Biochemical Basis of Respiratory Disease 855

Array of hope: expression profiling identifies

disease biomarkers and mechanism
Soumyaroop Bhattacharya and Thomas J. Mariani1
Division of Neonatology and Center for Pediatric Biomedical Research, University of Rochester, 601 Elmwood Avenue, Box 850, Rochester, NY 14642, U.S.A.

Abstract
High-throughput, genome-wide analytical technologies are now commonly used in all fields of medical
research. The most commonly applied of these technologies, gene expression microarrays, have been
shown to be both accurate and precise when properly implemented. For over a decade, microarrays have
provided novel insight into many complex human diseases. Microarray-based discovery can be classified
www.biochemsoctrans.org

into three components, biomarker detection, disease (sub)classification and identification of causal
mechanism, in order of accomplishment. Within the respiratory system, the application of microarrays has
achieved significant success in all components, particularly with respect to lung cancer. Numerous studies
over the last half-decade have applied this technology to the characterization of non-malignant respiratory
diseases, animal models of respiratory disease and normal developmental processes. Studies of obstructive
lung diseases by many groups, including our own, have yielded not only disease biomarkers, but also some
novel putative pathogenic mechanisms. We have successfully used an integrative genomics approach,
combining microarray analysis with human genetics, to identify susceptibility genes for COPD (chronic
obstructive pulmonary disease). Interestingly, we find that the assessment of quantitative phenotypic
variables enhances gene discovery. Our studies contribute to the identification of obstructive lung disease
biomarkers, provide data associated with disease phenotypes and support the use of an integrated
approach to move beyond marker identification to mechanism discovery.

Introduction level, the basic principle of microarray technology is com-

The completion of genome sequencing of specific organisms,
combined with technological advances in the capability to
detect changes in genome-wide expression at both the mRNA
and protein levels, has heralded a new era in our quest for
understanding the complex pathways and mechanisms in
Biochemical Society Transactions
plementary hybridization of nucleotides as explained by the
Watson–Crick double helical model of DNA. Microarrays
measure transcriptomic modifications that, either at the single
gene level or collectively in multiple genes, lead to changes
in protein expression. In fact, it is unusual for changes in the

living organisms. In the last decade, there has been significant
advancement in microarray technology with the development
of many different platforms which are being used for
analysing gene expression, genotyping and other applications
[1,2] (Figure 1). According to the central dogma of molecular
biology, genomic DNA is first transcribed into mRNA,
which thereafter is translated into protein. Proteins play crit-
ical roles in most intra- and extra-cellular activities, including
enzymatic, regulatory and structural functions. However,
relative difficulties of expression measurement capabilities
at the protein level and availability of technologies of high-
throughput methods (expression microarrays) for detection
of individual mRNA have led to the wide use of microarrays
to simultaneously measure the sum of all mRNA expression
in a sample, also called the transcriptome [3]. Like most clas-
sical methods for analysis of gene expression at the mRNA
Key words: biomarker, chronic obstructive pulmonary disease (COPD), disease mechanism,
expression profiling, lung, microarray.
Abbreviations used: COPD, chronic obstructive pulmonary disease; MAQC, MicroArray Quality
Control; NSCLC, non-small-cell lung carcinoma; SERPINE2, serpin peptidase inhibitor, clade E
(nexin, plasminogen activator inhibitor type 1), member 2.
1
To whom correspondence should be addressed (email tom_mariani@urmc.
rochester.edu).
level of a specific mRNA to not be accompanied by changes in
the protein level for that gene. Furthermore, although not all
changes in protein expression and function levels are captured
at the steady-state level of that particular mRNA, their
downstream effects are captured by the full transcriptome.
In the early part of the decade, when the use of gene
expression microarrays was growing exponentially (see Fig-
ure 2), there was considerable concern and debate regarding
the precision of the technology. For instance, Tan et al. [4]
reported divergence in microarray-based gene expression
measurements. Similar observations, and the experiences
of many investigators, resulted in concerns being raised
regarding the potential utility of microarrays. This was met by
an independent and thorough evaluation of the technology
by the MAQC (MicroArray Quality Control) [5] project.
The MAQC project was developed by the FDA (Food and
Drug Administration), the EPA (Environmental Protection
Agency) and the NIST (National Institute of Standardization
and Technology), in association with commercial stakehold-
ers and academic laboratories. The purpose was to evaluate
the accuracy of microarray technology, to provide quality-
control tools and to develop guidelines for microarray data
analysis. The MAQC project involved quantification of gene

