Loss of myoepithelial calponin-1 characterizes high-risk ductal carcinoma in situ cases, which are further stratified by T cell composition.

A hallmark of ductal carcinoma in situ (DCIS) progression is a loss of the surrounding ductal myoepithelium. However, whether compromise in myoepithelial differentiation, rather than overt cellular loss, can be used to predict the risk of DCIS progression is unknown. Here we address this question utilizing pure and mixed DCIS cases (N = 30) as surrogates for DCIS at low and high risk for progression, respectively. We used multiplex immunohistochemical staining to evaluate the relationship between myoepithelial cell differentiation and lymphoid immune cell types associated with poor prognostic DCIS. Our results show that myoepithelial calponin-1 discriminates between pure and mixed DCIS lesions better than histological subtype, presence of necrosis, or nuclear grade. Additionally, focal loss of myoepithelial cells associated with increased PD-1+CD8+ T cells, which suggests a link between the myoepithelium and immune surveillance. To identify associations between calponin-1 expression and immune response, we performed unsupervised hierarchical clustering of myoepithelial and immune cell biomarkers on 219 DCIS lesions from 30 cases. Notably, the majority of pure (low-risk) DCIS lesions clustered in a high calponin-1, T cell low group, whereas the majority of mixed (high-risk) DCIS lesions clustered in a low calponin-1, T cell high group, specifically with CD8+ and PD-1+CD8+ T cells. However, a subset of pure DCIS lesions had a similar calponin-1 and immune signature as the majority of mixed DCIS lesions, which have low calponin-1 and T cell enrichment-raising the possibility that these pure DCIS lesions might be at a high risk for progression.

A METHOD FOR QUANTIFICATION OF CALPONIN EXPRESSION IN MYOEPITHELIAL CELLS IN IMMUNOHISTOCHEMICAL IMAGES OF DUCTAL CARCINOMA IN SITU.

Ductal carcinoma in situ (DCIS) is breast cancer confined within mammary ducts, surrounded by an intact myoepithelial cell layer that prevents local invasion. A DCIS diagnosis confers increased lifetime risk of developing invasive breast cancer (IBC) and results in surgical excision with radiation, and possibly endocrine- or chemo-therapy. DCIS is known to be over treated, with associated co-morbidities. Biomarkers are needed that delineate patients at low risk of DCIS progression from patients requiring more aggressive treatment. Investigating the role of myoepithelial cell differentiation in barrier function is anticipated to provide insight into DCIS progression and delineate between low and high risk lesions. Here, we develop a high throughput technique to assess loss of myoepithelial differentiation markers. This method facilitates automated analysis of a clinically relevant histopathologic feature, as demonstrated by a high correlation with pathologist annotation (r = 0.959), and further, contributes analytical foundations to a multiplexed immunohistochemistry (IHC) approach.

Differential Roles for the Coagulation Factors XI and XII in Regulating the Physical Biology of Fibrin.

In the contact activation pathway of the coagulation, zymogen factor XII (FXII) is converted to FXIIa, which triggers activation of FXI leading to the activation of FIX and subsequent thrombin generation and fibrin formation. Feedback activation of FXI by thrombin has been shown to promote thrombin generation in a FXII-independent manner and FXIIa can bypass FXI to directly activate FX and prothrombin in the presence of highly negatively charged molecules, such as long-chain polyphosphates (LC polyP). We sought to determine whether activation of FXII or FXI differentially regulate the physical biology of fibrin formation. Fibrin formation was initiated with tissue factor, ellagic acid (EA), or LC polyP in the presence of inhibitors of FXI and FXII. Our data demonstrated that inhibition of FXI decreased the rate of fibrin formation and fiber network density, and increased the fibrin network strength and rate of fibrinolysis when gelation was initiated via the contact activation pathway with EA. FXII inhibition decreased the fibrin formation and fibrin density, and increased the fibrinolysis rate only when fibrin formation was initiated via the contact activation pathway with LC polyP. Overall, we demonstrate that inhibition of FXI and FXII distinctly alter the biophysical properties of fibrin.