Evaluation of the Distribution of Stem Cell Markers in Human Breast Cancer Reveals Correlation with Clinical Progression and Metastatic Disease in Ductal Carcinoma.

Abstract

Abstract Background: Tumour stem cell theory suggests that a small percentage of cells in a tumour harbour intrinsic characteristics making them resistant to treatment. This could explain how patients with metastatic disease show clinical relapse several months after starting treatment due to the survival of a small group of cells with unique characteristics, including the ability to give rise to a new population of cells with resistant phenotype. We examined the distribution and expression of a panel of stem cell markers in human breast cancer primary tumours.Methods: Breast cancer primary tumours (n=114) and matched background tissue (n=30) were processed for frozen sections and RNA extraction. Frozen sections from matched tissues (tumour and background) were immuno-stained with PSCA, CD44, CD49b and CD133 antibodies and staining intensity analysed by computer image analysis. RNA was reverse transcribed and quantified before analysis by Q-PCR for the stem cell markers. Results were expressed as copy numbers of transcript/50 ng RNA (standardised by GAPDH/CK19).Results: Immunohistochemical assay revealed that there was a loss of expression of PSCA in tumour sections (149.85±24.89) compared to background tissue (222.85±9.88, n=20, p<0.0001). CD44 protein was expressed strongly in background endothelia and epithelia but was weaker and diffuse in tumour cells (134.5±29.89 and 187.9±21.28 respectively, p<0.0001). CD133 expression was lost in tumour-associated endothelial cells with diffuse and weak staining in tumours compared to background (194.9±35.76 and 222.2±20.46 respectively, p<0.006). Conversely, CD49b strongly stained in tumour and associated vessels and ducts but was weakly stained in background epithelia, albeit strongly staining background ducts and vessels (166.7±29.78 and 211.75±15.32 respectively, p<0.0001). Quantitative analysis of the gene transcripts further illuminated the expression of the stem cell markers: CD44 and PSCA were reduced in patients with poor outcome (metastatic disease and death from breast cancer), with a marked reduction in ductal carcinoma, particularly with metastasis to bone although these did not reach significant difference. However, CD49b and CD133 were significantly reduced in patients with metastatic disease (0.0001±0.0002 and 3.63±3.4 respectively) compared with those remaining alive and well after 10 year follow-up (31±15, p=0.03 and 553±261, p=0.039). CD133 was also significantly reduced in patients with ductal carcinoma/bone metastasis. Conversely, CD49F was increased in patients with a poor outcome and those with ductal cancer and bone metastases.Conclusion: This is the first study to determine the distribution and expression pattern of these stem cell markers in human breast cancer. We have shown that there is a significant association between loss of expression and metastatic disease in patients with breast cancer. Such differential expression may play a part in breast cancer disease progression, and suggests that the current stem cell theory may not hold true for all cancer types. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1157.