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A Three-Gene Signature for Outcome in Soft Tissue Sarcoma

  • KMG Klinik Silbermühle GmbH
  • Liquid Genomics, Inc.

Abstract and Figures

Finding markers or gene sets that would further classify patients into different risk categories and thus allow more individually adapted multimodality treatment regimens in soft tissue sarcomas is necessary. In this study, we investigated the prognostic values of hypoxia-inducible factor 1a (HIF1a), heparin-binding epidermal growth factor-like growth factor (HB-EGF), vascular endothelial growth factor (VEGF), and other angiogenesis-related gene expressions, as well as their interrelationships. Formalin-fixed paraffin-embedded tissue samples were obtained from 45 patients with soft tissue sarcoma (median age 57 years, range 16-85 years). After laser capture microdissection direct quantitative real-time reverse transcription-PCR (TaqMan) assays were done in triplicates to determine HIF1a, HB-EGF, VEGF, and other gene expression levels. Multivariate Cox [corrected] regression analysis revealed significant independent associations of HB-EGF, HIF1a, and VEGF-C gene expression to the overall survival (P < 0.0001). A combined factor of these three genes showed a relative risk for shorter survival of 5.5, more than twice higher as in an increasing International Union against Cancer Stage. Receiver operating characteristic curve analysis showed a significant sensitivity of 73% and specificity of 82% of this factor for the diagnosis of short (<3 years) versus long (3-9 years) survival (P = 0.0002). VEGF-A showed significant gender differences in the association to survival. Measuring HIF1a, HB-EGF, and VEGF-C expression may contribute to a better understanding of the prognosis of patients with soft tissue sarcoma and may even play a crucial role for the distribution of patients to multimodal therapeutic regimens. Prospective studies investigating the response to different adjuvant or palliative therapies seem to be warranted.
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Imaging, Diagnosis, Prognosis
A Three-Gene Signature for Outcome in Soft Tissue Sarcoma
Andreas-Claudius Hoffmann,
Kathleen D. Danenberg,
Helge Taubert,
Peter V. Danenberg,
and Peter Wuerl
Abstract Purpose: Finding markers or gene sets that would further classify patients into different
risk categories and thus allow more individually adapted multimodality treatment regi-
mens in soft tissue sarcomas is necessary. In this study, we investigated the prognostic
values of hypoxia-inducible factor 1a (HIF1a), heparin-binding epidermal growth factor
like growth factor (HB-EGF), vascular endothelial growth factor (VEGF), and other
angiogenesis-related gene expressions, as well as their interrelationships.
Experimental Design: Formalin-fixed paraffin-embedded tissue samples were obtained
from 45 patients with soft tissue sarcoma (median age 57 years, range 1685 years).
After laser capture microdissection direct quantitative real-time reverse transcription-
PCR (TaqMan) assays were done in triplicates to determine HIF1a, HB-EGF, VEGF,
and other gene expression levels.
Results: Multivariate Cox regression analysis revealed significant independent asso-
ciations of HB-EGF,HIF1a,andVEGF-C gene expression to the overall survival (P<
0.0001). A combined factor of these three genes showed a relative risk for shorter sur-
vival of 5.5, more than twice higher as in an increasing International Union against
Cancer Stage. Receiver operating characteristic curve analysis showed a significant
sensitivity of 73% and specificity of 82% of this factor for the diagnosis of short
(<3 years) versus long (3-9 years) survival (P= 0.0002). VEGF-A showed significant
gender differences in the association to survival.
Conclusions: Measuring HIF1a,HB-EGF,andVEGF-C expression may contribute to a
better understanding of the prognosis of patients with soft tissue sarcoma and may
even play a crucial role for the distribution of patients to multimodal therapeutic regi-
mens. Prospective studies investigating the response to different adjuvant or palliative
therapies seem to be warranted. (Clin Cancer Res 2009;15(16):51918)
Although soft tissue sarcomas have an incidence rate of <1%
per year in the United States, they range among the five leading
causes of death in adolescent and young adult (1). The group of
soft tissue sarcomas consists of several histologic types, with
malignant fibrous histiocytomas being very common in pa-
tients older than 40 years and neurogenic sarcomas being more
common in younger ones (2).
Soft tissue sarcomas are considered to be relatively aggressive.
Every second diagnosed patient will ultimately die from the dis-
ease. The tumors are relatively resistant to radio and chemother-
apy (3, 4). Whenever possible patients receive surgery in
combination with radiotherapy, second-line treatment, or treat-
ment of metastatic disease, mainly consists of ifosfamide or da-
carbazine and doxorubicin, but with limited success and no
relevant survival benefit for the combination regimes (5, 6).
The locally invasive growth and the prognosis determining high
rate of metastases often lead to difficulties accomplishing a
complete resection. Therefore, the necessity to find new targets
for tumor therapy is obvious (7).
Imatinib mesylate has been shown to be a successful treat-
ment option in Gastrointestinal Stromal Tumors (GIST-Tumors)
and could also be potentially used in soft tissue sarcomas
(8, 9). Johnson and others recently described the strong induc-
tion of epidermal growth factorlike growth factor (HB-EGF)
release through imatinib mesylate (10). HB-EGF is able to bind
to EGF receptors, the main target of the tyrosine kinase inhibi-
tors (11), with a higher affinity than EGF itself and has recently
been discussed as a potential molecular target (12, 13). Hepar-
anase (HPSE) functions as an endoglycosidase that cleaves he-
paran sulfate chains of proteoglycans (14, 15). Enclosed in the
heparan sulfate glycosaminoglycans are growth factors and an-
giogenic factors, like basic fibroblast growth factor (bFGF) and
Authors' Affiliations:
Department of Medicine (Cancer Research), West
German Cancer Center, Molecular Oncology Risk-Profile Evaluation,
University Hospital Essen, Essen, Germany;
Department of Biochemistry
and Molecular Biology and Norris Comprehensive Cancer Center,
University of Southern California;
Response Genetics, Inc., Los Angeles,
Department of Pathology, University of Halle-Wittenberg,
Halle, Germany; and
Department of Surgery, Malteser St. Franziskus-
Hospital, Flensburg, Germany
Received 10/2/08; revised 3/13/09; accepted 4/24/09; published OnlineFirst
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Andreas C. Hoffmann, Department of Medicine (Cancer
Research), West GermanCancer Center, Molecular OncologyRisk-ProfileEval-
uation,University HospitalEssen, Hufelandstrasse 55,Essen, 45122, Germany.
Phone: 49- 201-723-85036; Fax: 49-201-723-5733; E-mai l: ach
F2009 American Association for Cancer Research.
5191 Clin Cancer Res 2009;15(16) August 15,
HB-EGF that are released upon degradation with HPSE (16).
Thus far, these genes and the interrelationship have not been
further evaluated in soft tissue sarcoma.
Hypoxia-inducible factor 1a (HIF1a) has been described as
one of the main drivers of angiogenesis and has been shown
to correlate with prognosis in many cancers (17, 18). Lately,
Shintani and others used immunohistochemical staining to
show, for the first time, that HIF1a protein overexpression in
malignant fibrous histiocytoma correlates with poor overall
survival (19). Thus far, there has been no approach to measure
mRNA expression levels of HIF1a and the dependent angiogen-
ic markers in soft tissue sarcoma. It is well known that HIF1a
regulates vascular endothelial growth factor (VEGF) expression
under hypoxic conditions (20). The correlation of bFGF and
platelet-derived growth factor (PDGF) to HIF1a and VEGF
was previously described in various cancers but never scruti-
nized in soft tissue sarcoma (21, 22).