Biochem. Soc. Trans. (2009) 37, 855–862; doi:10.1042/BST0370855 

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856 Biochemical Society Transactions (2009) Volume 37, part 4
Figure 1 Overview of the utility of gene expression microarray
technology in lung disease biomarker and therapeutic target
discovery
Multiple factors such as experimental design, laboratory methods
and analytical approach contribute to data accuracy. Application of
the technology can be broadly distinguished as class discovery, class
prediction and mechanism discovery as defined by distinct analytical
methods and experimental goals. Integrating the study of normal
developmental processes, animal models of disease and human genetic
epidemiology, with an emphasis upon quantitative variable analyses,
can assist in the goal of identifying causal genes/pathways as we have
described for SERPINE2 in COPD [55].
expression levels using seven microarray platforms tested at
three independent sites, with five replicates at each location.
Although each microarray platform studied had different
performance characteristics, they generated comparable
results with up to 95 % concordance with regard to defining
differential expression. We have recently completed a study
showing similar cross-platform concordance with time-series
data (R. Du, K. G. Tantisira, V. J. Carey, S. Bhattacharya, S.
Metje, B. J. Klanderman, R. Gaedigk, R. Lazarus, A.T. Kho,
T. J. Mariani, J. S. Leeder and S.T. Weiss, unpublished work).
These data unequivocally demonstrated extremely high levels
of gene expression microarray measurement precision.
There is no doubt that the technology has suffered
setbacks due to rapid growth. Even though all microarray
platforms apply the same basic principle of complementary
hybridization, diverse probe designs have led to a multitude of
problems in quantitative data acquisition and management. In
retrospect, we can conclude that significant limitations in the

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implementation of microarray technology have led to poor
performance. We can categorize these limitations as primarily
including deficiencies in: (i) experimental design/study size,
(ii) analytical methods and (iii) probe sequences.
Possibly the most important, and most overlooked,
aspect of successful expression array analysis is appropriate
experimental design including adequately powered sample
size. A question often asked by investigators is what number
of samples to study. Optimal sample sizes can be determined
using modified power calculations [7]. The primary objectives
of a microarray experiment are usually class comparison, class
discovery or class prediction [8]. For class comparison and
class prediction studies, a large number of biological replicates
(not technical replicates) are recommended, whereas for
class discovery, technical replicates from the same indi-
vidual provide a better assessment of disease classification.
Conversely, for inbred animal models (e.g. mouse), where
genetic heterogeneity is limited and exposures are controlled,
technical replicates (and pooling of biological replicates) are
preferred. Irrespective of the study objectives, the use of im-
proper control samples that are derived from tissues of origin
other than that of the treatment samples lead to erroneous
predictive classifications due to confounding of the samples
[9].
Although there are established protocols for laboratory
procedures leading to the generation of data, there is no
clear consensus on a ‘best’ method for data analysis. Early
analytical approaches relied heavily on a non-statistical
measure of expression differences (fold change) [10] that
was repeatedly shown to lack sensitivity and specificity. For
instance, we showed that a statistical approach to accurately
define differential expression, using measurement precision
in technical replicates, is not directly proportional to fold
change [11]. Other statistical methods, such as standard t tests,
were recognized as being subject to problems of multiple
testing resulting from repeated measures in a limited number
of samples. Numerous complex and/or specific mathematical
and statistical approaches have been subsequently deve-
loped and applied to microarray data (see [12] and references
therein). Like experimental design, the choice of methods
for statistical analysis should be chosen based on the
structure and distribution of the data and the objective of the
study. Fortunately, the MAQC project and user consensus
have pointed to a limited number of preferred analytical
approaches that are recognized as being robust and effective.
Another long-appreciated source of ‘noise’ or technical
variability within expression arrays is inaccuracy of probe
sequences, which may not match the transcripts they are
intended to measure. This was first widely appreciated
due to a ‘manufacturing’ (annotation) error made by the
leading commercial source of the technology, which led to
a large number of arrays/experiments that inadvertently
measured sense transcripts [13]. However, this proved to
be no more than a minor setback in the evolution of a
powerful and complex technology. More recently, it has
been appreciated that sequence databases evolve, resulting
in transient (in)accuracy of probes [14]. We have previously
 Biochemical Basis of Respiratory Disease 857

