Abstract.

Monitoring the protein expression pattern in tumour

cells by proteomics technologies offers opportunities to discover
potentially new biomarkers for the early detection and
diagnosis of cancer. Different proteomic tools such as 2D-
PAGE, 2D-DIGE, SELDI-ToF-MS technology, protein arrays,
ICAT, iTRAQ and MudPIT have been used for differential
analysis of various biological samples, including cell lysates,
cell secretome (conditioned medium), serum, plasma, tumour
tissue and nipple aspirate fluid, to better understand the
molecular basis of cancer pathogenesis and the validation and
characterisation of disease-associated proteins. In recent years,
there has been a large increase in cancer-related publications
dealing with new biomarker discovery for cancer, therefore, in
this review we have focused on the contribution of proteomics
technologies in serum and conditioned medium-based
oncology research particularly for lung, breast, melanoma and
pancreatic cancer.
Although many effective therapies are present for early
detection and diagnosis, cancer remains a major cause of
death and disease. Cancer is a complex disease that reflects
the genetic, as well as protein changes within a cell. Gene
expression data gives us limited relevant information since
proteins are the main functional units performing all
biological process in the cell or organism and may have
post-transcriptional event(s) and post-translational
modification(s) that contribute to the biological activity of
proteins. Protein expression patterns are also changed
specifically and significantly in response to every disease (1).
The first protein cancer marker, carcinoembryonic antigen
(CEA), was identified in 1965 in patient serum for the
detection of colorectal cancer (2). Other biomarkers
discovered in the 1970s and 1980s include prostate-specific
antigen (PSA) for prostate cancer, CA-19 for colorectal and
pancreatic cancer, CA-15-3 for breast cancer and CA-125
for ovarian cancer. However, not all biomarkers are
effective in all clinical situations. For example, PSA is well
established in clinical practice, but approximately one third
of patients with an elevated PSA level often undergo
unnecessary medical procedures because they do not have
a malignant form of prostate cancer (3). Many types of
cancer, such as lung carcinoma and melanoma do not have
any significant biomarkers available to screen at the early
stage of disease. Identification of new tumour biomarkers
with predictive value is necessary to allow early detection
and treatment of cancer.
Proteomics Technologies
With the recent developments in electrophoresis, imaging,
protein labelling, protein array-based approaches and mass
spectrometric technologies, along with developments in
genomic and protein bioinformatics, proteomics may
provide powerful information for improved biomarker
discovery. Several proteomics technologies including two-
dimensional polyacrylamide gel electrophoresis (2D-
PAGE), surface enhanced laser desorption/ionisation time
of flight (SELDI-ToF), protein arrays, isotope coded affinity
tags (ICAT), iTRAQ and multidimensional protein
identification technology (MudPIT) are the approaches
being implemented in cancer research. 2D-PAGE and
SELDI-ToF are the main technologies used in serum cancer
research, however other technologies such as protein arrays,
ICAT, iTRAQ and MudPIT also offer great potential for
future biomarker discovery in cancer.
Two-dimensional electrophoresis. 2D-PAGE is the most
widely used proteomics technique to study the proteome as
well as cancer biomarkers (4-8). 2D-PAGE remains
1247
Correspondence to: Paula Meleady, National Institute for Cellular
Biotechnology, Dublin City University, Glasnevin, Dublin 9,
Ireland. Tel: +353 1 7005700, Fax: +353 1 7005484, e-mail:
Paula.Meleady@dcu.ie
Key Words: Serum, cancer, proteomics, SELDI, 2D PAGE, 2D
DIGE, review.