In this study, we used a multigene panel to evaluate the
expression of HIF1a,HB-EGF,VEGF-C, and other angiogenic
markers; the prognostic values of these gene expressions; and
their interrelationships in soft tissue sarcoma. We measured
the mRNA expression levels of these genes with quantitative
real-time reverse transcription-PCR (RT-PCR) in formalin-
fixed paraffin-embedded tissue and then further analyzed
the abovementioned genes and their correlation with clinical
and histopathologic variables.
Materials and Methods
Study population, demographic data, and staging procedures. Formalin-
fixed paraffin-embedded samples were gathered from 45 patients with
either malignant fibrous histiocytoma (MFH; 19 of 45, 42.2%) or neu-
rogenic sarcoma (NS; 26 of 45, 57.8%) with a median age of 57 (MFH
70, 39-85; NS 53, 16-72) years at time of operation who were scheduled
for primary surgical resection and were treated with postoperative radio-
therapy (patient characteristics; Table 1). All patients were treated at the
University Hospital of Leipzig, Germany. Tumor-node-metastasis stag-
ing was done according to the criteria of the International Union against
Cancer (23).
Microdissection. After a review of representative H&E-stained slides
of the formalin-fixed paraffin-embedded blocks by a pathologist to es-
timate the tumor load per sample, section slides of 10-μmthickness
were obtained for laser captured microdissection (P.A.L.M. Microlaser
Technologies AG). All tumor slides were prepared as described exten-
sively by Vallbohmer and others in 2005 (24).
Isolation of RNA and cDNA synthesis. The isolation of RNA from
tumor tissue isolated by the microdissection was done in accordance
with a patented procedure at Response Genetics, Inc. (U.S. Patent
6248,535). The cDNA preparation steps were accomplished as de-
scribed previously (25).
Quantitative real-time PCR. To quantify HIF1a,HPSE,bFGF,
PDGFA, PDGFRA, HB-EGF,VEGF,VEGF-C, vascular endothelial growth
factor receptor 1 (VE GFR1), VEGFR2,andVEGFR3 mRNA expression
levels, we used an endogenous reference gene (β-actin) and our gene
set on a method based on real-time fluorescence detection of amplified
cDNA [ABI PRISM 7900 Sequence Detection System (TaqMan) Perkin-
Elmer Applied Biosystem]. The RT-PCR was implemented as previously
described by Kuramochi and others in 2006 (26). All genes were run on
all samples in triplicates. The detection of amplified cDNA results in a
cycle threshold (C
) value, which is inversely proportional to the
amount of cDNA. The higher the ensuing cycle threshold (C
the more PCR cycles were necessary to attain detection limit, which
means less cDNA. Colon, liver, and St. Universal Mix RNAs (Stratagene)
were used as control calibrators on each plate. All primers were selected
using the Gene Express software (Applied Biosystems) but were adapted
to the needs of RNA/cDNA as extracted out of paraffin-embedded tis-
sue. All primers were validated before use, analogical to the described
method of Schneider and others in 2005 (27). All results are expressed
as ratios between two absolute measurements (gene of interest/endog-
enous reference gene) to account for loading differences. We used a log
transformation before statistical analysis, including a multiplier which
accounts the average CT values maintained for each gene during the
validation process on the calibrators and therefore allows comparing
samples which were run on different RT-PCR well plates.
The primary sequences of the herein used genes were designed as
previously published by Azuma and colleagues (28).
Statistical analysis. The interrelationship among gene expression
levels were tested with Spearman's test for bivariate correlations. The
Mann-Whitney Utest for not normally distributed samples was used
to test whether the gene expression levels were influenced by the differ-
ent clinicopathologic parameters. Every gene was tested with the
Kaplan-Meier method to estimate the different associations of gene
expression levels with overall survival. Differences in survival between
the high- and low-expression group were analyzed with the log-rank test.
To evaluate independent prognostic factors associated with survival
multivariate cyclooxygenase (Cox) proportional hazards, regression
analysis with stepwise selection was used with the gene set and the tu-
mor stage [International Union against Cancer (UICC)] as covariates
Translational Relevance
This manuscript describes important relationships
between hypoxia-inducible factor 1a and down-
stream angiogenesis genes with potential use for
targeted therapy in soft tissue sarcoma.
Table 1. Patient characteristics
Parameter No. patients (%)
Median age all 57; MFH: median 70, range 39-85; NS: median 53,
range 16-72
Male 18 (40.0%)
Female 27(60.0%)
Malignant fibrous histiocytoma 19 (42.2%)
Neurogenic sarcoma 26 (57.8%)
pT category
pT1 10 (22.2%)
pT2 35 (77.8%)
pN category
pN1 38 (84.4%)
pN2 7 (15.6%)
20 (44.4%)
25 (55.6%)
Residual tumor category
45 (100%)
UICC stage
IIA/B 15 (33.3%)
IIIA/B 20 (44.4%)
IVA/B 10 (22.3%)
Abbreviations: UICC, International Union Against Cancer; UICC
1997; pTNM, TNM pathologic classification; pN, regional lymph
node metastasis; G, grade of differentiation.
5192Clin Cancer Res 2009;15(16) August 15, 2009
Imaging, Diagnosis, Prognosis
after adjustment for potential confounders (tumor staging, type of
tumor resection, and age of patients).
A data mining technique provided by the SAS Institute was used to
split gene expression in high- and low-level groups based on a platform
that recursively partitions data according to the relationship between
the Xand Yvalues, creating a tree of partitions (recursive descent par-
tition analysis). By searching all possible cuts, it finds a set of cut points
of Xvalues (gene expression) that best predict the Yvalue (survival
time). These data splits are done recursively forming a tree of decision
rules until the desired fit is reached; the most significant split is deter-
mined by the largest likelihood ratio χ
statistic. In either case, the
split is chosen to maximize the difference in the responses between
the two branches of the split. This method was previously used by Lu
and others (29).
We used receiver operating characteristic (ROC) curve analysis to test
the ability of the chosen cut points to discriminate short survivors
(<3 y) from long survivors (3-9 y; refs. 30, 31). The level of significance
was set to P< 0.05. All Pvalues reported were based on two-sided tests.
All statistical tests were done using the Software Packages SPSS for
Windows, Version 16.0 and JMP 7.0 Software (SAS).
Spearman's test for bivariate correlations. Spearman's test on
the log-transformed δCT values showed significant correlations
between some of the gene expressions. HIF1a was correlated to
nearly every gene we tested (Fig. 1).
Comparison of gene expression levels throughout subgroups. To
test whether gene expression levels were significantly different
in-between clinicopathologic subgroups like primary tumor
expansion (pT), Grading, and UICC stage, we used the
Mann-Whitney Utest for not normally distributed samples.
There was no significant difference in gene expression levels
of HIF1a (P= 0.36), HB-EGF (P= 0.8), VEGF-C (P= 0.19),
bFGF (P= 0.89), PDGFA (P=0.68),PDGFRA(P= 0.23),
VEGF (P= 0.58), VEGFR1 (P=0.45),VEGFR2 (P= 0.82),
VEGFR3 (P=0.99),orHPSE (P= 0.31) between pT1 and
pT2 tumors.
The gene expression levels of HIF1a,VEGF-C,HPSE,bFGF,
VEGF,VEGFR1,VEGFR3, and PDGFRA did not differ signifi-
cantly between low- and high-grade tumors (P=0.37;P=
0.79; P= 0.55; P= 0.28; P= 0.45; P= 0.1; P= 0.85); they were,
however, close to significance regarding HB-EGF and PDGFA
(P=0.06;P= 0.06) and even significant concerning VEGFR2
and grading (P= 0.04).
