Surgical procedures and postsurgical tissue processing significantly affect expression of genes and EGFR-pathway proteins in colorectal cancer tissue.
Author(s): David KA, Unger FT, Uhlig P, Juhl H, Moore HM, Compton C, Nashan B, Dörner A, de Weerth A, Zornig C
Publication: Oncotarget, 2014, Vol. 5, Page 11017-28
PubMed ID: 25526028 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of warm ischemia, cold ischemia, and preservation method on the quantification of phosphorylated protein, immunohistochemical (IHC) staining, and gene expression profiles of colorectal cancer (CRC), liver metastases, and normal adjacent tissue.
Conclusion of Paper
Gene expression profiles were shown to change significantly with a warm ischemia time of 0 versus 10 min and an even greater number of genes showed significant expression level changes after 10-45 min of cold ischemia time. Normal adjacent colon and liver tissue specimens were less affected by cold ischemia time than their cancerous counterparts. The authors highlight changes in the epidermal growth factor receptor (EGFR) pathway gene expression in response to cold ischemia time. Phosphorylation levels of proteins in the protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) pathways tended to decrease in normal and tumor tissue specimens subjected to 10-45 min cold ischemia time compared pre-surgery biopsies. IHC staining for phosphorylated epidermal growth factor receptor (pEGFR), phosphorylated protein kinase B (pAKT), phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), and pAKT decreased in CRC specimens with increased cold ischemia time. On the other hand, heat shock protein 27 (HSP27) phosphorylation was found to increase with longer cold ischemia times.
Studies
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Study Purpose
The purpose of this study was to determine the effects of warm ischemia and cold ischemia on the quantification of phosphorylated protein, IHC staining, and gene expression profiles of CRC, liver metastases, and normal adjacent tissue. Colon specimens from 50 patients and liver specimens from 43 patients were collected for the study. 40 snap frozen specimens (vapor phase of liquid nitrogen) were used for the quantification of phosphorylated protein, while IHC analysis was performed on an unspecified number of formalin-fixed, paraffin-embedded (FFPE) specimens (fixed for 16-72 hours). RNA was extracted using phenol chloroform and the Qiagen RNeasy MinElute Cleanup Kit. Gene expression analysis was performed using snap frozen specimens with RNA integrity numbers >7 and GeneChip Human Genome U133 Plus 2.0 arrays.
Summary of Findings:
118 genes changed expression levels by more than 2-fold between normal adjacent liver specimens (colon and cancerous specimens were not analyzed) obtained with 0 (prior to hepatic pedicle clamping) and 10 min warm ischemia (10 minutes post-clamping). An average of 830 genes showed significant expression level changes between 10 and 45 min of cold ischemia time for metastatic liver CRC. For primary CRC, averages of 1,234 and 1,553 genes showed significant expression level changes after 10 and 45 min cold ischemia time, respectively compared to biopsies obtained with 0 min warm ischemia and assumed minimal cold ischemia time (details are not reported). Normal adjacent colon and liver tissue specimens were similarly less affected by 10-45 min cold ischemia time, with averages of less than 500 genes showing significant expression level changes compared to biopsies. The authors highlight changes in the EGFR pathway gene expression in response to cold ischemia time, with the most notable changes occurring in CRC tissue as opposed to normal adjacent liver or colon. While the total levels of most proteins within the AKT and MAPK pathways were not affected by cold ischemia time in normal liver and colon tissue, AKT, mechanistic target of rapamycin (MTOR), ERK1/2, and glycogen synthase kinase 3 beta (GSK3B) increased after 10 minutes of cold ischemia time compared to biopsies in tumor tissue. At the same time, phosphorylation levels of proteins in these pathways tended to decrease in normal and tumor tissue specimens subjected to 10-45 min cold ischemia time compared pre-surgery biopsies. IHC staining for pEGFR, pAKT, pERK1/2, and pAKT decreased in CRC specimens. On the other hand, HSP27 phosphorylation was found to increase with longer cold ischemia times. After 45 min cold ischemia, pHSP27 had increased 8-fold in cancer tissue and 2-fold in normal adjacent liver specimens.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Normal Adjacent
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein ELISA Protein Immunohistochemistry RNA Automated electrophoresis/Bioanalyzer RNA DNA microarray Protein Colorimetric assay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) ELISA Specific Targeted peptide/protein HIF1alpha
HSP27/pHSP27
EGFR/pEGFR
AKT/pAKT
MTOR/pMTOR
p70-S6K/pp70-S6K
GSK3B/pGSK3B
MEK1/2/pMEK1/2
ERK1/2/pERK1/2
Immunohistochemistry Specific Targeted peptide/protein pAkt
pERK1/2
pEGFR
pmTOR
pHer3
Biospecimen Acquisition Biospecimen location Colon
Liver
Preaquisition Diagnosis/ patient condition Primary CRC
Metastatic liver CRC
Normal adjacent colon
Normal adjacent liver
Preaquisition Warm ischemia time 0 min
10 min
Biospecimen Acquisition Cold ischemia time 0 min
10 min
20 min
45 min
Biospecimen Acquisition Method of tissue acquisition Biopsy
Surgical resection