ANTICANCER RESEARCH 27: 1247-1256 (2007)
Review
Proteomic Approaches for Serum
Biomarker Discovery in Cancer
PRIYANKA MAURYA, PAULA MELEADY, PAUL DOWLING and MARTIN CLYNES
National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland
0250-7005/2007 $2.00+.40
challenging mainly because of its low sensitivity and
reproducibility. Modified 2D electrophoresis by fluorescent
tagging to proteins, differential gel electrophoresis (DIGE),
offers increased throughput, ease of use, reproducibility,
and accurate quantitation of protein expression differences
(9). This system enables the separation of two or three
fluorescently labelled protein samples (Cy2, Cy3 and Cy5)
on the same gel. Differential analysis software identifies the
differentially expressed protein targets that can be trypsin-
digested and readily identified using mass spectrometry by
generating peptide mass fingerprints (PMF) using matrix
assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-ToF MS), a technique that is both
relatively easy to use and reasonably sensitive for identifying
proteins. Additionally other mass spectrometry techniques
such as electrospray ionization (ESI-MS/MS) are capable of
providing amino acid sequence information on peptide
fragments of the parent protein (10).
Although 2D-PAGE-based techniques have a reasonable
level of throughput, there are a number of difficulties
inherent to the technique such as separation of low abundant
proteins as it is difficult to enrich for these proteins.
Membrane proteins are also difficult to separate due to poor
solubility. Efforts have been made to overcome these
limitations. For example, low abundant proteins can be
identified using higher protein concentrations, and applying
fractionation methods (7). Moreover, membrane proteins
can be identified to some extent by using commercially
available mild detergents such as oligooxyethylene,
sulfobetaine, dodecyl maltoside or decaethylene glycol mono
hexadecyl, as use of strong detergents like SDS interfere with
the isoelectric focusing of proteins (11). Additional problems
with 2D-electrophoresis include insufficient resolution to
separate multiple species originating from a single protein
with post-translational modifications, such as those with
carbohydrates, difficulties in detecting proteins with
molecular masses <120 kDa and those with pI values <4 or
>9, low visualisation of less-abundant proteins, and co-
migration of proteins to the same spots (12). Conventional
2D-electrophoresis shows only protein expression and cannot
detect protein-protein interactions or protein function
without using particular methods such as affinity
electrophoresis (13).
SELDI-ToF MS. This technique allows proteins/peptides to
be profiled from different biological samples on a variety of
chemically (e.g. anionic, cationic, hydrophobic, hydrophilic,
metal affinity capture) or biochemically (e.g. immobilized
antibody, receptor, DNA, enzyme) defined chromatographic
surfaces. A small amount of sample of interest is loaded onto
ProteinChip arrays that selectively bind different subsets of
proteins in crude samples by adsorption, partition,
electrostatic interaction or affinity chromatography according
to their surface chemistries. After a short incubation period,
unbound proteins and unspecific substances are washed away
with an appropriate buffer and water. The ToF reader
records the time-of-flight and calculates the accurate
molecular weight of proteins/peptides in the form of a
spectral map containing mass to charge ratios (m/z) and
intensities corresponding to each bound protein/peptide.
Biomarker Wizard software analyses the spectral map and
detects differentially expressed protein/peptides with
statistical significance. An increase in published research
using SELDI-ToF in the past few years itself has
demonstrated its potential for the early detection of cancer,
especially for low molecular weight cancer-associated
proteins. For example, applications of SELDI-ToF have been
demonstrated for the early detection of prostrate (14, 15),
breast (16, 17), bladder (18) ovarian (19, 20), pancreatic (21),
and lung (22) cancer biomarkers. However, there is some
controversy over this technology such as its reproducibility,
the bioinformatics used, the possibility of over-fitting, the
potential bias in the samples, as well as how this could
possibly fit into a routine diagnostic lab (23-25).
