protein is
highly expressed in normal
human lung tissue and it was recently demonstrated to regulate
lung surfactant
homeostasis.35 The second ranked gene, endomucin (EMCN), is an
endothelial sialomucin
and an endothelial-specific marker, involved in cell-cell and
cell-extracellular matrix
interactions.36 Integrin α9 (ITGA9), which forms a heterodimeric
receptor with activated β1
integrin, has been demonstrated to bind directly to VEGFA, and
to contribute to
angiogenesis.37 α9 β1 integrin ligand, tenascin-C, enhances
secretion of S1P (sphingosine 1-
phosphate) in endothelial cells. In turn, S1P promotes
endothelial cell barrier integrity,38
acting as an anti-permeability agent39 and modulating vessel
integrity through its cognate
receptor S1PR1. S1PR1 inhibits VEGFR2 signaling and suppresses
endothelial hypersprouting
via stabilization of junctional VE-cadherin, which leads to
enhanced cell-cell adhesion.40
PECAM-1 and JAM2 are adhesive proteins that accumulate in
adherens junctions and
maintain the restrictiveness of the endothelial barrier. RhoJ,
an endothelial-enriched Rho
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
13
GTPase, regulates angiogenesis and vessel integrity.41 GNG11 is
a member of the γ subunit
family of heteromeric G-protein, which regulates cellular
senescence in response to
environmental stimuli.42 To obtain the ranking of the
discriminating power for each gene, we
performed optimized between-group classification (OBC)43
sensitivity analysis for this
signature. OBC suggests that for angiogenesis-associated
signature S1PR1, GPR116, and
PECAM1 have the highest discriminating power for TTP under BE
(Supplementary Figure 5a ).
The hypoxia-response genes with predictive value for TTP under
BE were mostly genes
involved in the glycolytic and other metabolic pathways44: PFKP
(phosphofructokinase,
platelet), LDHA (lactate dehydrogenase A), GPI
(glucose-6-phosphate isomerase), ALDOA
(aldolase A), ACOT7 (acyl-CoA thioesterase 7), PGK1
(phosphoglycerate kinase), SLC25A32
(solute carrier family 25, mitochondrial folate carrier, member
32) and SLC2A1 (solute carrier
family 2, glucose transporter, member 1). The first-ranked
hypoxia-response gene DDIT4
(DNA-damage-inducible transcript 4 protein, also known as REDD1)
has been shown to be
implicated in inhibition of the mTORC1 (mammalian target of
rapamycin kinase) signaling
pathway, which is relevant in tumor suppression;45 MIF
(macrophage migration inhibitory
factor) has been revealed to act within the tumor
microenvironment to stimulate
angiogenesis and promote immune evasion;46 ADM (adrenomedullin)
is involved in
promoting tumor progression by sustaining proliferation and
angiogenesis.47 For this
signature, OBC indicated that DDIT4 and MIF have the highest
discriminatory power
(Supplementary Figure 5b). Importantly, the hypoxia-response
signature shows both high
predictive and prognostic value and could potentially guide
future clinical decisions.
Our gene expression analyses suggest that the low-risk patients
(most likely to respond to
antiangiogenic combined BE therapy and have better outcome) have
a tumor
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
14
microenvironment that sustains controlled angiogenesis,
developing blood vessels with
increased levels of integrity and reduced permeability. The
controlled angiogenesis is
associated with lower risk of hypoxia within the tumors (lower
levels of hypoxia response
genes). On the other hand, the high-risk patients (less likely
to respond to BE treatment and
have worse outcome) have a tumor microenvironment that sustains
aberrant angiogenesis
with reduced vascular stability and increased vascular
permeability. This phenotype is
further associated with increased tumor hypoxia and an earlier
onset of disease progression.
Moreover, we observed that the 10-gene hypoxia-response
signature has a higher
prognostic power than the angiogenesis-associated signature.
This most likely indicates that
CT has a relatively high contribution to OS, which is expected
taking into account that
hypoxic tumors are less responsive to CT than normoxic
tumors.48
Additionally, we performed LOOCV from the original dataset in
order to test the robustness
of the gene signatures associated with TTP under BE and OS
endpoints. The perturbations
resulting from LOOCV had an insignificant impact on our findings
demonstrating the
robustness of the discriminatory power of our gene signatures
(Supplementary Table 3).
