Gene Expression Profiling Predicts Survival
in Conventional Renal Cell Carcinoma
Hongjuan Zhao1, Bo ¨rje Ljungberg2, Kjell Grankvist2, Torgny Rasmuson2, Robert Tibshirani3, James D. Brooks1*
1 Department of Urology, Stanford University School of Medicine, Stanford, California, United States of America, 2 Departments of Surgical and Perioperative Sciences,
Urology, and Andrology, Medical Biosciences, Clinical Chemistry, and Radiation Sciences, Oncology, Umea ˚ University, Umea ˚, Sweden, 3 Department of Health Research and
Policy, Stanford University School of Medicine, Stanford, California, United States of America
Competing Interests: The authors
have declared that no competing
Author Contributions: BL and JDB
designed the study. HZ, BL, KG, and
TR performed the experiments. HZ,
RT, and JDB analyzed the data. HZ,
BL, RT, and JDB contributed to
writing the paper.
Academic Editor: Francesco
Marincola, National Institutes of
Health, United States of America
Citation: Zhao H, Ljungberg B,
Grankvist K, Rasmuson T, Tibshirani
R, et al. (2006) Gene expression
profiling predicts survival in
conventional renal cell carcinoma.
PLoS Med 3(1): e13.
Received: July 18, 2005
Accepted: October 12, 2005
Published: December 6, 2005
Copyright: ? 2006 Zhao et al. This is
an open-access article distributed
under the terms of the Creative
Commons Attribution License, which
permits unrestricted use,
distribution, and reproduction in any
medium, provided the original
author and source are credited.
Abbreviations: cRCC, conventional
renal cell carcinoma; RCC, renal cell
carcinoma; SPC, supervised principal
* To whom correspondence should
be addressed. E-mail: jdbrooks@
A B S T R A C T
Conventional renal cell carcinoma (cRCC) accounts for most of the deaths due to kidney
cancer. Tumor stage, grade, and patient performance status are used currently to predict
survival after surgery. Our goal was to identify gene expression features, using comprehensive
gene expression profiling, that correlate with survival.
Methods and Findings
Gene expression profiles were determined in 177 primary cRCCs using DNA microarrays.
Unsupervised hierarchical clustering analysis segregated cRCC into five gene expression
subgroups. Expression subgroup was correlated with survival in long-term follow-up and was
independent of grade, stage, and performance status. The tumors were then divided evenly
into training and test sets that were balanced for grade, stage, performance status, and length
of follow-up. A semisupervised learning algorithm (supervised principal components analysis)
was applied to identify transcripts whose expression was associated with survival in the training
set, and the performance of this gene expression-based survival predictor was assessed using
the test set. With this method, we identified 259 genes that accurately predicted disease-
specific survival among patients in the independent validation group (p , 0.001). In
multivariate analysis, the gene expression predictor was a strong predictor of survival
independent of tumor stage, grade, and performance status (p , 0.001).
cRCC displays molecular heterogeneity and can be separated into gene expression
subgroups that correlate with survival after surgery. We have identified a set of 259 genes
that predict survival after surgery independent of clinical prognostic factors.
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P PL Lo oS S MEDICINE
Nearly half of the patients diagnosed with renal cell
carcinoma (RCC) succumb to their disease, and RCC accounts
for 95,000 deaths per year worldwide . In the United States,
approximately 36,160 cases will be diagnosed this year alone,
and 12,660 patients will die of their disease . Conventional
renal cell carcinoma (cRCC) accounts for approximately 75%
of all RCC and accounts for the majority of kidney cancer
mortality. Surgery (nephrectomy) can cure 60%–70% of
patients with localized disease and prolong survival in
patients with metastatic disease, although survival rates after
treatment have not changed appreciably in the past 30 y [2,3].
Cytokine therapy, which is reserved for patients with
advanced disease, can produce partial responses in 10%–
15% of patients and durable remissions in 5% .
