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Cancer curing immune responses against heterogeneous solid cancers require that a coordinated immune activation is initiated in the antigen avid but immunosuppressive tumor microenvironment (TME). The plastic TME, and the poor systemic tolerability of immune activating drugs are, however, fundamental barriers to generating curative anticancer immune responses. Here, we introduce the CarboCell technology to overcome these barriers by forming an intratumoral sustained drug release depot that provides high payloads of immune stimulatory drugs selectively within the TME. The CarboCell thereby induces a hot spot for immune cell training and polarization and further drives and maintains the tumor-draining lymph nodes in an anticancer and immune activated state. Mechanistically, this transforms cancerous tissues, consequently generating systemic anticancer immunoreactivity. CarboCell can be injected through standard thin-needle technologies and has inherent imaging contrast which secure accurate intratumoral positioning. In particular, here we report the therapeutic performance for a dual-drug CarboCell providing sustained release of a Toll-like receptor 7/8 agonist and a transforming growth factor-β inhibitor in preclinical tumor models in female mice., (© 2024. The Author(s).)

Liposomes carrying chemotherapeutic drugs can accumulate passively in solid tumors at high levels. However, additional targeting of the liposomes towards e.g. receptors expressed on cancer cells may improve their interaction and therapeutic properties. In this study, we designed a liposomal delivery system, which utilizes the intrinsic characteristics of HER2-positive tumors to ensure efficient delivery of oxaliplatin to the cancer cells. On the liposome surface, trastuzumab, an antibody specific to the HER2 receptor, was shown to facilitate internalization by the cancer cells. A polyethylene glycol (PEG) layer on the liposome surface provides protection from mononuclear phagocyte system uptake. To optimize the interaction between liposomes and cancer cells, a protease-sensitive cleavable peptide linker was inserted at the base of each PEG. The PEG layer is then cleaved off by intra- and extracellular matrix metalloproteinases (MMPs) upon accumulation in the tumor. Our data demonstrate that the removal of PEG significantly destabilizes the liposomes and leads to substantial oxaliplatin release. The proposed beneficial effect of combining antibody-mediated internalization with MMP sensitivity was confirmed in a series of in vivo studies using ovarian cancer xenograft models. The results demonstrated that HER2-targeted MMP-sensitive liposomes have superior anticancer activity compared to non-targeted and non-cleavable liposomes., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Cationic liposomes specifically target monocytes in blood, rendering them promising drug-delivery tools for cancer immunotherapy, vaccines, and therapies for monocytic leukaemia. The mechanism behind this monocyte targeting ability is, however, not understood, but may involve plasma proteins adsorbed on the liposomal surfaces. To shed light on this, we investigated the biomolecular corona of three different types of PEGylated cationic liposomes, finding all of them to adsorb hyaluronan-associated proteins and proteoglycans upon incubation in human blood plasma. This prompted us to study the role of the TLR4 co-receptors CD44 and CD14, both involved in signalling and uptake pathways of proteoglycans and glycosaminoglycans. We found that separate inhibition of each of these receptors hampered the monocyte uptake of the liposomes in whole human blood. Based on clues from the biomolecular corona, we have thus identified two receptors involved in the targeting and uptake of cationic liposomes in monocytes, in turn suggesting that certain proteoglycans and glycosaminoglycans may serve as monocyte-targeting opsonins. This mechanistic knowledge may pave the way for rational design of future monocyte-targeting drug-delivery platforms., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Thomas L. Andresen and Ladan Parhamifar reports a relationship with MonTa Biosciences that includes: founding. Thomas L. Andresen, Ladan Parhamifar and Rasmus Münter have patent #WO2019012107 issued to Technical University of Denmark., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Current clinical markers overestimate the recurrence risk in many lymph node negative (LNN) breast cancer (BC) patients such that a majority of these low-risk patients unnecessarily receive systemic treatments. We tested if differential microRNA expression in primary tumors allows reliable identification of indolent LNN BC patients to provide an improved classification tool for overtreatment reduction in this patient group., Methods: We collected freshly frozen primary tumors of 80 LNN BC patients with recurrence and 80 recurrence-free patients (mean follow-up: 20.9 years). The study comprises solely systemically untreated patients to exclude that administered treatments confound the metastasis status. Samples were pairwise matched for clinical-pathological characteristics to minimize dependence of current markers. Patients were classified into risk-subgroups according to the differential microRNA expression of their tumors via classification model building with cross-validation using seven classification methods and a voting scheme. The methodology was validated using available data of two independent cohorts (n = 123, n = 339)., Results: Of the 80 indolent patients (who would all likely receive systemic treatments today) our ultralow-risk classifier correctly identified 37 while keeping a sensitivity of 100% in the recurrence group. Multivariable logistic regression analysis confirmed independence of voting results from current clinical markers. Application of the method in two validation cohorts confirmed successful classification of ultralow-risk BC patients with significantly prolonged recurrence-free survival., Conclusion: Profiles of differential microRNAs expression can identify LNN BC patients who could spare systemic treatments demanded by currently applied classifications. However, further validation studies are required for clinical implementation of the applied methodology., (© 2024 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Resistance to aromatase inhibitor (AI) treatment and combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy (ET) are crucial clinical challenges in treating estrogen receptor-positive (ER+) breast cancer. Understanding the resistance mechanisms and identifying reliable predictive biomarkers and novel treatment combinations to overcome resistance are urgently needed. Herein, we show that upregulation of CDK6, p-CDK2, and/or cyclin E1 is associated with adaptation and resistance to AI-monotherapy and combined CDK4/6i and ET in ER+ advanced breast cancer. Importantly, co-targeting CDK2 and CDK4/6 with ET synergistically impairs cellular growth, induces cell cycle arrest and apoptosis, and delays progression in AI-resistant and combined CDK4/6i and fulvestrant-resistant cell models and in an AI-resistant autocrine breast tumor in a postmenopausal xenograft model. Analysis of CDK6, p-CDK2, and/or cyclin E1 expression as a combined biomarker in metastatic lesions of ER+ advanced breast cancer patients treated with AI-monotherapy or combined CDK4/6i and ET revealed a correlation between high biomarker expression and shorter progression-free survival (PFS), and the biomarker combination was an independent prognostic factor in both patients cohorts. Our study supports the clinical development of therapeutic strategies co-targeting ER, CDK4/6 and CDK2 following progression on AI-monotherapy or combined CDK4/6i and ET to improve survival of patients exhibiting high tumor levels of CDK6, p-CDK2, and/or cyclin E1., (© 2022. The Author(s).)

