Moreira1,2, Irina Gromova1,2, Teresa Cabezon1,2, Ulrik Ralfkiaer1,2, Per Guldberg1,2, Per thor Straten1,2, Henning Mouridsen1,3, Esbern Friis1,4, Dorte Holm1,5, Fritz Rank1,5and Pavel Gr
Trang 1Towards discovery-driven translational research
in breast cancer
Julio E Celis1,2, Jose´ M A Moreira1,2, Irina Gromova1,2, Teresa Cabezon1,2, Ulrik Ralfkiaer1,2, Per Guldberg1,2, Per thor Straten1,2, Henning Mouridsen1,3, Esbern Friis1,4, Dorte Holm1,5,
Fritz Rank1,5and Pavel Gromov1,2
1 The Danish Centre for Translational Breast Cancer Research, Copenhagen, Denmark
2 Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark
3 Department of Oncology, Copenhagen University Hospital, Denmark
4 Department of Breast and Endocrine Surgery, Copenhagen University Hospital, Denmark
5 Department of Pathology, The Centre of Diagnostic Investigations, Copenhagen University Hospital, Denmark
Introduction
The completion of the human genome project, as well
as the current availability of novel and powerful
tech-nologies within genomics, proteomics and functional
genomics, promise to have a major impact on clinical
practice, as these developments are likely to change
the way in which diseases will be diagnosed, treated
and monitored in the near future We are moving
increasingly from the study of single molecules to the analysis of complex biological systems, and one of the main challenges we face is how best to apply these powerful technologies to clinically relevant sam-ples in a well-defined clinical and pathological frame-work [1–6]
Cancer, being a complex disease that impinges on a significant proportion of the world population, has become a prime target for the application of novel
Keywords
breast cancer; proteomics; functional
genomics; signaling pathways; systems
biology; individualised medicine; translational
research
Correspondence
J E Celis, The Danish Centre for
Translational Breast Cancer Research and
Institute of Cancer Biology, Danish Cancer
Society, Strandboulevarden 49, DK-2100
Copenhagen, Denmark
Fax: +4535 25 73 76
Tel: +4535 25 73 63
E-mail: jec@cancer.dk
(Received 10 September 2004, revised 29
September 2004, accepted 29 September
2004)
doi:10.1111/j.1432-1033.2004.04418.x
Discovery-driven translational research in breast cancer is moving steadily from the study of cell lines to the analysis of clinically relevant samples that, together with the ever increasing number of novel and powerful tech-nologies available within genomics, proteomics and functional genomics, promise to have a major impact on the way breast cancer will be diag-nosed, treated and monitored in the future Here we present a brief report
on long-term ongoing strategies at the Danish Centre for Translational Breast Cancer Research to search for markers for early detection and tar-gets for therapeutic intervention, to identify signalling pathways affected in individual tumours, as well as to integrate multiplatform ‘omic’ data sets collected from tissue samples obtained from individual patients The ulti-mate goal of this initiative is to coalesce knowledge-based complementary procedures into a systems biology approach to fight breast cancer
Abbreviations
FIF, fat interstitial fluid; GSK-3, p-glycogen synthase kinase-3; IHC, immunohistochemistry; IL, interleukin; NIF, nonmalignant interstitial fluid; TIF, tumour interstitial fluid.