Tumor stage is the most powerful predictor of outcome in
patients with cRCC, although it provides a relatively crude
estimate of survival that limits its use in clinical decision
making . Several prognostic algorithms have been devel-
oped that incorporate tumor stage, grade, and patient
performance status, and they predict survival better than
stage alone [5–7]. Based on these algorithms, fewer radio-
graphic imaging and blood tests have been proposed for
patients predicted to have a low risk of recurrence after
surgery, and adjuvant therapy has been suggested for high-
risk patients. Unfortunately, many patients fall into inter-
mediate-risk categories, and these algorithms do not predict
survival or response to therapy in patients with advanced
The limitations of the prognostic algorithms and the varied
response to surgery and immunotherapy suggest that cRCCs
are molecularly diverse and that capturing relevant molecular
features could improve outcome prediction. In support of
this idea, several small series used DNA microarray analysis to
identify genes whose expression levels correlated with
survival in RCC, although the prognostic gene sets did not
overlap, and neither study has been validated independently
[8–10]. To identify gene expression correlates of survival in
cRCC, we used DNA microarrays to explore systematically the
molecular variations underlying the biologic and clinical
heterogeneity in a set of 177 tumors with associated detailed
clinical information, including long-term follow-up.
Tumors from 177 consecutive patients who underwent
radical nephrectomy for cRCC collected between 1985 and
2003 were selected from the fresh-frozen tissue bank in the
Department of Urology, Umea ˚ University Hospital (Umea ˚,
Sweden). Written informed consent was obtained from all
patients, and the study was approved by the institutional
review board of each participating center. Patients in the
study included 102 men and 75 women with cRCC diagnosed
on the nephrectomy specimens by pathologists at Umea ˚
University Hospital (summarized in Table 1). Mean age of the
patients was 65 y (range, 34 to 85 y), and performance status,
assessed using World Health Organization criteria, ranged
from 0 (65 patients), 1 (64 patients), 2 (37 patients), 3 (ten
patients), to 4 (one patient). Pathologic stage grouping of
patients in the study, based on preoperative radiographic
studies and pathological assessment of the surgical specimens
was I (49 patients), II (29 patients), III (40 patients), and IV (59
patients). No patient received neoadjuvant therapy prior to
surgery. Adjuvant interferon therapy was given to seven
patients and adjuvant hormonal therapy to 12 patients, and
all had stage IV disease at the time of surgery. Thirteen
patients who recurred after surgery received salvage inter-
feron therapy, nine had resection of metastases, and 19
received hormonal therapy. Patient follow-up status was
assessed at least yearly by routine clinical follow-up at Umea ˚
University Hospital or by contacting patients directly.
Median follow-up of censored patients was 76 mo (range 19
to 224 mo). During the follow-up period, 87 patients died of
their disease, 25 died of other causes, nine were alive with
disease, and 56 were alive and free of disease.
Gene Expression Profiling
Total RNA was isolated from the cRCC tissue samples using
TRIzol reagent (Invitrogen, Carlsbad, California, United
States), according to the manufacturer’s recommendations.
The integrity of the total RNA was assessed using a 2100
Bioanalyzer (Agilent Technologies, Palo Alto, California,
United States). Cy5-labeled total RNA from cRCC samples
was mixed with Cy3-labeled Universal Human Reference
RNA (Stratagene, La Jolla, California, United States) and
hybridized to cDNA microarrays (manufactured by the
Stanford Functional Genomics Facility) that contained over
40,000 cDNA clones, representing 27,290 unique UniGene
clusters as described previously . Arrays from ten differ-
ent print runs were used in the study, and all arrays passed a
set of quality control criteria defined by GenePix software,
including mean of median background less than 500, feature
variation less than 0.5, background variation less than 0.5, and
features with saturated pixels less than 0.1%. For an
explanation of each of these quality control measures, see
gn_genepix_pro.html. Microarrays were imaged using an
Axon GenePix 4000B scanner (Axon Instruments, Union City,
California, United States), and fluorescence ratios of the
tumor RNA specimens compared to the reference RNA were
determined using GenePix software. Data was entered into
Stanford Microarray Database for subsequent analysis .
The complete microarray dataset is available at http://smd.
pl?pub_no¼484. The data also have been deposited in
National Center for Biotechnology Information’s Gene
Expression Omnibus (see Accession Numbers section).