Whereas adoptive T cell therapy has been extensively studied for cancer treatment, the response is still limited primarily due to immune dysfunction related to poor cell engraftment, tumor infiltration and engagement, and lack of a target. In addition, the modification of therapeutic T cells often suffers from being complex and expensive. Here, we present a strategy to load T cells with SHP099, an allosteric SHP2 inhibitor, to enhance the therapeutic efficacy of the T cells. Remote-loading of SHP099 into lipid nanoparticles decorated with triarginine motifs resulted in nanocrystal formation of SHP099 inside the lipid vesicles and allowed high loading efficiency and prolonged retention of SHP099 nanocrystals within T cells. Cell-loaded SHP099 enabled sustained inhibition of the PD-1/PD-L1 signaling and increased cytolytic activity of the T cells. We show in a mouse model that tumor-homing T cells can circulate with the cargos, improving their tumor accumulation compared to systemically administered lipid nanoparticles. On an established solid tumor model, adoptively transferred SHP099 loaded T cells induced complete tumor eradication and durable immune memory against tumor rechallenging on all treated mice by effectively inhibiting the PD-1/PD-L1 checkpoint signal. We demonstrate that the combination of T cell therapy with SHP2 inhibition is a promising therapeutic strategy, and the lipid nanocrystal platform could be generalized as a promising approach for T cell loading of immunomodulatory drugs.