With respect to the distinct UICC stages, HIF1a,HB-EGF,
P= 0.26; P=0.74;P=0.55;P=0.78;P= 0.11; P=0.23;P=
0.32) did not show a significantly different expression; VEGFR1,
VEGFR3, and PDGFA gene expression were however significant
differently (P= 0.04; P= 0.01; P= 0.01) expressed in the discrete
UICC stages.
Partition tree analysis of genes based on survival time. HB-
EGF,HIF1a, and VEGF-C were the most significant divisors in
the recursive partitioning tree for all patients for survival. The
cut point for HB-EGF was the 80th percentile, HIF1a and
VEGF-C expression had their cut-point at the 40th percentile.
Survival analysis using the Kaplan-Meier method. Kaplan-
Meier analysis showed that high expression of HIF1a was corre-
lated to a significantly more favorable prognosis (P= 0.0036;
Fig. 2). The range of expression was 0.408 to 1.529 in the low-
expression group with a median of 1.061 and 1.535 to 8.689 in
the high-expression group with a median of 3.144. The survival
difference between the low-expression group (1 year, 16 of 45
patients, 36.6%) and the high-expression group (3 years, 29 of
45 patients, 64.4%) was 24 months.
In contrast, high expression of HB-EGF was significantly cor-
related to a shorter overall survival and therefore to an unfavor-
able prognosis (P= 0.005, Fig. 3). The cut point was at the 80th
percentile and divided the patients into a group with 9 months
median survival (high expression, 8 of 41 patients, 19.5%,
range 0.396-2.175, median 0.795) and a group of patients
with 27 months of median survival (low expression, 33 of 41
patients, 80.5%, range 0.000434-0.355, median 0.0137).
VEGF-C expression higher than the 40th percentile cut-point
was significantly associated with longer survival (P=0.0023;
Fig. 4). The low-expression group (range 0.000888-0.0985, me-
dian 0.00516) survived a median time of 15 months (17 of 42
patients, 40.5%) and the high-expression group survived 34
months in median (25 of 42, 59.5%), a survival difference of
19 months.
Multivariate Cox proportional hazard regression analysis. Cox
proportional hazard regression analysis with stepwise selection
used to test for the significance of independent association be-
tween the three genes, the UICC stage, and survival revealed
that HB-EGF was the strongest independent factor (P= 0.003)
with a relative risk of 4.4 [exp(b)] of higher HB-EGF for shorter
survival. The relative risk of a higher UICC stage for shorter sur-
vival was 2.5 [exp(b)]. HIF1a and VEGF-C were also significant-
ly included in the model, but their negative coefficient showed
a smaller exponent of 2.6 and 3.9, respectively. The overall
model fit was P< 0.0001.
We designed a combined group with patients showing high-
HIF1a expression (>40th percentile), highVEGF-C expression
(>40th percentile), and lowHB-EGF expression (<80th per-
centile), assuming that this group should have the best chance
for a long survival according to the Kaplan-Meier and Cox re-
gression analysis; the group consisted of 14 of 45 patients
(31.1%). Stepwise multivariate Cox regression analysis re-
vealed that the combined variable was the strongest indepen-
dent significantly associated factor for survival and superior to
Fig. 1. The used genes and their interrelationship with Spearman's test for
bivariate correlation (P< 0.05). The grayscale represents the number of
connections to other genes. The lighter the grayscale of the gene the fewer
correlations were found on this particular sample set.
5193 Clin Cancer Res 2009;15(16) August 15,
Three-gene signature in sarcoma
every other factor (gene expression, as well as UICC stage) by
itself [P= 0.0002; exp(b) = 5.48]. Figure 5 illustrates the sur-
vival curves for the combined factor out of the three genes
HB-EGF,HIF1a, and VEGF-C against the combined UICC clas-
sifications (IIA/B, IIIA/B, IVA/B) in the Cox proportional ha-
zards regression model.
All three genes, HIF1a,HB-EGF, and VEGF-C did show signif-
icant gender differences in the association to survival. In the
subgroup of female patients, the association of HB-EGF mRNA
expression with outcome was significantly stronger than in
male patients with a relative risk of 10.19 (P= 0.0007). Using
the combined factor of HIF1a,VEGF-C, and HB-EGF, there were
no significant gender differences.
ROC curve analysis. ROC curve analysis was used to assess
the sensitivity and the specificity of the combined gene expres-
sion of HIF1a,HB-EGF,andVEGF-C to distinguish short (3
years and less) from long survivors (3-9 years). The sensitivity
(true positive rate) was 72.73% and the specificity (true nega-
tive rate) was 82.35% for the diagnosis of short versus long sur-
vival in all patients. The area under the curve was 0.775
(confidence interval 0.626-0.886) with a significance level of
P= 0.0002. The positive likelihood ratio (true positive rate/false
positive rate) was 4.12, and the negative likelihood ratio (false
negative rate/true negative rate) was 0.33 (Fig. 6). Using each of
the single genes at the chosen cut points, HB-EGF,HIF1a, and
VEGF-C failed to properly classify patients according to the sur-
vival criteria used for the combination. The time point of 3
years had the highest sensitivity and specificity, but the combi-
nation of the three genes was significant also at 1, 2, and 5 years
to distinguish short from long survivors.
terestingly, analysis of the used VEGF genes revealed that, espe-
cially VEGF-A, showed significant gender differences in survival
of the examined patients. The 75th percentile cut point of
VEGF-A splits male patients in a high-expression group with a
median survival of 83.5 months and a low-expression group
with a median survival of nearly 5 years less (26 months,
P= 0.03). The female patients showing higher VEGF-A expres-
sion had a significantly worse outcome with a median survival
time of 10 months than the female patients with a low expres-
sion (median survival 27 months, P= 0.02). As mentioned ear-
lier VEGF-C showed significance for survival using Kaplan-
Meier log-rank test at the 40th percentile (P= 0.002). VEGF-
R2 and VEGF-R3 showed significance for survival in Kaplan-
Meier log-rank tests at the 60th and 70th percentile, respectively
(P= 0.03; P= 0.02). Nonetheless, in a multivariate Cox regres-
sion model containing the five VEGF genes used in this study
with the cut points defined by the recursive partitioning tree,
only VEGF-C evinced as independently and significantly associ-
ated with outcome, i.e., low expression as an unfavorable prog-
nostic factor (P= 0.0049).
In this study, we determined the gene expressions of HIF1a,
HB-EGF, and other members of the angiogenic pathway in
formalin-fixed paraffin-embedded samples of soft tissue sarco-
ma patients, malignant fibrous histiocytoma, and neurogenic
sarcoma who did not receive any chemotherapy before surgery.
By using laser capture microdissection to isolate tumor tissue
from the clinical specimens along with quantitative RT-PCR,
we hoped to achieve a more precise characterization of the as-
sociations of these gene expressions with each other and with
the outcome of the patients than was previously available.
By multivariate Cox regression analysis, we revealed signifi-
cant independent associations of HB-EGF,HIF1a, and VEGF-C
gene expression to the overall survival. By using the three most
significant genes, we were able to build a combined factor with
a relative risk for shorter survival of 5.5 that is twice higher
than a one-step increasing UICC stage. With ROC curve analy-
sis, we tested each of the three genes for the ability of the cho-
sen cut points to distinguish short (<3 years) and long survivors
(3-9 years). None of the genes had significant sensitivity or
Fig. 2. Kaplan-Meier plot, estimating overall survival and relapse-free
survival. Differences in survival between the high- and low-HIF1a
expression group were analyzed with the log-rank test. The black line
represents the high-expression group (cut point 40th percentile) with a
median survival of 36 mo (29 of 45 patients, 64.4%), whereas the light gray
curve represents the low-expression group with a median survival of
12 mo (16 of 45 patients, 36.6%).