Hierarchical clustering analysis. Fluorescence ratios were
normalized by mean-centering genes for each microarray,
mean-centering each gene across all microarrays, and center-
ing within each of ten microarray print runs (to minimize
potential print-run-specific bias). We selected 3,674 genes
represented by 5,560 clones on the microarrays whose
expression was both well measured and highly variable
among samples (a complete list is available at http://smd.
pl?pub_no¼484). We defined well-measured genes as those
with a ratio of signal intensity to background noise of more
than 1.5 for either the Cy5-labeled cRCC sample or the Cy3-
labeled reference sample, in at least 70% of the samples
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Gene Expression and Survival in Renal Cancer
hybridized. Genes with highly variable expression were
defined as those whose expression was higher or lower by a
factor of at least three than the average expression of all
cRCC samples in at least ten cRCC samples. We applied two-
way (genes-against-samples) average-linkage hierarchical clus-
tering and used TreeView to visualize the results . We
compared the survival times of the five gene expression
subgroups using Kaplan-Meier survival analysis and the log-
Supervised principal components analysis. For outcome
prediction, we randomly divided samples—that had been
prestratified to ensure that a similar proportion of samples in
each group were from patients who had died and with similar
clinical parameters, including tumor stage grouping, grade,
performance status, and length of follow-up—into a separate
training set (88 samples) and test set (89 samples) (Table 2). In
the training set, we calculated modified univariate Cox
proportional-hazard scores for all genes (n ¼ 14,814) that
were well measured to identify genes whose expression
correlated with the duration of survival. (The modification
adds a constant to the denominator, as described in .) We
selected a set of genes whose absolute Cox score statistic
exceeded a threshold that was chosen using multiple 2-fold
cross-validation. To determine the threshold, the training
samples were divided randomly and principal components
were derived from half of the samples and then used in a Cox
model to predict survival in the other half. We repeated this
entire process five times and found that a threshold of 61.5
yielded the highest average partial log-likelihood ratio
statistic. Principal components analysis was then performed
on all cases in the training set, using 340 transcripts
representing 259 genes whose absolute Cox score equaled
or exceeded the threshold. Only the first principal compo-
nent was associated significantly with survival. For patients in
the test set, a continuous risk score (that is, the supervised
principal components [SPC] risk score) was calculated for
each patient, based on transcript levels across the 340
transcripts and the weights assigned to each transcript
derived from SPC analysis of the training set. Multivariate
proportional-hazards analysis was performed on the test set
with the SPC risk score as a continuous variable, along with
stage grouping, grade, performance status, and gene expres-
sion subgroup derived from hierarchical clustering analysis.
To evaluate the gene set as a categorical predictor of
survival, we divided the training set into tertiles based on the
SPC risk scores. The test set then was divided into three
groups based on the tertiles of the SPC risk scores of the
training set. We compared the survival times of the three
subgroups in the training and test sets using Kaplan-Meier
survival analysis and the log-rank test.
SPC analysis and multivariate proportional-hazards analy-
sis were performed with the use of the R software package
(available at www.r-project.org) and the superpc R package
(available at http://www-stat.stanford.edu/;tibs/superpc). Ka-
plan-Meier survival analysis was performed with WinStat
software (R. Fitch Software, Staufen, Germany).
Gene Expression Profiles of cRCCs
Hierarchical clustering analysis of the 177 patient samples
described in Table 1 was performed using 5,560 clones
representing 3,674 unique genes whose expression varied
more than 3-fold from the mean expression ratio (specimen
RNA/reference RNA) in at least ten samples (Figure 1). A
detailed view of the sample cluster dentodrogram is displayed
in Figure S1. Tumors were partitioned into two main groups
and five subgroups based on the differential expression of
these 3,674 genes (Figure 1A). The grouping of the tumors in
Table 1. Description of Patients
Patient sex: n (%) Male: 102 (58)
Female: 75 (42)
65 (range 34 to 85)
0: 65 (37)
1: 64 (36)
2: 37 (21)
3: 10 (5.5)
4: 1 (0.5)
I: 49 (28)
II: 29 (16)
III: 40 (23)
IV: 59 (33)
1: 9 (5)
2: 34 (19)
3: 94 (53)
4: 40 (23)
76 (range 19 to 224)
Mean patient age (y)
WHO performance status: n (%)
Tumor stage: n (%)
Tumor grade: n (%)
Mean follow-up (mo)
Patient treatment: n (%)
Adjuvant interferon therapy
Adjuvant hormonal therapy
Salvage interferon therapy
Resection of metastases
Salvage hormonal therapy
Patient outcome: n (%)
Died of disease
Died of other causes
Alive with disease
Alive and free of disease
WHO, World Health Organization.
