Intense basolateral membrane staining indicates HER2 positivity in invasive micropapillary breast carcinoma

UT-A1 is regulated by vasopressin and is localized to the apical membrane and intracellular compartment of inner medullary collecting duct (IMCD) cells. UT-A3 is also expressed in the IMCD and is regulated by forskolin in heterologous systems. The goal of the present study is to investigate mechanisms by which vasopressin regulates UT-A3 in rat IMCD. In fresh suspensions of rat IMCD, forskolin increases the phosphorylation of UT-A3, similar to UT-A1. Biotinylation studies indicate that UT-A3 is located in the plasma membrane. Forskolin treatment increases the abundance of UT-A3 in the plasma membrane similar to UT-A1. However, these two transporters do not form a complex through a protein-protein interaction, suggesting that transporter function is unique to each protein. While immunohistochemistry localized UT-A3 to the basal and lateral membranes, a majority of the staining was cytosolic. Immunohistochemistry of vasopressin-treated rat kidney sections also localized UT-A3 primarily to the cytosol with basal and lateral membrane staining but also showed some apical membrane staining in some IMCD cells. This suggests that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter may move from the cytosol to all plasma membranes to increase urea flux in the IMCD. In summary, this study confirms that UT-A3 is located in the inner medullary tip where it is expressed in the basolateral membrane, shows that UT-A3 is a phosphoprotein in rat IMCD that can be trafficked to the plasma membrane independent of UT-A1, and suggests that vasopressin may induce UT-A3 expression in the apical plasma membrane of IMCD.

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Immunohistochemical analysis of pathway HER2 4B5 performance in breast carcinoma expressing HER4.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.27_suppl.16 ◽

2012 ◽

Vol 30 (27_suppl) ◽

pp. 16-16

Author(s):

Julie I Jay ◽

Patrick S Brunhoeber ◽

Margie H Smith ◽

Ryan R Williams ◽

Michael Sugarman ◽

...

Keyword(s):

Breast Carcinoma ◽

Immunohistochemical Analysis ◽

Cross Reactivity ◽

Target Antigen ◽

Her2 Expression ◽

Medical Systems ◽

Hek 293 ◽

Membrane Staining ◽

Positive Score ◽

Her2 Antigen

16 Background: The ability of pharmacodiagnostic antibodies to specifically detect their target antigen is essential to their performance. Non-clinical protein based models for specificity of two human epidermal growth factor 2 (HER2) pharmacodiagnostic antibodies, Oracle CB11 and PATHWAY HER2 4B5 (4B5) (Ventana Medical Systems Inc., Tucson, AZ, USA), have been reported to demonstrate affinity to both HER2 and HER4. To date there has been no evidence showing that affinity of 4B5 for HER4 impacts the IHC clinical HER2 assay. We therefore sought to investigate whether there exists any risk of the 4B5 antibody detecting HER4-positive but HER2-negative cases as falsely positive due to a hypothetical cross reactivity to HER4. Methods: A HER4 IHC assay was developed by screening ten HER4 antibodies for specificity by staining FFPE blocks of recombinant HEK-293 cells expressing HER1, HER2, HER3 and HER4. Specific HER4 antibodies were then evaluated in breast carcinoma to identify a clone that provided distinct membrane staining in tumour cells. This pattern was used as a reference in order to detect any potential interference of HER4 detection in determining the HER2 score. The HER4 antibody E200 (Epitomics) was selected and used to evaluate a cohort of breast carcinoma cases. The HER2 expression in these cases was then assessed using anti-HER2 clone PATHWAY HER2 4B5 and a HER2 reference antibody, clone SP3 (Spring Bioscience). Results: 117 out of 241 (48%) breast tissue cases evaluated were HER4 positive for membrane staining by IHC using E200. The scoring criteria for determining the HER4 score was based on the system used to score HER2, however a positive score indicated any membrane staining and therefore a 1+, 2+ or 3+ score was considered HER4-positive. 4B5 exhibited similar staining patterns as SP3 in co-expressing HER4 and HER2 tissues indicating that 4B5 was detecting only the HER2 antigen. Eighteen cases were HER4-positive by E200 and level 0 by 4B5 including a HER4 level 3+ case. Conclusions: PATHWAY HER2 4B5, a clinically relevant pharmacodiagnostic antibody for detecting HER2 expression in breast carcinoma, does not demonstrate cross reactivity to HER4 in IHC of FFPE clinical tissues.

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