Fig. 3. Kaplan-Meier plot, estimating overall survival and relapse-free
survival. Differences in survival between the highand the lowHB-EGF
expression group were analyzed with the log-rank test. The black line
represents the high-expression group (cut point 80th percentile) with a
median survival of 9 mo (8 of 41 patients, 19.5%), whereas the light gray
curve represents the low-expression group with a median survival of 27 mo
(33 of 41 patients, 80.5%).
5194Clin Cancer Res 2009;15(16) August 15, 2009
Imaging, Diagnosis, Prognosis
specificity but when using the combined factor the true positive
rate was 73% and the true negative rate was 82% with a signif-
icance of P= 0.0002.
There only have been very few studies thus far to further eval-
uate the associations of the angiogenic genes to outcome of pa-
tients with soft tissue sarcoma, although the association of
HIF1a with outcome has recently been discussed in a variety
of other entities (19, 21, 22, 3241). All studies showed a sig-
nificant independent association of HIF1a expression to surviv-
al, although the results were different as to the fact whether
high or low levels are associated with an unfavorable prognosis.
Shintani and others reported that using semiquantitative
immunohistochemistry to detect HIF1a in 49 samples of soft
tissue sarcomas they were able to reveal a significantly indepen-
dent association of HIF1a overexpression with an unfavorable
prognosis. As previously published, we found a significantly
independent association between HIF1a overexpression and
decreased survival time in pancreatic ductal adenocarcinoma
(41). Although we were not able to confirm the linkage of
high-HIF1a expression to an aggravated prognosis in soft tissue
sarcoma, we were able to substantiate the finding that HIF1a
seems to be significantly associated with outcome in patients
with soft tissue sarcoma. Furthermore, we were able to show
this correlation with mRNA-based quantitative RT-PCR in soft
tissue sarcoma and therefore hoped to get a more precise and
objective result than is possible with immunohistochemistry.
Volm and Koomagi examined HIF1a expression in samples
of patients with squamous lung cancer for their correlation to
outcome (42). They analyzed HIF1a in formalin-fixed paraffin-
embedded nonsmall cell lung carcinoma samples (n= 96) by
means of immunohistochemistry to clarify the relationship of
HIF1a overexpression to survival. Despite the findings of Shin-
tani, they found HIF1a overexpression to be significantly asso-
ciated with a favorable prognosis, a result that was confirmed in
oral floor squamous cell and renal cell carcinoma (38, 39). It
has to be mentioned that HIF1a is known to undergo a rapid
posttranscriptional degradation under normoxic conditions by
the ubiquitin-proteasome system, which is restricted by an
oxygen-dependent degradation domain within HIF-1, but un-
der hypoxic conditions, the accumulation of HIF-1a involves
stabilization of the protein (43). For several genes, such as thy-
midylate synthase and dihydropyrimidine dehydrogenase, there
have been studies that provided information on a generally
close linear correlation between the expression of mRNA and
the protein expression (44, 45). Although the regulation of
HIF1a RNA expression seems to respond rapidly to the oxygen
concentrations in the cell (46), meaning that the mechanisms of
HIF1a regulation are transcriptional, as well as a posttranscrip-
tional, it is controversially discussed whether this mechanisms
correlate well with each other (47). Therefore, there might be
quite different findings in correlative studies dependent on
whether they are based on protein or mRNA expression. Further-
more, alternative splicing of HIF1a and upstream genes might
influence the different expression patterns (4851).
The association of HB-EGF with outcome and tumor aggres-
siveness has been recently discussed in bladder, breast, pancre-
atic, and hepatocellular cancer (5258) and is even discussed as
a molecular drug target (12, 13). However, its relationship with
the outcome of patients with soft tissue sarcoma has not been
elucidated. In our study group, we found HB-EGF to be the
strongest independent factor significantly associated with sur-
vival. Using stepwise multivariate Cox proportional hazards re-
gression models, we were able to show that HB-EGF mRNA
expression was stronger associated with outcome than the UICC
stage. Moreover, we were able to use the single factors HIF1a,
VEGF-C, and HB-EGF that showed independent association with
survival as a combined factor that had a relative risk for a exac-
erbated prognosis of 5.48 with a significance of P= 0.0002. In-
terestingly only the combined factor was feasible to distinguish
short (<3 years) from long survivors (3-9 years) with a sensitivity
(true positive rate) of 73% and a specificity (true negative rate)
of 82%. The difference in significance cannot so much be
Fig. 4. Kaplan-Meier plot, estimating overall survival and relapse-free
survival. Differences in survival between the highand lowVEGF-C
expression group were analyzed with the log-rank test. The black line
represents the high-expression group (cut point 40th percentile) with a
median survival of 15 mo (17 of 42 patients, 40.5%), whereas the light gray
curve represents the low-expression group with a median survival of 34 mo
(25 of 42, 59.5%).
Fig. 5. Survival plot from multivariate Cox regression analysis estimating
overall survival and relapse-free survival. The upper light gray solid line
represents the patients with high-HIF1a expression (>40th percentile),
highVEGF-C expression (>40th percentile), and lowHB-EGF expression
(<80th percentile). The lower black solid line represents patients with
low-HIF1a expression (<40th percentile), lowVEGF-C expression (<40th
percentile), and highHB-EGF expression (>80th percentile). The dashed
lines represent the different combined UICC stages (from top to bottom)
5195 Clin Cancer Res 2009;15(16) August 15,
Three-gene signature in sarcoma
explained by differently sized groups (11 of 30 versus 11 of 34)
but rather from the possibly higher association of three genes
out of the angiogenesis pathway to tumor size and vitality of
the tumor cells and therefore survival of the patient. This strong
association of HB-EGF with outcome in soft tissue sarcoma is
especially noteworthy because imatinib mesylate has a strong
physiologic effect on HB-EGF release (10). Imatinib itself is
frequently discussed as a new treatment option for soft tissue
sarcomas (59, 60).
The mRNA expression levels of the genes used in the signa-
ture, HIF1a,HB-EGF, and VEGFC, did not seem to have a sig-
nificant association with tumor size, grading, or the different
UICC stages. These results potentially underline, together with
Cox regression analysis, that gene expression of HIF1a,HB-EGF,
and VEGFC in this patient group was an independent factor. We
did, however, find significant differences for some of the other
used genes like VEGFR1,VEGFR2,VEGFR3,andPDGFA
throughout the clinicopathologic subgroups. It has to be further
examined whether this could lead to markers that help in the
preoperative staging process, meaning that a preoperative biop-
sy and analysis could provide essential information about po-
tential treatment relevant facts earlier than it is currently
available. This could eventually lead to an altered staging and
treatment process with the possibility of neoadjuvant regimens
for certain patient groups.
Due to the emerging discussion about the use of Bevacizumab
in soft tissue sarcoma, we wanted to evaluate the expression pro-
file of VEGF-A, VEGF-C, and their receptors. Interestingly
VEGF-A showed gender-related differences in Kaplan-Meier
based log-rank tests, a fact especially interesting because VEGF
is known to have strong estrogen dependence (6163). Using
multivariate analysis, we can not substantiate this finding, only
VEGF-C seemed to be independently associated with survival.
Also HB-EGF seemed to be much stronger associated with sur-
vival in female patients than in male patients. It might however
be interesting to pursue this hypothesis in a larger patient set to
elucidate whether this possible gender differences can be sub-
stantiated and might therefore be a possible explanation for
the previously described difficulties of implementing new anti-
angiogenic drugs in soft tissue sarcoma (6).
