Cathy Eng M.D., Salil Sethi M.D., George J.Chang, M.D., Miguel A. Rodriguez-Bigas, M.D., John M. Skibber, M.D., Jianjun Shen Ph.D., Jijiang Zhu Ph.D., Aki Ohinata P.A., Stanley R. Hamilton M.D., Donghui Li, Ph.D. The University of Texas M.D. Anderson Cancer Center, Houston, Texas. Proteomic Analysis of Potential Tumor Markers in AJCC Stage II and III Colon Cancer.

20%-60% of stage II and stage III colon cancer patients may develop recurrent disease, 80% occurring within the first 3 years of diagnosis despite surgical resection. 1 Adjuvant oxaliplatin-based chemotherapy is approved for stage III colon cancer patients and has been determined to result in a 23% decreased risk of recurrence relative to 5- fluorouracil alone.2 Despite the use of adjuvant chemotherapy < 10% absolute benefit in overall survival has been established. 2,3 CEA is the only recommended tumor marker to detect disease recurrence during surveillance but is not elevated in approximately 20% of patients. High throughput techniques are widely available but are not yet universally validated in detecting disease development or recurrence in colorectal cancer. Background

Identify differentially expressed protein biomarkers in tumor tissue relative to adjacent normal tissue (control). Establish protein biomarkers to predict disease development or recurrence. Objectives

A retrospective study of tissue samples from AJCC high-risk stage II (T 3-4 N 0 M 0 ) or stage III (T X N 1-3 M 0 ) colon cancer patients who underwent surgical resection at M.D. Anderson from Medical records were reviewed for demographics and clinical information. Protein was extracted from a total of 55 frozen paired tumor and adjacent normal tissue. Individual samples were subjected to differential CyDye labeling and separated by differential gel electrophoresis. Differentially expressed protein spots were identified by image analysis using DeCyder 2-D gel image analysis software. Proteins were identified in the first 10 patients by in gel digestion and matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) profiling. Differentially expressed proteins were validated by Western blot in the same protein extract and by immunohistochemistry in a commercially available colon tumor tissue array ( Methods

A typical 2D gel image and quantification of intensity of each protein spot are shown in Figure 1. The intensity of 83 differentially expressed protein spots were analyzed by image analysis (Table 1); 23 proteins demonstrated a > 4-fold difference in expression when tumors were compared to adjacent normal tissues. 84 proteins were identified from 143 protein spots analyzed by MALDI-TOF (Table 2). These proteins include many metabolic enzymes, detoxification enzymes, signal transduction proteins, immuno-response or inflammatory response proteins, cytoskeleton proteins, or calcium-binding proteins. Three proteins, i.e. alpha-acid glycoprotein (alpha-AGP), EF-Tu (elongation factor Tu, mitochondrial precursor), and VDAC-2 (voltage-dependent anion-selective channel protein 2) had >4 fold difference in expression in the tumor tissue relative to control (Table 3). Protein expression of alpha-AGP, EF-Tu and VDAC-2 was validated by immunohistochemistry in a tissue array of 48 colon adenocarcinomas and was detected in 91%, 100%, and 90% of tumor tissue, respectively (Figure 2). Results

Typical 2-D Images Figure 1

Typical 2-D Images Figure 1 contd.

Summary of Image Analysis of 2-D gels Fold change (T/N) No. of spots with T/N ratio ≥ 4 Single spots Increased Decreased Multiple spots Increased Decreased At least one ↑ and one ↓ 22 Total 8323 Table 1

Identification of differentially expressed proteins Protein SpotsClassification Fold Change T/N Table 2

Identification of differentially expressed proteins Protein SpotsClassification Fold Change T/N Table 2 contd.

Identification of differentially expressed proteins Protein SpotsClassification Fold Change T/N Table 2 contd.

Identification of differentially expressed proteins Protein SpotsClassification Fold Change T/N Table 2 contd.

Identification of differentially expressed proteins Protein SpotsClassification Fold Change T/N Table 2 contd.

Identification of differentially expressed proteins Protein SpotsClassification Fold Change T/N Table 2 contd.

Identification of differentially expressed proteins Protein SpotsClassification Fold Change T/N Table 2 contd.

Highly differentially expressed proteins Name of the ProteinExpressiont-test result Elongation factor Tu (EF-TU) ↑ (8.44 fold)0.011 Voltage-dependent anion- selective channel protein 2 (VDAC2) ↑ (9.48 fold)0.034 Alpha-1-acid glycoprotein ( α -1-AGP) ↓ (4.16 fold) Table 3

Validation of EF-Tu by IHC EF-Tu expression in normal tissue (lower left) vs. colon cancer (upper right) EF-Tu expression in colon cancer (magnified 200x’s) Figure 2

Proteomics analysis in paired tumor and normal adjacent tissues has identified 3 novel colon tumor biomarkers, i.e. EF-Tu, VDAC2, and alpha-1-AGP. Alpha-1-AGP is an acute phase protein and a marker for inflammation and cancer. 4 Serum alpha-1-acid glycoprotein has been demonstrated to be an index of dissemination in breast cancer. Elongation factor is required for the elongation of polypeptide chain. EF-Tu over- expression, representing mitochondrial changes, has been previously detected in esophageal squamous cell carcinoma. 5 Voltage-dependent anion-selective channel protein 2 is an ion channel protein that plays a role in apoptosis.6 Over- expression of this protein has been detected in other human cancers. Identification of differential proteins in both tumor and adjacent normal tissue may provide further insight into colon cancer carcinogenesis as well as disease recurrence. The predictive value of these protein markers for tumor recurrence and a companion prospective analysis of AJCC stage II and stage III colon cancer patients is currently underway. Discussion

1.Sargent et al: JCO, Vol 23, No 34 (December 1), 2005: pp Andre et al: N Engl J Med Jun 3;350(23): Quasar Collaborative Group: Lancet Dec 15;370(9604): Croce et al: Histol Histolpathol, 2005; 20: Qi et al: Proteomics, 2005, 5: Simamura et al: J Bioenerg Biomembr 2008 Jun;40(3): References

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