“Shedding” light on HER4 signaling in normal and malignant breast tissues

Receptor Tyrosine Kinases of the Epidermal Growth Factor Receptor Family play a pivotal role as drivers of carcinogenesis and uncontrolled cell growth for a variety of malignancies, not least for breast cancer. Besides the estrogen receptor, the HER2 receptor was and still is a representative marker for advanced taxonomic sub-differentiation of breast cancer and emerged as one of the first therapeutic targets for antibody based therapies. Since the approval of trastuzumab for the therapy of HER2-positive breast cancer in 1998 anti-HER2 treatment strategies are being modified, refined, and successfully combined with complementary treatments, nevertheless there is still potential for improvement. The HER2 relatives, namely HER1 (i.e., EGFR), HER3 and HER4 share a high degree of molecular homology and together form a functional unit for signal transmission. Under regular conditions, receptor coexpression patterns and receptor interaction represent key parameters for signaling robustness, which ensures cellular growth control and enables tissue differentiation. In addition, treatment efficiency of e.g., an anti-HER2 targeting is substantially determined by the expression pattern of HER receptors on target cells. Within the receptor family, the HER4 plays a particular role and is engaged in exceptional signaling activities. A favorable prognostic impact has been attributed to HER4 expression in breast cancer under specific molecular conditions. HER4-specific cellular effects are initially determined by a ligand-dependent or -independent receptor activation. Essential processes as cell growth and proliferation, cell differentiation, and apoptotic cell death can be initiated by this receptor. This review gives an overview of the role of HER4 in normal and malignant breast epithelial cells and tissues. Specific mechanism of HER4 activation and subsequent intracellular signaling will be described by taking a focus on effects provoked by receptor shedding. HER4 activities and specific effects will be correlated to breast cancer subtypes and the impact of HER4 on course and outcome of disease will be considered. Moreover, current and potential therapeutic approaches will be discussed

Multiplextest zum Nachweis verschiedener Tumorantigene an einer Karzinomzelllinie durch zeitaufgelöste Einzelphotonenzählung

Tumorzellen tragen für sie charakteristische Antigene, sogenannte Tumorantigene, die sie von anderen Zellen unterscheiden und daher ein Merkmal für die Diagnostik darstellen. Für die Therapie spielen diese Tumorantigene zunehmend eine wichtige Rolle, weil sie eine spezifische Angriffsstelle für Antikörper bieten, die diese Zellen über eine Antikörper-Antigenkopplung markieren und somit für das Immunsystem erkennbar machen. Je detaillierter die Charakterisierung eines Tumors (Tumorheterogenität, Expressionsdichte der Tumorantigene) in Bezug auf solche Antigene z. B. membranständige Rezeptoren erfolgt, desto spezifischer könnten zukünftige Therapien auf den Patienten individuell abgestimmt werden. Für einen möglichst effizienten und exakten quantitativen Nachweis verschiedener Antigene an einem Tumorgewebeschnitt bietet die Methode der zeitaufgelösten Einzelphotonenzählung TCSPC (Time Correlated Single Photon Counting) in Kombination mit verschiedenen Farbstofflabels eine interessante Alternative zu herkömmlichen Verfahren. Diese Art des fluoreszenten Nachweises kombiniert die Vorteile des konfokalen Laserscanning-Mikroskops mit denen eines Multilabeltests. Für den Fluoreszenznachweis eines Antigens wird der dazu komplementäre Antikörper mit einem Farbstoff gekoppelt. Möchte man verschiedene Antigene an einem biologischen System gleichzeitig detektieren, so muss jeder spezifisch bindende Antikörper mit einem anderen Farbstoff konjugiert werden. Dieser kann sich in seiner Emissionswellenlänge von den anderen unterscheiden, der Fluoreszenznachweis erfolgt dann über geeignete optische Filtersysteme. Bei der TCSPC-Messung ist die charakteristische Messgröße die Fluoreszenzlebensdauer des am Antikörper gekoppelten Farbstoffs. Alle Farbstoffe werden mit derselben monochromatischen Lichtquelle zur Fluoreszenz angeregt und emittieren in dasselbe Wellenlängenfenster