Although the genes on themselves showed significantly differ-
ent strong associations with outcome in the gender subgroups of
the combined factor of HIF1a,VEGF-C and HB-EGF was inde-
pendent from gender significantly associated with survival. We
can only speculate that high HIF1a and VEGF-C indicate that the
vascularization of the tumors was increased, a fact that would
surely provide a better response to therapy.
It has to be mentioned that the herein used subentities, ma-
lignant fibrous histiocytoma, and neurogenic sarcoma may con-
sist of different histologic subtypes, like malignant peripheral
nerve sheath tumors, schwannomas, dedifferentiated liposarco-
mas, or other sarcomas. Therefore these results should not be
generalized at the moment. However, also due to our results
from other entities like pancreatic cancer, we think that further
studies examining these angiogenic genes on larger patient col-
lectives seem to be warranted.
The significant independent association of HIF1a,VEGF-C,
and HB-EGF expression with survival probability (P< 0.0001)
and the significantly higher relative risk of patients when using
a combined factor of these three genes [exp(b) = 5.5] compared
with the UICC stages suggest that these three genes may con-
tribute to a better understanding of the prognosis of patients
with soft tissue sarcoma and may even play a crucial role
for the distribution of patients to multimodal therapeutic regi-
mens. The gender differences in drug-related genes like HB-
EGF and VEGF-A lend support to the idea that hormone levels
might influence angiogenesis and therefore potentially also
affect drug efficiency in patients treated with tyrosine kinase
inhibitors or antiangiogenic drugs. Larger studies including
patients treated with actual chemotherapeutics seem to be
Disclosure of Potential Conflicts of Interest
A. Hoffman is a Consultant for RGI, Inc.; K. Danenberg is CEO of
Response Genetics, Inc.; P. Danenberg is Scientific Advisor for RGI, Inc.
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Imaging, Diagnosis, Prognosis
... For laser-captured microdissection (PALM Microlaser Technologies AG, Munich, Germany), slides of 10-μm thickness were obtained. All tumor slides were prepared as described previously [23]. ...
... 6248,535). The resulting tumor RNA was reversetranscribed into complementary DNA (cDNA) as described previously [23]. Expression of MDR1, ERCC1, and ACTB (β-actin, endogenous reference) was quantified by real-time fluorescence detection of amplified cDNA (ABI PRISM 7900 Sequence Detection System [TaqMan]; Perkin-Elmer Applied Biosystems, Foster City, CA). ...
... Expression of MDR1, ERCC1, and ACTB (β-actin, endogenous reference) was quantified by real-time fluorescence detection of amplified cDNA (ABI PRISM 7900 Sequence Detection System [TaqMan]; Perkin-Elmer Applied Biosystems, Foster City, CA). The reverse transcription-polymerase chain reaction (RT-PCR) assay was implemented as described previously [23]. All primers were selected using the Gene Express software (Applied Biosystems) but were adapted to the requirements of cDNA generated from RNA, which was extracted from the FFPE tissue. ...
Full-text available
PURPOSE: The role of adjuvant chemotherapy in patients with locally advanced bladder cancer still remains to be defined. We hypothesized that assessing the gene expression of the chemotherapy response modifiers multidrug resistance gene 1 (MDR1) and excision repair crosscomplementing 1 (ERCC1) may help identify the group of patients benefiting from cisplatin-based adjuvant chemotherapy. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded tumor samples from 108 patients with locally advanced bladder cancer, who had been enrolled in AUO-AB05/95, a phase 3trial randomizing a maximum of three courses of adjuvant cisplatin and methotrexate (CM) versus methotrexate, vinblastine, epirubicin, and cisplatin (M VEC), were included in the study. Tumor cells were retrieved by laser-captured microdissection and analyzed for MDR1 and ERCC1 expression using a quantitative real-time reverse transcription-polymerase chain reaction assay. Gene expression levels were correlated with clinical outcomes by multivariate Cox proportional hazards regression analysis. RESULTS: Expressions of MDR1 and ERCC1 were independently associated with overall progression-free survival (P = .001, relative risk = 2.9 and P = .01, relative risk = 2.24, respectively). The correlation of high MDR1 expression with inferior outcome was stronger in patients receiving M-VEC, whereas ERCC1 analysis performed equally in the CM and M-VEC groups. CONCLUSIONS: High MDR1 and ERCC1 gene expressions are associated with inferior outcome after cisplatin-based adjuvant chemotherapy for locally advanced bladder cancer. Prospective studies are warranted to define a role for MDR1 and ERCC1 analysis in individualizing multimodality treatment in locally advanced bladder cancer.
... There are only a few studies evaluating the relationship between HIF-1α mRNA expression and the outcome of soft tissue sarcoma patients [23]. In a previous study on a subgroup of 45 patients, of the 114 STS samples we studied, an association between low HIF-1α mRNA expression and an unfavorable outcome was found [23]. ...
... There are only a few studies evaluating the relationship between HIF-1α mRNA expression and the outcome of soft tissue sarcoma patients [23]. In a previous study on a subgroup of 45 patients, of the 114 STS samples we studied, an association between low HIF-1α mRNA expression and an unfavorable outcome was found [23]. However, a study in esophageal squamous cell carcinomas found no association between prognosis and HIF-1α mRNA expression [24]. ...
Full-text available
In various tumors, the hypoxia inducible factor-1α (HIF-1α) and the epidermal growth factor-receptor (EGFR) have an impact on survival. Nevertheless, the prognostic impact of both markers for soft tissue sarcoma (STS) is not well studied. We examined 114 frozen tumor samples from adult soft tissue sarcoma patients and 19 frozen normal tissue samples. The mRNA levels of HIF-1α, EGFR, and the reference gene hypoxanthine phosphoribosyltransferase (HPRT) were quantified using a multiplex qPCR technique. In addition, levels of EGFR or HIF-1α protein were determined from 74 corresponding protein samples using ELISA techniques. Our analysis showed that a low level of HIF-1α or EGFR mRNA (respectively, relative risk (RR) = 2.8; p = 0.001 and RR = 1.9; p = 0.04; multivariate Cox´s regression analysis) is significantly associated with a poor prognosis in STS patients. The combination of both mRNAs in a multivariate Cox’s regression analysis resulted in an increased risk of early tumor-specific death of patients (RR = 3.1, p = 0.003) when both mRNA levels in the tumors were low. The EGFR protein level had no association with the survival of the patient’s cohort studied, and a higher level of HIF-1α protein associated only with a trend to significance (multivariate Cox’s regression analysis) to a poor prognosis in STS patients (RR = 1.9, p = 0.09). However, patients with low levels of HIF-1α protein and a high content of EGFR protein in the tumor had a three-fold better survival compared to patients without such constellation regarding the protein level of HIF-1α and EGFR. In a bivariate two-sided Spearman’s rank correlation, a significant correlation between the expression of HIF-1α mRNA and expression of EGFR mRNA (p < 0.001) or EGFR protein (p = 0.001) was found, additionally, EGFR mRNA correlated with EGFR protein level (p < 0.001). Our results show that low levels of HIF-1α mRNA or EGFR mRNA are negative independent prognostic markers for STS patients, especially after combination of both parameters. The protein levels showed a different effect on the prognosis. In addition, our analysis suggests a possible association between HIF-1α and EGFR expression in STS.