Table 2. Patient Distribution between Training and Test Set
Parameters Training SetTest Setp-Value
Performance status 0.37
Median follow-up (range) months
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Gene Expression and Survival in Renal Cancer
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Gene Expression and Survival in Renal Cancer
the dendrogram did not appear to be an artifact of the genes
used to generate the cluster because varying data-filtering
criteria (and the number of genes used in the hierarchical
clustering analysis) resulted in a similar pattern of specimen
clustering. A large and diverse set of genes distinguished the
two main groups of tumors, all of which showed relatively
high expression in tumors in subgroups 1 and 2 compared to
subgroups 3, 4, and 5 (black bar in Figure 1A). These genes
are involved in a variety of biological processes, including
angiogenesis (FLT1, EPAS1, and JAG1), the Wnt signaling
pathway (FZD1, FZD4, and TCF4), cell adhesion (CDH13,
PECAM1, and VCAM1), and cellular metabolism (UGT2B7,
UGT2B4, and GSTA2).
Each of the five subgroups of tumors displayed distinct
gene expression patterns. Examples of clusters whose
expression patterns distinguished between the subgroups
are shown in Figure 1B. Expression patterns in subgroups 1
and 2 were largely similar, although they differed in a set of
genes involved in diverse biological processes, including the
transcriptional regulators MLL3, EYA3, JMJD1C, CNOT4,
CNOT6L, SP3, and TEAD1 (Figure 1I). Compared to the other
cRCCs, those in subgroup 4 showed lower expression of many
hypoxia-regulated genes (e.g., HIG2, EGLN3, CA9, and STC2)
(Figure 1C). Conventional RCCs commonly harbor VHL gene
mutations that result in increased expression of hypoxia-
regulated genes, suggesting that subgroup 4 cancers either
lack inactivating VHL mutations or downregulate hypoxia
signaling pathways [15,16]. Subgroup 4 tumors also showed
increased expression of many genes that characterize
chromophobe carcinomas and oncocytomas, including KIT
and the mitochondrial genes NNT, FH, GOT1, GOT2,
SLC25A5, ATP2B1, ATP5G3, ATP5B, and ATP6V1A (Figure
1H). We have previously observed similar expression patterns
in a subset of cRCCs that have granular cytoplasm , and a
review of the pathological specimens revealed that 11 of 13
tumors in subgroup 4 were conventional carcinomas with
granular cytoplasm. Subgroup 3 showed much higher
expression of proliferation-associated genes compared to
other tumors (CDCA3, CDC2, CENPE, CENPF, RRM2, and
CCNB2), suggesting a higher proliferative activity in these
tumors [18,19] (Figure 1E). Interestingly, there was little
correlation between expression levels of the hypoxia-regu-
lated genes and proliferation-associated genes (Pearson’s
correlation coefficient of 0.22), suggesting that higher
proliferation activity does not render cRCCs hypoxic and
highlighting that expression of hypoxia-regulated genes is an
intrinsic feature of most cRCCs. Subgroup 3 tumors (and
some subgroup 5 tumors) also showed high expression of
several collagen genes (COL12A1, COL3A1, COL6A1, COL1A1,
and COL5A2), and high expression of collagen genes has been
associated with poor prognosis in several tumor types 
(Figure 1D). Subgroup 5 uniquely displayed decreased
expression of a large set of genes that prominently included
several membrane transporters (NUP54, VPS54, STAM2,
MAPK8IP3, G3BP2, and SLC30A9) (Figure 1J). The distinct
gene expression profiles of each of the subgroups suggest that
cRCCs are molecularly heterogeneous despite their similar
Gene Expression Subgroups of cRCC Differ in Their Clinical
The gene expression subgroups did not simply reflect
differences in stage, grade, or performance status since none
of these clinical parameters was significantly associated with
tumor subtype (p . 0.5 by the chi-square test) (Figure 2A and
2B). The two main groups of cRCC defined by unsupervised
hierarchical clustering analysis showed a small but significant
difference in survival (subgroups 1 and 2 compared to
subgroups 3, 4, and 5, p ¼ 0.002 by the log-rank test) (Figure
2C). The five expression subgroups better defined classes of
tumors that differed in their long-term survival. Kaplan-
Meier analysis showed that patients with tumors in subgroup
3 had the worst outcome and those in subgroups 1 and 2 the
best compared to other subgroups (p , 0.001 by the log-rank
test) (Figure 2D). Furthermore, multivariate analysis showed
that the expression subgroup was a powerful predictor of
survival and was independent of grade, stage grouping, and
performance status (p ¼ 0.005, by the Cox model likelihood
ratio test). Therefore, gene expression profiles separate cRCC
into five subgroups that differ prognostically and that reflect
differences in the behavior of the tumors not captured by
stage, grade, and performance status.
