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transplantation the changes in gene expression in donor organs can be correlated with outcomes for a given recipient. These findings can assist the transplant community in deter- mining which recipients should receive a given organ to yield the greatest benefit. Gene expression may also reflect the environmental past to which a patient may have been exposed and the impacts of those changes can be correlated with clinical signs and symptoms.

Some centers have developed comprehensive genomic programs for a specific disease process in order to define novel genetic therapeutic strategies that may work in con- junction with surgical approaches to improve long-term out- comes. Through collaborative efforts at Baylor College of Medicine, researchers have established a genomic program for the study of hepatocellular carcinoma (HCC) in order to improve both patient and allograft survival after undergoing either liver resection or liver transplantation for HCC. They have established an effective biobanking protocol and after adequate tissue is confirmed by independent pathologists, genomic sequencing is performed.Their genomic sequencing includes whole genome sequencing, whole exome sequenc- ing, gene-specific analysis, gene expression, and epigenetic analysis. After all data are analyzed, and true genetic mutations are identified, it is hoped that that they will identify those genetic modifications that are pivotal to HCC progression so they can prevent, stop the growth, or shrink the dis- ease. Therapies can then be targeted to patients with specific genomic characteristics.4

Proteomics

Proteomics is the large-scale global analysis of proteins.

While genomics seeks to sequence the genes and transcrip- tomics seeks to understand the expression of all of genes, proteomics seeks to identify all of the proteins of the pro- teome. The general platform for proteomic research for the identification of novel biomarkers of disease involves the fol- lowing. Proteins are extracted from biological samples and a

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matched control. The proteins are enzymatically digested into peptides; the peptides are ionized and then introduced into the mass spectrometer. Mass spectrometry (MS) yields the mass of the peptide whereas tandem mass spectrometry

(MS/MS) yields fragment masses for sequencing. The pro- teins need to be digested because the majority of the MS instruments cannot yield sequence data on whole proteins.

Once the MS and MS/MS data are obtained, the peptide is identified by comparing its parent mass and sequence against a database, such as in silico virtual digest of all of the proteins in the proteome. The virtual fragment peptides from those virtual peptides that match the mass of the parent peptide within a certain tolerance are compared against the real frag- ment ions obtained from the MS/MS stage. From this com- parison, a statistical probability is generated which reflects the likelihood that the virtual and real peptides are the same.

One of the difficulties with proteomics is the challenge of identifying all proteins in a given sample when some may be more abundant than others. Since the mass spectrometer can only perform MS/MS to obtain sequence information on one peptide at a time, the likelihood of sequencing a low abun- dant protein is very low. Therefore, the peptides need to be separated before they are introduced into the mass spectrom- eter. The most common way of doing this is with a high-per- formance liquid chromatography (HPLC) coupled directly to the mass spectrometer. This technique is commonly referred to as LC MS/MS. In some instances two-dimensional HPLC separation is coupled to electrospray ionization and mass spectroscopy (ESI-MS). Typically, the first dimension is per- formed “offline” in which the HPLC is not connected to the MS, and instead the eluting proteins or peptides are collected in fractions. Those collected fractions are then run individu- ally in the second dimension that is most commonly a reverse phase HPLC separation that is coupled directly to the mass spectrometer.The advantages of this technique are increased sampling depth, increased ability to detect low abundant pro- teins, and a higher yield of total protein identifications.5

Chapter 9.  Translational Research and New Approaches 141

Another technique of identifying the quantity of a protein in a given biological sample is the Luminex 100 xMAP (multi-analyte profiling) System (Austin, TX). This has been used to identify cytokine levels in human plasma when assessing the systemic inflammatory state in patients under- going lower extremity revascularization.6 This bead-based assay system is a flow cytometric analysis using novel fluores- cent beads that are covalently linked to antibodies specific for individual analytes. By coupling the specificity of anti- body-based capture of specific cytokines using chromophorelabeledantibodieswithflowcytometricanalyses,theanalytical system can multiplex the analysis of theoretically an unlim- ited number of cytokines simultaneously from a single sam- ple. The Luminex technology simultaneously identifies the quantity of a given analyte, as well as its identity. Similar tech- nology has become the gold standard for the detection and identification of deleterious alloantibodies of a kidney recipi- ent to a specific kidney donor.The specific proteomic analyti- cal technique that is chosen usually depends on the expertise of the collaborative team.

The introduction of peptide analysis by mass spectrometry in combination with bioinformatics for data processing has revolutionized the field of proteomics. There are multiple potential applications of proteomics to clinical medicine.

These techniques can allow for protein sequencing, relative protein quantification, posttranslational modifications (e.g., glycosylation or phosphorylation), protein–protein interac- tions, and the identification of biomarkers for a given disease.

Although the development of the proteomic field has lagged behind that of genomics, there are an increasing number of published studies demonstrating important clinical applica- tions of these techniques.

In a preliminary proteomic study, Tweedle et al.7 studied the levels of heat shock protein 27 (HSP27) in colorectal can- cer samples. The expression of HSP27 in a cohort of 404 patients with colorectal cancer with a predominantly poor prognosis was characterized. HSP27 levels in diagnostic rec- tal biopsies were compared with matched surgical samples to

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determine whether changes in expression occurred in the time between biopsy and surgery and to investigate whether preoperative radiation therapy affected expression. The authors found that HSP27 overexpression was strongly asso- ciated with poor-cancer-specific survival in rectal cancer but not in colon cancer in those with a poor prognosis. HSP27 levels remained unchanged in the majority of cases between diagnostic biopsies and matched surgical controls, regardless of whether patient had undergone preoperative radiotherapy.

They authors concluded that HSP27 is an independent marker of poor outcome in rectal cancer and its expression is not affected by neoadjuvant radiotherapy.This study can lead to future studies to evaluate whether HSP27 levels should be considered as a stratification factor for the treatment of rectal cancer.

In another translational research study Ren et al.8 performed a two-dimensional analysis of human hepatocellular carcinoma (HCC) line, HepG2, and an immortal hepatic cell line, LO2. They identified that phosphoglycerate mutase 1 (PGAM1) was markedly upregulated in the HepG2 line compared to controls.This finding led to determining the role of PGAM1 in patients with HCC. Immunohistochemistry (IHC) was performed on excised HCC specimens.Weak IHC staining for PGAM1 correlated with a 5-year patient survival of 55.6% compared to 18.2% in patients with tumors that demonstrated strong PGAM1 staining. In addition, shRNAsmediated repression of PGAM1 expression resulted in sig- nificant inhibition in liver cancer cell growth both in vitro and in vivo. Findings from this study that “translated” laboratory findings into the clinical setting may lead to novel therapies targeting PGAM1 in the setting of HCC.

Another application for the use of proteomics is in the identification of novel biomarkers in the setting of traumatic brain injury (TBI). TBI has received increased publicity recently due to its high incidence among disabled soldiers returning from both Iraq and Afghanistan. In addition, TBI has also impacted both amateur and professional athletics as physicians struggle to determine when players, who sustain significant concussions, can return to full contact. The ability