Unfortunately, we could not investigate the performance of our
gene expression signatures
using an identical independent data set, as an additional gene
expression data set from
pretreatment biopsies of treatment-naïve NSCLC patients
receiving the same therapeutic
scheme is not available for validation. However, we tested our
10-gene signatures
correlating with TTP under BE using a data set comprising GELs
of biopsies from a pretreated
NSCLC population (BATTLE-1 study,49 trial registration ID:
NCT00409968, raw data GEO series
accession number: GSE33072). We analyzed the association between
our 10-gene
expression signatures and PFS for two treatment arms: erlotinib
(25 patients, all EGFR-WT)
and sorafenib (31 patients, all EGFR-WT; the patients with
squamous cell or adenosquamous
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
15
carcinoma were excluded). This analysis revealed that our 10
gene angiogenesis-associated
signature had no predictive value for either erlotinib or
sorafenib response as second line
therapy. Likewise, the 10-gene hypoxia-response signature had no
predictive value for
erlotinib response. However, for the second line
sorafenib-treated patient group (majority
erlotinib resistant), we found that the 10-gene hypoxia-response
signature discriminated
between low-risk (responders) and high-risk patients
(non-responders): PFS 3.65 months
(95%CI: 1.87 ─ 8.74) and 2.61 months (95%CI: 1.81 ─ 3.61),
respec vely (P = 0.0186,
Supplementary Figure 6). Sorafenib, a multikinase inhibitor,
achieves antiangiogenic effects
by blocking VEGFR and PDGFR. In addition, several studies
suggest that sorafenib exerts a
negative regulatory effect on angiogenesis by suppressing
expression of VEGF via inhibition
of HIF-1α accumulation and activation.50 Although caution in
extrapolating data from one
clinical trial to the other is required, our findings suggest
that the hypoxia-response
signature may be a predictive biomarker of anti-VEGF(R)
treatment response (such as
bevacizumab and sorafenib) and has no predictive power for
erlotinib activity. The lack of
predictive value of the 10-gene angiogenesis in the context of
second line sorafenib
treatment could indicate that the expression of the
angiogenesis-associated genes
significantly changes from treatment naïve tumors to CT
resistant tumors, whereas hypoxia-
response GELs are affected to a lesser extent. Importantly, only
18 patients in sorafenib-
treated group had biopsies originating from the lung; the gene
expression signatures of
tumor-associated vascular endothelial cells originating from
other organs could vary greatly.
Nonetheless, our findings suggest that the 10-gene
hypoxia-response has a high predictive
value of treatment response even in the context of second line
antiangiogenic therapy and a
great potential for future clinical use.
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
16
The positive correlation between pretreatment
angiogenesis-associated and hypoxia-
response GELs from tumor biopsies and clinical outcomes
following BE treatment derived
from our analyses supports further evaluation of these candidate
gene signatures as
potential biomarkers for the selection of the patient
subpopulation most likely to obtain
benefit from antiangiogenic therapy. There are, however, several
limitations that accompany
our study. Our study comprises a relatively low number of
patients, and control groups (no
treatment, and bevacizumab-only and erlotinib-only treatment)
are absent. Further
validation with larger number of patients and adequate control
arms is needed.
Nevertheless, we found highly statistically significant
differences in the hypoxia-response
GELs of responders vs. non-responders to antiangiogenic therapy
in both our data set and an
additional independent data set. This is very promising and
suggests that the identified
signatures may be clinically useful for further stratifying
non-squamous NSCLC patients and
allow for personalized treatment to avoid unnecessary costs and
patient exposure to
toxicity.
Conclusions
We identified 10-gene angiogenesis and hypoxia signatures, which
can predict the subgroup
of patients with higher likelihood of responding to angiogenic
therapy. These patients had
higher GELs of the genes mainly involved in maintaining the
vascular barrier integrity and
lower levels of hypoxia-response genes. Moreover, these patients
showed improved OS and
75 % of them experienced a high tumor shrinkage level (between
16 - 76 % TS) at 12 weeks
after the beginning of treatment. Although there is a very
important implication to patient
selection for antiangiogenic therapy, the results of this study
are preliminary and need to be
further validated.
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
17
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: A. Franzini, M. H. Brutsche
Development of methodology: A. Franzini, M. H. Brutsche
Acquisition of data (provided animals, acquired and managed
patients, provided facilities, etc.): A. Franzini, I. I. Macovei,
C. Droege, D. Betticher, F. Zappa
Analysis and interpretation of data (e.g., statistical analysis,
biostatistics, computational analysis): A. Franzini, F. Baty, I. I.
Macovei, O. Dürr, D. Klingbiel, M. H. Brutsche
Writing, review, and/or revision of the manuscript: A. Franzini,
F. Baty, B. D. Grigoriu, D. Klingbiel, M. H. Brutsche
Administrative, technical, or material support (i.e., reporting
or organizing data, constructing databases): A. Franzini, B. D.
