Immunomodulatory Synthetic Glycocluster Molecule Prevents Melanoma Growth in vivo.

Triacedimannose (TADM) is a synthetic trivalent acetylated glycocluster and a transmembrane macrophage activator independent of the mannose receptor. TADM induces Th1-type immune responses and suppresses Th2-type cytokines in acute and chronic allergic inflammation models in vivo. We, therefore, wanted to test whether TADM could also facilitate anti-tumour tissue responses similar to what has been observed for the immune checkpoint inhibitors, such as anti-PD-1 and anti-CTLA-4. A syngeneic mouse melanoma model was selected since metastatic melanoma has been successfully targeted by checkpoint inhibitors in the clinic. TADM inhibited the growth of B16 mouse melanoma tumours at levels comparable to an anti-PD-1 antibody. TADM-treated tumours encompassed significantly more apoptotic cells as measured by TUNEL staining, and interferon-gamma (IFN-γ) expression was increased in the spleens of TADM-treated mice compared to untreated controls. TADM-treated mice also demonstrated increased Ly6C low monocytes and neutrophils in the spleens. However, TADM-treated tumours showed no discernible differences in infiltrating immune cells. TADM can alone suppress the growth of melanoma tumours. TADM likely activates M1 type macrophages, type N1 neutrophils, and CD8+ and Th1 T cells, suppressing the type 2 immune response milieu of melanoma tumour with a strong type 1 immune response.

Joint online distance learning to complement postgraduate pathology training in preparation for national board examinations.

AIMS: To meet the flexible learning needs of pathology residents preparing for national board examinations, a joint distance learning approach was developed using both asynchronous and synchronous activities with whole slide images, drawing on empirical educational research on online distance learning. METHODS: In a case study of an implementation of the designed joint distance learning approach with a geographically dispersed group of pathology residents in Finland, the participants' perceptions were measured with a 12-item questionnaire covering the value of the learning opportunity, the quality of the sociocognitive processes and their emotional engagement and social cohesion. Communication during the online session was also recorded and analysed to provide objectivity to the self-report data. RESULTS: The effectiveness of joint online learning for knowledge acquisition and preparation for national board examinations was highly rated. However, despite strong emotional engagement during synchronous activities, participants reported minimal interpersonal interaction, which was also reflected in the recordings of the online session. CONCLUSION: Using a technology integration framework and guided by the principles of self-determination theory, joint distance learning is emerging as a beneficial addition to postgraduate pathology programmes in preparation for national examinations. However, to realise the full potential of interpersonal interaction, participants should be prepared for an appropriate mindset.

Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

PURPOSE: Human epidermal growth factor receptor 2 (HER2)-targeted therapies, such as trastuzumab, benefit patients with HER2-positive metastatic breast cancer; however, owing to traditional pathway activation or alternative signaling, resistance persists. Given the crucial role of the formin family in shaping the actin cytoskeleton during cancer progression, these proteins may function downstream of the HER2 signaling pathway. Our aim was to uncover the potential correlations between formins and HER2 expression using a combination of public databases, immunohistochemistry, and functional in vitro assays. METHODS: Using online databases, we identified a negative prognostic correlation between specific formins mRNA expression in HER2-positive cancers. To validate these findings at the protein level, immunohistochemistry was performed on HER2 subtype breast cancer tumors to establish the links between staining patterns and clinical characteristics. We then knocked down individual or combined formins in MDA-MB-453 and SK-BR-3 cells and investigated their effects on wound healing, transwell migration, and proliferation. Furthermore, we investigated the effects of erb-b2 receptor tyrosine kinase 2 (ERBB2)/HER2 small interfering RNA (siRNA)-mediated knockdown on the PI3K/Akt and MEK/ERK1 pathways as well as on selected formins. RESULTS: Our results revealed that correlations between INF2, FHOD1, and DAAM1 mRNA expression and ERBB2 in HER2-subtype breast cancer were associated with worse outcomes. Using immunohistochemistry, we found that high FHOD1 protein expression was linked to higher histological grades and was negatively correlated with estrogen and progesterone receptor positivity. Upon formins knockdown, we observed effects on wound healing and transwell migration, with a minimal impact on proliferation, which was evident through single and combined knockdowns in both cell lines. Notably, siRNA-mediated knockdown of HER2 affected FHOD1 and INF2 expression, along with the phosphorylated Akt/MAPK states. CONCLUSION: Our study highlights the roles of FHOD1 and INF2 as downstream effectors of the HER2/Akt and HER2/MAPK pathways, suggesting that they are potential therapeutic targets in HER2-positive breast cancer.