Stimulation of monocytes with immunomodulating agents can harness the immune system to treat a long range of diseases, including cancers, infections and autoimmune diseases. To this end we aimed to develop a monocyte-targeting delivery platform based on cationic liposomes, which can be utilized to deliver immunomodulators and thus induce monocyte-mediated immune responses while avoiding off-target side-effects. The cationic liposome design is based on functionalizing the liposomal membrane with a cholesterol-anchored tri-arginine peptide (TriArg). We demonstrate that TriArg liposomes can target monocytes with high specificity in both human and murine blood and that this targeting is dependent on the content of TriArg in the liposomal membrane. In addition, we show that the mechanism of selective monocyte targeting involves the CD14 co-receptor, and selectivity is compromised when the TriArg content is increased, resulting in complement-mediated off-target uptake in granulocytes. The presented mechanistic findings of uptake by peripheral blood leukocytes may guide the design of future drug delivery systems utilized for immunotherapy. STATEMENT OF SIGNIFICANCE: Monocytes are attractive targets for immunotherapies of cancers, infections and autoimmune diseases. Specific delivery of immunostimulatory drugs to monocytes is typically achieved using ligand-targeted drug delivery systems, but a simpler approach is to target monocytes using cationic liposomes. To achieve this, however, a deep understanding of the mechanisms governing the interactions of cationic liposomes with monocytes and other leukocytes is required. We here investigate these interactions using liposomes incorporating a cationic arginine-rich lipopeptide. We demonstrate that monocyte targeting can be achieved by fine-tuning the lipopeptide content in the liposomes. Additionally, we reveal that the CD14 receptor is involved in the targeting process, whereas the complement system is not. These mechanistic findings are critical for future design of monocyte-targeting liposomal therapies., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: T.L. Andresen, L. Parhamifar and R. Münter have IP on cationic liposomes for immunotherapy (Patent number WO2019012107). The authors declare no other relevant affiliations with financial interests., (Copyright © 2022. Published by Elsevier Ltd.)

Several gene expression signatures based on mRNAs and a few based on long non-coding RNAs (lncRNAs) have been developed to provide prognostic information beyond clinical evaluation in breast cancer (BC). However, the comparison of such signatures for predicting recurrence is very scarce. Therefore, we compared the prognostic utility of mRNAs and lncRNAs in low-risk BC patients using two different classification strategies. Frozen primary tumor samples from 160 lymph node negative and systemically untreated BC patients were included; 80 developed recurrence-i.e., regional or distant metastasis while 80 remained recurrence-free (mean follow-up of 20.9 years). Patients were pairwise matched for clinicopathological characteristics. Classification based on differential mRNA or lncRNA expression using seven individual machine learning methods and a voting scheme classified patients into risk-subgroups. Classification by the seven methods with a fixed sensitivity of ≥90% resulted in specificities ranging from 16-40% for mRNA and 38-58% for lncRNA, and after voting, specificities of 38% and 60% respectively. Classifier performance based on an alternative classification approach of balanced accuracy optimization also provided higher specificities for lncRNA than mRNA at comparable sensitivities. Thus, our results suggested that classification followed by voting improved prognostic power using lncRNAs compared to mRNAs regardless of classification strategy.

CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy have shown impressive efficacy in estrogen receptor-positive advanced breast cancer. However, most patients will eventually experience disease progression on this combination, underscoring the need for effective subsequent treatments or better initial therapies. Here, we show that triple inhibition with fulvestrant, CDK4/6i and AKT inhibitor (AKTi) durably impairs growth of breast cancer cells, prevents progression and reduces metastasis of tumor xenografts resistant to CDK4/6i-fulvestrant combination or fulvestrant alone. Importantly, switching from combined fulvestrant and CDK4/6i upon resistance to dual combination with AKTi and fulvestrant does not prevent tumor progression. Furthermore, triple combination with AKTi significantly inhibits growth of patient-derived xenografts resistant to combined CDK4/6i and fulvestrant. Finally, high phospho-AKT levels in metastasis of breast cancer patients treated with a combination of CDK4/6i and endocrine therapy correlates with shorter progression-free survival. Our findings support the clinical development of ER, CDK4/6 and AKT co-targeting strategies following progression on CDK4/6i and endocrine therapy combination, and in tumors exhibiting high phospho-AKT levels, which are associated with worse clinical outcome., (© 2021. The Author(s).)