Grigoriu, C. Droege, D. Betticher, F. Zappa, M. H. Brutsche
Study supervision: A. Franzini, M. H. Brutsche
Supplementary Information
Note: Supplementary data for this article are available at
Clinical Cancer Research Online
(http://clincancerres.aacrjournals.org/).
Grant Support
This work was supported by the Swiss Cancer League & Swiss
Cancer Center (KLS-2880-02-2012), KSSG Medical Research Center
(MFZF_2014_001) and Romanian–Swiss Research Program
(IZERZO_142235/1).
Acknowledgements
We thank all the investigators involved in the SAKK 19/05 trial
for collecting the samples and SAKK for funding the Affymetrix
arrays.
References
1. Horimoto Y, Polanska UM, Takahashi Y, Orimo A. Emerging roles
of the tumor-associated stroma in promoting tumor metastasis. Cell
Adh Migr 2012;6:193-202. 2. Hanahan D, Weinberg RA. Hallmarks of
cancer: the next generation. Cell 2011;144:646-74. 3. Brabek J,
Mierke CT, Rosel D, Vesely P, Fabry B. The role of the tissue
microenvironment in the regulation of cancer cell motility and
invasion. Cell Commun Signal 2010;8:22. 4. McMillin DW, Negri JM,
Mitsiades CS. The role of tumour-stromal interactions in modifying
drug response: challenges and opportunities. Nat Rev Drug Discov
2013;12:217-28. 5. Meads MB, Gatenby RA, Dalton WS.
Environment-mediated drug resistance: a major contributor to
minimal residual disease. Nat Rev Cancer 2009;9:665-74. 6. Hanahan
D, Folkman J. Patterns and emerging mechanisms of the angiogenic
switch during tumorigenesis. Cell 1996;86:353-64.
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
18
7. Weis SM, Cheresh DA. Tumor angiogenesis: molecular pathways
and therapeutic targets. Nat Med 2011;17:1359-70. 8. Ellis LM,
Hicklin DJ. VEGF-targeted therapy: mechanisms of anti-tumour
activity. Nat Rev Cancer 2008;8:579-91. 9. Ferrara N, Hillan KJ,
Gerber HP, Novotny W. Discovery and development of bevacizumab, an
anti-VEGF antibody for treating cancer. Nat Rev Drug Discov
2004;3:391-400. 10. Hurwitz H, Fehrenbacher L, Novotny W,
Cartwright T, Hainsworth J, Heim W, et al. Bevacizumab plus
irinotecan, fluorouracil, and leucovorin for metastatic colorectal
cancer. N Engl J Med 2004;350:2335-42. 11. Escudier B, Bellmunt J,
Negrier S, Bajetta E, Melichar B, Bracarda S, et al. Phase III
trial of bevacizumab plus interferon alfa-2a in patients with
metastatic renal cell carcinoma (AVOREN): final analysis of overall
survival. J Clin Oncol 2010;28:2144-50. 12. Perren TJ, Swart AM,
Pfisterer J, Ledermann JA, Pujade-Lauraine E, Kristensen G, et al.
A phase 3 trial of bevacizumab in ovarian cancer. N Engl J Med
2011;365:2484-96. 13. Kreisl TN, Kim L, Moore K, Duic P, Royce C,
Stroud I, et al. Phase II trial of single-agent bevacizumab
followed by bevacizumab plus irinotecan at tumor progression in
recurrent glioblastoma. J Clin Oncol 2009;27:740-5. 14. Bergers G,
Hanahan D. Modes of resistance to anti-angiogenic therapy. Nat Rev
Cancer 2008;8:592-603. 15. Dang DT, Chun SY, Burkitt K, Abe M, Chen
S, Havre P, et al. Hypoxia-inducible factor-1 target genes as
indicators of tumor vessel response to vascular endothelial growth
factor inhibition. Cancer Res 2008;68:1872-80. 16. Rapisarda A,
Melillo G. Overcoming disappointing results with antiangiogenic
therapy by targeting hypoxia. Nat Rev Clin Oncol 2012;9:378-90. 17.
