Expression of the Hypoxia-Inducible and Tumor-Associated Carbonic Anhydrases in Ductal Carcinoma in Situ of the Breast Charles C. Wykoff, Nigel Beasley,

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Expression of the Hypoxia-Inducible and Tumor-Associated Carbonic Anhydrases in Ductal Carcinoma in Situ of the Breast Charles C. Wykoff, Nigel Beasley, Peter H. Watson, Leticia Campo, Stephen K. Chia, Ruth English, Jaromir Pastorek, William S. Sly, Peter Ratcliffe, Adrian L. Harris The American Journal of Pathology Volume 158, Issue 3, Pages 1011-1019 (March 2001) DOI: 10.1016/S0002-9440(10)64048-5 Copyright © 2001 American Society for Investigative Pathology Terms and Conditions

Figure 1 Immunoblotting for CA IX and CA XII. A: CA IX and CA XII expression detected by Western blot correlates with the respective immunostaining (IHC) score in invasive breast carcinomas. CA IX and CA XII are detected in tumor extracts that are strongly positive for CA IX or CA XII by IHC in adjacent paraffin sections (IHC score for each CA is indicated). B: CA IX and CA XII expression and response to hypoxia in two breast tumor cell lines exposed to either normoxia (N; 20% O2) or hypoxia (H; 0.1% O2) for 16 hours. The American Journal of Pathology 2001 158, 1011-1019DOI: (10.1016/S0002-9440(10)64048-5) Copyright © 2001 American Society for Investigative Pathology Terms and Conditions

Figure 2 CA IX and CA XII have different expression profiles in non-neoplastic and neoplastic breast tissues. Low levels of CA IX expression are detected in low-grade DCIS (A) and more prominently in high-grade DCIS associated with central necrosis (B). CA XII expression in normal breast lobules and ducts (C), in ductal hyperplasia (D), in high-grade DCIS with accentuation adjacent to central luminal necrosis (E), and at higher levels in low-grade DCIS (F). Original magnifications, ×10 (B–E) and ×20 (A andF). The American Journal of Pathology 2001 158, 1011-1019DOI: (10.1016/S0002-9440(10)64048-5) Copyright © 2001 American Society for Investigative Pathology Terms and Conditions

Figure 3 CA IX and CA XII expression are inversely related to grade and the presence of necrosis in DCIS. CA IX expression shown relative to grade (upper left) and necrosis (upper right) and CA XII expression shown relative to grade (lower left) and necrosis (lower right). Columns (CA IX, black; CA XII, hatched) represent the mean IHC score with bars showing SD relative to DCIS grade (low, intermediate, high) and necrosis (absent, present). The American Journal of Pathology 2001 158, 1011-1019DOI: (10.1016/S0002-9440(10)64048-5) Copyright © 2001 American Society for Investigative Pathology Terms and Conditions

Figure 4 CA IX and CA XII expression is unrelated to MIB1 expression. The pattern of expression of CA IX (A andB), CA XII (C andD), and MIB1 (E andF), was assessed in serial sections from intermediate-grade DCIS (left column) and high-grade DCIS (right column). CA IX expression is restricted to the inner zone of luminal epithelium adjacent to central necrosis. CA XII expression is present throughout the duct wall, but accentuated adjacent to necrosis in high-grade DCIS, and also in the adjacent portion of non-neoplastic ductal epithelium (C, upper left). In contrast, MIB1-positive nuclei are distributed throughout the duct in both intermediate- and high-grade DCIS, and are absent from normal epithelium (E, upper left). Arrows withinF indicate MIB1-positive nuclei. Original magnifications, ×20. The American Journal of Pathology 2001 158, 1011-1019DOI: (10.1016/S0002-9440(10)64048-5) Copyright © 2001 American Society for Investigative Pathology Terms and Conditions

Figure 5 MIB1 expression relative to CA IX or CA XII expression, DCIS grade, and the presence of necrosis. Columns (CA IX, black; CA XII, hatched; MIB1, clear) represent the mean MIB1 IHC score with bars showing SD relative to CA IX or CA XII expression (negative, positive), DCIS grade (low, intermediate, high), and necrosis (absent, present). The American Journal of Pathology 2001 158, 1011-1019DOI: (10.1016/S0002-9440(10)64048-5) Copyright © 2001 American Society for Investigative Pathology Terms and Conditions