Significance analysis of microarrays (SAM) and gene ontology analysis
SAM analysis was performed to identify genes that were significantly different between Claudin-low tumors compared with the remaining samples[5]. Expression analysis systemic explorer (EASE) was used to identify gene ontology categories overrepresented in the Claudin-low gene list compared to the genes present on the array.
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissue sections (~5um) were processed using standard immunostaining methods. Following deparaffinization in xylenes, slides were rehydrated through a graded series of alcohol and rinsed in phosphate buffered saline. Endogenous peroxidase activity was blocked with 3% hydrogen peroxidase. Samples were steamed for antigen retrieval with 10 mM citrate buffer (pH 6.0) for 30 min. Slides were then incubated for 20 minutes with diluted normal blocking serum. The sections were incubated for 60 minutes at room temperature with primary antibody to claudin 3 (Zymed 18-7340 1:100) or e-cadherin (Cell Marque ECH-6 pre-diluted) . The slides were incubated for 45 minutes with
ABC reagent (Vector Laboratories). Sections were incubated in peroxidase substrate solution for visualization.. Slides were counterstained with hematoxylin and examined by light
microscopy. Tumor immunoreactivity was scored 0=negative, 1=weak positive, 2=moderate positive, and 3=strong positive.
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CHAPTER V
LOSS OF THE RETINOBLASTOMA TUMOR SUPPRESSOR IS A COMMON EVENT IN BASAL-LIKE AND LUMINAL B BREAST CARCINOMAS
PREFACE
This chapter represents a manuscript that is being prepared for submission. I
performed data analysis, scoring of IHC staining, development of the figures, and the writing of the manuscript. Xiaping He prepared DNA from tumors and normal patient lymphocytes, and IHC staining and scoring. Cheng Fan assisted with the data analysis. Charles Perou was the Principal Investigator, conceived and designed the study, and helped draft the paper.
Jason I Herschkowitz, Xiaping He, Cheng Fan, and Charles M. Perou.(2007) Loss of the
retinoblastoma tumor suppressor is a common event in Basal-like and Luminal B breast carcinomas. [in preparation]
SUMMARY
Breast cancers can be classified using whole genome expression into distinct subtypes that show differences in patient prognosis. One of these groups, the basal-like carcinomas, are poorly differentiated, highly metastatic, and genomically unstable. These tumors also contain specific genetic alterations with one example being frequent p53 mutations. The loss of the tumor suppressor gene encoded by the retinoblastoma (RB1) locus is a well-
characterized occurrence in many tumor types. However, its role in breast cancer is less clear with many reports demonstrating a Loss of Heterozygosity (LOH), but which does not correlate with loss of RB1 protein expression. Here we report that LOH of the RB1 locus was observed at a high frequency in basal-like and luminal B tumors. These tumors also concurrently have low expression of RB1 mRNA as assessed by DNA microarray. As in previous reports, we did not see a significant correlation between RB1 LOH and protein
expression as measured by immunohistochemistry (IHC). p16INK4a, however, was highly
expressed both by microarray and IHC, in basal-like tumors presumably due to a previously reported feedback loop caused by RB1 loss. These results suggest that the functional loss of RB1 is a common event in the progression of basal-like and luminal B breast tumors, which may play a key role in dictating therapeutic responses.