Ebos JM, Kerbel RS. Antiangiogenic therapy: impact on invasion,
disease progression, and metastasis. Nat Rev Clin Oncol
2011;8:210-21. 18. Miles DW, Chan A, Dirix LY, Cortes J, Pivot X,
Tomczak P, et al. Phase III study of bevacizumab plus docetaxel
compared with placebo plus docetaxel for the first-line treatment
of human epidermal growth factor receptor 2-negative metastatic
breast cancer. J Clin Oncol 2010;28:3239-47. 19. Robert NJ, Dieras
V, Glaspy J, Brufsky AM, Bondarenko I, Lipatov ON, et al. RIBBON-1:
randomized, double-blind, placebo-controlled, phase III trial of
chemotherapy with or without bevacizumab for first-line treatment
of human epidermal growth factor receptor 2-negative, locally
recurrent or metastatic breast cancer. J Clin Oncol
2011;29:1252-60. 20. Gyanchandani R, Kim S. Predictive biomarkers
to anti-VEGF therapy: progress toward an elusive goal. Clin Cancer
Res 2013;19:755-7. 21. Baty F, Rothschild S, Fruh M, Betticher D,
Droge C, Cathomas R, et al. EGFR exon-level biomarkers of the
response to bevacizumab/erlotinib in non-small cell lung cancer.
PLoS One 2013;8:e72966. 22. Baty F, Facompre M, Kaiser S,
Schumacher M, Pless M, Bubendorf L, et al. Gene profiling of
clinical routine biopsies and prediction of survival in non-small
cell lung cancer. Am J Respir Crit Care Med 2010;181:181-8. 23.
Edlund K, Lindskog C, Saito A, Berglund A, Ponten F,
Goransson-Kultima H, et al. CD99 is a novel prognostic stromal
marker in non-small cell lung cancer. Int J Cancer
2012;131:2264-73. 24. Larsen AK, Ouaret D, El Ouadrani K, Petitprez
A. Targeting EGFR and VEGF(R) pathway cross-talk in tumor survival
and angiogenesis. Pharmacol Ther 2011;131:80-90. 25. Maity A, Pore
N, Lee J, Solomon D, O'Rourke DM. Epidermal growth factor receptor
transcriptionally up-regulates vascular endothelial growth factor
expression in human glioblastoma cells via a pathway involving
phosphatidylinositol 3'-kinase and distinct from that induced by
hypoxia. Cancer Res 2000;60:5879-86. 26. Fong GH. Mechanisms of
adaptive angiogenesis to tissue hypoxia. Angiogenesis
2008;11:121-40. 27. Zappa F, Droege C, Betticher D, von Moos R,
Bubendorf L, Ochsenbein A, et al. Bevacizumab and erlotinib (BE)
first-line therapy in advanced non-squamous non-small-cell lung
cancer (NSCLC) (stage
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
19
IIIB/IV) followed by platinum-based chemotherapy (CT) at disease
progression: a multicenter phase II trial (SAKK 19/05). Lung Cancer
2012;78:239-44. 28. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay
YD, Antonellis KJ, Scherf U, et al. Exploration, normalization, and
summaries of high density oligonucleotide array probe level data.
Biostatistics 2003;4:249-64. 29. Bair E, Tibshirani R.
Semi-supervised methods to predict patient survival from gene
expression data. PLoS Biol 2004;2:E108. 30. Subramanian A, Tamayo
P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set
enrichment analysis: a knowledge-based approach for interpreting
genome-wide expression profiles. Proc Natl Acad Sci USA
2005;102:15545-50. 31. Livak KJ, Schmittgen TD. Analysis of
relative gene expression data using real-time quantitative PCR and
the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-8. 32. Masiero
M, Simoes FC, Han HD, Snell C, Peterkin T, Bridges E, et al. A core
human primary tumor angiogenesis signature identifies the
endothelial orphan receptor ELTD1 as a key regulator of
angiogenesis. Cancer Cell 2013;24:229-41. 33. Buffa FM, Harris AL,
West CM, Miller CJ. Large meta-analysis of multiple cancers reveals
a common, compact and highly prognostic hypoxia metagene. Br J
Cancer 2010;102:428-35. 34. Zhu C-Q, Tsao M-S. Prognostic markers
in lung cancer: is it ready for prime time? Transl Lung Cancer Res
2014;3:149-58. 35. Yang MY, Hilton MB, Seaman S, Haines DC,
Nagashima K, Burks CM, et al. Essential regulation of lung
surfactant homeostasis by the orphan G protein-coupled receptor
GPR116. Cell Rep 2013;3:1457-64. 36. Ueno M, Igarashi K, Kimura N,
Okita K, Takizawa M, Nobuhisa I, et al. Endomucin is expressed in
embryonic dorsal aorta and is able to inhibit cell adhesion.
Biochem Biophys Res Commun 2001;287:501-6. 37. Vlahakis NE, Young
BA, Atakilit A, Hawkridge AE, Issaka RB, Boudreau N, et al.
