Background

1. Proteomic Analysis of Potential Tumor Markers in AJCC Stage II and III Colon Cancer. 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.

2. Background 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.

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

4. Methods • A retrospective study of tissue samples from AJCC high-risk stage II (T3-4N0M0) or stage III (TXN1-3M0) colon cancer patients who underwent surgical resection at M.D. Anderson from 1999-2005. • 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 (www.thermo.com/labvision).

5. Results • 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).

6. Typical 2-D Images Figure 1

7. Typical 2-D Images Figure 1 contd.

8. Summary of Image Analysis of 2-D gels Table 1

9. Identification of differentially expressed proteins Table 2

10. Identification of differentially expressed proteins Table 2 contd.

11. Identification of differentially expressed proteins Table 2 contd.

12. Identification of differentially expressed proteins Table 2 contd.

13. Identification of differentially expressed proteins Table 2 contd.

14. Identification of differentially expressed proteins Table 2 contd.

15. Identification of differentially expressed proteins Table 2 contd.

16. Highly differentially expressed proteins Table 3

17. 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

18. Discussion • 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.

19. References • Sargent et al: JCO, Vol 23, No 34 (December 1), 2005: pp. 8664-8670 • Andre et al: N Engl J Med. 2004 Jun 3;350(23):2343-51 • Quasar Collaborative Group: Lancet. 2007 Dec 15;370(9604):2020-9 • Croce et al: Histol Histolpathol, 2005; 20: 91-97 • Qi et al: Proteomics, 2005, 5: 2960-2971. • Simamura et al: J Bioenerg Biomembr 2008 Jun;40(3):213-7.

20. Making Cancer History COLON CANCER THANK YOU