Trang 2technologies – often referred to as the ‘omic’ platforms
– as these may help to identify subgroups of cancer
patients having specific molecular features associated
with clinical outcomes [7–14] This development is
expected to lead to a predictive, individualized
approach to cancer care, and will facilitate the selection
of treatment modalities that are most likely to benefit
the individual patient Currently, patient classification
is based on clinical parameters and cellular
morphol-ogy, as well as immunohistochemical analysis using a
restricted number of prognostic⁄ predictive markers
related to different cell functions such as cell
prolifer-ation, angiogenesis, invasion and metastasis These
parameters can only classify patients into subgroups
with various prognoses, but it is expected that the
com-bination of known risk factors with new expression and
genomic data will be instrumental to clinicians
confron-ted with treatment options
At present, we are experiencing a transition in
cancer research as we move from the use of single
‘omic’ platforms to their integrated use to achieve
‘systems biology’ [15] This integrated approach
should lead to a better understanding of the
under-lying biology of living cells and organisms, resulting
in turn in a more effective translation of basic
dis-coveries into clinical applications [1–5,16] However,
the implementation of discovery-driven translational
cancer research requires the coordination of basic
research activities, facilities and infrastructures, as
well as the creation of an integrated and
multidisci-plinary environment with the participation of all the
stakeholders in the cancer ordeal, i.e basic
research-ers, surgeons, oncologists, pathologists,
epidemiolo-gists, patients, patient advocacy groups, funding
agencies and industrial partners Issues related to
sample collection, handling and storage,
standardiza-tion of protocols, common references, number of
patients, availability of normal controls, access to
bio-banks, tissue arrays, clinical information,
follow-up clinical data, computational and statistical
analy-sis, as well as ethical considerations are critical, and
must be carefully considered and dealt with from the
beginning [1,2,4]
Discovery-driven translational cancer research has
only recently gathered momentum among the basic and
clinical research community, and as a consequence
there are currently few long-term programmes that use
tissue biopsies and bio-fluids as their main sources for
generating multiple data sets [1,2,4,5] The application
of high throughput technologies to the analysis of
tis-sue biopsies is far more demanding than the analysis of
bio-fluids, due to the heterogeneous nature of the
tis-sues and istis-sues related to sample preparation, as well
as to problems associated with managing long-term prospective⁄ retrospective programmes [1,2,5] Tumours usually contain malignant cells showing different degrees of differentiation as well as other cell types, which together compose the ‘tumour microenviron-ment’ [17–29] The cellular heterogeneity problem has generally been addressed using microdissection tech-niques that allow the dissection of a defined set of puri-fied cell populations [30,31] This technology, however, cannot solve the problem altogether, as heterogeneity can be observed even in a small number of cells when several markers are assessed simultaneously using im-munohistochemistry The application of cDNA micro-array-based expression profiling allows the analysis of single cells through elegant amplification strategies, and several studies reporting expression profiles generated from laser-captured cells have demonstrated the feasi-bility of the approach [32–34] Similar single cell analy-sis of cultured cells using gel-based proteomics [35–37],
on the other hand, does not provide a viable alternative
at the moment, as more sensitive protein detection methods are needed to identify a significant number of proteins [38]
Studies of bio-fluids using various proteomic tech-nologies far exceed those of tissues, as the samples are easier to collect and handle One type of analysis in particular, serum proteomics pattern diagnostic analy-sis, pioneered by Liotta, Petricoin and coworkers [39], has shown promise in identifying features that differen-tiate normal from malignant conditions in ovarian, prostate and breast cancer [39–42] This approach uses
a combination of low resolution mass spectra gener-ated by surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) and artificial intelligence-based informatics algorithms, to search for protein patterns or features in serum that may detect cancer at
an early stage [39] Liotta and coworkers have hypo-thesized that degradation and cleavage of the proteins that perfuse the tumour microenvironment is a major source of the peptides that appear in the blood Once