ADAM17-mediated EGFR ligand shedding directs macrophage-promoted cancer cell invasion.

Macrophages in the tumor microenvironment have a substantial impact on tumor progression. Depending on the signaling environment in the tumor, macrophages can either support or constrain tumor progression. It is therefore of therapeutic interest to identify the tumor-derived factors that control macrophage education. With this aim, we correlated the expression of A Disintegrin and Metalloproteinase (ADAM) proteases, which are key mediators of cell-cell signaling, to the expression of protumorigenic macrophage markers in human cancer cohorts. We identified ADAM17, a sheddase upregulated in many cancer types, as a protein of interest. Depletion of ADAM17 in cancer cell lines reduced the expression of several protumorigenic markers in neighboring macrophages in vitro as well as in mouse models. Moreover, ADAM17-/- educated macrophages demonstrated a reduced ability to induce cancer cell invasion. Using mass spectrometry-based proteomics and ELISA, we identified heparin-binding EGF (HB-EGF) and amphiregulin, shed by ADAM17 in the cancer cells, as the implicated molecular mediators of macrophage education. Additionally, RNA-Seq and ELISA experiments revealed that ADAM17-dependent HB-EGF ligand release induced the expression and secretion of CXCL chemokines in macrophages, which in turn stimulated cancer cell invasion. In conclusion, we provide evidence that ADAM17 mediates a paracrine EGFR-ligand-chemokine feedback loop, whereby cancer cells hijack macrophages to promote tumor progression.

Overexpression of Human Estrogen Biosynthetic Enzyme Hydroxysteroid (17beta) Dehydrogenase Type 1 Induces Adenomyosis-like Phenotype in Transgenic Mice.

Hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) is an enzyme that converts estrone to estradiol, while adenomyosis is an estrogen-dependent disease with poorly understood pathophysiology. In the present study, we show that mice universally over-expressing human estrogen biosynthetic enzyme HSD17B1 (HSD17B1TG mice) present with adenomyosis phenotype, characterized by histological and molecular evaluation. The first adenomyotic changes with endometrial glands partially or fully infiltrated into the myometrium appeared at the age of 5.5 months in HSD17B1TG females and became more prominent with increasing age. Preceding the phenotype, increased myometrial smooth muscle actin positivity and increased amount of glandular myofibroblast cells were observed in HSD17B1TG uteri. This was accompanied by transcriptomic upregulation of inflammatory and estrogen signaling pathways. Further, the genes upregulated in the HSD17B1TG uterus were enriched with genes previously observed to be induced in the human adenomyotic uterus, including several genes of the NFKB pathway. A 6-week-long HSD17B1 inhibitor treatment reduced the occurrence of the adenomyotic changes by 5-fold, whereas no effect was observed in the vehicle-treated HSD17B1TG mice, suggesting that estrogen is the main upstream regulator of adenomyosis-induced uterine signaling pathways. HSD17B1 is considered as a promising drug target to inhibit estrogen-dependent growth of endometrial disorders. The present data indicate that HSD17B1 over-expression in TG mice results in adenomyotic changes reversed by HSD17B1 inhibitor treatment and HSD17B1 is, thus, a potential novel drug target for adenomyosis.

GIT1 protects against breast cancer growth through negative regulation of Notch.