... It has been confirmed by a number of studies that expression of HIF-1α is correlated with poor prognosis in various cancers, including gastric, esophageal and lung cancers (Zhang et al. 2013;Ping et al. 2014;Wang et al. 2014). However, the prognostic role of HIF-1α expression in bone and soft tissue sarcoma has not reached a consensus since inconsistent results were reported in previous studies Kim et al. 2015;Hu et al. 2015;Guo et al. 2014;Smeland et al. 2012;Chen et al. 2012aChen et al. , b, 2011Zeng et al. 2010;Huang et al. 2010;Boeuf et al. 2010;Hoffmann et al. 2009;Mizobuchi et al. 2008;Kubo et al. 2008;Chen et al. 2008;Yang et al. 2007;Shintani et al. 2006). To date, there has been no comprehensive meta-analysis to clarify its prognostic role in sarcoma. ...
... One study in Chinese language was also excluded, for its unfamiliarity for non-Chinese speakers. Eventually, 16 articles published from 2006 to 2015 were included in the current meta-analysis ( Fig. 1) Kim et al. 2015;Hu et al. 2015;Guo et al. 2014;Smeland et al. 2012;Chen et al. 2008Chen et al. , 2012aChen et al. , b, 2011Zeng et al. 2010;Huang et al. 2010;Boeuf et al. 2010;Hoffmann et al. 2009;Mizobuchi et al. 2008;Kubo et al. 2008;Yang et al. 2007;Shintani et al. 2006). The NOS scores of the included studies are counted and shown in Table 1. ...
Full-text available
The prognostic significance of Hypoxia-inducible factor-1α (HIF-1α) in patients with bone and soft tissue sarcoma remains controversial. To investigate the impact of its expression on survival outcomes, we performed a meta-analysis. Comprehensive literature searches were conducted in PubMed, Web of Science, Embase and Cochrane Library. A total of 16 studies published from 2006 to 2015 were included. We found that expression of HIF-1α was significantly associated with higher rate of metastasis (RR 3.21, 95 % CI 2.12–4.84, P < 0.001), poorer overall survival (HR 2.05, 95 % CI 1.51–2.77, P < 0.001) and poorer disease-free survival (HR 2.05, 95 % CI 1.55–2.70, P < 0.001). In addition, when subgroup analysis was conducted according to histology type, the significant correlations to poor overall survival and disease-free survival were also observed in patients with osteosarcoma, chondrosarcoma and soft tissue sarcoma. Publication bias was not found and sensitivity analysis showed the results were stable. In conclusion, HIF-1α expression might be an effective predicative factor of poor prognosis for bone and soft tissue sarcoma.
... Mutations in IDH1 and 2 lead to the production of 2-hydroxyglutarate [201], an inducer of HIF1α stabilization. HIF1a expression and hypoxia are associated with poor survival of sarcoma patients [68][69][70][202][203][204]. ...
Full-text available
Metabolic rewiring offers novel therapeutic opportunities in cancer. Until recently, there was scant information regarding soft tissue sarcomas, due to their heterogeneous tissue origin, histological definition and underlying genetic history. Novel large-scale genomic and metabolomics approaches are now helping stratify their physiopathology. In this review, we show how various genetic alterations skew activation pathways and orient metabolic rewiring in sarcomas. We provide an update on the contribution of newly described mechanisms of metabolic regulation. We underscore mechanisms that are relevant to sarcomagenesis or shared with other cancers. We then discuss how diverse metabolic landscapes condition the tumor microenvironment, anti-sarcoma immune responses and prognosis. Finally, we review current attempts to control sarcoma growth using metabolite-targeting drugs.
... In addition, our analyses indicate that both EGF and HB-EGF are present systemically at higher concentrations in sarcoma subjects than healthy controls (Fig. 1). HB-EGF has been identified previously as a potential biomarker in certain subtypes of sarcoma (30), and high levels of HB-EGF transcripts have been correlated with decreased overall survival (31). EGF is expressed in soft-tissue sarcomas, and initial studies into EGFR blockade plus chemotherapy have shown decreased tumor growth both in vitro and in a mouse model of fibrosarcoma (32), but EGFR levels may contribute to starvation-and chemotherapeutic-resistance in osteosarcoma (33). ...
Full-text available
Sarcomas are a rare but fatal tumor type that accounts for <1% of adult solid malignancies and ~15% of childhood malignancies. Although the use of immunotherapy is being actively investigated for other solid tumors, advances in immunotherapy for sarcoma patients are lacking. To better understand the systemic immune environment in sarcoma patients, we performed a detailed multiplex analysis of serum cytokines, chemokines, and protumorigenic factors from treatment-naive subjects with localized, high-grade sarcoma. Because obesity is a major healthcare issue in the United States, we additionally examined the effects of obesity on serum protein profiles in our sarcoma subject cohort. We found that the systemic host environment is profoundly altered to favor tumor progression, with epidermal growth factor, angiopoietin-2, vascular endothelial growth factor A, IL-6, IL-8, and MIP-1β all increased relative to tumor-free controls (all p < 0.05). Surprisingly, we found that obesity did not exacerbate this protumorigenic profile, as epidermal growth factor and IL-8 decreased with increasing subject body mass index (both p < 0.05 versus normal or overweight subjects). The Th2-related cytokines IL-4, IL-5, and IL-13 were also decreased in the presence of obesity. Thus, although the systemic environment in sarcoma subjects favors tumor progression, obesity does not further aggravate the production of protumorigenic factors.
... Microarray technology, applied to tumors, has the potential to overcome this difficulty and to provide a probe to monitor average hypoxia in the tumor mass [60]. The use of gene expression signatures to measure hypoxia has been reported [36,56,61] and their potential as prognostic factors was shown, for example, in soft tissues sarcomas [62] and hepatocellular carcinoma [63]. ...
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Background More than fifty percent of neuroblastoma (NB) patients with adverse prognosis do not benefit from treatment making the identification of new potential targets mandatory. Hypoxia is a condition of low oxygen tension, occurring in poorly vascularized tissues, which activates specific genes and contributes to the acquisition of the tumor aggressive phenotype. We defined a gene expression signature (NB-hypo), which measures the hypoxic status of the neuroblastoma tumor. We aimed at developing a classifier predicting neuroblastoma patients’ outcome based on the assessment of the adverse effects of tumor hypoxia on the progression of the disease. Methods Multi-layer perceptron (MLP) was trained on the expression values of the 62 probe sets constituting NB-hypo signature to develop a predictive model for neuroblastoma patients’ outcome. We utilized the expression data of 100 tumors in a leave-one-out analysis to select and construct the classifier and the expression data of the remaining 82 tumors to test the classifier performance in an external dataset. We utilized the Gene set enrichment analysis (GSEA) to evaluate the enrichment of hypoxia related gene sets in patients predicted with “Poor” or “Good” outcome. ResultsWe utilized the expression of the 62 probe sets of the NB-Hypo signature in 182 neuroblastoma tumors to develop a MLP classifier predicting patients’ outcome (NB-hypo classifier). We trained and validated the classifier in a leave-one-out cross-validation analysis on 100 tumor gene expression profiles. We externally tested the resulting NB-hypo classifier on an independent 82 tumors’ set. The NB-hypo classifier predicted the patients’ outcome with the remarkable accuracy of 87 %. NB-hypo classifier prediction resulted in 2 % classification error when applied to clinically defined low-intermediate risk neuroblastoma patients. The prediction was 100 % accurate in assessing the death of five low/intermediated risk patients. GSEA of tumor gene expression profile demonstrated the hypoxic status of the tumor in patients with poor prognosis. Conclusions We developed a robust classifier predicting neuroblastoma patients’ outcome with a very low error rate and we provided independent evidence that the poor outcome patients had hypoxic tumors, supporting the potential of using hypoxia as target for neuroblastoma treatment.
