- Email: [email protected]
22. Geminin: A prognostic marker in breast cancer
1140 HSP 70 proteins promotes breast cancer cell survival, yet the role of BAG1 in HER2+ cancer is not yet defined. Methods: Proliferation assays, Western blot analysis, and co-immunoprecipitation were performed in breast cancer cell lines. A library of compounds predicted to inhibit BAG-1 protein interactions was tested by invitro GST-BAG-1/HSC70 pull-down assay. Results: HER2-inhibiting drugs caused upregulation of BAG-1L expression in HER2+ cell lines. An increase in proliferation was observed in HER2+ cells overexpressing BAG-1L and BAG-1M compared to BAG-1S or pcDNA controls. siRNA knockdown of BAG-1 showed a decrease in proliferation. These effects were abrogated following treatment with trastuzumab. Thioflavin S inhibition of BAG-1 resulted in synergistic killing of HER2+ cells treated with trastuzumab. We have developed an antibody shown to be specific for BAG-1L by Western blot and co-immunoprecipitation assays. Conclusion: Ongoing work involves correlation of our findings with BAG-1L expression patterns divulged from immunohistochemical studies of whole sections and tissue microarrays using a previously described breast cancer cohort. Identification of compounds inhibiting BAG-1/ HSC70 protein interaction in HER2+ cells may lead to the development of novel anti-cancer therapies. 22. Geminin: A prognostic marker in breast cancer Sreekumar Sundara Rajan 1,2 , Andrew Hanby1,2 , Kieran Horgan 2 , Val Speirs1 1 University of Leeds, Leeds Institute of Molecular Medicine, Leeds, West Yorkshire, UK 2 Leeds Teaching Hospitals NHS Trust, Leeds, West Yorkshire, UK Introduction: Geminin is an endogenous inhibitor of DNA replication and has been shown to indicate the rate of cell cycle progression in sporadic breast cancer. The aim of our study was to evaluate the prognostic role of geminin expression in breast cancer patients. Methods: Breast cancer tissue microarrays (TMA) containing 291 tumours were stained using anti-geminin antibody (NCL-L; Mouse monoclonal; Novocastra). Slides were scanned using AperioScanScopeÔ and labelling index [LI] was calculated for geminin expression. ROC was used to determine the optimum cut-off value for geminin LI (>2%) and Kaplan-Meier curves were plotted to determine disease free survival (DFS) and breast cancer specific survival (BCSS). Results: Our results showed poor BCSS amongst patients with geminin positivity (56/186) in comparison to those who were negative (12/ 105) (Log Rank c2 ¼ 11.89; HR 2.85 (1.53, 5.32); Mean ¼ 146 months, SD 68.13). Similarly, DFS was worse in patients with geminin positivity (Log Rank c2 ¼11.47; p¼0.001; HR¼2.63 (1.47, 4.71)). The tumour size, nodal status, geminin LI (>2%), grade, mitotic index, ER and LVI were all significant prognostic factors predicting poor DFS and BCSS. However, on Cox multivariate regression analysis, only geminin LI (DFS, p¼0.000 (CI 1.67, 6.26); BCSS, p¼0.000 (CI 1.79, 7.53)), nodal status (N3 vs. N0, DFS, p¼0.000 (CI 2.38, 10.05); BCSS, p¼0.000 (CI 2.88, 12.6)) and tumour size (DFS, p¼.001(1.006, 1.026); BSCC, p¼0.003 (CI 1.006, 1.026)) were found to be independent predictors. Conclusion: Expression of geminin in breast cancer is a strong and independent predictor of adverse outcome. 23. MTSS1 expression in human colorectal cancer and its significance e A cohort study Afaq Siddiqu, Ye Lin, Rachael Hargest, Mahir Al-Rawi, Weng Jiang 1 University Hospital of Wales, Cardiff, UK 2 Glan Clwyd Hospital, Rhyl, UK Introduction: Colorectal cancer is the third most common cancer in the UK. Each year, 38,000 patients are diagnosed with colorectal cancer. Death among these patients is due to metastasis both locally and systemically. Recently, a new molecule has been reported to be a potential metastasis related gene, namely MTSS1. The gene was found to be reduced in
ABSTRACTS aggressive tumour cell lines. Clinical studies, though limited, have indicated that it may be a metastasis suppressing molecule. This study is to investigate the MTSS1 expression in normal and cancerous tissue and its correlation with patient outcome.