Hyperactive Notch signalling is frequently observed in breast cancer and correlates with poor prognosis. However, relatively few mutations in the core Notch signalling pathway have been identified in breast cancer, suggesting that as yet unknown mechanisms increase Notch activity. Here we show that increased expression levels of GIT1 correlate with high relapse-free survival in oestrogen receptor-negative (ER(-)) breast cancer patients and that GIT1 mediates negative regulation of Notch. GIT1 knockdown in ER(-) breast tumour cells increased signalling downstream of Notch and activity of aldehyde dehydrogenase, a predictor of poor clinical outcome. GIT1 interacts with the Notch intracellular domain (ICD) and influences signalling by inhibiting the cytoplasm-to-nucleus transport of the Notch ICD. In xenograft experiments, overexpression of GIT1 in ER(-) cells prevented or reduced Notch-driven tumour formation. These results identify GIT1 as a modulator of Notch signalling and a guardian against breast cancer growth.

Tumor margins that lead to reoperation in breast cancer: A retrospective register study of 4,489 patients.

BACKGROUND AND OBJECTIVES: Optimal margins for ductal carcinoma in situ (DCIS) remain controversial in breast-conserving surgery (BCS) and mastectomy. We examine the association of positive margins, reoperations, DCIS and age. METHODS: A retrospective study of histopathological reports (4489 patients). Margin positivity was defined as ink on tumor for invasive carcinoma. For DCIS, we applied 2 mm anterior and side margin thresholds, and ink on tumor in the posterior margin. RESULTS: The incidence of positive side margins was 20% in BCS and 5% in mastectomies (p < 0.001). Of these patients, 68% and 14% underwent a reoperation (p < 0.001). After a positive side margin in BCS, the reoperation rates according to age groups were 74% (<49), 69% (50-64), 68% (65-79), and 42% (80+) (p = 0.013). Of BCS patients with invasive carcinoma in the side margin, 73% were reoperated on. A reoperation was performed in 70% of patients with a close (≤1 mm) DCIS side margin, compared to 43% with a wider (1.1-2 mm) margin (p = 0.002). The reoperation rates were 55% in invasive carcinoma with close DCIS, 66% in close extensive intraductal component (EIC), and 83% in close pure DCIS (p < 0.001). CONCLUSIONS: Individual assessment as opposed to rigid adherence to guidelines was used in the decision on reoperation.

Cargo-specific recruitment in clathrin- and dynamin-independent endocytosis.

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.

Stromal interaction molecule 1 (STIM1) knock down attenuates invasion and proliferation and enhances the expression of thyroid-specific proteins in human follicular thyroid cancer cells.

Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.

CIP2A Interacts with TopBP1 and Drives Basal-Like Breast Cancer Tumorigenesis.

Basal-like breast cancers (BLBC) are characterized by defects in homologous recombination (HR), deficient mitotic checkpoint, and high-proliferation activity. Here, we discover CIP2A as a candidate driver of BLBC. CIP2A was essential for DNA damage-induced initiation of mouse BLBC-like mammary tumors and for survival of HR-defective BLBC cells. CIP2A was dispensable for normal mammary gland development and for unperturbed mitosis, but selectively essential for mitotic progression of DNA damaged cells. A direct interaction between CIP2A and a DNA repair scaffold protein TopBP1 was identified, and CIP2A inhibition resulted in enhanced DNA damage-induced TopBP1 and RAD51 recruitment to chromatin in mammary epithelial cells. In addition to its role in tumor initiation, and survival of BRCA-deficient cells, CIP2A also drove proliferative MYC and E2F1 signaling in basal-like triple-negative breast cancer (BL-TNBC) cells. Clinically, high CIP2A expression was associated with poor patient prognosis in BL-TNBCs but not in other breast cancer subtypes. Small-molecule reactivators of PP2A (SMAP) inhibited CIP2A transcription, phenocopied the CIP2A-deficient DNA damage response (DDR), and inhibited growth of patient-derived BLBC xenograft. In summary, these results demonstrate that CIP2A directly interacts with TopBP1 and coordinates DNA damage-induced mitotic checkpoint and proliferation, thereby driving BLBC initiation and progression. SMAPs could serve as a surrogate therapeutic strategy to inhibit the oncogenic activity of CIP2A in BLBCs. SIGNIFICANCE: These results identify CIP2A as a nongenetic driver and therapeutic target in basal-like breast cancer that regulates DNA damage-induced G2-M checkpoint and proliferative signaling.

Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies.

Microscopy analysis of tumour samples is commonly performed on fixed, thinly sectioned and protein-labelled tissues. However, these examinations do not reveal the intricate three-dimensional structures of tumours, nor enable the detection of aberrant transcripts. Here, we report a method, which we name DIIFCO (for diagnosing in situ immunofluorescence-labelled cleared oncosamples), for the multimodal volumetric imaging of RNAs and proteins in intact tumour volumes and organoids. We used DIIFCO to spatially profile the expression of diverse coding RNAs and non-coding RNAs at the single-cell resolution in a variety of cancer tissues. Quantitative single-cell analysis revealed spatial niches of cancer stem-like cells, and showed that the niches were present at a higher density in triple-negative breast cancer tissue. The improved molecular phenotyping and histopathological diagnosis of cancers may lead to new insights into the biology of tumours of patients.

A prognostic model based on cell-cycle control predicts outcome of breast cancer patients.

BACKGROUND: A prognostic model combining biomarkers of metaphase-anaphase transition of the cell cycle was developed for invasive breast cancer. The prognostic value and clinical applicability of the model was evaluated in comparison with the routine prognosticators of invasive breast carcinoma. METHODS: The study comprised 1135 breast cancer patients with complete clinical data and up to 22-year follow-up. Regulators of metaphase-anaphase transition were detected immunohistochemically and the biomarkers with the strongest prognostic impacts were combined into a prognostic model. The prognostic value of the model was tested and evaluated in separate patient materials originating from two Finnish breast cancer centers. RESULTS: The designed model comprising immunoexpressions of Securin, Separase and Cdk1 identified 8.4-fold increased risk of breast cancer mortality (p < 0.0001). A survival difference exceeding 15 years was observed between the majority (> 75%) of patients resulting with favorable as opposed to unfavorable outcome of the model. Along with nodal status, the model showed independent prognostic impact for all breast carcinomas and for subgroups of luminal, N+ and N- disease. CONCLUSIONS: The impact of the proposed prognostic model in predicting breast cancer survival was comparable to nodal status. However, the model provided additional information in N- breast carcinoma in identifying patients with aggressive course of disease, potentially in need of adjuvant treatments. Concerning N+, in turn, the model could provide evidence for withholding chemotherapy from patients with favorable outcome.

UBR5 Is Coamplified with MYC in Breast Tumors and Encodes an Ubiquitin Ligase That Limits MYC-Dependent Apoptosis.

For maximal oncogenic activity, cellular MYC protein levels need to be tightly controlled so that they do not induce apoptosis. Here, we show how ubiquitin ligase UBR5 functions as a molecular rheostat to prevent excess accumulation of MYC protein. UBR5 ubiquitinates MYC and its effects on MYC protein stability are independent of FBXW7. Silencing of endogenous UBR5 induced MYC protein expression and regulated MYC target genes. Consistent with the tumor suppressor function of UBR5 (HYD) in Drosophila, HYD suppressed dMYC-dependent overgrowth of wing imaginal discs. In contrast, in cancer cells, UBR5 suppressed MYC-dependent priming to therapy-induced apoptosis. Of direct cancer relevance, MYC and UBR5 genes were coamplified in MYC-driven human cancers. Functionally, UBR5 suppressed MYC-mediated apoptosis in p53-mutant breast cancer cells with UBR5/MYC coamplification. Furthermore, single-cell immunofluorescence analysis demonstrated reciprocal expression of UBR5 and MYC in human basal-type breast cancer tissues. In summary, UBR5 is a novel MYC ubiquitin ligase and an endogenous rheostat for MYC activity. In MYC-amplified, and p53-mutant breast cancer cells, UBR5 has an important role in suppressing MYC-mediated apoptosis priming and in protection from drug-induced apoptosis. SIGNIFICANCE: These findings identify UBR5 as a novel MYC regulator, the inactivation of which could be very important for understanding of MYC dysregulation on cancer cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1414/F1.large.jpg.