Gene Expression-Based Survival Predictor
Having identified gene expression signatures in tumors at
the time of diagnosis that predict outcome by unsupervised
methods (i.e., based purely on gene expression signatures
intrinsic to the tumors), we attempted to define a gene
expression-based survival predictor by correlating survival
time with the gene expression signatures. We have found that
supervised analyses that correlate gene expression with
disease recurrence or survival (as binary outcome variables)
or duration of survival are overly simplistic models of these
complex datasets and in general not very accurate at
predicting clinical outcomes. We have instead used ‘‘semi-
supervised’’ learning approaches to identify gene sets
associated with survival in adult acute myeloid leukemia
and diffuse B-cell lymphoma and have shown that they better
identify gene expression signatures that are correlated with
outcome compared to unsupervised and supervised methods
Figure 1. Unsupervised Hierarchical Clustering Analysis of 177 cRCCs
(A) Overview of the gene expression patterns of 3,674 genes whose expression varied more than 3-fold in at least ten samples across the 177 samples.
Each row represents a single gene, and each column an experimental sample. Colored bars identify the locations of the inserts in (C–J). The degree of
color saturation corresponds with the ratio of gene expression shown at the top of the image.
(B) Dendrogram representing similarities in the expression patterns between experimental samples. Samples were separated into two main groups and
five subgroups (one in purple, two in blue, three in dark green, four in orange, and five in light blue) by the clustering algorithm.
(C) Hypoxia-induced gene cluster.
(D) Collagen gene cluster.
(E) Proliferation gene cluster.
(F–I) Genes distinguishing the two main groups (subgroups 1 and 2 from subgroups 3, 4, and 5).
(H) Energy generation gene cluster.
(J) Genes downregulated uniquely in subgroup 5.
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Gene Expression and Survival in Renal Cancer
of data analysis [11,21]. Whereas unsupervised methods assign
tumors to a class based solely on gene expression, and
supervised approaches use clinical outcome data to select
genes associated with prognosis, semisupervised methods
combine the advantages of both.
To identify genes highly correlated with survival in cRCC,
we used SPC analysis, a novel semisupervised approach we
have developed recently [14,22]. Patient samples were divided
randomly into a training set of 88 cases and a test set of 89
cases that were balanced for stage grouping, grade, patient
performance status, and length of follow-up (Table 2). In the
training set, a modified Cox score was calculated for all well-
measured genes, and genes whose Cox score exceeded a
threshold that best predicted survival were used to carry out
unsupervised principal components analysis (Figure 3). To
determine the Cox threshold, we split the training set,
performed principal components analysis in one half of the
samples and used the model to predict survival in the other
half. By varying the threshold of Cox scores and using 2-fold
cross-validation, we found that a threshold of 61.5 (averaged
over five separate repeats of this procedure) best predicted
survival (i.e., yielded the highest average partial log-likelihood
There were 340 transcripts (representing 259 genes whose
Cox score equaled or exceeded this threshold), and they were
used to perform principal components analysis on the entire
training set. (For a full list of transcripts with unigene cluster
ID, locus link ID, gene symbol, and gene ontology annota-
tions, see Table S1.) As can be seen in Figure 4, only the first
principal component was strongly correlated with survival. In
247 genes, high expression levels were associated with
prolonged survival (Figure 4A), and in only 12 genes was
high expression associated with shorter survival, including
BAG2, DCBLD2, EDG2, GNAS, IGLC2, NCF1, NME2, PFN2,
PRPS2, REG4, SLC7A5, and TFAP2C. There did not appear to
be enrichment of gene ontology annotations in this prog-
nostic gene set. However, some expression features suggested
biological processes that underlie differences in tumor
behavior. For instance, three genes involved in adhesion
and diapedesis of lymphocytes, CD34, PECAM1, and VCAM1,
show higher expression in tumors with good prognosis, and a
lymphocytic-mediated immune response can alter the clinical
Figure 2. Relationship of Gene Expression Subgroups to Clinical Parameters and SPC Risk Score in 177 cRCCs
(A) Dendrogram from the hierarchical cluster, with the clinical information for each of the samples. Subgroups are color-coded as in Figure 1. Color
shade corresponds to the ranges of each of the clinical parameters displayed. Expected survival times for the censored observations were estimated
from the Kaplan-Meier curve for all patients.