Table 1.1 Staging of cancer
Nodal metastasis Recurrence Distant metastasis
T2 T3 TNM1 TNM4 N0 N2 Non recurrence Recurrence No distant Distant metastasis
Mean
Standard error
P value
233 34.82 29.8 3.55 54.4 3.22 156 39.9 181 16.8
206 25.7 13.8 2.34 41.5 2.22 109 34.9 126 13.0
p¼0.86 P¼0.09
P¼0.33 P¼0.31 P¼0.20
Method: The expression of MTSS1 was examined in colorectal cancer cohort using quantitative gene transcript analysis and immunohistochemical method. Results: Analysis of MTSS1 expression levels in IHC colon cancer tumour tissue (n¼94) cohort was significantly reduced compared to non-tumour tissue (n¼80) (normal mean 131.3165+/-SD33.2086 vs. tumour mean 84.79012+/-SD28.98874 p<0.05). Further MTSS1 transcript levels were found to be reduced in advance stage of disease, nodal, distant metastasis and local recurrence. (Table1.1) These results are not of statistical significance because of the small numbers in our cohort. Kaplan eMeier survival analysis showed patients with higher expression levels of MTSS1 had longer disease-free survival (p<0.05). Discussion: In our cohort, low level of MTSS1 expression was found to be reduced in colorectal tumour samples, in aggressive tumours, nodal metastasis, recurrence and distant metastasis. The current data suggest MTSS1 may be a putative tumour suppressor gene involved in metastasis of the colon cancer. 24. KRAS and BRAF mutations are rare in anal squamous cell carcinoma, justifying the use of EGFR targeted therapies Paul Ziprin, Kathleen Elliott, Temi Lampejo, Ben Poskitt, Robert Goldin, Susan Cleator, Michael Osborn Imperial College, London, UK Introduction: The incidence of anal squamous cell carcinoma (SCC) has increased markedly and it continues to have a poor prognosis. 36% of patients with loco-regional disease experience a recurrence, while only 10% of patients with metastases survive for 2 years or more; thus new treatment approaches are required. Assessment of the frequency of KRAS and BRAF mutation in anal SCC is essential when assessing the potential therapeutic value of epidermal growth factor receptor (EGFR) inhibition with monoclonals. Methods: Patients treated for anal cancer at a regional centre were identified from an anal cancer database. Analysis of KRAS and BRAF mutation status was performed using pyrosequencing on formalin-fixed, paraffin-embedded clinical material. Codons 12, 13 and 61 on the KRAS gene and 599, 600 and 601 on the BRAF gene were analysed. Results: Twenty-seven samples were analysed. Analysis for KRAS failed for codon 61 in one sample and for codons 12/13 in a second sample. In 1/26 (3.8%) a N/c.34G>A KRAS mutation was detected on sequencing and a small amount of mutant product detected on conventional PCR methods confirmed the mutation. 4/27 (15.4%) samples were found to have BRAF mutations, all V600E. Conclusion: KRAS and BRAF mutations are uncommon in anal SCC, justifying the potential use of anti-EGFR receptor therapy. Such a treatment approach requires assessment in clinical trials. KRAS assessment is not routinely undertaken prior to treatment of head and neck SSC with antiEGFR monoclonal antibodies and we have not demonstrated a definite need in the context of anal SCC.
ABSTRACTS aggressive tumour cell lines. Clinical studies, though limited, have indicated that it may be a metastasis suppressing molecule. This study is to investigate the MTSS1 expression in normal and cancerous tissue and its correlation with patient outcome.
Table 1.1 Staging of cancer
Nodal metastasis Recurrence Distant metastasis
T2 T3 TNM1 TNM4 N0 N2 Non recurrence Recurrence No distant Distant metastasis
Mean
Standard error
P value
233 34.82 29.8 3.55 54.4 3.22 156 39.9 181 16.8
206 25.7 13.8 2.34 41.5 2.22 109 34.9 126 13.0
p¼0.86 P¼0.09
P¼0.33 P¼0.31 P¼0.20
Method: The expression of MTSS1 was examined in colorectal cancer cohort using quantitative gene transcript analysis and immunohistochemical method. Results: Analysis of MTSS1 expression levels in IHC colon cancer tumour tissue (n¼94) cohort was significantly reduced compared to non-tumour tissue (n¼80) (normal mean 131.3165+/-SD33.2086 vs. tumour mean 84.79012+/-SD28.98874 p<0.05). Further MTSS1 transcript levels were found to be reduced in advance stage of disease, nodal, distant metastasis and local recurrence. (Table1.1) These results are not of statistical significance because of the small numbers in our cohort. Kaplan eMeier survival analysis showed patients with higher expression levels of MTSS1 had longer disease-free survival (p<0.05). Discussion: In our cohort, low level of MTSS1 expression was found to be reduced in colorectal tumour samples, in aggressive tumours, nodal metastasis, recurrence and distant metastasis. The current data suggest MTSS1 may be a putative tumour suppressor gene involved in metastasis of the colon cancer. 24. KRAS and BRAF mutations are rare in anal squamous cell carcinoma, justifying the use of EGFR targeted therapies Paul Ziprin, Kathleen Elliott, Temi Lampejo, Ben Poskitt, Robert Goldin, Susan Cleator, Michael Osborn Imperial College, London, UK Introduction: The incidence of anal squamous cell carcinoma (SCC) has increased markedly and it continues to have a poor prognosis. 36% of patients with loco-regional disease experience a recurrence, while only 10% of patients with metastases survive for 2 years or more; thus new treatment approaches are required. Assessment of the frequency of KRAS and BRAF mutation in anal SCC is essential when assessing the potential therapeutic value of epidermal growth factor receptor (EGFR) inhibition with monoclonals. Methods: Patients treated for anal cancer at a regional centre were identified from an anal cancer database. Analysis of KRAS and BRAF mutation status was performed using pyrosequencing on formalin-fixed, paraffin-embedded clinical material. Codons 12, 13 and 61 on the KRAS gene and 599, 600 and 601 on the BRAF gene were analysed. Results: Twenty-seven samples were analysed. Analysis for KRAS failed for codon 61 in one sample and for codons 12/13 in a second sample. In 1/26 (3.8%) a N/c.34G>A KRAS mutation was detected on sequencing and a small amount of mutant product detected on conventional PCR methods confirmed the mutation. 4/27 (15.4%) samples were found to have BRAF mutations, all V600E. Conclusion: KRAS and BRAF mutations are uncommon in anal SCC, justifying the potential use of anti-EGFR receptor therapy. Such a treatment approach requires assessment in clinical trials. KRAS assessment is not routinely undertaken prior to treatment of head and neck SSC with antiEGFR monoclonal antibodies and we have not demonstrated a definite need in the context of anal SCC.