Salivary gland mucoepidermoid carcinoma is a clinically, morphologically and genetically heterogeneous entity: a clinicopathological study of 40 cases with emphasis on grading, histological variants and presence of the t(11;19) translocation

International audienceAims: To correlate World Health Organization (WHO) grade, patient's outcome and presence of t(11;19) to histological tumor variants in 40 well characterized mucoepidermoid carcinomas (MECs) out of a series of 290 salivary gland carcinomas. Methods and Results: MECs were classified as classical based on the presence of equal proportions of the three cell types or the dominance (≥50%) of mucous cells beside at least one other cell type, and as variant if composed of ≥80% single cell type. Classical MECs were more common (n=23). Variant MECs had predominant squamoid (n=9), eosinophilic (n=5), or clear cell (n=3) morphology. 27 tumors were WHO grade 1, 3 grade 2 and 10 grade 3. The t(11;19) was detected in 82%, 35% and 0% of classical MEC, variant MEC and non-MEC, respectively. Classical MECs were significantly associated with age ≤60 years (p<0.001), grade 1 (p<0.001), and t(11;19) (p=0.003). Short overall survival was significantly associated with age >60 years (p=0.001) and UICC stage >I (p=0.031), residual tumor (p<0.001), tumor grade >1 (p=0.001) and squamoid variant (p=0.002) in Kaplan-Meier analysis. Conclusions: The results underscore the great histological diversity of MEC, the reproducibility of the WHO grading and the value of histological subtypes as an additional prognostic factor

IL-15 enhances the anti-tumor activity of trastuzumab against breast cancer cells but causes fatal side effects in humanized tumor mice (HTM)

Cancer immunotherapy has been shown to enhance established treatment regimens. We evaluated the potential reinforcing effect of IL-15 in trastuzumab treated humanized tumor mice (HTM) which were generated by concurrent transplantation of neonatal NOD-scid IL2R.null mice with human hematopoietic stem cells (HSC) and HER2 positive breast cancer cells (metastasizing SK-BR-3, solid tumor forming BT474). We found that trastuzumab treatment efficacy mainly depends on the immediate anti-tumorigenic cellular effect which is significantly enhanced by tumor interacting immune cells upon cotransplantion of HSC. However, trastuzumab treatment caused elevated CD44 expression on tumor cells that metastasized into the lung and liver but did not hinder tumor cell dissemination into the bone marrow. Moreover, in a number of SK-BR-3-transplanted animals disseminated CD44(high)/CD24(low) tumor cells lost trastuzumab sensitivity. Concerning the FcYRIIIa polymorphism, trastuzumab treatment efficiency in HTM was higher in mice with NK-cells harboring the high affinity FcYRIIIa compared to those with low affinity Fc.RIIIa. In contrast, IL-15 caused the strongest NK-cell activation in heterozygous low affinity Fc.RIIIa animals. Although IL-15 enhanced the trastuzumab mediated tumor defense, an unspecific immune stimulation resulted in preterm animal death due to systemic inflammation. Overall, treatment studies based on "patient-like" HTM revealed critical and adverse immune-related mechanisms which must be managed prior to clinical testing

CX3CL1 Overexpression Prevents the Formation of Lung Metastases in Trastuzumab-Treated MDA-MB-453-Based Humanized Tumor Mice (HTM)