Figure 2 Analysis of growth in the application of microarray technology as defined by published research articles (A) or publicly
deposited datasets (B)
(A) The number of all research articles (black line) and lung-specific research articles (grey line) published each year, together
with the relative number of articles focusing on lung cancer (black bars) or non-cancerous lung tissue (grey bars). (B) The
number of all datasets (black line) and lung-specific datasets (grey line) deposited in the National Center for Biotechnology
Information Gene Expression Omnibus each year, together with the relative number of the datasets focusing on lung cancer
(black bars) or non-cancerous lung tissue (grey bars). Although the number of lung-related research articles initially grew at
the same pace as all microarray research articles, there seems to have been a slowing in the pace of lung-related publications
over the last 5 years. Likewise, the number of lung-specific datasets publicly deposited prior to 2004 was representative of
all datasets as a whole, but the rate of growth of lung-related datasets has slowed substantially since then. Note publicly
deposited datasets may not be entirely representative of all data being studied and published, dependent on journal-specific
rules for data deposition.

shown improved accuracy and cross-platform comparisons
when accounting for these inaccuracies [15].
Technological accomplishments
Having survived these temporary setbacks and limitations,
microarrays have developed into standard tools for high-
throughput analysis of gene expression, and continue to grow
in information quality and in new applications. The evolution
of microarray technology has been a gradual process. The
technological principles involving combinatorial chemistry
have been in development since the late 1960s with the works
of R. Bruce Merrifield (Nobel Prize in Chemistry, 1984), with
arrays in their current forms first appearing in the early-to-
mid-1990s. Both Stephen Fodor and Mark Schena developed
the early prototypes of cDNA and oligonucleotide arrays


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858 Biochemical Society Transactions (2009) Volume 37, part 4
almost simultaneously [1,2]. While Fodor in collaboration
with Lubert Stryer of Stanford University received a small
business innovative research grant from the NIH (National
Institutes of Health) and went on to establish Affymetrix
Inc., Schena pioneered the cDNA technology under the
guidance of Pat Brown at Stanford University [16].
By the late 1990s, the power and potential of microarray
technology were fully appreciated and it was being applied
in an effort to develop novel descriptions of diseased states.
Novel genes and pathways, previously not implicated in
the pathophysiology of a certain disease, may emerge from
microarray studies to provide new theories regarding the
disease process and potential therapeutic drug targets.
The spread in use of the technology was unprecedented
(Figure 2A), with exponential growth in the number of
publications reporting results from its application in the early
part of this decade. Parallel growth was initially observed
in the lung biology and disease research community, pre-
dominantly by those focused on lung cancer (50–60 % of
publications each year), although over the last few years,
its use in lung research has apparently lagged. Regardless
of the sample studied, expression microarray application to
disease can be classified under three broad topics: biomarker
discovery or class prediction, disease subclassification or
class discovery and uncovering the disease mechanism.
Biomarker discovery
Expression arrays have been widely used to predict the
state or ‘class’ of an unknown sample using pre-existing
information. One of the most studied human diseases by
expression-profiling technology is cancer (Figure 2). Initial
microarray studies were heavily focused on identifying gene
expression markers for human cancer phenotypes distin-
guishing them from normal samples. Golub et al. [8] described
the potential of expression array data to predict disease
using the distinctions between human AML (acute myeloid
leukaemia) and ALL (acute lymphoblastic leukaemia) as their
experimental model [8]. Although these diseases are capable
of being discriminated by other means (such as cytology),
this study served as proof-of-principle that the technology
could define expression markers informative for the diseased
state. This approach has been widely applied to many diseases
or previously recognized disease subtypes including breast
cancers, cutaneous malignant melanoma, diffuse large B-cell
lymphoma, colon cancer, leukaemia and ovarian carcinomas
(see [17] and references therein). In the pulmonary system,
numerous human disease states and animal models of disease
have been subjected to expression profiling in an effort to
define disease biomarkers and/or class predictors [18].
Detection of genomic signatures from individual studies
has provided a wealth of information, but these data are
limited for pathological diagnosis unless validated externally.
One major limitation to this goal is the small number
of samples in individual studies, particularly for human
studies where there is a high degree of both intra- and
inter-population variability. This ultimately results in disease
biomarkers that are population dependent, rather than having