Integrin alpha9beta1 directly binds to vascular endothelial growth
factor (VEGF)-A and contributes to VEGF-A-induced angiogenesis. J
Biol Chem 2007;282:15187-96. 38. Garcia JG, Liu F, Verin AD,
Birukova A, Dechert MA, Gerthoffer WT, et al. Sphingosine
1-phosphate promotes endothelial cell barrier integrity by
Edg-dependent cytoskeletal rearrangement. J Clin Invest
2001;108:689-701. 39. Argraves KM, Gazzolo PJ, Groh EM, Wilkerson
BA, Matsuura BS, Twal WO, et al. High density
lipoprotein-associated sphingosine 1-phosphate promotes endothelial
barrier function. J Biol Chem 2008;283:25074-81. 40. Gaengel K,
Niaudet C, Hagikura K, Lavina B, Muhl L, Hofmann JJ, et al. The
sphingosine-1-phosphate receptor S1PR1 restricts sprouting
angiogenesis by regulating the interplay between VE-cadherin and
VEGFR2. Dev Cell 2012;23:587-99. 41. Kim C, Yang H, Fukushima Y,
Saw PE, Lee J, Park JS, et al. Vascular RhoJ is an effective and
selective target for tumor angiogenesis and vascular disruption.
Cancer Cell 2014;25:102-17. 42. Hossain MN, Sakemura R, Fujii M,
Ayusawa D. G-protein gamma subunit GNG11 strongly regulates
cellular senescence. Biochem Biophys Res Commun 2006;351:645-50.
43. Baty F, Bihl MP, Perriere G, Culhane AC, Brutsche MH. Optimized
between-group classification: a new jackknife-based gene selection
procedure for genome-wide expression data. BMC Bioinformatics
2005;6:239. 44. Doherty JR, Cleveland JL. Targeting lactate
metabolism for cancer therapeutics. J Clin Invest 2013;123:3685-92.
45. Ben Sahra I, Regazzetti C, Robert G, Laurent K, Le
Marchand-Brustel Y, Auberger P, et al. Metformin, independent of
AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1.
Cancer Res 2011;71:4366-72. 46. Girard E, Strathdee C, Trueblood E,
Queva C. Macrophage migration inhibitory factor produced by the
tumour stroma but not by tumour cells regulates angiogenesis in the
B16-F10 melanoma model. Br J Cancer 2012;107:1498-505.
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
20
47. Nikitenko LL, Fox SB, Kehoe S, Rees MC, Bicknell R.
Adrenomedullin and tumour angiogenesis. Br J Cancer 2006;94:1-7.
48. Harrison L, Blackwell K. Hypoxia and anemia: factors in
decreased sensitivity to radiation therapy and chemotherapy?
Oncologist 2004;9 Suppl 5:31-40. 49. Kim ES, Herbst RS, Wistuba,
II, Lee JJ, Blumenschein GR, Jr., Tsao A, et al. The BATTLE trial:
personalizing therapy for lung cancer. Cancer Discov 2011;1:44-53.
50. Liu LP, Ho RL, Chen GG, Lai PB. Sorafenib inhibits
hypoxia-inducible factor-1alpha synthesis: implications for
antiangiogenic activity in hepatocellular carcinoma. Clin Cancer
Res 2012;18:5662-71.
Figure legends:
Figure 1: Treatment course for patients with no prior therapy
included in the SAKK 19/05 phase II trial, study endpoints and
microenvironment compartments analyzed by GSEA; TTP ─ me to
progression, TS ─ tumor shrinkage, OS ─ overall survival.
Figure 2: Enrichment plot from GSEA shows statistically
significant enrichment of the angiogenesis-associated genes
(P=0.004) (A) and in the hypoxia-response genes (P=0.001) (B) in
the gene set predictive of TTP under BE. Top 12-ranked
angiogenesis-associated genes (C) and hypoxia-response genes (D)
derived from GSEA.
Figure 3: Hierarchical clustering of top 10-ranked
angiogenesis-associated genes (A) and hypoxia-response genes (B)
significantly associated with TTP under BE showing gene expression
variation among patients. The heat maps represent relative
intensity values of gene expression levels. Kaplan–Meier TTP under
BE curves of the three patients groups (low-risk, medium-risk and
high-risk) defined by the 10-gene angiogenesis-associated
signature, P = 0.013 for Cluster A1 vs. Cluster A2, (C) and two
patient groups (low-risk and high-risk) defined by the 10-gene
hypoxia-response signature, P = 0.016 (D). The P values of these
associations were determined by log-rank test.