Formin Proteins FHOD1 and INF2 in Triple-Negative Breast Cancer: Association With Basal Markers and Functional Activities.

Basal-like breast cancer is an aggressive form of breast cancer with limited treatment options. The subgroup can be identified immunohistochemically, by lack of hormone receptor expression combined with expression of basal markers such as CK5/6 and/or epidermal growth factor receptor (EGFR). In vitro, several regulators of the actin cytoskeleton are essential for efficient invasion of basal-like breast cancer cell lines. Whether these proteins are expressed in vivo determines the applicability of these findings in clinical settings. The actin-regulating formin protein FHOD1 participates in invasion of the triple-negative breast cancer cell line MDA-MB-231. Here, we measure the expression of FHOD1 protein in clinical triple-negative breast cancers by using immunohistochemistry and further characterize the expression of another formin protein, INF2. We report that basal-like breast cancers frequently overexpress formin proteins FHOD1 and INF2. In cell studies using basal-like breast cancer cell lines, we show that knockdown of FHOD1 or INF2 interferes with very similar processes: maintenance of cell shape, migration, invasion, and proliferation. Inhibition of EGFR, PI3K, or mitogen-activated protein kinase activity does not alter the expression of FHOD1 and INF2 in these cell lines. We conclude that the experimental studies on these formins have implications in the clinical behavior of basal-like breast cancer.

Proliferation-associated miRNAs-494, -205, -21 and -126 detected by in situ hybridization: expression and prognostic potential in breast carcinoma patients.

PURPOSE: To visualize by in situ hybridization (ISH) the levels of a set of proliferation-associated miRNAs and to evaluate their impact and clinical applicability in prognostication of invasive breast carcinoma. METHODS: Tissue specimen from breast carcinoma patients were investigated for miRNAs-494, -205, -21 and -126. Prognostic associations for levels of miRNAs were analyzed based on complete clinical data and up to 22.5-year follow-up of the patient material (n = 285). For detection of the miRNAs, an automated sensitive protocol applying in situ hybridization was developed. RESULTS: MiRNA-494 indicated prognostic value for patients with invasive breast carcinoma. Among node-negative disease reduced level of miRNA-494 predicted 8.5-fold risk of breast cancer death (p = 0.04). Altered levels and expression patterns of the studied miRNAs were observed in breast carcinomas as compared to benign breast tissue. CONCLUSIONS: The present paper reports for the first time on the prognostic value of miRNA-494 in invasive breast cancer. Particularly, detection of miRNA-494 could benefit patients with node-negative breast cancer in identifying subgroups with aggressive disease. Based on our experience, the developed automatic ISH method to visualize altered levels of miRNAs-494, -205, -21 and -126 could be applied to routine pathology diagnostics providing that conditions of tissue treatment, especially fixation delays, are managed.

HSD17B1 expression induces inflammation-aided rupture of mammary gland myoepithelium.

