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1.  Can we accurately report PTEN status in advanced colorectal cancer? 
BMC Cancer  2014;14:128.
Loss of phosphatase and tensin homologue (PTEN) function evaluated by loss of PTEN protein expression on immunohistochemistry (IHC) has been reported as both prognostic in metastatic colorectal cancer and predictive of response to anti-EGFR monoclonal antibodies although results remain uncertain. Difficulties in the methodological assessment of PTEN are likely to be a major contributor to recent conflicting results.
We assessed loss of PTEN function in 51 colorectal cancer specimens using Taqman® copy number variation (CNV) and IHC. Two blinded pathologists performed independent IHC assessment on each specimen and inter-observer variability of IHC assessment and concordance of IHC versus Taqman® CNV was assessed.
Concordance between pathologists (PTEN loss vs no loss) on IHC assessment was 37/51 (73%). In specimens with concordant IHC assessment, concordance between IHC and Taqman® copy number in PTEN loss assessment was 25/37 (68%).
Assessment PTEN loss in colorectal cancer is limited by the inter-observer variability of IHC, and discordance of CNV with loss of protein expression. An understanding of the genetic mechanisms of PTEN loss and implementation of improved and standardized methodologies of PTEN assessment are required to clarify the role of PTEN as a biomarker in colorectal cancer.
PMCID: PMC3941793  PMID: 24564252
PTEN; Colorectal; Immunohistochemistry; Copy number; Mutation
2.  KRAS G13D Mutation and Sensitivity to Cetuximab or Panitumumab in a Colorectal Cancer Cell Line Model 
The treatment of metastatic colorectal cancer (mCRC) includes drugs targeting the epidermal growth factor receptor (EGFR). Mutation in codon 12 or 13 in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene, downstream of the EGFR, evokes constitutive activation of the RAS/RAF/MAPK signaling pathway and correlates with resistance to anti-EGFR monoclonal antibody (mAb) therapies. However, a retrospective study reported that a proportion of patients with the KRAS G13D mutation may respond to cetuximab. A similar analysis for panitumumab was not as conclusive. We sought to determine the sensitivity of CRC cell lines to cetuximab or panitumumab treatment and to investigate the correlation of the KRAS mutational status of the CRC cell lines to the responsiveness to cetuximab or panitumumab.
To determine the responsiveness of CRC cell lines to cetuximab or panitumumab, cell lines were treated with an optimized concentration of each mAb, and proliferation assays were conducted.
After treatment with cetuximab or panitumumab, at the optimum concentration of 8 μg/well, the KRAS G13D mutant cell lines HCT-116, LoVo, and T84 showed intermediate sensitivity to both treatments, between the resistant KRAS G12V mutant cell line SW480 and the sensitive KRAS wild-type cell line LIM1215. One of the G13D cell lines was significantly more sensitive to panitumumab than to cetuximab (P = .02).
The specific KRAS mutation determines the responsiveness to anti-EGFR monoclonal antibody treatment, corresponding to reported clinical observations.
PMCID: PMC3930148  PMID: 24558511
3.  Prognostic impact and the relevance of PTEN copy number alterations in patients with advanced colorectal cancer (CRC) receiving bevacizumab* 
Cancer Medicine  2013;2(3):277-285.
Loss of phosphatase and tensin homologue (PTEN) expression may be prognostic in colorectal cancer (CRC) and may have a correlation with vascular endothelial growth factor (VEGF) expression via hypoxia-inducible factor 1 (HIF-1) alpha, and the PI3K/mTOR pathways. We therefore have explored the prognostic association of PTEN loss and the potential that PTEN loss may be predictive of outcome with bevacizumab. Patients enrolled in the AGITG MAX trial, a randomized Phase III trial of capecitabine (C) +/− bevacizumab (B) (+/− mitomycin C [M]) with available tissues were analyzed for PTEN expression (loss vs. no loss) as assessed using a Taqman® copy number assay (CNA). Of the original 471 patients enrolled, tissues from 302 (64.1%) patients were analyzed. PTEN loss was observed in 38.7% of patients. There was no relationship between PTEN loss and KRAS or BRAF mutation. PTEN status was not prognostic for progression-free survival (PFS) or overall survival (OS) in multivariate analyses adjusting for other baseline factors; loss versus no loss PFS hazard ratio (HR) 0.9 (0.7–1.16), OS HR 1.04 (0.79–1.38). PTEN was not prognostic when assessed by KRAS and BRAF status. By using the comparison of C versus CB+CBM, PTEN status was not significantly predictive of the effectiveness of B for PFS or OS. PTEN status was not prognostic for survival in advanced colorectal cancer, irrespective of KRAS or BRAF status. PTEN status did not significantly predict different benefit with bevacizumb therapy.
PMCID: PMC3699839  PMID: 23930204
Bevacizumab; colorectal; KRAS; prognosis; PTEN; VEGF
4.  Desmin expression in colorectal cancer stroma correlates with advanced stage disease and marks angiogenic microvessels 
Clinical proteomics  2011;8(1):16.
Biomarkers that improve stratification of colorectal cancer patients for adjuvant therapy versus resection alone, or that are predictive of response to therapeutic agents, have the potential to greatly improve patient selection for such therapies. The aim was to determine proteins differentially expressed within the malignant epithelial glands and closely associated stromal elements compared to matched normal mucosa, and to characterise the over-expression of one such protein as a potential biomarker.
Protein from laser microdissected tumor and normal mucosa was analysed by two dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry to determine differentially over expressed tumor proteins. Tumor over-expression of one such protein, desmin, was quantified using immunofluorescence staining in a larger cohort. Dual staining for desmin and vimentin, or desmin and von Willebrand factor, was performed to determine the cell type of interest.
Desmin expression was significantly increased between stage I and III tumors, (P < 0.0001), and stage II and III tumors, (P < 0.0001). Strong focal desmin expression was found in stroma directly adjacent to carcinomatous glands and microvessels. These cells showed co-localisation of desmin and vimentin in close association with cells expressing VWF, indicating they were pericytes. Significantly higher levels of desmin-positive pericytes were observed in late stage tumors, consistent with increased angiogenesis.
Pericyte coverage of vasculature is a marker of vessel maturation, hence desmin expression may have use as a marker for microvessel maturation. Clinical trials will be needed to determine its use in identifying tumors that will be less responsive to anti-angiogenic therapy.
PMCID: PMC3259060  PMID: 22141345
Colorectal cancer; 2D DIGE; desmin; biomarker; pericyte; angiogenesis
5.  Receptor protein tyrosine kinase EphB4 is up-regulated in colon cancer 
We have used commercially available cDNA arrays to identify EphB4 as a gene that is up-regulated in colon cancer tissue when compared with matched normal tissue from the same patient.
Quantitative RT-PCR analysis of the expression of the EphB4 gene has shown that its expression is increased in 82% of tumour samples when compared with the matched normal tissue from the same patient. Using immunohistochemistry and Western analysis techniques with an EphB4-specific antibody, we also show that this receptor is expressed in the epithelial cells of the tumour tissue and either not at all, or in only low levels, in the normal tissue.
The results presented here supports the emerging idea that Eph receptors play a role in tumour formation and suggests that further elucidation of this signalling pathway may identify useful targets for cancer treatment therapies.
PMCID: PMC64642  PMID: 11801186

Results 1-5 (5)