Figure 4: Angiogenesis and hypoxia-associated gene signatures
associate with tumor shrinkage in NSCLC. Enrichment plot from GSEA
shows statistically significant enrichment of the
angiogenesis-associated genes (P=0.002) (A) and in the
hypoxia-response genes (P=0.038) (B) in the gene signature
correlating with TS. Top 12-ranked angiogenesis-associated genes
(C) and hypoxia-response (D) derived from GSEA, which correlate
with TS. Correlation between the two patients groups defined by the
10-gene angiogenesis-associated signature (E) and hypoxia-response
signature (F) (high-risk and low-risk; n = 28) and tumor shrinkage
at 12 weeks after BE treatment (box plots). The boxes represent the
median ± interquartile range (IQR). Whiskers delimit the highest
and lowest non-outlier data points (defined as greater/less than
1.5 × IQR).
Figure 5: Angiogenesis and hypoxia-associated gene signatures
predicting OS in NSCLC. Enrichment plot from GSEA shows
statistically significant enrichment of the angiogenesis-associated
genes (P = 0.031), (A) and hypoxia-response genes (P = 0.001) (B)
in the gene signature predictive of OS. Top 12-ranked
angiogenesis-associated genes (C) and top 12-
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Gene signatures predictive of response to therapy in NSCLC
21
ranked hypoxia-response genes (D) derived from GSEA, which
significantly correlate with OS. Kaplan–Meier OS curves of the two
patients groups (low-risk and high-risk) defined by the 10-gene
angiogenesis-associated signature (P = 0.035) (E) and 10-gene
hypoxia signature (P = 0.001) (F). The P values of the associations
were determined by log-rank test.
Figure 6: Comparison between microarray and RT-qPCR GELs.
Spearman's correlations were performed between relative gene
expression levels (GELs) determined by RT-qPCR (x-axis) and RNA
microarray (y-axis) for nine angiogenesis-associated genes for 40
out of the 42 patients. The mRNA levels for each gene of interest
were determined by RT qPCR and correlated with microarray
expression scores determined after data processing, both calculated
relative to HPRT1 gene. r, Spearman’s rank correlation
coefficient.
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Bevacizumab/Erlotinib (BE) therapy
n × 3 weeks until progression
Endpoints: TS TTP under BE OS
Standard chemotherapy (CT)
6 × 6 weeks or until progression
Biopsies from patients with stage
IIIB or IV non-squamous
NSCLC with no prior therapy
Compartments: Angiogenesis Hypoxia
12 weeks
Figure 1
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
0.1
0.3
0.5
Angiogenesis
Enri
chm
en
t sco
re (
ES
)
0 5000 10000 15000
01
23
45
Rank in Ordered Dataset
Ranked lis
t m
etr
ic
0.1
0.3
0.5
Enri
chm
en
t sco
re (
ES
)
0 5000 10000 15000
01
23
45
Rank in Ordered Dataset
Ranked lis
t m
etr
ic
HypoxiaA BFigure 2
C DGene symbol Rank in gene list Rank metric score Enrichment
score (ES)
GPR116 403 1.924 0.027
EMCN 611 1.700 0.060
ITGA9 648 1.667 0.103
LDB2 707 1.629 0.143
MEF2C 805 1.561 0.179
GNG11 829 1.546 0.219
CALCRL 865 1.522 0.257
KDR 987 1.455 0.289
PECAM1 1019 1.435 0.326
S1PR1 1117 1.391 0.357
JAM2 1288 1.313 0.382
RHOJ 1364 1.289 0.412
Gene symbol Rank in gene list Rank metric score Enrichment score
(ES)
DDIT4 18 3.539 0.082
MIF 33 3.276 0.157
PFKP 53 2.991 0.226
ADM 69 2.835 0.291
LDHA 132 2.513 0.346
GPI 137 2.494 0.404