Protein array technology. Protein arrays are being used for
drug discovery, biomarker identification and molecular
profiling of cellular material (26-29). Protein arrays are
generated by spotting antibodies (26) or other affinity
reagents, such as aptamers, purified proteins (30), peptides
(28) or fractionated proteins (27), onto some sort of matrix
either on flat solid phases or in capillary systems to generate
protein arrays. The sample is applied to the array to allow
specific binding of the sample to the array and then the
arrays are washed to remove the unbound fraction. The
process can also be reversed whereby the protein samples
of interest are spotted onto the matrix and then probed with
different affinity reagents (31). The wider application of
protein arrays in biomedical research is still limited, partly
because of the cost of producing antibodies and the limited
availability of antibodies with high specificity and high
affinity for the target. Additionally, the difficulties
associated with preserving proteins in their biologically
active conformation before analysis with protein arrays
further limits the application of this technology as a routine
proteomic strategy.
Isobaric Tag Labelling Technology
ICAT. Isotope-coded affinity tags (ICAT) use stable isotope
labelling to perform quantitative analysis of paired protein
samples. They contain a protein-reactive group, an ethylene
glycol linker and a biotin tag (32). Two different isotope
tags are generated by using linkers that contain either eight
hydrogen atoms (d0, light reagent) or eight deuterium
atoms (d8, heavy reagent) which bind covalently to cysteine
moieties of amino acid within protein(s). Both samples are
ANTICANCER RESEARCH 27: 1247-1256 (2007)
1248
mixed, digested with trypsin, fractionated by avidin affinity
chromatography and then these differentially tagged
peptides are scanned in a mass spectrometer. Spectral peak
analysis in single mass spectrometric (MS) mode of the
isotopically resolved peptides from the two different sources
enables quantitation of the relative amounts of the peptide
and hence the protein levels. Differentially expressed
proteins are then identified by tandem MS (MS/MS)
sequencing. One weakness of ICAT is that only cysteine-
containing peptides can be labelled. Approximately 10% of
proteins do not have cysteine, therefore they will not be
detected by ICAT.
iTRAQ. A similar isobaric labelling technology to ICAT,
called iTRAQ, that labels amine residues in peptides has
been recently developed (33). iTRAQ contains a set of four
isobaric reagents and therefore can analyze up to four
protein samples at one time. After trypsin digestion,
samples are labelled with four independent iTRAQ
reagents. The reporter groups of the iTRAQ reagents will
split from the peptide and generate small fragments for
each sample with mass/charges (m/z) of 114, 115, 116, and
117. The intensity of each of these peaks represents the
quantity of small reporter group fragments and thus
represents the quantity of a peptide sample. Peaks in the
spectrum graph are used to identify peptide sequences and
therefore protein sequences. By comparing the amounts of
peptides labelled with each iTRAQ reagent, quantitative
differences can be readily measured. A comparative analysis
of iTRAQ and ICAT suggests that the information
generated by the two methods is complementary. Both
methods have some advantages and disadvantages over the
other. Unlike iTRAQ, ICAT is preferred for low abundant
proteins including signalling molecules, however
overlapping peaks in the MS spectrum can compromise the
quality of results. On the other hand, apart from non-
specific nature of labelling, iTRAQ requires lengthy sample
processing separately that increases the chances of
experimental variation (34).
MudPIT. MudPIT is a non-gel approach that uses
multidimensional high-pressure liquid chromatography
(LC/LC) separation, tandem mass spectrometry and
database searching (35). MudPIT permits a rapid and
simultaneous separation and identification of proteins and
peptides in a complex mixture without the need for pre- or
post-separation labelling, which is not possible in 2D-
electrophoresis, ICAT or iTRAQ (36). The complex protein
mixture is digested with a specific protease, then peptide
fragments are allowed to separate in parallel with two-
dimensional liquid chromatography using a strong cation
exchange (SCX) column that separate peptides based on
charge, and then by a reverse phase (RP) column based on
hydrophobicity. Eluted peptides are identified by tandem
mass spectrometry and the technique is extremely sensitive
and reproducible. One of the major weaknesses of MudPIT
is in identifying quantitative differences in protein
expression across protein mixtures (37). In a comparative
study with 2D-electrophoresis, MudPIT has been reported
to demonstrate superior detection efficiency (36).