in the blood circulation, these peptides bind to abun-dant carrier proteins such as albumin that accumulate and transport them [43] Other studies, however, have highlighted several problems associated with the tech-nology, particularly related to the fact that the identity
of the peptides included in the serum features is not known Also, reproducibility, portability and sample source have been a matter of concern and much dis-cussion [44–46] The use of high resolution mass spectrometry platforms to collect the data, on the other hand, is expected to yield superior classification
of serum protein features of a number of cancers in the near future [47]
Trang 3Breast cancer
Breast cancer is the most common malignancy among
women in the Western world and constitutes 18% of
all cancers in women In Denmark approximately 3800
women develop breast cancer per year and an
estima-ted 1200 die from the disease In addition, the number
of registered cases has tripled over the past 40 years,
and breast cancer is now the number one cause of
can-cer mortality in women
Today, routine mammography, i.e screening for
breast cancer by X-ray examination, is the only
accep-ted method for early detection of breast cancer and a
recent meta-analysis of seven large-scale studies has
confirmed its value as a screening tool [48] Although
screening-detected cancers are significantly smaller
than nonscreening ones [49], there is a clear need to
improve the efficiency and sensitivity of the method as
it misses about 10% of the cases and gives a certain
percentage of false positive results Parameters such as
axillary lymph node status, tumour size, histological
grade and age in combination with predictive factors
such as oestrogen and progesterone receptors are
cur-rently used for selecting the appropriate systemic
ther-apy [50] The status of immunohistochemistry (IHC) in
diagnostic breast pathology has recently been
thor-oughly reviewed [51] Although pitfalls associated with
performance and interpretation of IHC results are
plentiful, current routine diagnostic breast pathology is
well equipped to provide modern ‘omics’ research with
important complementary information
Patients with primary breast cancer are offered
sur-gery, often followed by adjuvant therapeutics, i.e
che-motherapy, radiotherapy and⁄ or endocrine therapy
Despite these treatments, approximately 40% of
patients with lymph node-positive disease will
experi-ence a relapse, and the majority of these patients will
die from disseminated cancer [52,53] For patients with
lymph node-negative disease, the 5-year recurrence rate
is 25%, suggesting that the risk of recurrence and
subsequent death is closely related to the stage of the
disease at the time of primary surgery It is reasonable
to assume that the survival rate of breast cancer can
be improved, if the number of patients being
diag-nosed with early stage disease, i.e, node-negative
dis-ease, is increased
Chemotherapy and⁄ or endocrine therapy is offered
to patients at low, moderate and high risk of
recur-rence and death, i.e to a prognostically heterogeneous
group of patients with a range of risk from a few
per-cent up to 80% This group, which constitutes about
70% of all new breast cancer patients [54], is
charac-terized according to the following classical prognostic
factors: nodal status (positive); size of the primary tumour (‡ 20 mm); malignancy grade (2–3) and steroid receptor status (negative) With adjuvant systemic ther-apy being offered to this patient group, 30–40% of the expected deaths can be avoided However, in absolute terms, the mortality reduction amounts to only a few percent (i.e from 5 to 3%) in the low risk group, and
to 25% in the high risk group (i.e from 80% to 60%) Thus, although adjuvant systemic therapy has led to a considerable improvement of the prognosis of the breast cancer population, it also carries the signifi-cant adverse effect of overtreatment [55]
It is well known from the treatment of advanced breast cancer that patients nonresponsive to one
speci-fic type of therapy may react to another type, indica-ting that the response to a specific treatment relates
to specific characteristics (predictive factors) of the tumour Thus, there is a need to develop new inde-pendent prognostic and predictive indicators in pri-mary breast cancer to improve patient selection for specific and individual treatments Moreover, it is important to develop new diagnostic methods to detect breast cancer at a very early stage, as early detection increases survival rate Today, many cancers are detec-ted late, when spreading to the surrounding tissue and metastases has taken place
The Danish Centre for Translational Breast Cancer Research
Responding to the above challenges, the Danish Can-cer Society catalysed in 2002 the creation of a multidis-ciplinary, multi-centre research environment, The Danish Centre for Translational Breast Cancer Research (DCTB), to fight breast cancer DCTB hosts scientists working in various areas of preclinical cancer research (cell cycle control, invasion and microenvi-ronmental alterations, apoptosis, cell signalling and immunology) with clinicians (surgeons, oncologists), pathologists and epidemiologists in an integrated, mis-sion-oriented discovery-driven translational research environment The DCTB places the patient at the cen-tre and the ultimate goal is to conduct research to improve survival and quality of life of breast cancer patients
The underlying concept behind this long-term, con-certed approach is the use of multiple experimental paradigms from genomics, proteomics and functional genomics, to the prospective analysis of clinically rele-vant fresh samples obtained from the same patient, along with the systematic integration of the biological and clinical data sets [2,4] The aim of this systematic knowledge-based approach is (a) to understand the
Trang 4molecular mechanisms underlying disease pathogenesis,
(b) facilitate early detection and prognosis, and (c) to
provide novel targets for therapeutic intervention For
retrospective studies, the Centre efforts are supported
by the Danish Breast Cancer Cooperative Group
(DBCG), a group that manages a tumour repository
bank containing frozen tissue samples from
approxi-mately 10 000 breast cancer patients with clinical
fol-low-up of up to 10 years In due course, the DBCG is
expected to facilitate the implementation of therapeutic
modalities on a nationwide basis
Below we give a brief account of our long-term
strategies on ongoing long-term strategies within
DCTB to search for markers for early detection and
targets for therapeutic intervention, identify signalling
pathways affected in individual tumours, as well as to
integrate multiplatform ‘omic’ data sets collected from
tissue samples obtained from individual patients
Biomarkers for early detection and
targets for therapeutic intervention
Biomarkers for early detection
Success in detecting markers for early breast cancer
detection depends very much on the sources that are
used to conduct the search To date, two strategies
have been used to search for these biomarkers One is
based on the comparative analysis of the proteome of
peripheral fluids, and the other on similar analysis of
diseased tissues Even though appealing, the first
approach suffers from the drawback that markers are
likely to be present in lower amounts in the blood
cir-culation and, as a result, may be difficult to detect
Diseased tissues on the other hand may express the
markers at relatively higher levels, but it is not an easy
task to identify those that ultimately will appear in the
blood circulation The use of nipple-aspirated fluid as
a source of biomarkers has been explored in a few
cases, but no systematic studies have been reported so
far [56,57]
Our group has recently devised an alternative
approach that is based on the analysis of near fluids,
as these may be enriched in externalized biomarkers
and may justify a systematic long-term search [4,58]
Compelling evidence indicates that tumour growth
and progression is dependent on the malignant
poten-tial of the tumour cells as well as on the
multidirec-tional interactions of local factors produced by all
the cell types – tumour, stroma and endothelial cells,
in addition to immune and inflammatory cells –
pre-sent in the local microenvironment [17–29] All these
cells secrete, shed or release proteins to the interstitial
fluid that bathes the tumour microenvironment and some of these proteins, including biomarkers, may eventually reach the blood circulation through the lymphatic vascular system This ‘near fluid’, the tumour interstitial fluid (TIF) [58], may provide a rich source of biomarkers for early detection as well
as novel targets for therapeutic intervention as its protein composition can be readily compared with its nonmalignant counterpart (NIF) using proteomic technologies Figure 1 illustrates the steps we have undertaken to retrieve this fluid from fresh tumour biopsies, nonmalignant tissue, axillary nodal metasta-sis (MIF), and fat interstitial fluid (FIF) obtained immediately following surgery So far, we have ana-lyzed the protein composition of TIFs recovered from
30 high-risk patients The criteria for high-risk cancer applied by DBCG are age below 35 years old, and⁄ or tumour diameter of more than 20 mm, and⁄ or histo-logical malignancy 2 or 3, and⁄ or negative oestrogen and progesterone receptor status and⁄ or positive axil-lary status Figure 2A shows a two-dimensional iso-electrofocusing (IEF) gel of a representative fluid The protein composition of the TIF is strikingly dif-ferent to that of serum, although both fluids share some of their major components (Fig 2B) The TIF
is highly enriched in proteins that are either secreted via the classical endoplasmic reticulum (ER)⁄ Golgi pathway, shed by membrane vesicles (membrane blebbing), or externalized by plasma membrane trans-porter ([59] and references therein) Quantitation of the ratio of thioredoxin (externalized by an ER⁄ Golgi independent route)⁄ cytokeratin 18 (CK18) in whole tumour lysates and its corresponding TIF, yielded values that differ by a factor of 10 or more (data not shown), suggesting that nonspecific protein release due to cell death is not a major contributor
to TIF
So far, 284 primary translation products, as well as hundreds of post-translational modifications, have been identified using a combination of procedures that include mass spectrometry, 2D gel immunoblotting and cytokine-specific antibody arrays (RayBiotech, Atlanta, GA, USA) [4,58] Cellular function categories assigned to the known proteins include – but are not limited to – ion transport, cell motility, transporter activity, protein transport, signal transduction, response to oxidative stress, immune response, energy pathways, regulation of gene expression, proteolytic pathways, protein metabolism, maintainers of cytoske-leton organization, cell–cell signalling, regulation of cell–cell communication and regulation of cell growth [58] A protein database will soon be available through our web site (http://proteomics.cancer.dk)
Trang 5A systematic search for potential biomarkers starts
by comparing the protein profiles of nonmalignant
(NIF) and tumour (TIF) fluids, and the identification
of proteins that are highly deregulated in the latter
(Fig 2C,D) This is a long-term endeavour that will
require the qualitative and quantitative comparison of
hundreds of sample pairs using gel-based proteomics,
the preparation of specific antibodies against putative
markers for validation, as well as the analysis of the
corresponding serum⁄ plasma and other control
sam-ples for the presence of these markers
As the above approach mainly detects proteins
pre-sent at moderate to high levels and is set on a
discov-ery mode, we are establishing in parallel more sensitive
antibody microarray platforms to detect known, lesser
abundant components that may be potential
biomark-ers themselves For example, elevated levels of
interleu-kin (IL)-6 have been observed in the serum of patients
with breast cancer [60], and the levels of this cytokine
have been shown to predict the survival of patients
with metastatic breast cancer [61] These observations
prompted us to assess the levels of IL-6 both in NIF
and TIF Figure 3A shows cytokine-specific antibody
arrays (120 cytokines; RayBiotech) reacted with NIF
and TIF proteins retrieved from the same patient
Increased levels of several cytokines, including IL-6
were observed in the TIF, and some of these changes
could be independently confirmed using the Bio-Plex
system from Bio-Rad (Hercules, CA, USA) which can
quantitatively measure 17 human cytokines
simulta-neously (Fig 3B) Clearly, there is a good correlation
between the levels of IL-6 as determined by the two
platform technologies, a fact that was further validated
by performing IHC on nonmalignant and tumour tis-sues using an IL-6 specific antibody known to work with paraffin-embedded sections As exemplified in Fig 4B, the tumour tissue stains strongly with the antibody as compared to its nonmalignant tissue coun-terpart (Fig 4A, patient 46), indicating that indeed the elevated levels of IL-6 observed in TIF are in part due
to the production of this cytokine by the tumour cells The high levels of IL-6 observed in the sera of cancer patients, however, may not only reflect production of this cytokine by tumour cells, as fat tissue (Fig 4C) – which is very abundant in the mamma (Fig 1) – pro-duces significant levels of IL-6 as judged by antibody array analysis of FIF (Fig 4D) The bona fide origin
of FIF is supported by the presence of leptin, a protein known to be produced by adipose tissue (Fig 4D) Whether IL-6 produced by fat cells reaches the blood circulation is at present unknown [62–65], but this pos-sibility must be taken into consideration when search-ing for specific biomarkers This example illustrates the difficulties one faces in biomarker discovery, and underscores how careful one must be when interpreting data generated from complex samples
Targets for therapeutic intervention – towards identifying and building up pathways affected
in breast tumours The goal of individualized treatment is to provide ther-apies to which the patients may best respond This will require a thorough understanding of the molecular mechanism underlying the particular phenotype in order to identify potential therapeutic agents with a
Fig 1 Recovering the TIF from fresh breast tumours Fresh tissue (about 0.25 g) washed twice in phosphate buffered saline (NaCl ⁄ P i ), is cut in small pieces of about 1–3 mm 3 and placed in a 10-mL conical plastic tube containing 0.8–1 mL of NaCl⁄ P i Samples are then incuba-ted for 1 h at 37 C in a humidified CO 2 incubator Following incubation the samples are centrifuged at 300 g, at room temperature for
1 min and the supernatant is aspirated with the aid of an elongated Pasteur pipette Samples are then further centrifuged at 4000 g, at 4 C
in a refrigerated centrifuge Aliquots for gel analysis are freeze dried while the rest is kept at )80 C until further use.
Trang 6high degree of specificity Key biological processes that
harbour potential targets for intervention include cell
proliferation, apoptosis, invasion and metastasis,
angiogenesis and genomic instability [66] Today,
tar-geted therapies are aimed mainly at growth factor
sig-nalling pathways, and tyrosine-kinase receptors such
as Her2⁄ neu and epidermal growth factor receptor
(EGFR) Antibodies and rationally designed
small-molecule drugs developed to target specific small-molecules
involved in key processes of tumour progression
such as Herceptin (trastuzumab), Erbitux
(IMC-C225, cetuximab), Tarceva (erlotinib HCP), Avastin
(bevacizumab), Gleevec (imatinib mesylate), and Iressa (gefitinib) are currently in clinical trials Based on results obtained in preclinical studies with animal mod-els, which showed that systemic administration of growth factor inhibitors could restrain the growth and metastatic potential of human TCC xenografts [67], and the promising results obtained with EGFR inhibi-tors in other tumour types, several trials are now ongoing to test these agents, alone or in combination,
in patients with various cancers [68]
The potential therapeutic applications of proteins present in the TIF is clear as this fluid contains many
Fig 2 Protein profiling of interstitial fluids Isoelectrofocusing (IEF) 2D gels of proteins from (A) TIF 41, (B) serum, (C) NIF 46 and (D) TIF 46.
Trang 7growth factors and signalling molecules (Fig 3A)
[58], and it is probable that many additional
interest-ing novel proteins involved in the regulation of the
tumour ecosystem may be found, in particular using
shotgun proteomics [69] and references therein The
TIF can be recovered from tissues using
nondenatur-ing conditions, and as such it can be readily analyzed
using protein biochip technologies that provide
resourceful tools for target identification and
valid-ation, as well as for studying protein interactions
(enzyme substrates, drugs, lipids, etc.) In particular,
antibody-based arrays can detect protein
phosphoryla-tion as a means to assess the funcphosphoryla-tion of a given
sig-nalling pathway [70,71] Phosphorylation is a key
regulatory factor in many aspects of cell proliferation,
and as a result the phosphorylation status of novel
proteins is eagerly being pursued using mass
spectro-metry [72–74]
In an effort to identify signalling pathways affec-ted in individual breast tumours and to focus our search for potential targets for intervention, we have started comprehensive IHC analysis of tumours using phospho-specific antibodies against key signalling molecules [75] In parallel, these studies are being complemented using reverse tissue lysate arrays [76,77] in collaboration with Zeptosens (Zeptosens
AG, Witterswil, Switzerland) In the long run, these studies are expected to provide a framework in which to integrate data on known signalling mole-cules, as well as on forthcoming ‘omics’ information generated from the same tumours (see below) As an example, Fig 5 shows IHCs of two breast tumours probed with antibodies against p53 (Fig 5A,D), p-p53 (Fig 5B,E) and p-glycogen synthase kinase-3 (GSK-3; Fig 5C,F), a serine⁄ threonine kinase involved in various pathways including the
A
B
Fig 3 IL-6 levels in NIF and TIF (A) Cyto-kine-specific antibody arrays (RayBio human cytokine array series 1000) were incubated with 0.5 mL of NIF and TIF accor-ding to manufacturer’s instructions (B) Quantification of cytokines using the BioPlex system (Bio-Rad).
Trang 8A B
Fig 4 IHC of nonmalignant epithelial cells
(A) and tumour cells (B) from patient 46
usi-ng IL-6 antibodies (C) Haematoxylin and
eosin staining of fat tissue located far from
a tumour (D) Cytokine-specific antibody
array (RayBio) incubated with FIF as
described in Fig 3.
Fig 5 Aberrant Wnt ⁄ b-catenin pathway in tumour 14 (A–F) immunohistochemistry of paraffin sections of tumours 14 and 7 incubated with antibodies against p53 (A and D), phosphorylated p53 (p-p53, serine 15) (B and E) and phosphorylated GSKb (p-GSKb, C and F).