ALDOA 238 2.211 0.450
ACOT7 373 1.975 0.488
SLC25A32 976 1.460 0.487
PGK1 1110 1.394 0.512
TUBA1B 1238 1.331 0.535
SLC2A1 1420 1.268 0.554Research. on June 12, 2021. © 2015
American Association for Cancerclincancerres.aacrjournals.org
Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
002
078
102
055
103
056
094
060
070
091
090
058
077
051
080
065
038
099
057
063
088
064
082
083
097
081
096
098
074
095
049
093
069
068
067
076
023
101
061
084
075
087
CALCRL
EMCN
GNG11
GPR116
PECAM1
ITGA9
MEF2C
S1PR1
LDB2
KDR
−2 0 2
Row Z−Score
Color Key
Cluster A1 Cluster A2 Cluster A3
Angiogenesis
103
102
094
093
002
078
058
083
082
077
023
060
074
056
099
055
084
098
080
091
038
096
057
063
097
061
051
049
075
095
081
067
101
070
076
087
068
065
090
088
064
069
SLC25A32
PFKP
ACOT7
DDIT4
ADM
PGK1
LDHA
ALDOA
MIF
GPI
−2 0 2 4
Row Z−Score
Color Key
Cluster H1 Cluster H2
Hypoxia
0 2 4 6 8 10 12 14
0
10
20
30
40
50
60
70
80
90
100
Time (months)
Pro
po
rtio
no
fp
ati
en
tsw
ith
ou
tp
rog
resio
n Cluster H1
Cluster H2
Cluster H2 18 17 13 10 7 2 1 0
Cluster H1 24 15 9 3 2 2 1 0
Patients at risk:
A B
C D
Figure 3
0 2 4 6 8 10 12 14
0
10
20
30
40
50
60
70
80
90
100
Time (months)
Pro
po
rtio
no
fp
ati
en
tsw
ith
ou
tp
rog
resio
n Cluster A1
Cluster A2
Cluster A3
Cluster A1 9 9 7 5 4 2 1 0
Cluster A2 12 6 3 2 1 0 0 0
Cluster A3 21 17 12 6 4 2 1 0
Patients at risk:
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
0.1
0.3
0.5
En
rich
me
nt sco
re (
ES
)
0 5000 10000 15000
01
23
4
Rank in Ordered Dataset
Ra
nke
d lis
t m
etr
ic
Angiogenesis
0.1
0.2
0.3
0.4
0.5
En
rich
me
nt sco
re (
ES
)
0 5000 10000 15000
01
23
4
Rank in Ordered Dataset
Ra
nke
d lis
t m
etr
ic
Hypoxia
−20
0
20
40
60
80
Patients at risk (metagene score)
TS
(%)
FALSE. TRUE.
18 10 −20
0
20
40
60
80
Patients at risk (metagene score)
TS
(%)
FALSE. TRUE.
18 10
A B
C
E
D
F
Figure 4
Gene symbol Rank in gene list Rank metric score Enrichment score
(ES)
LDB2 622 1.818 0.010
VWF 664 1.785 0.053
MYCT1 669 1.784 0.098
TEK 700 1.763 0.141
EMCN 734 1.745 0.183
CDH5 749 1.735 0.227
TIE1 788 1.716 0.268
JAM2 1072 1.559 0.291
ZNF423 1139 1.533 0.326
ROBO4 1197 1.506 0.361
ADCY4 1398 1.425 0.386
PTPRB 1690 1.328 0.402
Gene symbol Rank in gene list Rank metric score Enrichment score
(ES)
MRPL15 61 2.960 0.090
PSMA7 82 2.836 0.179
ANKRD37 170 2.465 0.252
MIF 228 2.285 0.321
LDHA 473 1.951 0.368
GPI 605 1.829 0.418
TUBA1B 1212 1.501 0.430
YKT6 1522 1.382 0.456
PFKP 2118 1.206 0.459
ACOT7 3154 0.991 0.429
ALDOA 3247 0.975 0.455
PGK1 3600 0.922 0.463
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
0.1
0.3
0.5
En
rich
me
nt sco
re (
ES
)
0 5000 10000 15000
01
23
45
Rank in Ordered Dataset
Ra
nke
d lis
t m
etr
ic
Angiogenesis
0.0
0.2
0.4
0.6
En
rich
me
nt sco
re (
ES
)
0 5000 10000 15000
01
23
45
Rank in Ordered Dataset
Ra
nke
d lis
t m
etr
ic
Hypoxia
0 6 12 18 24 300
10
20
30
40
50
60
70
80
90
100
Time (months)
Cu
mu
lati
ve
su
rviv
al(%
)
Low risk
High risk
Low risk 21 18 13 8 4 3 2
High risk 21 17 12 6 4 2 1
Patients at risk:
0 6 12 18 24 300
10
20
30
40
50
60
70
80
90
100
Time (months)
Cu
mu
lati
ve
su
rviv
al(%
)
Low risk
High risk
Low risk 21 19 16 12 5 2 1
High risk 21 14 10 3 2 2 2
Patients at risk:
A B
C D
E F
Figure 5
Gene symbol Rank in gene list Rank metric score Enrichment score
(ES)
GPR116 504 1.798 0.030
MYCT1 811 1.563 0.064
