cathy eng m.d., salil sethi m.d., george j.chang, m.d., miguel a. rodriguez-bigas, m.d., john m....
TRANSCRIPT
![Page 1: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/1.jpg)
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.
![Page 2: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/2.jpg)
• 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
![Page 3: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/3.jpg)
• Identify differentially expressed protein biomarkers in tumor tissue relative to adjacent normal tissue (control).
• Establish protein biomarkers to predict disease development or recurrence.
Objectives
![Page 4: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/4.jpg)
•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).
Methods
![Page 5: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/5.jpg)
•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
![Page 6: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/6.jpg)
Typical 2-D Images
Figure 1
![Page 7: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/7.jpg)
Typical 2-D Images
Figure 1 contd.
![Page 8: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/8.jpg)
Summary of Image Analysis of 2-D gels
Fold change (T/N)
No. of spots
No. of spotswith T/N ratio ≥ 4
Single spots
IncreasedDecreased
5811
141
Multiple spots
IncreasedDecreased
93
42
At least one ↑
and one ↓
2 2
Total 83 23
Table 1
![Page 9: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/9.jpg)
Identification of differentially expressed proteins
Protein Spots
Classification Fold Change T/N
Table 2
![Page 10: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/10.jpg)
Identification of differentially expressed proteins
Protein Spots
Classification Fold Change T/N
Table 2 contd.
![Page 11: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/11.jpg)
Identification of differentially expressed proteins
Protein Spots
Classification Fold Change T/N
Table 2 contd.
![Page 12: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/12.jpg)
Identification of differentially expressed proteins
Protein Spots
Classification Fold Change T/N
Table 2 contd.
![Page 13: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/13.jpg)
Identification of differentially expressed proteins
Protein Spots
Classification Fold Change T/N
Table 2 contd.
![Page 14: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/14.jpg)
Identification of differentially expressed proteins
Protein Spots
Classification Fold Change T/N
Table 2 contd.
![Page 15: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/15.jpg)
Identification of differentially expressed proteins
Protein Spots
Classification Fold Change T/N
Table 2 contd.
![Page 16: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/16.jpg)
Highly differentially expressed proteins
Name of the Protein Expression t-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)
0.0038
Table 3
![Page 17: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/17.jpg)
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
![Page 18: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/18.jpg)
• 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
![Page 19: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/19.jpg)
1. Sargent et al: JCO, Vol 23, No 34 (December 1), 2005: pp. 8664-8670
2. Andre et al: N Engl J Med. 2004 Jun 3;350(23):2343-51
3. Quasar Collaborative Group: Lancet. 2007 Dec 15;370(9604):2020-9
4. Croce et al: Histol Histolpathol, 2005; 20: 91-97
5. Qi et al: Proteomics, 2005, 5: 2960-2971.
6. Simamura et al: J Bioenerg Biomembr 2008 Jun;40(3):213-7.
References
![Page 20: 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.,](https://reader033.vdocuments.site/reader033/viewer/2022051315/56649e895503460f94b8eae4/html5/thumbnails/20.jpg)
THANK YOU
COLON CANCER