CELL SUSPENSION FROM ADJACENT NORMAL BREAST TISSUE TGFalpha erb B2 erb B2 hoechst 10 10 1 10 2 10 3 10 4 10 10 1 10 2 10 3 10 4 10 10 1 10 2 10 3 10 4 10 10 1 10 2 10 3 10 4 cytokeratin anti-cytokeratin -> cytokeratin anti-cytokeratin -> TGFalpha anti-TGF alpha -> cytokeratin anti-cytokeratin -> RB121324 RB121324 RB121324 RB121324 CK+TGF+erb- CK-TGF+erb- ALL CELLS It is well established that normal hematopoietic cells exhibit stable differentiation antigen expression, but malignant cells exhibit aberrant antigen expression. This figure shows the expression of cytokeratin, erb B2 and TGF ligand in normal breast epithelium (cytokeratin positive) adjacent to a lesion. Needle biopsies are obtained approximately 2 cm from the lesion to provide tissue for evaluating the immunophenotypic pattern of non malignant breast tissue. Biopsy of normal epithelium from 37 patients show this same profile. The average erb B2 expression was 1% (range 0 - 8%). In contrast, TGF expression was greater than 10% in 13/38 specimens. The average percentage of positive cells was 9% (range 0 - 60%). When stromal cells and leukocytes were evaluated (cytokeratin negative), none were found expressing erb B2, but 27/37 patients had >10% TGF+ cells with a mean of 52% (range 0 - 100%). Thus, marker expression is stable in normal breast tissue even if it is adjacent to a malignant tumor. The DNA content can be measured for each population. 256 512 768 1024 256 512 768 1024 256 512 768 1024 hoechst -> DNA hoechst -> DNA hoechst -> DNA RB121324 RB121324 RB121324

TGFalpha erb B2 erb B2 hoechst cytokeratin cytokeratin TGFalpha Like leukemic cells, malignant breast epithelium exhibits aberrant antigen expression. Furthermore, distinct subsets of malignant cells can be resolved and their DNA distribution obtained. A complex malignant tumor exhibition four distinct aneuploid subpopulations is shown. Subpopulations resolved are erb+TGF- (yellow), erb+TGF+ (green) erb-TGF+ (red) and erb-TGF- (pink). The malignant populaion with the highest S phase fraction is ertb+TGF+. The DNA distribution of the normal diploid cells (CK-, cyan) resolved by marker pattern can be also obtained; note the high S phase fraction of the normal stromal cells within the tumor.

TGFalpha erb B2 erb B2 hoechst cytokeratin cytokeratin TGFalpha Not all breast tumors look alike in their aberrant antigen expression. In this example, there are no cells expressing erb B2 and a significant fraction are also TGF negative (red). This tumor exhibits heterogeneous expression of cytokeratin so that diploid stromal cells (cyan) cannot be resolved from CK+/-TGF+ (green) tumor cells.