... The involvement of angiogenesis factors in soft-tissue sarcoma (vascular endothelial growth factor [VEGF] [2], fibroblastic growth factor [FGF] [3], placenta-derived growth factor [PIGF] [4], hypoxia-inducible factor 1-α [HIF 1-α] [5], plateletderived growth factor receptors [PDGF] [2]) has been presented in further publications underlying the relationship between these mediators and tumor grade [6], response [4], worse metastasis-free and overall survival (OS) rates in localized disease [7]. For patients with advanced disease, palliative chemotherapy, based on high-dose doxorubicin and ifosfamide regimens, is the backbone of treatment. ...
Introduction: In the past decade, treatment options for metastatic renal cell carcinoma and soft-tissue sarcoma have expanded. Pazopanib was discovered during the screening of compounds that suppressed vascular endothelial growth factor receptor-2 (VEGFR-2). As other tyrosine kinase inhibitors (TKI), pazopanib is not totally specific for one target since it also inhibits stem-cell factor receptor (cKIT), platelet-derived growth factor receptors (PDGFRα, β), VEGFR-1 and -3. Areas covered: Clinical pharmacology, drug-drug interactions and safety data published on pazopanib, between January 2006 and April 2016, are reviewed. Expert opinion: This new therapy has been shown to improve progression-free survival compared with previous approaches, in renal cell cancer and soft-tissue sarcoma. However, some specific sub-populations, such as elderly patients, patients with brain metastases or with Eastern Cooperative Oncology Group Performance Status (ECOG PS) 2 or comorbidities, are poorly represented in pivotal pazopanib phase III studies. Pazopanib meets criteria defining therapies as candidates for therapeutic drug monitoring: large intra- and inter-patient pharmacokinetic variability, potential pharmacokinetic drug-drug interactions, pharmacokinetic/pharmacodynamic relationship and narrow therapeutic index. Knowledge of predictors that can be used to guide dosing regimens in the target population and in special populations needs to be improved.
Clinical research, being patient-oriented, is based predominantly on clinical data – symptoms reported by patients, observations of patients made by health-care providers, radiological images, and various metrics, including laboratory measurements that reflect physiological functions. Recently, however, a new type of data – genes and their products – has entered the picture, and the expectation is that given clinical conditions can ultimately be linked to the function of specific genes. The postgenomic era is characterized by the availability of the human genome as well as the complete genomes of numerous reference organisms. How genomic information feeds into clinical research is the topic of this chapter. We first review the molecules that form the “blueprint of life” and discuss the surrounding research methodologies. Then we discuss how genetic data are clinically integrated. Finally, we relate how this new type of data is used in different clinical research domains.
Background: Soft-tissue sarcomas (STS) are a group of rare, heterogeneous and aggressive tumors, with high metastatic risk and relatively few efficient systemic therapies. We hypothesized that the Genomic Grade Index (GGI), a 108-gene signature previously developed in early-stage breast cancer, might improve the prognostic assessment of patients with early-stage STS. Patients and methods: We collected gene expression and clinicopathological data of 678 operated STS, and searched for correlations between the GGI-based classification and clinicopathological variables, including the metastasis-free survival (MFS). Results: Based on GGI, 275 samples (41%) were classified as "GGI-low" and 403 (59%) as "GGI-high". The "GGI-high" class was more associated with poor-prognosis features than the "GGI-low" class: pathological grade 3 (p=9.50E-11), undifferentiated sarcomas and leiomyosarcomas (p<1.00E-06), location in extremities (p<1.00E-06), and complex genetic profile (p=2.1E-20). The 5-year MFS was 53% (95%CI 47-59) in the "GGI-high" class vs. 78% (95%CI 72-85) in the "GGI-low" class (p=3.02E-11), with a corresponding Hazard Ratio for metastatic relapse equal to 2.92 (95%CI 2.10-4.07; p=2.23E-10). In multivariate analysis, the GGI-based classification remained significant, whereas the pathological grade did not. In fact, the GGI-based classification stratified the patients with pathological grade 1-2 and those with pathological grade 3 in two classes with different 5-year MFS. Comparison of the GGI and CINSARC multigene signatures revealed similar correlations with clinicopathological variables, which were however stronger with GGI than with CINSARC, a strong concordance (71%) in term of low-risk or high-risk classifications, and independent prognostic value for MFS in multivariate analysis, suggesting complementary prognostic information. Conclusion: GGI refines the prediction of MFS in operated STS and might improve the tailoring of adjuvant chemotherapy. Further clinical validation is warranted in larger retrospective, then prospective series, as well as the functional validation of relevant genes that could provide new therapeutic targets.
Background and aim: Sarcomas are of mesenchymal origin and typically show abundant tumour stroma and presence of necrosis. In search for novel biomarkers for personalised therapy, we determined the prognostic impact of stromal markers, hypoxia and neovascularity in high-grade soft tissue leiomyosarcoma and pleomorphic undifferentiated sarcoma. Method: We evaluated CD163, colony-stimulating factor (CSF)-1, CD16 and hypoxia-inducible factor 1 (HIF-1)α using immunohistochemical staining and assessed microvessel density using CD31 in 73 high-grade leiomyosarcomas and undifferentiated pleomorphic sarcomas of the extremities and the trunk wall. The results were correlated to metastasis-free and overall survival. Results: Expression of HIF-1α was associated with the presence of necrosis and independently predicted shorter metastasis-free survival (HR 3.2, CI 1.4 to 7.0, p=0.004), whereas neither expression of the stromal markers CD163, CD16 and CSF-1 nor microvessel density was prognostically relevant in this series. Conclusions: There is increasing evidence for the prognostic role of hypoxia in high-grade soft tissue sarcoma, and these data suggest that HIF-1α expression represents a candidate prognostic biomarker for clinical application in high-grade leiomyosarcoma and undifferentiated pleomorphic sarcoma.
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Thymidylate synthase (TS) is the target enzyme for 5-fluorouracil (5-FU). We have correlated TS protein and gene expression with the response in patients with colorectal (n = 9) and gastric cancer (n = 12) treated with infusional 5-FU plus leucovorin (LV) or infusional 5-FU/LV and cisplatin, respectively. TS protein expression was analyzed by Western blot using TS106 monoclonal antibody and densitometry scanning. TS gene expression was measured by PCR analysis using beta-actin as an internal standard and expressed as a TS:beta-actin mRNA ratio. A close linear relationship was noted between TS protein expression and TS gene expression (r2 = 0.60) for the 21 tumor samples analyzed. TS immunohistochemical staining on 15 of the 21 samples revealed that the TS staining intensity correlated closely with TS protein and mRNA expression. In two biopsy samples, TS protein levels and TS gene expression did not correlate; however, one of these exhibited a focal TS staining pattern. Both the TS protein level and TS gene expression were significantly associated with response to 5-FU-based therapy. Patients with responsive disease had a mean TS protein level of 0.17 +/- 0.03 arbitrary units (range, 0.05 to 0.38), whereas in patients whose tumors did not respond, the mean TS protein level was significantly higher 0.60 +/- 0.09 (range, 0.06 to 1.01; P < 0.01). A similar pattern was noted with TS gene expression. In patients with responsive disease, the mean TS:beta-actin gene ratio was 1.36 +/- 0.3 (range, 0.5-3.3 x 10(-3). In contrast, biopsies from patients with unresponsive disease had a mean TS:beta-actin gene ratio of 15.4 +/- 2.6 x 10(-3) (range, 2.7-35.9; P < 0.01). TS protein and TS mRNA expression are highly correlated, and each predict for response to 5-FU/LV-based chemotherapy in patients with colorectal and gastric cancer.