Hydroxysteroid (17-beta) dehydrogenase type 1 (HSD17B1) converts low-active estrogen estrone to highly active estradiol. Estradiol is necessary for normal postpubertal mammary gland development; however, elevated estradiol levels increase mammary tumorigenesis. To investigate the significance of the human HSD17B1 enzyme in the mammary gland, transgenic mice universally overexpressing human HSD17B1 were used (HSD17B1TG mice). Mammary glands obtained from HSD17B1TG females at different ages were investigated for morphology and histology, and HSD17B1 activity and estrogen receptor activation in mammary gland tissue were assessed. To study the significance of HSD17B1 enzyme expression locally in mammary gland tissue, HSD17B1-expressing mammary epithelium was transplanted into cleared mammary fat pads of wild-type females, and the effects on mammary gland estradiol production, epithelial cells and the myoepithelium were investigated. HSD17B1TG females showed increased estrone to estradiol conversion and estrogen-response element-driven estrogen receptor signaling in mammary gland tissue, and they showed extensive lobuloalveolar development that was further enhanced by age along with an increase in serum prolactin concentrations. At old age, HSD17B1TG females developed mammary cancers. Mammary-restricted HSD17B1 expression induced lesions at the sites of ducts and alveoli, accompanied by peri- and intraductal inflammation and disruption of the myoepithelial cell layer. The lesions were shown to be estrogen dependent, as treatment with an antiestrogen, ICI 182,780, starting when lesions were already established reversed the phenotype. These data elucidate the ability of human HSD17B1 to enhance estrogen action in the mammary gland in vivo and indicate that HSD17B1 is a factor inducing phenotypic alterations associated with mammary tumorigenesis.

Publisher Correction: Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity.

In this Article originally published, owing to a technical error, author affiliations were incorrectly assigned in the HTML version; the PDF was correct. These errors have now been corrected.

PTTG1-interacting protein (PTTG1IP/PBF) predicts breast cancer survival.

BACKGROUND: PTTG1-interacting protein (PTTG1IP) is an oncogenic protein, which participates in metaphase-anaphase transition of the cell cycle through activation of securin (PTTG1). PTTG1IP promotes the shift of securin from the cell cytoplasm to the nucleus, allowing the interaction between separase and securin. PTTG1IP overexpression has been previously observed in malignant disease, e.g. in breast carcinoma. However, the prognostic value of PTTG1IP in breast carcinoma patients has not previously been revealed. METHODS: A total of 497 breast carcinoma patients with up to 22-year follow-up were analysed for PTTG1IP and securin immunoexpression. The results were evaluated for correlations with the clinical prognosticators and patient survival. RESULTS: In our material, negative PTTG1IP immunoexpression predicted a 1.5-fold risk of breast cancer death (p = 0.02). However, adding securin immunoexpression to the analysis indicated an even stronger and independent prognostic power in the patient material (HR = 2.5, p < 0.0001). The subcellular location of securin was found with potential prognostic value also among the triple-negative breast carcinomas (n = 96, p = 0.052). CONCLUSIONS: PTTG1IP-negativity alone and in combination with high securin immunoexpression indicates a high risk of breast cancer death, resulting in up to 14-year survival difference in our material.

Separase is a marker for prognosis and mitotic activity in breast cancer.

BACKGROUND: Cancer cell proliferation is a critical feature in classifying and predicting the outcome of breast carcinoma. Separase has a central role in cell cycle progression in unleashing sister-chromatids at anaphase onset. Abnormally functioning separase is known to lead to chromosomal instability. METHODS: The study comprises 349 breast carcinoma patients treated in Central Hospital of Central Finland. The prognostic value, role as a proliferation marker and regulatory interactions of separase are evaluated by immunohistochemical and double- and triple-immunofluorescence (IF) detections based on complete clinical data and >22-year follow-up of the patient material. RESULTS: In our material, abnormal separase expression predicted doubled risk of breast cancer death (P<0.001). Up to 11.3-year survival difference was observed when comparing patients with and without separase expressing cancer cell mitoses. Particularly, abnormal separase expression predicted impaired survival for luminal breast carcinoma (P<0.001, respectively). In multivariate analyses, abnormal separase expression showed independent prognostic value. The complex inhibitory interactions involving securin and cyclin B1 were investigated in double- and triple-IFs and revealed patient subgroups with aberrant regulation and expression patterns of separase. CONCLUSIONS: In our experience, separase is a promising and clinically applicable proliferation marker. Separase expression shows strong and independent prognostic value and could be developed into a biomarker for treatment decisions in breast carcinoma, particularly defining prognostic subgroups among luminal carcinomas.