(B) Distribution of stage, grade, and patient performance status among five subgroups.
(C) Kaplan-Meier estimates of disease-specific survival in the two main gene expression groups of patients (subgroups 1 and 2 shown by the red bar
below the dendrogram, compared to subgroups 3, 4, and 5 designated by the green bar).
(D) Kaplan-Meier estimates of disease-specific survival in the five subgroups of patients.
The X symbols in (C) and (D) denote censored data.
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Gene Expression and Survival in Renal Cancer
course of cRCCs . Furthermore, high expression of VCAM1
has been shown previously to predict survival in cRCC
patients with metastatic disease . Elevated expression of
FZD2 and TCF4, members of the Wnt signaling pathway, also
correlated with longer survival.
For each case, SPC analysis computed a risk score (SPC risk
score) that represents the sum of the weighted expression
levels for each of the 340 prognostic transcripts. Not
surprisingly, the SPC risk score was highly correlated with
survival in the training set (p , 0.001 by the log-rank test). To
validate the SPC predictor, we computed risk scores for each
of the 89 cases in the test set, using the model developed in
the training set, and tested whether these scores were
correlated with survival (Figure 4B). When the SPC risk score
was used as a continuous variable, it was a strong predictor of
survival in the independent test set (p , 0.001 by the log-rank
test). Fewer genes from the SPC set also could predict
outcome since genes were identified based on their correla-
tion with survival. For instance, the top four genes in the SPC
predictor could be used to predict survival in the test set at p
¼ 0.02. Moreover, multivariate analysis showed that the SPC
risk score provided powerful prognostication independent of
stage, grade, and performance status (p , 0.001, by the Cox
model likelihood ratio test) (Table 3). Further investigation
showed that the log-relative risk was fairly linear in the SPC
score. When cases in the test set were split into localized
(stages I and II) and advanced disease (stages III and IV), SPC
risk score as a continuous variable continued to be highly
correlated with survival and was independent of grade and
performance status (p ¼ 0.011, and p ¼ 0.045, respectively, by
the Cox model likelihood ratio test).
To illustrate the performance of the SPC risk score in
predicting survival, we divided the training and test sets into
tertiles based on the SPC risk scores of the training set. In
both the training and test sets, Kaplan-Meier analysis showed
that the group with the highest SPC risk score had
significantly worse survival compared to the other two groups
(Figure 4C and 4D). When used as a categorical predictor, the
SPC risk score again predicted survival independent of grade,
stage, and performance status in all tumors in the test set and
in the high-stage tumors (stage groups III and IV; see Figure
4F), although not in the low-stage tumors (stage groups I and
II; see Figure 4E), likely due to low numbers of high-risk cases
in the test set. It should be emphasized, however, that the SPC
risk score is continuous, and survival is directly correlated
with the SPC risk score. Although cases can be assigned to risk
categories based on the SPC risk score, outcome is better
predicted when the SPC risk score is used continuously,
rather than categorically (Figure 4C–4F).
Relationship of SPC Risk Score to the Gene Expression
Most of the 259 prognostic genes comprising the SPC risk
score were found in clusters that distinguished between the
two major groups of cRCC that were defined by unsupervised
hierarchical clustering analysis (i.e., between subgroups 1 and
2 and subgroups 3, 4, and 5) (see Figures 1A [black bar] and
2C). Despite this overlap, the SPC risk score predicted
outcome independent of tumor subgroup in the test set (p
¼0.0013, by the Cox model likelihood ratio test), even though
the tumor subgroup had been assigned in the original
hierarchical cluster of all 177 tumors (comprising both the
training and test sets) and was based on differences in
expression over 3,674 genes. Tumor subgroup, on the other
hand, did not predict survival independent of the SPC risk
score (p ¼ 0.12 by the Cox model likelihood ratio test).
We have identified gene expression patterns that correlate
with survival after nephrectomy in cRCC. We used unsuper-
vised hierarchical clustering analysis to identify five distinct
subgroups that differed in their expression patterns over
3,674 genes. These subgroups were correlated with survival
time after surgery that was independent of tumor stage,
grade, and patient performance status. The consistency of
gene expression within the subgroups, regardless of tumor
stage, suggests that distinct molecular genetic changes
present at early stages of tumor development determine the
fate of the cancer and can be used to predict clinical
outcome. The identification of these five new subgroups
supports the use of gene expression profiling for prognosti-
cation in cRCC and highlights the value of unsupervised
analytic methods to provide insights into the clinical and
biological heterogeneity of cRCC.
We used a novel, semisupervised analytic strategy to
identify 259 genes that better predicted survival than the
gene expression subgroups, and we have validated this
prognostic gene set on an independent group of patients.
We used SPC analysis to compute a continuous risk score that
predicted survival in the test set independent of stage, grade,
performance status, and gene expression subgroup. Combin-
ing the SPC risk score with tumor grade, stage, and patient
Training set N=88
Test set N=89
Cox score calculated for
each well measured gene
Extract principal components
(PCs) using only genes with
a Cox score that exceeds the
Fit Cox model to survival
Determine Cox score
threshold by cross-validation
Evaluate predictive value
of the SPC risk score
by Cox model
Calculate SPC risk score
for each test case using the
formula derived from the
Figure 3. Overview of the Strategy Used for the Development and
Validation of a Prognostic Gene List
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Gene Expression and Survival in Renal Cancer
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Gene Expression and Survival in Renal Cancer
performance status may help identify patients with cRCC who
have a high probability of being cured of their disease and
need less intensive follow-up testing after surgery, or high-
risk individuals who might be referred for adjuvant treat-
ments even though their disease is clinically occult. The SPC
method employed in this paper could be used generally in
microarray studies to correlate gene expression with survival
time. Other approaches, such as the model-based mixture
proposal of Jones et al., could also be tried .
An interesting feature of the SPC prognostic gene set is
that 95% of the genes show relatively high levels of
expression in patients with good outcome and low expression
in those with poor outcome. Notably, this pattern of gene
expression was observed in a set of 51 prognostic genes
identified in 29 cRCCs by Takahashi et al., and 15 of these
genes were found in the SPC gene set . Unfortunately, we
were not able to evaluate the usefulness of the SPC gene set in
predicting survival in their patients because their dataset is
not publicly available. Another study by Vasselli and cow-
orkers identified 45 genes associated with survival based on
the Cox proportional-hazards score, using 58 stage IV tumors
from patients with good performance status . Those genes
share minimal overlap (one out of 45) with the SPC gene set,
possibly because this study included a highly selected group
of patients with tumors of conventional and nonconventional
histology. We and others have reported striking differences in
transcript profiles of renal cancers of different histologies,
and these differences could significantly influence the gene
identified that correlate with prognosis [17,24].
The 259 prognostic genes do not show enrichment for any
single biological pathway and are not localized to a single
region of the genome. The diversity of the pathways
represented in the SPC gene set argues that expression of
different functional groups of genes contributes to cRCC
growth, metastasis, and lethality.
Several molecules have been shown to correlate with
prognosis in cRCC; however, most of them were not selected
by SPC analysis as strong predictors of survival in our dataset
[25,26]. While some of these molecules might have important
biological roles in cRCC progression, they could be excluded
from the SPC gene list because they are relatively weak
predictors of survival. For instance, some transcripts, like
ADFP (a hypoxia-induced gene) did not provide a strong
enough correlation with survival to make the SPC gene set.
However, in our dataset, ADFP was correlated with a number
of the SPC genes in a cluster that defined main tumor groups
I and II (see Figure 1) by unsupervised clustering analysis.
When the 177 cases were separated into two groups based on
their median expression level of ADFP, they displayed
significantly different cancer-specific survival (p ¼ 0.03).
Carbonic anhydrase 9, another potential prognostic marker
for advanced RCC showed uniformly low expression in
subgroup 4, which has the worst survival rate, although its
expression did not correlate with survival in the whole
dataset. Therefore, predictions of outcome that are based on
single genes will be less robust that that for multiple genes,
such as those of the SPC predictor.
Gene expression profiling can improve outcome prediction
in patients with cRCC beyond that provided by stage, grade,
and patient performance status. Application of the SPC risk
score in the clinical setting will depend on independent
confirmation of our findings and could occur through custom
DNA microarrays or quantitative reverse-transcriptase poly-
merase chain reaction assays [27–29]. Since as few as four
genes in the SPC gene set can estimate prognosis, it should be
possible to develop clinically useful predictors of survival
based on these technologies. Regardless, our study demon-
strates the molecular heterogeneity of cRCC and opens
opportunities for improved biological understanding of the
molecular subgroups of the disease and their response to
Figure S1. Dendrogram Representing the Similarities in the Gene
Expression Patterns between Experimental Samples
Found at DOI: 10.1371/journal.pmed.0030013.sg001 (451 KB PDF).