Trang 9Wnt⁄ b-catenin signalling cascade [78–80] The latter
plays an important role in development by
augment-ing the signallaugment-ing activity of b-catenin, a structural
component of the cell-cell adhesions that is known
to be deregulated in various cancers, including breast
cancer [81–85] Both p53 and GSK-3b have been
implicated in regulating the levels of b-catenin [86]
As shown in Fig 5A, tumour 14 depicts activation
of p53 in a region of the lesion in which GSK-3b is
also activated (Fig 5C), while the other tumour does
not When activated, GSK-3b phosphorylates
(tyro-sine phosphorylation) b-catenin, a fact that precludes
its binding to a-catenin and E-cadherin, thus
pre-venting cell adhesion and facilitating spreading
Phosphorylation of b-catenin targets it for
degrada-tion by the proteasome [87], and the p53 activadegrada-tion
is known to feed back and down-regulates b-catenin synthesis [86]
The presence in tumours of known signalling mole-cules involved in various pathways is being performed using antibody specific arrays (Panorama antibody microarray) that contain probes against hundreds of signalling molecules Arrays are incubated with pro-teins extracted from tumours using low concentrations
of Triton X-100 yielding semiquantitative data for many proteins simultaneously Figure 6 shows a rep-resentative protein antibody array analysis of a tumour In addition, quantitative gel-based proteomic (Fig 7) and transcriptomic data are being generated from the same tumour samples in selected cases in an effort to identify groups of coregulated proteins and mRNAs that may provide an integrated view of
Fig 6 Detection of signalling molecules in tumours using the Panorama antibody array (Sigma-Aldrich) Fresh tumours were minced and homogenized at room temperature with 0.1% Triton in phosphate buffered saline (NaCl ⁄ P i ) Samples were centrifuged at 300 g, at room tem-perature for 1 min and the supernatant was aspirated with the aid of an elongated Pasteur pipette The supernatant was then further centrifuged
at 4000 g, at 4 C in a refrigerated centrifuge Aliquots for gel analysis were freeze dried while the rest is kept at )80 C until further use.
Trang 10signalling cascades, extend the number of possible
tar-get candidates, as well as provide a more detailed
molecular phenotype of the tumour based on multiple
parameters
Future directions/conclusions
The analysis of tumour samples using single ‘omic’
platform technologies such as microarrays has
exempli-fied the value of this technology to classify tumours as
well as to derive signatures for prognosis and response
to treatment, particularly in lymphomas [7,8,88],
leuk-aemia [89,90] and breast cancer [12–14,91,92] Today,
however, it is becoming increasingly clear that we must
use multiplatform technologies to classify subgroups of
patients for more precise and predictive, individualized
approaches to cancer treatment [93] These efforts must
be accompanied by the development of bioinformatics
tools for integrating and mining the data as well as by
systematic, knowledge-based long-term approaches that
are supported by proper clinical infrastructures
There are several issues, however, that must be care-fully and promptly addressed if we are going to fulfil the dream of bringing individualized cancer care closer
to reality First of all, we must acknowledge the value
of long-term research and provide the appropriate legal and ethical framework to encourage the collabor-ation among all the stakeholders in the cancer ordeal Bridging the gap between basic and clinical research, facilitating the engagement of the industry, creating new infrastructures and bio banks, as well as the cre-ation of innovative clinical trials are among the items that require urgent action The aim of cancer research
is to improve the life expectancy and quality of life of patients and we must make every effort to coordinate current activities in order to achieve this goal
Acknowledgements
We would like to thank Gitte Lindberg Stort, Dorrit Lu¨tzhøft, Hanne Nors, Michael Radich Johansen, Britt Olesen and Signe Trentemøller for expert
Fig 7 Quantitative gel-based proteomics Twenty tumour cryo-sections (8 microns) were dissolved in 100 lL of Zeptosens lysis solution and 40 lL were applied to the first dimension isoelectrofocusing gel The first section of each tumour is routinely kept for histology analysis
in order to facilitate the interpretation of the data.