CX3CL1 is a multifunctional chemokine that is involved in numerous biological processes, such as immune cell attraction and enhanced tumor immune cell interaction, but also in enhancing tumor cell proliferation and metastasis. The multifarious activity is partially determined by two CX3CL1 isoforms, a membrane-bound and a soluble version generated by proteolytic cleavage through proteases. Here, we investigated the impact of CX3CL1 overexpression in MDA-MB-453 and SK-BR-3 breast cancer cells. Moreover, we evaluated the therapeutic capacity of Matrix-Metalloproteinases-inhibitors TMI-1 and GI254023X in combination with the anti-HER2 antibody trastuzumab in vitro and in vivo. TMI-1 and GI254023X caused a reduced shedding of CX3CL1 and of HER2 in vitro but without effects on tumor cell proliferation or viability. In addition, trastuzumab treatment did not retard MDA-MB-453 cell expansion in vitro unless CX3CL1 was overexpressed upon transfection (MDA-MB-453CX3CL1). In humanized tumor mice, which show a coexistence of human tumor and human immune system, CX3CL1 overexpression resulted in a slightly enhanced tumor growth. However, trastuzumab treatment attenuated tumor growth of both MDA-MB-453CX3CL1 and empty vector transfected MDA-MB-453 transplanted mice but showed enhanced efficiency especially in preventing lung metastases in CX3CL1 overexpressing cancer cells. However, TMI-1 did not further enhance the trastuzumab treatment efficacy

The presence of PD-1 positive tumor infiltrating lymphocytes in triple negative breast cancers is associated with a favorable outcome of disease

Triple negative breast cancer patients have a poor course of disease not least because of limited treatment options however immunotherapy by targeting the PD-1/PD-L1 checkpoint system is a promising strategy to improve the outcome. Here we systematically investigated the expression of PD-1 on tumor infiltrating lymphocytes and PD-L1 on both tumor and infiltrated immune cells. Moreover, the PD-L1 gene status in tumor cells was assessed. 103 tissue microarray samples derived from triple negative breast cancer specimens were immunohistochemically stained against PD-1 and PD-L1. Dual marker fluorescence in-situ hybridization was applied to the PD-L1 gene and centromere region of chromosome 9. The disease free and overall survival rates were determined as a function of the PD-1/PD-L1 status. A slight gain of the PD-L1 gene region was found in 55% of all samples but an elevated PD-L1/cen9 ratio was rather rare (7%). An increased gene dose is not associated with an enhanced protein expression and the PD-L1 expression only weakly correlates with the amount of immune cell infiltration. Instead, we found an association of PD-L1 expression on tumor and immune cells, respectively. Notably, the PD-1 expression on immune cells is associated with a favorable disease free and overall survival. PD-1 expression indicates an enhanced immunological anti-tumor activity and represents a favorable prognostic impact. A deeper understanding of factors that affect the regulation and function of the PD-1/PD-L1 system is required to establish predictive variables and to utilize the system for therapeutic intervention of triple negative breast cancer patients

Immune Checkpoint Profiling in Humanized Breast Cancer Mice Revealed Cell-Specific LAG-3/PD-1/TIM-3 Co-Expression and Elevated PD-1/TIM-3 Secretion

Checkpoint blockade is particularly based on PD-1/PD-L1-inhibiting antibodies. However, an efficient immunological tumor defense can be blocked not only by PD-(L)1 but also by the presence of additional immune checkpoint molecules. Here, we investigated the co-expression of several immune checkpoint proteins and the soluble forms thereof (e.g., PD-1, TIM-3, LAG-3, PD-L1, PD-L2 and others) in humanized tumor mice (HTM) simultaneously harboring cell line-derived (JIMT-1, MDA-MB-231, MCF-7) or patient-derived breast cancer and a functional human immune system. We identified tumor-infiltrating T cells with a triple-positive PD-1, LAG-3 and TIM-3 phenotype. While PD-1 expression was increased in both the CD4 and CD8 T cells, TIM-3 was found to be upregulated particularly in the cytotoxic T cells in the MDA-MB-231-based HTM model. High levels of soluble TIM-3 and galectin-9 (a TIM-3 ligand) were detected in the serum. Surprisingly, soluble PD-L2, but only low levels of sPD-L1, were found in mice harboring PD-L1-positive tumors. Analysis of a dataset containing 3039 primary breast cancer samples on the R2 Genomics Analysis Platform revealed increased TIM-3, galectin-9 and LAG-3 expression, not only in triple-negative breast cancer but also in the HER2+ and hormone receptor-positive breast cancer subtypes. These data indicate that LAG-3 and TIM-3 represent additional key molecules within the breast cancer anti-immunity landscape