Table S1. The 259 Genes Predicting Survival Identified using SPC
Found at DOI: 10.1371/journal.pmed.0030013.st001 (65 KB XLS).
The complete microarray dataset has been deposited in National
Center for Biotechnology’s Gene Expression Omnibus (http://www.
ncbi.nlm.nih.gov/geo/) and is accessible through accession number
Table 3. Prognostic Significance of SPC Risk Score Compared to
Clinical Features (p-Values) by the Log-Rank Test Using the Test
SPC risk score
Likelihood ratio test statistics: clinical parameters 55.5 (3 df), SPC 12 (1 df), clinical parameters and SPC together 69.4
Figure 4. Outcome Prediction Using the SPC Risk Score
(A) Overview of the gene expression patterns of the 259 prognostic genes in the training set, with their SPC risk scores arranged in ascending order and
the survival time in descending order. Each row represents a single gene, and each column a patient sample. The degree of color saturation
corresponds to the ratio of gene expression in each sample compared to the mean expression across all samples.
(B) Gene expression profiles of the 259 prognostic genes in the test set.
(C) Kaplan-Meier estimates of disease-specific survival in low-, intermediate-, and high-risk groups of patients in the training set defined by the tertiles of
SPC risk scores.
(D) Kaplan-Meier estimates of disease-specific survival of low, intermediate and high-risk groups of patients in the test set defined based on the tertiles
of the SPC risk scores of the training set.
(E) Kaplan-Meier estimates of disease-specific survival in stage group I and II patients in the test set.
(F) Kaplan-Meier estimates of disease-specific survival in stage III and IV patients in the test set.
PLoS Medicine | www.plosmedicine.org January 2006 | Volume 3 | Issue 1 | e130123
Gene Expression and Survival in Renal Cancer
Acknowledgments Download full-text
The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript. The
authors would like to thank the patients who participated in this
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Background. The kidneys filter the blood and eliminate waste in the
urine through a complex system of filtration tubules. All of the blood in
the body passes through the kidneys approximately 20 times an hour.
Conventional renal cell carcinoma (cRCC) is the most common type of
kidney cancer and arises from the cells that line the filtration tubules of
the kidney. Nearly half of the people who get RCC die from the disease.
Gene expression profiling, a laboratory technique, offers promise for
guiding the diagnosis and treatment of cancers.
Why Was This Study Done? The current method for estimating survival
using clinical markers (such as tumor stage and grade) has limitations. As
has been found for other cancer types, the hope is that gene expression
profiling could identify molecular markers that could be used for more
accurate diagnosis, prognosis, and possibly serve as drug targets for
effective therapies. Several small gene expression studies of cRCC done
so far have each identified prognostic gene sets, but these genes did not
overlap, and studies have not been validated independently. This larger
study looked systematically for variations in gene expression that were
correlated with the clinical heterogeneity of cRCCs.
What Did the Researchers Do and Find? The researchers studied a set
of 177 tumors from patients for whom they had detailed clinical
information, including data on long-term survival. They found a set of
259 genes whose activity in the tumor correlated with long-term survival
independent of the standard clinical predictors. Most of the genes
showed high levels of expression in patients with good outcome and
low expression in those with poor outcome. They then used this
information to show that they could accurately predict survival in an
independent group of patients.
What Do These Findings Mean? The researchers identified a set of
genes whose activity predicted survival after surgery independent of
clinical prognostic factors. This suggests that expression profiles could
help to distinguish between more aggressive and less aggressive types
of cRCCs. If confirmed by other studies, such expression profiles could be
used with information on tumor grade, stage, and patient performance
to help identify patients with cRCC who have a high probability of being
cured and need less intensive treatment and follow-up testing after
surgery and others whose cancers should be treated more aggressively.
Where Can I Get More Information Online? The following Web sites
have information on kidney cancer.
Cancer Research UK:
US National Cancer Institute:
Kidney Cancer Association:
PLoS Medicine | www.plosmedicine.orgJanuary 2006 | Volume 3 | Issue 1 | e130124
Gene Expression and Survival in Renal Cancer