Implementation of Proteomics Technologies in
Serum Cancer Research
Lung cancer. Lung cancer is clinically divided into two major
histological types, non-small cell (NSCLC) and small cell
lung cancer (SCLC). NSCLC accounts for 75-85% of lung
cancer patients and consists of several subtypes,
predominantly squamous cell carcinomas, adenocarcinomas
and large cell carcinomas, which are treated in the same
manner. Small cell lung cancer accounts for 15-25% of lung
cancer patients, often has neuroendocrine components and
is primarily treated with chemotherapy and/or radiotherapy.
Many lung cancers are composed of histologically mixed
tumour types consisting of non-small cell and small cell
components. Dyspnoea, cough and thoracic pain are early
signs of lung cancer, however they are not specific to all
cases, while haemoptysis often indicates advanced disease.
There is no satisfactory marker for the early detection of
lung cancer. However, many widely used biomarkers are
available for differential diagnosis and sub typing, i.e. CEA,
CYFRA 21-1, SCC, NSE and ProGRP (38-41), but offer
low specificity and/or sensitivity.
Various putative biomarkers from serum and conditioned
medium have been identified using proteomics as a
searching tool. Dihydrodiol dehydrogenase (DDH) was
shown to be secreted by the adenocarcinoma cell line, A549,
by performing 2D electrophoresis on conditioned medium
followed by mass spectrometry (42). Following this
observation, the levels of DDH mRNA and protein
expression were found to be significantly higher in 15
NSCLC cancer tissues and protein levels were also
significantly increased in the serum of the NSCLC patients
compared to non-malignant lung tumour and healthy
controls. Since mRNA expression of DDH was also
reported to be elevated in NSCLC tumour specimens, this
raises the possibility of using DDH as a tissue marker and a
novel serological marker for NSCLC detection (42, 43).
Protein gene product 9.5 (PGP 9.5) is a neurospecific
polypeptide and was proposed as a marker for non-small
cell lung cancer, based on its expression in tumour tissue
(44, 45). PGP 9.5 and autoantibodies against PGP 9.5 were
observed in sera from lung cancer patients through an
antibody-based reactivity assay against lung adenocarcinoma
proteins, resolved by 2D-PAGE (46). However PGP 9.5 is
not limited to lung cancer, as it has also been reported in
Maurya et al: Serum Proteomics and Cancer (Review)
1249
pancreatic cancer (47). PGP 9.5 was also shown to be
present at the cell surface, as well as secreted in conditioned
medium by A549 (46).
In another study, approximately 250 and 100 different
proteins were detected in HSA- and IgG-depleted serum
samples from lung cancer patients using 2D-LCMS/MS and
1D-LCMS/MS respectively (48). Similarly, the serum
samples from lung cancer patients were analyzed with 2D-
PAGE after depletion of highly abundant plasma proteins
by immunoaffinity chromatography and fractionation by
anion-exchange chromatography; 58 gene products,
including the classic plasma proteins and the tissue-leakage
proteins catalase, clusterin, ficolin, gelsolin, lumican,
tetranectin, triosephosphate isomerase and vitronectin, were
identified from this study (49).
SELDI proteomic patterns of lung cancer patient serum
have also been assessed to distinguish lung cancer patients
from healthy individuals. Three protein peaks from the
profiles of 30 lung cancer patients and 51 age- and sex-
matched healthy individuals achieved 93.3% sensitivity in
detection of lung cancer patients (50). Similarly, five protein
peaks at 11493, 6429, 8245, 5335 and 2538 Da were chosen
automatically as a biomarker pattern from the profile of a
training set composed of 208 serum samples, including 158
lung cancer patients and 50 healthy individuals, and this
pattern provided sensitivity of 91.4% in the detection of
non-small cell lung cancers (NSCLC) (51).
