Cancer-immune interactions in ER-positive breast cancers: PI3K pathway alterations and tumor-infiltrating lymphocytes.

INTRODUCTION: The presence of tumor-infiltrating lymphocytes (TILs) is correlated with good prognosis and outcome after (immuno)therapy in triple-negative and HER2-positive breast cancer. However, the role of TILs in luminal breast cancer is less clear. Emerging evidence has now demonstrated that genetic aberrations in malignant cells influence the immune landscape of tumors. Phosphatidylinositol 3-kinase (PI3K) is the most common altered pathway in ER-positive breast cancer. It is unknown whether changes in the PI3K pathway result in a different composition of the breast tumor microenvironment. Here we present the retrospective analysis of a prospective randomized trial in ER-positive breast cancer on the prognostic and predictive value of specific tumor-associated lymphocytes in the context of PI3K alterations. METHODS: We included 563 ER-positive tumors from a multicenter trial for stage I to III postmenopausal breast cancer patients, who were randomized to tamoxifen or no adjuvant therapy. The amount of CD8-, CD4-, and FOXP3-positive cells was evaluated by immunohistochemistry and quantified by imaging-analysis software. We analyzed the associations between PIK3CA hotspot mutations, PTEN expression, phosphorylated proteins of the PI3K and MAPK pathway (p-AKT, p-ERK1/2, p-4EBP1, p-p70S6K), and recurrence-free interval after adjuvant tamoxifen or no adjuvant treatment. RESULTS: CD8-positive lymphocytes were significantly more abundant in PIK3CA-mutated tumors (OR = 1.65; 95% CI 1.03-2.68). While CD4 and FOXP3 were not significantly associated with prognosis, patients with tumors classified as CD8-high had increased risk of recurrence (HR = 1.98; 95% CI 1.14-3.41; multivariable model including PIK3CA status, treatment arm, and other standard clinicopathological variables). Lymphocytes were more often present in tumors with increased PI3K downstream phosphorylation. This was most pronounced for FOXP3-positive cells. CONCLUSION: These exploratory analyses of a prospective trial in luminal breast cancer suggest high CD8 infiltration is associated with unfavorable outcome and that PI3K pathway alterations might be associated with the composition of the tumor microenvironment.

Mechanical forces used for cell fractionation can create hybrid membrane vesicles.

The ability to understand the inner works of the cell requires methods for separation of intracellular membrane-enclosed compartments. Disruption of the plasma membrane (PM) by mechanical forces to investigate the content of the cell is common practice. Whether vesicles or membranes of different sources can fuse as a result is unclear. If such contamination occurs, conclusions based on these techniques should consider these. Utilizing an endoplasmic reticulum (ER) membrane marker and a PM marker, we were able to detect the source of membranes following the breakup of cells using flow cytometry and immuno Electron Microscopy (immuno EM). Fractionation processes produced a small fraction of new membrane entities from two distinctively different origins generated during the initial disruption steps in a temperature independent manner, stressing that defining organelles or intrinsic fusion events based on such procedures and markers are valid when exceeding the small number of vesicles fused during the fractionation process.

Targeting the function of IFN-gamma-inducible protein 10 suppresses ongoing adjuvant arthritis.

IFN-gamma-inducible protein 10 (IP-10) is a CXC chemokine that is thought to manifest a proinflammatory role because it stimulates the directional migration of activated T cells, particularly Th1 cells. It is an open question whether this chemokine is also directly involved in T cell polarization. We show here that during the course of adjuvant-induced arthritis the immune system mounts a notable Ab titer against self-IP-10. Upon the administration of naked DNA encoding IP-10, this titer rapidly accelerates to provide protective immunity. Self-specific Ab to IP-10 developed in protected animals, as well as neutralizing Ab to IP-10 that we have generated in rabbits, could inhibit leukocyte migration, alter the in vivo and in vitro Th1/Th2 balance toward low IFN-gamma, low TNF-alpha, high IL-4-producing T cells, and adoptively transfer disease suppression. This not only demonstrates the pivotal role of this chemokine in T cell polarization during experimentally induced arthritis but also suggests a practical way to interfere in the regulation of disease to provide protective immunity. From the basic science perspective, this study challenges the paradigm of in vivo redundancy. After all, we did not neutralize the activity of other chemokines that bind CXCR3 (i.e., macrophage-induced gene and IFN-inducible T cell alpha chemoattractant) and yet significantly blocked not only adjuvant-induced arthritis but also the in vivo competence to mount delayed-type hypersensitivity.