ITGA9 1228 1.346 0.084
RHOJ 1369 1.297 0.118
KDR 2137 1.059 0.108
CDH5 2260 1.031 0.135
GNG11 2354 1.009 0.163
CALCRL 2379 1.004 0.195
LDB2 2485 0.987 0.222
ESAM 2601 0.962 0.247
JAM2 3091 0.879 0.247
ROBO4 3220 0.859 0.268
Gene symbol Rank in gene list Rank metric score Enrichment score
(ES)
MIF 100 2.699 0.061
ALDOA 265 2.177 0.105
MRPL15 290 2.124 0.156
LDHA 333 2.059 0.204
ADM 387 1.957 0.249
GPI 388 1.955 0.297
DDIT4 442 1.880 0.341
ACOT7 466 1.844 0.385
SHCBP1 757 1.598 0.407
PFKP 931 1.489 0.434
PSMA7 977 1.460 0.467
NDRG1 1307 1.320 0.480
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
0 5 10 150.0
0.5
1.0
1.5
2.0
GELs, RT-qPCR/HPRT1
GE
L,
mic
roa
rra
y/H
PR
T1
r = 0.6437
P < 0.0001
GNG11
0.0 0.2 0.4 0.6 0.80.0
0.5
1.0
1.5
2.0
GELs, RT-qPCR/HPRT1
GE
L,
mic
roa
rra
y/H
PR
T1
r = 0.7032
P < 0.0001
EMCN
0.0 0.5 1.0 1.50.8
1.0
1.2
1.4
1.6
1.8
2.0
GELs, RT-qPCR/HPRT1
GE
L,
mic
roa
rra
y/H
PR
T1
r = 0.6283
P < 0.0001
ITGA9
0.0 0.1 0.2 0.30.0
0.5
1.0
1.5
2.0
2.5
GELs, RT-qPCR/HPRT1
GE
L,
mic
roa
rra
y/H
PR
T1
r = 0.7131
P < 0.0001
KDR
0 2 4 6 8 100.0
0.5
1.0
1.5
2.0
2.5
GELs, RT-qPCR/HPRT1
GE
L,
mic
roa
rra
y/H
PR
T1
r = 0.6534
P < 0.0001
PECAM1
0 1 2 3 40.0
0.5
1.0
1.5
2.0
GELs, RT-qPCR/HPRT1
GE
L,
mic
roa
rra
y/H
PR
T1
r = 0.5102
P = 0.0008
S1PR1
0.0 0.5 1.0 1.5 2.00.0
0.5
1.0
1.5
2.0
2.5
GELs, RT-qPCR/HPRT1
GE
L,
mic
roa
rra
y/H
PR
T1
r = 0.7030
P < 0.0001
JAM2
0.00 0.02 0.04 0.06 0.08 0.100.0
0.5
1.0
1.5
2.0
2.5
GELs, RT-qPCR/HPRT1
GE
L,
mic
roa
rra
y/H
PR
T1
r = 0.6588
P < 0.0001
RHOJ
0.0 0.5 1.0 1.5 2.00.0
0.5
1.0
1.5
2.0
2.5
GELs, RT-qPCR/HPRT1
GE
L,
mic
roa
rra
y/H
PR
T1
r = 0.6912
P < 0.0001
GPR116Figure 6
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/
-
Published OnlineFirst April 28, 2015.Clin Cancer Res Anca
Franzini, Florent Baty, Ina I Macovei, et al. lung cancer patients
(SAKK 19/05 trial)therapeutic benefit in advanced non-squamous
non-small cell Gene expression signatures predictive of
bevacizumab/erlotinib
Updated version
10.1158/1078-0432.CCR-14-3135doi:
Access the most recent version of this article at:
Material
Supplementary
http://clincancerres.aacrjournals.org/content/suppl/2015/04/29/1078-0432.CCR-14-3135.DC1
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and
accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to
receive free email-alerts
Subscriptions
Reprints and
[email protected] at
To order reprints of this article or to subscribe to the
journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take
you to the Copyright Clearance Center's (CCC)
.http://clincancerres.aacrjournals.org/content/early/2015/04/28/1078-0432.CCR-14-3135To
request permission to re-use all or part of this article, use this
link
Research. on June 12, 2021. © 2015 American Association for
Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for
publication but have not yet been edited. Author Manuscript
Published OnlineFirst on April 28, 2015; DOI:
10.1158/1078-0432.CCR-14-3135
http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-14-3135http://clincancerres.aacrjournals.org/content/suppl/2015/04/29/1078-0432.CCR-14-3135.DC1http://clincancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://clincancerres.aacrjournals.org/content/early/2015/04/28/1078-0432.CCR-14-3135http://clincancerres.aacrjournals.org/
Article FileFigure 1Figure 2Figure 3Figure4Figure 5Figure 6