Abstract The hypoxia-inducible factor (HIF-1α), a basic helix-loop-helix transcription factor, is known to heterodimerize with ARNT1, a nuclear translocator, to trigger the overexpression in many cells of genes involved in resistance to hypoxia. Although HIF-1α and ARNT1 are both expressed in brain, their cellular localization and function therein are unknown. Here, using in situ hybridization and immunocytochemistry, we show that HIF-1α is expressed in normoxic cerebral neurons together with not only ARNT1 but also ARNT2, a cerebral translocator homologous to ARNT1 but displaying, unlike ARNT1, a selective neuronal expression. In contrast, other potential partners of the translocators, i.e. the aryl hydrocarbon receptor (AHR) and the single-minded protein 2 (SIM2), are not expressed in the adult brain. We also identify two splice variants of HIF-1α in brain, one of which dimerizes with ARNT2 even more avidly than with ARNT1. The resulting heterodimer, in contrast with the HIF-1α/ARNT1 complex, does not recognize the HIF-1-binding site of the hypoxia-induced erythropoietin (Epo) gene, suggesting that it controls transcription of a distinct set of genes. We therefore propose that HIF-1α and ARNT2 function as preferential dimerization partners in neurons to control specific responses, some of which may not be triggered by hypoxia. In support of this proposal, in nonhypoxic PC12 cells constitutively coexpressing HIF-1α, ARNT1 and ARNT2, downregulation of either HIF-1α or ARNT2, obtained with selective antisense nucleotides, resulted in inhibition of [3H]thymidine incorporation.
The expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was investigated for 76 cases of breast carcinoma. HB-EGF was expressed in 71.8% of the carcinoma cases but only slightly in normal mammary glands. Interestingly, its expression was inversely related to biological aggressiveness of the breast carcinoma. These results suggest that HB-EGF may play a crucial role in the early stage of this carcinoma.
Cleavage of membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields a soluble HB-EGF isoform (sHB-EGF), which is an activating epidermal growth factor receptor (EGFR) ligand and a C-terminal fragment HB-EGF-C acting directly in the nucleus. In bladder cancer, overexpression of both HB-EGF and EGFR have been observed, but to the authors' knowledge the prognostic significance of different modes of HB-EGF signaling have remained unclear. Expression and intracellular localization of HB-EGF and EGFR were examined by immunohistochemistry in paraffin-embedded specimens from 121 patients who underwent cystectomy for bladder cancer. Tumor stage was pTis/pT1 in 7 patients, pT2 in 41 patients, pT3 in 55 patients, and pT4 in 18 patients. Lymph node metastases were present in 32 patients. Using an antibody directed against the C-terminal domain, HB-EGF expression was detected in the cytoplasm or in the nucleus of tumor cells. EGFR staining was uniform at the plasma membrane. The actuarial 5-year cancer-specific survival of patients with tumors with predominant nuclear HB-EGF staining was 28% compared with 57% if HB-EGF staining was predominantly cytoplasmic (P = .027). Disease outcome of patients with a 'mixed' HB-EGF staining pattern was found to be between that of the 2 former groups. In agreement with previous studies, strong EGFR expression was associated with poor prognosis. Despite strong EGFR expression, predominant cytoplasmic HB-EGF staining was associated with a more favorable outcome, whereas a predominant nuclear pattern defined a subgroup with extremely poor prognosis (5-year tumor-specific survival of 55% vs 13%, respectively; P = .026). The current study results confirm that EGFR expression is significantly correlated with disease-specific mortality but that the outcome is also influenced by the mode of HB-EGF signaling. Additional nuclear HB-EGF signaling, indicative of increased cleavage of proHB-EGF, appears to enhance the adverse activities. Cytoplasmic HB-EGF staining likely reflects proHB-EGF, which may also exert antiproliferative effects.
Vascular endothelial growth factor (VEGF), an endothelial cell mitogen and permeability factor, participates in tumor angiogenesis, but less is known about its regulation or function in normal vascular homeostasis. In the uterus, which undergoes cyclic changes in its vasculature, VEGF is induced by estrogen. Since the pituitary gland contains highly permeable capillaries and is estrogen-responsive, our objectives were to localize VEGF expression within the pituitary and to determine whether it is regulated by estrogen in both the pituitary and the somatolactotrope cell line, GH(3). Ovariectomized rats were injected with estradiol, and pituitaries and uteri were subjected to in situ hybridization or quantitative reverse transcription-polymerase chain reaction (RT-PCR). VEGF expression was strong and punctate in the neural lobe, weaker and diffuse in the anterior lobe and undetectable in the intermediate lobe. Two VEGF isoforms, 164 and 120, were detected in all tissues. In the posterior pituitary, VEGF expression was 3- to 6-fold higher than in the anterior pituitary or uterus and was unaltered by estrogen. In contrast, anterior pituitary VEGF was induced by estrogen within 1 h, peaked at 3 h, and returned to basal levels by 24 h. Similar dynamics, albeit 10-fold higher, were seen in the uterus. Translated VEGF proteins were detected by Western blot in both the anterior pituitary and uterus. GH(3) cells also showed a dose- and time-dependent induction of VEGF expression by estrogen. In conclusion: (1) VEGF expression is higher in the neural lobe than in the anterior lobe and is undetectable in the intermediate lobe, (2) the expression of VEGF164 and VEGF120 is rapidly upregulated by estrogen in the anterior pituitary but is unchanged in the posterior pituitary, and (3) the pituitary lactotrope cell line, GH(3), also increases VEGF expression in response to estradiol.
Pancreatic cancer still has one of the worst prognoses of all cancers with a 5-year survival rate of 5%, making it necessary to find markers or gene sets that would further classify patients into different risk categories and thus allow more individually adapted multimodality treatment regimens. Especially heparanase (HPSE) has recently been discussed as a key factor in pancreatic cancer. Paraffin-embedded tissue samples were obtained from 41 patients with pancreatic adenocarcinoma who were scheduled for primary surgical resection. Direct quantitative real-time reverse transcriptase polymerase chain reaction (TaqMan) assays were performed in triplicates to determine HPSE, hypoxia inducible factor-1 alpha (HIF1a), platelet-derived growth factor alpha (PDGFA), heparin-binding EGF-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF) gene expression levels. HPSE was significantly correlated to PDGFA (p = 0.04) and HIF1a (p = 0.04). The correlation of HIF1a to bFGF and HB-EGF was significant (p = 0.04, p = 0.02). Stepwise multiple linear regression models showed a significant independent association of HPSE with lymph node metastasis (p = 0.025) and with dedifferentiation (p = 0.042). Heparanase seems to be significantly associated with lymph node metastasis (p = 0.025) as well as dedifferentiation (p = 0.042). We assume that HPSE plays a crucial role for the aggressiveness of pancreatic cancer. Larger studies including more patients seem to be warranted.
The limitations of diagnostic "accuracy" as a measure of decision performance require introduction of the concepts of the "sensitivity" and "specificity" of a diagnostic test. These measures and the related indices, "true positive fraction" and "false positive fraction," are more meaningful than "accuracy," yet do not provide a unique description of diagnostic performance because they depend on the arbitrary selection of a decision threshold. The receiver operating characteristic (ROC) curve is shown to be a simple yet complete empirical description of this decision threshold effect, indicating all possible combinations of the relative frequencies of the various kinds of correct and incorrect decisions. Practical experimental techniques for measuring ROC curves are described, and the issues of case selection and curve-fitting are discussed briefly. Possible generalizations of conventional ROC analysis to account for decision performance in complex diagnostic tasks are indicated. ROC analysis is shown to be related in a direct and natural way to cost/benefit analysis of diagnostic decision making. The concepts of "average diagnostic cost" and "average net benefit" are developed and used to identify the optimal compromise among various kinds of diagnostic error. Finally, the way in which ROC analysis can be employed to optimize diagnostic strategies is suggested.
Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2, consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia.