Discovery of Biomarkers For Endometrial Cancer - Bioscience Technology Online

Applied Biosystems

850 Lincoln Centre Dr.
Foster City, CA, 94404
Website: http://www.appliedbiosystems.com

Discovery of Biomarkers For Endometrial Cancer

Dr. K.W. Michael Siu, Professor of Chemistry and NSERC/MDS SCIEX Industrial Research Chair, York University, Toronto, Canada.

By Mark Springer

A group of Canadian researchers led by Dr. K.W. Michael Siu, Professor of Chemistry and NSERC/MDS SCIEX Chair, York University, Toronto, Canada, has identified a panel of nine potential cancer biomarkers (PCMs) that may be used to eventually diagnose endometrial cancer (EmCa), the fourth most common cancer among women in North America.^(1,2)

In this study, the researchers reported that an important factor in the identification of the protein biomarkers was the use of two complementary protein labeling reagents from Applied Biosystems. The combined use of iTRAQ reagents and cleavable ICAT reagents resulted in the discovery and identification of nine differentially expressed proteins in normal and cancerous endometrial tissue samples. In the near future, the researchers hope to develop these newfound PCMs, and perhaps others, into a panel of markers that can be used by clinicians as a diagnostic test for endometrial cancer.⁽¹⁾

“To be able to detect cancer using biomarkers in a test, especially a blood test, is extremely attractive, because it means that you will be able to diagnose cancer, much more readily and in a less invasive manner,” notes Dr. Siu.

As the NSERC/MDS SCIEX Industrial Research Chair, Dr. Siu collaborates with MDS/SCIEX scientists to develop prototype instrumentation and new processes and to better understand the chemistries that underlie these systems and processes. Dr. Siu directs a team of 20 project scientists, postdoctoral fellows, research associates and graduate students.

One of Dr. Siu’s current non-instrumental research projects involves discovering, identifying and validating biomarkers for EmCa.

Potential panel of EmCa cancer biomarkers

Today, there are only a few single protein biomarker tests for cancer, one of the most well-known marker-based tests being the prostate specific antigen (PSA) test for prostate cancer. A biomarker (CA 125) also exists for ovarian cancer. However, according to Dr. Siu, PSA is prostate specific, but not prostate-cancer specific, and CA 125 is only highly expressed in a fraction of ovarian cancer patients. At present, there are no diagnostic tests based on multiple markers, which should increase specificity and diagnostic accuracies.

Each of the 9 proteins that the Canadian team recently identified showed differential expression in samples of cancerous and normal endometrial tissue. In EmCa tissue samples, six of the nine PCMs were overexpressed, whereas three were underexpressed. All nine PCMs have previously been linked with cancers other than EmCa.

“Right now,” notes Dr. Siu, “There are no markers specific for endometrial cancer, but we now have a collection of protein markers, and our hope is to be able to use all of them together as markers for the disease.”

Collectively, as a panel of markers, the newly discovered PCMs have the potential of being developed into a diagnostic test for EmCa.

“Our finding is significant because we have amassed a number of potential cancer markers,” says Dr. Siu. “These markers are considered potential until after we have completed validation, done the appropriate clinical tests, and determined that they are effective. Over time, we hope that we will have many more potential markers and will be able to use them to make a diagnostic test based on the best biomarkers.”

Panels preferable to single markers

Because cancer is a complex disease with a number of different types and subtypes, the accuracy of a diagnosis of any kind of cancer will increase with the number of biomarkers available to clinicians. In this finding, nine proteins met the criteria of differential expression upon comparison of protein expression profiles of both cancerous and normal endometrium tissue samples. Moreover, there was at least a two-fold change in protein expression when cancer endometrium tissue samples were compared with normal tissue samples, including both the proliferative and secretory phases.⁽¹⁾

“It is very important for us to find more cancer markers in the future, and to make use of as many of the best markers as possible,” explains Dr. Siu.

As all cancers involve uncontrolled cell differentiation, Dr. Siu notes that different cancer forms share some common biochemical pathways, and induce and suppress common proteins. However, it is unlikely that the extent of induction and suppression will be identical among different cancers, so the use of multiple markers in a panel will likely increase the chance of differentiating cancers based on expression pattern.

The nature of EmCa

Typically endometrial cancer afflicts post-menopausal women. The cancer usually begins in the lining of the uterus (endometrium). As is the case with almost all cancers, early detection of EmCa greatly increases the success of treatment. If detected early, this slow growing cancer is successfully treated about 90% of the time. However, if left undetected, it can metastasize and spread past the pelvic region to affect the other parts of the body.⁽²⁾

“Early detection in any cancer is important, because an early detection would mean that there is a better chance of a longer-term survival. The idea is to actually diagnose as early as possible and before the cancer has metastasized,” explains Dr. Siu.

Hysterectomy, the surgical removal of the uterus is the most common treatment for endometrial cancer.⁽²⁾

click here to enlarge
Figure 1. The iTRAQ reagents consist of a charged reporter group, a peptide reactive group and a neutral balance group. The peptide reactive group reacts with primary amines and labels all peptides. An increase in the overall protein coverage results from all peptides being labeled.

Multiplexing of reagents with EmCa samples

In general, Dr. Siu notes, it is challenging to find protein biomarkers for cancer. An additional challenge that researchers faced in their search for EmCa protein markers is the inherent physiologic variations in protein expression levels that result from normal menstrual cycling.⁽¹⁾

In most women, the endometrium undergoes a 28-day cyclic change in hormone levels. The first 14 days is known as the proliferative stage, and the second 14 days is known as the secretory phase.

“For our proteomics work we compare the expression levels of potential cancer marker proteins in EmCa tissues against these two phases of normal tissues.” notes Dr. Siu.

In their recent paper, the researchers noted that the multi-sample capability of the iTRAQ reagent technology is useful for this kind of comparative study.

Multiplex reagents allow one to compare the concentrations of protein markers in up to four samples, so that’s the beauty of it. Before, we couldn’t really do it,” says Dr. Siu.

Reagents in the iTRAQ line are multiplexed sets of four isobaric reagents which are amine-specific and yield labeled peptides that are identical in mass (Figure 1). The multiplexing capabilities of the reagents made it possible for the researchers to perform a proteomic analysis of both the major phases of the normal endometrium, while simultaneously comparing them against two cancer samples.

“We were able to compare the four samples simultaneously. So, the reagents allowed us a more efficient experimental attack,” explains Dr. Siu.

Use of the reagents allows both quantitation and identification of peptides from MS/MS spectra. The labeling technique uses a specific reagent that selectively modifies the N-terminus and lysine residues of every peptide. Peptide samples labeled with different forms of the reagent are pooled then fractionated first by strong cation exchange chromatography; the fractions are then further separated by on-line capillary LC and analyzed directly by tandem mass spectrometry.

“The ability to quantify expressed proteins, or compare expression levels is important. A yes or no answer is a start, but it is not as powerful as having the ability to compare the concentrations” notes Dr. Siu.

Complementary reagents

In this study, the researchers also employed another protein labeling reagent from Applied Biosystems/MDS SCIEX, cleavable ICAT reagents, to allow them to examine different protein pools.⁽⁴⁾

The ICAT reagent consists of three key components: a protein group that reacts with cysteine amino acids; a linker, which is labeled with either heavy hydrogen (d8) isotopes or light (d0) hydrogen isotopes; and a biotin affinity tag that makes possible isolation and detection of peptides labeled with either the heavy or light version of the ICAT reagent. Through use of both light and heavy reagents, researchers can label all the proteins containing cysteine residues in two different samples, combine them and, then, simultaneously identify and determine the relative quantity of the proteins present in both samples.

In contrast, the cleavable ICAT reagent accomplishes peptide labeling using carbon 13 as opposed to hydrogen isotopes for peptide labeling. The reagent also contains a cleavable biotin group, which can be cleaved prior to MS/MS.

“The carbon 13 is fantastic because it allows co-elutions of labeled peptides,” explains Dr. Siu. “Co-elutions mean that the differentially labeled peptides will actually elute as a single peak as opposed to having slightly different retention times. This will facilitate quantitation.”

For applications of the cleavable reagents, the researchers determined relative quantification of proteins from TOF MS scans. They then calculated the relative areas between the pairs of light and heavy label series of peaks.

LC/MS/MS analyses were performed using an Applied Biosystems/MDS SCIEX QSTAR Pulsar i Hybrid LC/MS/MS System, a QqTOF mass spectrometer. Data analysis for the iTRAQ reagent experiments was performed with Pro QUANT Software, while results for cleavable reagent experiments were analyzed with Pro ICAT Software, both from Applied Biosystems/MDS SCIEX.⁽¹⁾

“The two labeling reagents are complementary in that one tends to be better for more abundant proteins, and the other the less abundant proteins” says Dr. Siu. “There are obviously overlaps, but, together, the two reagents allow us to see a wider spectrum of proteins and cancer markers. We’re dealing with a complex problem, so we try to use all of the weapons in the arsenal to come up with as complete an answer as possible.”