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global applicability. This is true with regard to studies of lung
diseases, where biomarkers for acute and chronic diseases,
including severe asthma and COPD (chronic obstructive
pulmonary disease), and environmental exposures have been
identified [19]. We have recently presented an analysis of lung
tissue gene expression in subjects with COPD [20]. We used
a novel combination of discrete and quantitative variable
analysis to determine differential expression. Inclusion
of quantitative phenotypes helped in providing a set of
robust COPD markers, which successfully predicted disease
in an independent COPD population [20]. Admittedly,
this success is much more of an exception than the rule.
However, with an increasing number of microarray datasets
being deposited in the public domain [21,22] (Figure 2B),
real opportunity exists for more reliable information to be
generated through the integration of multiple, independently
generated datasets focusing on the same biological paradigm.
To facilitate this concept, MIAME (Minimum Information
About a Microarray Experiment) was developed, which
serves as the standard information required to accompany
microarray data to ensure correct interpretation of the data
and independent verification of analysis of results [23].
Many groups have proposed approaches for data
integration across platforms and laboratories (for example
see [24, 25] and references therein). Meta-analysis approaches
have also been applied to validate results from different
studies, which, in certain cases, have proved to be successful
[17]. With a large number of microarray studies on breast
cancer, great promise existed to leverage these data to identify
a disease biomarker that could be used as a true diagnostic
tool. Indeed, van’t Veer et al. [26] identified a gene expression
signature strongly predictive of patients with either poor or
good prognosis. The gene-expression biomarker has been
used as a predictor of the outcome of disease in young patients
with breast cancer in combination with standard prediction
tools based on clinical and histological criteria [27]. A 70-gene
marker chip has been developed and tested for diagnosis
of breast cancer that outperformed all clinical variables
in predicting the likelihood of distant metastases within
5 years [27]. This represents the first clinically approved gene
expression microarray test for molecular-based therapy.
Disease subclassification
The identification of cancer subtypes can sometimes be a
difficult process, as it relies on the subjective interpretation of
both clinical and histopathological observations with the aim
of classifying samples in currently accepted subtypes based on
the tissue of origin of the tumour. This sometimes is hampered
by a lack of clinical information or unclear classification
of samples based on histology. Alternatively, some diseases
present with similar histopathological features of clearly dis-
tinct origin or with variable prognoses. Most early microarray
studies involved marker distinction between normal and ab-
errant (diseased) tissues. However, much enthusiasm has been
generated regarding the ability of gene expression microar-
rays to clarify disease subclasses, and even identify previously
unappreciated classes with distinct aetiologies, prognoses

and/or therapeutic responses. This potential is particularly
relevant for cancer, as was first described by Alizadeh et al.
[28] for B-cell lymphoma. Microarray analysis has led to the
identification of molecular classification of several human
malignant tumours based on pathological parameters, namely
stage, recurrence, prognostic outcome or therapy response
[29] in breast cancer [26,27,30], cervical lymph node meta-
stasis in oral squamous cell cancer [31], non-small-cell lung
cancers and clear cell renal carcinoma [33]. One of the most
extensively analysed microarray datasets involves a compar-
ison of leukaemia subtypes [8], which has also been used for
identifying predictors for therapy response [34]. Every new
discovery of disease subtype molecular markers creates a path
for the development of molecular-targeted therapies.
The prognostic and discovery potential of microarrays to
perform disease subclassification are possibly best exempli-
fied as applied to NSCLC (non-small-cell lung carcinoma),
the most common form of the most frequent cause of cancer
death in the world. Lung carcinomas are a heterogeneous
collection of tumours characterized by a large number of
chromosomal and structural abnormalities. NSCLCs can
be subclassified into adenocarcinomas (the most common),
squamous cell carcinomas and large-cell carcinomas [35].
Bhattacharjee et al. [36] demonstrated the ability to define
NSCLC subtypes based on gene expression profiles and first
identified ‘molecular’ subclasses of adenocarcinoma. These
observations have been replicated in numerous other studies
[37,38], with the common limitation of the identification
of population-dependent markers (as described above).
In the absence of robust markers, meta-analysis has been
implemented, with limited success. Potti et al. [39] identified
meta-genes to predict therapy response in patients with early-
stage non-small-cell lung cancer. In contrast, Ramaswamy
et al. [40] re-analysed multiple datasets of tumour expression
profiles to identify a set of gene expression markers that can
be used as a multiclass cancer diagnosis tool.
In addition to lung cancer, disease subtype classification
has also been attempted in other lung disorders such as
COPD [41], pulmonary fibrosis [42] or asthma [43]. For
instance, two laboratories have recently described markers
for severe drug-resistant asthma in lung epithelial cells
[44] and in peripheral blood cells [45]. In an interesting
study, Kho et al. [46] used the organ-specific development
transcriptome as a basis for subclass discovery. Molecular
subclassification promises the hope of defining class-specific
mechanisms and routes for therapeutic intervention.
Causal mechanisms
Even though gene-based signature sets have been developed,
the causal mechanisms are still unclear. Initially, it was hoped
that defining expression biomarkers of disease would lead
directly to causality. We now appreciate that analytical meth-
ods and experimental designs focusing on class prediction
are typically not efficient at uncovering disease mechanismss.
One means used to apply microarrays to uncover the
disease mechanism was intuitive: the use of animal modelling.
Although such models have been developed in an effort to
Biochemical Basis of Respiratory Disease 859
determine the effects of candidate genes, they can also be used
for gene discovery. Such studies are common throughout the
literature including those concerning the lung, particularly
for models of lung inflammation and allergic hypersensitivity.
Many of these studies suffer from the limitations described
above for studies of clinical specimens (namely, small study
size, ambiguous analytical methods etc.), but have provided
tremendous insights nonetheless. For instance, expression
profiling from animal models has provided insights into
COPD pathogenesis [47]. Additionally, Novershtern et al.
[48] have recently developed a signature gene set for asthma
by integrating information from genome-wide expression
and protein studies of animal models. In particular, the
integration of multiple genomic approaches, such as integrat-
ing animal models and genetics, has been particularly useful
in uncovering mechanistic genes/pathways. In terms of re-
spiratory disease, we recognized the study by Karp et al. [49]
of an allergic hypersensitivity model of asthma as an early
example of this approach.
Most microarray studies are limited to comparison
analysis, aiming to identify genes with a change in expression
between two classes. In contrast, DeRisi et al. [50] published
a study of the yeast life cycle that initiated the use of
microarrays in time-series studies. These types of experiments
have typically been used for biological discovery in ‘normal’
samples, and not for the identification of disease biomarkers.
However, another successful approach for defining a causal
mechanism has been through the integration of human
genetics and genome-wide expression, such as that used by
Blackshaw et al. [51]. Here, they used transcriptomic profiles
of normal developing eye tissue to identify biologically
relevant candidate causal genes within loci linked to human
eye diseases. Collectively, results from these and similar
studies have provided novel insights into the mechanisms
of disease pathogenesis, and raise expectations of developing
therapeutic targets from the application of DNA microarrays
to complex diseases.
As described above, multiple groups have attempted to
identify candidate genes for COPD, a complex human disease
probably influenced by a genetic component (α 1 -antitrypsin
deficiency), an environmental component (cigarette smoking)
and gene-by-environment interactions [41,52–54]. We re-
cently reported the identification of a COPD susceptibility
gene through the integration of human genetics with gene
expression profiling of normal lung development and diseased
lung tissue. Like Blackshaw et al. [51], we used transcriptomic
profiles of organ development to inform us of biologically
relevant candidate genes within a disease-linked locus. We
identified SERPINE2 [serpin peptidase inhibitor, clade E
(nexin, plasminogen activator inhibitor type 1), member
2], a homologue of the only known COPD susceptibility
gene, and went on to show that expression of this gene
was aberrant in lung tissue from COPD subjects [55]. Of
particular note, SERPINE2 expression was not a robust class
prediction marker, but was highly correlated with multiple
quantitative measures of lung function in multiple datasets
where quantitative phenotypes for COPD were available

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860 Biochemical Society Transactions (2009) Volume 37, part 4
Figure 3 Using quantitative disease phenotypes for gene
expression biomarker discovery
We initiated gene discovery for COPD in a genetically linked locus
(chromosome 2q) in two independent datasets [41,58]. We focused
on identifying gene expression changes associated with quantitative
phenotypic variables characteristic of COPD such as forced airflow,
diffusing capacity of the lung for carbon monoxide (DLCO ) and total
lung capacity (TLC). A significant positive correlation is indicated in black,
while a significant negative correlation is indicated in grey. Note that TLC
is inversely proportional to the other phenotypic variables. While there
was evidence for association of multiple genes within the locus in each
dataset, there was a single gene, SERPINE2, consistently associated with
multiple quantitative phenotypic variables in both datasets. FEF 25–75%,
forced expiratory flow at 50% of vital capacity; FEV1 , forced expiratory
volume in 1s; FVC, forced vital capacity.
(Figure 3). Within the disease-linked locus, this was uniquely
true of SERPINE2. Multiple groups, including our own, have
subsequently demonstrated significant associations between
SERPINE2 gene variants and COPD phenotypes [55,56] and
aberrant SERPINE2 expression in COPD lung tissue [57].
We have further discovered that deficiency in SERPINE2 in
the mouse leads to the development of COPD-related lung
histopathology (S. Srisuma and T.J. Mariani, unpublished
work). We believe that such an integrative genomics approach
helps us to expedite candidate gene identification, and that
the use of quantitative variables may be particularly useful to
uncover causal disease mechanisms and pathways.
Summary
Gene expression microarrays have come a long way from
being a complex technology (sometimes poorly applied) in
bio-medical research and are now a critical component of
state-of-the-art research on disease discovery, therapeutic
responsiveness and pathogenesis. The tools used for statistical
analysis, data mining and archiving have steadily improved,
and objective criteria demonstrate a high level of precision
for the technology when applied appropriately. Numerous
studies in respiratory medicine have provided a wealth of data
describing biological paradigms from normal development
to lung cancer. Although the primary application of the tech-
nology has been to identify biomarkers for disease, the

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technology has been successful in the identification of disease
subtypes and molecular diagnostic predictions. We have
here listed only a select few instances of the wide ranging
capabilities of this high-throughput technology. With proper
planning and experimental design, it has been shown to dis-
cover or further resolve complex disease mechanisms to
levels previously unimaginable. When used in combination
with animal models and genetic studies, particularly focusing
on quantitative variable analysis, it has provided unexpected
power to identify disease mechanisms. Even though we
have seen considerable advancement in the technology, in
order to achieve its full potential, genome-wide expression
studies have to co-evolve in a multidisciplinary approach that
includes a combination of well-developed machine-learning
algorithms and systems biology approaches. This can
only be achieved when clinicians, surgeons, pathologists,
epidemiologists, bioinformaticians and molecular biologists
undertake a well-co-ordinated effort to properly plan and
conduct every step of the process starting from experimental
design to validation of the results.
Funding
This work was supported by grants from the National Institutes
of Health [grant numbers HL071885 and ES014372] and Flight
Attendant Medical Research Institute.
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Received 26 February 2009
doi:10.1042/BST0370855