Breast cancer. The classical pathological methods that are
used to predict survival, development of metastatic disease
or to guide selection of primary therapy (52, 53) in patients
with breast cancer rely on anatomical staging of cancer and
include the Nottingham Prognostic Index (54), Adjuvant
Online (AO) (55) and the St. Gallen criteria (56). The
current St. Gallen derived algorithm for selection of
adjuvant systemic therapy for early breast cancer patients
relies on tumour size and grade, nodal status, menopausal
status, peritumoural vessel invasion, endocrine status and
HER2 (epidermal growth factor receptor 2) status. HER2
is the most prominent and commonly used biomarker for
breast cancer detection (57). However MMP-2 (matrix
metalloproteinase-2 immuno-reactive protein), absence of
oestrogen and progesterone receptors and high expression
of Ki-67 (Mib-1) antigen, osteopontin (OPN), urikinase type
plasmonogen activator and its inhibitors (PAI-1 and 2) and
cathepsins (B and L) have also been indicated as prognostic
biomarkers for breast cancer (58-66).
In a proteomics study of breast cancer serum, two
proteins, HSP27 (up-regulated) and 14-3-3 sigma (down-
regulated) were identified using 2D-PAGE coupled with
MALDI-TOF-MS (67). Comparison of the expression
patterns of these two proteins correctly classified 97% of the
controls as not cancer and 100% of cancer samples as
malignant. This result yielded 100% sensitivity. The positive
predictive value for this sample set was 98%. In another
study 2D-DIGE analysis of serum samples obtained from 39
patients with breast cancer and 35 controls revealed that
proapolipoprotein A-I, transferrin, and hemoglobin were
up-regulated and three proteins, apolipoprotein A-I,
apolipoprotein C-III, and haptoglobin a2 were down-
regulated in cancer patients (42). However, in cross
validation with routine clinical immunochemical reactions,
the serum levels of apolipoprotein A-I and haptoglobin
could not be detected, indicating the superiority and
sensitivity of the 2D-DIGE technique.
A pattern of three serum biomarkers (two up-regulated
at 8.1 kDa and 8.9 kDa and one down-regulated at 4.3 kDa)
were identified from the SELDI profiles of 169 serum
samples from 103 breast cancer patients, 41 healthy women,
and from 25 benign breast cancer patients that distinguished
patients from controls (16). The sensitivity and specificity
after cross-validation within the sample group
(bootstrapping), using a random subset of the data to build
the model and testing it with the remaining data, were
found to be 93% and 91%, respectively. However, a new set
of samples in a later study confirmed the up-regulation of
the 8.1 kDa and 8.9 kDa biomarker species; subsequently
the 8.9 kDa species was identified to be a complement
component of C3a (desArg) and the 8.1 kDa species as a C-
terminal-truncated form of C3a (desArg) (68). In a later
study also using SELDI-TOF MS, the combination of an
independent cancer biomarker, Ca 15.3, with the serum
biomarkers, 4.286 kDa and 4.302 kDa possibly
corresponding to the 4.3 kDa peak identified by Li et al.
(16), and 8.919 kDa and 8.961 kDa possibly corresponding
to the 8.9 kDa peak also identified by Li et al. (16),
significantly improved breast cancer diagnosis (69). In
another study, four peaks, CA1 (17.3 kDa), CA2 (26.2 kDa),
CA3 (5.7 kDa), and CA4 (8.9 kDa), were chosen as
potential biomarkers from the SELDI profile of 49 breast
cancer patients, 51 patients with benign breast diseases and
33 healthy women to build a prediction model using
artificial neural networks and discriminant analysis (70).
One hundred percent sensitivity and specificity were
observed in a training set while a blind test set, showed
76.47% sensitivity and 90.00% specificity.
Two genes, BRCA-1 and BRCA-2, have been observed to
be strong tumour suppressors in men and women (71, 72).