Potential cancer biomarkers released into the bloodstream

Several of the PCMs discovered and identified in this study, including chaperonin 10 and pyruvate kinase, have previously been observed in blood, which opens up the possibility of an eventual development of a blood-based diagnostic test for EmCa.

“Obviously, the ideal situation would be to test for cancer markers in a bodily fluid, for example, blood or urine,” Dr. Siu notes.

Although elevation of levels of chaperonin 10 and pyruvate kinase have previously been associated with other kinds of cancer, when combined with other biomarkers in a panel, there is potential that the overall differential expression pattern of the entire panel of markers will be indicative of a particular type of cancer.

As Dr. Siu explains, “We hope that, if we have something like 10, 20, or 30 cancer markers, then the type of response -- which marker is expressed and in what concentration may be different depending on the cancer site,” says Dr. Siu. “For example, if we test a serum sample for breast cancer, we may look for a pattern of 10 cancer markers where number one, two, four and 10 are highly expressed. In another cancer, we may look for a different pattern of expression. So, from the pattern and the extent of the response, you can determine the type of cancer.”

Closing in on a biomarker test for EmCa

In the near future, Dr. Siu, and his colleagues hope to determine the absolute expression level of PCMs in normal and EmCa tissue. This will make it possible for the researchers to establish ranges of PCM concentrations in normal and EmCa tissues. For these kinds of experiments, iTRAQ reagents, which allow tryptic peptides of known amounts in absolute isotope-dilution experiments, provide a useful research tool.⁽¹⁾

Dr. Siu and the other authors of this study believe that the use of multiple protein biomarker labeling techniques may best be suited for initial marker discovery, followed by validation by other more traditional methods that are amenable to large-scale screening.

“Today, we are in a discovery phase, in which we analyze normal and cancerous samples, discover protein markers, and then we go through a validation phase in which we confirm the accuracy of our findings,” explains Dr. Siu.

Among the MS strategies for biomarker validation that Dr. Siu is currently investigating include methods of obtaining quantitative information about potential cancer biomarkers through a workflow involving multiple reaction monitoring (MRM)-initiated detection and sequencing performed using an Applied Biosystems/MDS SCIEX 4000 Q TRAP LC/MS/MS system. Called the MIDAS workflow, the first applications of this workflow were recently published and describe how the MIDAS workflow was used to help identify protein posttranslational modifications, including identifying sites of protein phosphorylation.^(5,6) With the ultimate goal of the researchers being the development of a diagnostic test that uses a panel of biomarkers to identify EmCa at the earliest stages of the disease, Dr. Siu and colleagues hope that a non-tissue based diagnostic test may be feasible sometime in the near future.

“I hope that we can actually accomplish a lot within the next year,” says Dr. Siu. “Our goal is to have a diagnostic test ready in three years.”

More information on the specific chemistries and instruments discussed in this article is available from: Applied Biosystems, 800-327-3002, www.appliedbiosystems.com

References
1. DeSouza, L., Diehl, G., Rodrigues, M.J., Guo, J., Romaschin, A.D, Colgan, T.J. and Siu, K.W.M. Search for Cancer Markers from Endometrial Tissues Using Differentially Labeled Tags iTRAQ and cICAT with Multidimensional Liquid Chromatography and Tandem Mass Spectrometry, Journal of Proteome Research 4(2):377-386 (April 2005).
2. Mayo Clinic staff, “Endometrial Cancer,” www.mayoclinic.com.
3. Ross, P.L., Huang, Y.N., Marchese, J.N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., Jacobson, A. and Pappin, D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents, Molecular & Cellular Proteomics 3(12):1154-1169 (December 2004).
4. Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H. and Aebersold, R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags, Nature Biotechnology 17(10): 994-9. (October 1999).
5. Unwin, R.D., Griffiths, J.R., Leverentz, M.K., Grallert, A., Hagan, I.M. and Whetton, A.D. Multiple Reaction Monitoring to Identify Sites of Protein Phosphorylation with High Sensitivity, Molecular & Cellular Proteomics 4:1134–1144 (August 2005).
6. Cox, D.M., Zhong, F., Du, M., Duchoslav, E., Sakuma, T. and McDermott, J.C. Multiple Reaction Monitoring as a Method for Identifying Protein Posttranslational Modifications. Journal of Biomolecular Techniques 16(2) :83-90, (June 2005).