Fifteen serum samples from women with BRCA-1 mutations
who developed breast cancer (BRCA-1 Ca) and 15 from
those who did not (Carrier), 16 from normal volunteers
(NL), and 16 from women with sporadic breast cancer
(SBC) were profiled with SELDI-TOF enabling the
differentiation between BRCA-1 Ca vs. Carrier with 87%
sensitivity and 87% specificity and BRCA-1 Ca vs. SBC
patients with 94% sensitivity and 100% specificity (73).
ANTICANCER RESEARCH 27: 1247-1256 (2007)
1250
Similar results with
~
86% discrimination between women
with BRCA-1 breast cancers from the 15 non-cancer
BRCA-1 carriers were later observed (74).
For optimization and individualization of therapeutic
decisions, a multiprotein complex (including haptoglobin,
C3a complement fraction, transferrin, apolipoprotein C1
and apolipoprotein A1)-based model developed on SELDI
profiles of denatured and fractionated early postoperative
serum samples from 81 high-risk early breast cancer patients
was shown to correctly predict the outcome in 83% of
patients for metastatic relapse (75).
Pancreatic cancer. Poor prognosis in pancreatic cancer is
attributed to the fact that most patients do not develop
overt symptoms until the disease has disseminated or caused
local organ dysfunction (76). Currently CA 19-9 is the
accepted serum marker for pancreatic cancer, but it was
approved only for monitoring treatment response by the
U.S. Food and Drug Administration (77, 78).
Approximately 80-90% of people with pancreatic cancer
have elevated levels of this marker in their blood (79).
Additionally, current methods of diagnosis, including CA
19-9, are ineffective for identifying small, surgically
resectable cancers.
To identify potential serum markers for pancreatic
cancer, serum samples from 3 pancreatic cancer patients
and 3 normal and healthy individuals were analysed using
2D-DIGE coupled with MALDI/TOF/TOF-MS and 24
unique up-regulated proteins and 17 unique down-
regulated proteins were identified in cancer serum (8).
Increased levels of apolipoprotein E, R-1-antichymotrypsin
and inter-R-trypsin inhibitor in serum proteome analysis of
20 patients with pancreatic cancer and 14 controls were also
found to be associated with pancreatic cancer by western
blot analysis; the use of these proteins resulted in a
sensitivity of 82.6% and a specificity of 100% in pancreatic
cancer diagnosis. In another study, samples from 32 normal
and 30 pancreatic cancer patients were analysed using 2D-
PAGE and 9 spots out of 154 commonly overexpressed
proteins discriminated 100% of pancreatic cancer samples
and 94% of normal samples (80). Fibrinogen-, a protein
associated with the hypercoagulable state of pancreatic
cancer, was identified as one of these nine spots and was
found to discriminate all cancer cases from normal sera,
successfully indicating the potential of fibrinogen- in
pancreatic cancer prediction.
Differential glycoprotein expression has also been
observed in human cancer serum (81). Sialylated
glycoproteins from highly abundant protein-depleted serum
samples of normal and pancreatic cancer individuals were
extracted using three different lectins [wheat germ
agglutinin (WGA), elderberry lectin (SNA), Maackia
amurensis lectin, (MAL)] after HPLC-based fractionation.
2D-PAGE was used for protein separation and
approximately 130 sialylated glycoproteins were identified
using LCMS/MS. The sialylated plasma protease C1
inhibitor was identified to be down-regulated in cancer
serum. Changes in glycosylation sites in cancer serum were
also observed by glycopeptide mapping using LC-ESI-
TOF-MS where the N83 glycosylation of R1-antitrypsin was
found to be down-regulated.
Patients with cancer have been found to frequently
develop autoantibodies and the identification of panels of
tumour autoantigens may have a role in the early diagnosis
of cancer and immunotherapy. DEAD-box protein 48
(DDX48), which is highly similar to eukaryotic initiation
factor 4A, was observed in 63.64% of patients with newly
diagnosed pancreatic cancer and in only 1.9% of normal
controls using an antibody-based reactivity assay (82).
Following on from this observation, a large sample set was
analysed to evaluate DDX48 as a diagnostic antigen and
33.33% of pancreatic cancer patients, 10% of colorectal
cancer patients, 6.67% of gastric cancer patients and 6.67%
of hepatocellular cancer patients were positive for anti-
DDX48 autoantibody reactivity using a purified DDX48
antigen-based ELISA assay, while none of the 20 chronic
pancreatitis patients, 30 lung cancer patients, and 60 normal
individuals were positive for this assay. Therefore, the
detection of autoantibodies to DDX48 in serum may
improve clinical diagnosis of pancreatic cancer (82).
Sera from 49 pancreatic cancer patients and 54 unaffected
individuals were profiled using SELDI-ToF-MS (83). Based
on the SELDI profiles, a classification model was generated
with classification and regression tree and logistic regression
methods and this resulted in differentiation of diseased
patients with 100% sensitivity. The specificity was 93.5%
using a decision tree algorithm and 100% using a logistic
regression model. In another study, the two most
discriminating protein peaks (m/z 3146 and 12861) from the
SELDI profiles of serum samples from 60 patients with
resectable pancreatic adenocarcinoma, 60 age- and sex-
matched patients with non-malignant pancreatic diseases and
60 age- and sex-matched healthy controls, differentiated
patients with pancreatic cancer from healthy controls with a
sensitivity of 78% and a specificity of 97%. This was found to
be significantly better than the current standard serum
marker, CA19-9, on the same sample set (with a 65%
sensitivity and 85% specificity) (84). The combination of the
two peaks and CA19-9 resulted in slightly improved
specificity. In a further study on pancreatic cancer, a set of
four mass peaks at 8766, 17272, 28080 and 14779 m/z, whose
mean intensities differed significantly in the SELDI profiles
of 245 plasma samples including diseased and normal
individuals, were selected as a biomarker profile using a
support vector machine learning algorithm which accurately
discriminated cancer patients from healthy controls in a
Maurya et al: Serum Proteomics and Cancer (Review)
1251
training cohort (sensitivity of 97.2% and specificity of 94.4%)
and in a validation cohort (sensitivity of 90.9% and a
specificity of 91.1%) (85). The combination of CA19-9 with
these differentially expressed peaks resulted in 100%
detection of pancreatic cancer cases (29/29 samples),
including early-stage (stages I and II) tumours.
Melanoma. Currently no protein serum marker is available
for surveillance of melanoma progression in early-stage
melanoma (86, 87). A study of serum specimens of 49 early-
stage (AJCC stage I and II) patients, including 25 patients
with melanoma recurrence and 24 without evidence of
disease, following resection were profiled using SELDI-ToF-
MS (86). The differential pattern of peaks among patients
with recurrence and without recurrence were used for
predicting the chances of cancer recurrence and resulted in
a sensitivity of 72% and a specificity of 75% that was
significant (86). Similarly, 205 serum samples from 101 early-
stage (American Joint Committee on Cancer (AJCC) stage
I) and 104 advanced stage (AJCC stage IV) melanoma
patients were analysed using SELDI-ToF and MALDI-ToF
MS. SELDI profiles were used to train artificial neural
networks (ANN). Based on these ANN algorithms, 88% of
stage assignment was correctly predicted; 80% of stage III
samples could be correctly as progressors or non-progressors
and 82% of stage III progressors were correctly identified.
The prediction accuracy was much improved compared to
the cases predicted by the conventional marker S-100 (88)
where only 21% of the stage III progressors were detected.
Future Directions
Significant progress has been made in proteomics
technology development in the last decade and this has
enabled researchers to move forward to a better
understanding of the disease. Several potential cancer type-
specific biomarkers for the early detection of the disease or
for therapy decision-making have been proposed using 2D-
electrophoresis or SELDI approaches on patient serum.
Newer technologies, such as protein arrays, ICAT, iTRAQ
and MudPIT, will increase this potential for biomarker
discovery in cancer.
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Received December 19, 2006
Accepted February 14, 2007
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