Resistance to endocrine therapy in estrogen receptor-positive (ER + ) breast cancer is a major clinical problem with poorly understood mechanisms. There is an unmet need for prognostic and predictive biomarkers to allow appropriate therapeutic targeting. We evaluated the mechanism by which minichromosome maintenance protein 3 (MCM3) influences endocrine resistance and its predictive/prognostic potential in ER + breast cancer. We discovered that ER + breast cancer cells survive tamoxifen and letrozole treatments through upregulation of minichromosome maintenance proteins (MCMs), including MCM3, which are key molecules in the cell cycle and DNA replication. Lowering MCM3 expression in endocrine-resistant cells restored drug sensitivity and altered phosphorylation of cell cycle regulators, including p53(Ser 315,33 ), CHK1(Ser 317 ), and cdc25b(Ser 323 ), suggesting that the interaction of MCM3 with cell cycle proteins is an important mechanism of overcoming replicative stress and anti-proliferative effects of endocrine treatments. Interestingly, the MCM3 levels did not affect the efficacy of growth inhibitory by CDK4/6 inhibitors. Evaluation of MCM3 levels in primary tumors from four independent cohorts of breast cancer patients receiving adjuvant tamoxifen mono-therapy or no adjuvant treatment, including the Stockholm tamoxifen (STO-3) trial, showed MCM3 to be an independent prognostic marker adding information beyond Ki67. In addition, MCM3 was shown to be a predictive marker of response to endocrine treatment. Our study reveals a coordinated signaling network centered around MCM3 that limits response to endocrine therapy in ER + breast cancer and identifies MCM3 as a clinically useful prognostic and predictive biomarker that allows personalized treatment of ER + breast cancer patients.

Purpose: In this cross-sectional study, we evaluated the association between morbidity and participation in the prevalence round of the Danish national mammography screening program., Patients and Methods: Morbidity was assessed by the Charlson Comorbidity Index (CCI) score (0, 1-2, and ≥3) and by 19 individual diagnoses. We retrieved data on participation from The Danish Quality Database of Mammography Screening and on diagnoses from The Danish National Patient Registry. We estimated prevalence proportion ratios (PR) with 95% confidence intervals (CI)., Results: In total, 519,009 (79.8%) women participated in the first national breast cancer screening round. Relative to women with a CCI score of 0, the adjusted PRs were 0.96 (95% CI: 0.95-0.96) for a CCI score of 1-2 and 0.80 (95% CI: 0.79-0.81) for a CCI score of ≥3. Compared with no disease, the PRs for a diagnosis of the most prevalent, but less severe diseases, chronic pulmonary disease, cerebrovascular disease, diabetes I and II were 0.93 (95% CI: 0.93-0.94), 0.96 (95% CI: 0.94-0.96), and 0.96 (95% CI: 0.95-0.97), respectively. Among women with low prevalent, but most severe diseases, the PRs were 0.69 (95% CI: 0.60-0.81) for AIDS and 0.73 (95% CI: 0.70-0.76) for metastatic solid tumor., Conclusion: Women with a high CCI score or one severe chronic condition are less likely to participate in breast cancer screening compared to women without disease. However, these women account for a small proportion of all non-participating women. Thus, it might be most beneficial to maximize breast cancer screening participation in women with less severe although more common morbidities., Competing Interests: The authors declare that there is no conflict of interest., (© 2020 Viuff et al.)

Transport of the majority of therapeutic molecules to the brain is precluded by the presence of the blood-brain barrier (BBB) rendering efficient treatment of many neurological disorders impossible. This BBB, nonetheless, may be circumvented by targeting receptors and transport proteins expressed on the luminal surface of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) has remained a popular target since its original description for this purpose, although clinical progression of TfR-targeted drug constructs or nanomedicines remains unsuccessful. One proposed issue pertaining to the use of TfR-targeting in nanomedicines is the efficient tuning of the ligand density on the nanoparticle surface. We studied the impact of TfR antibody density on the uptake and transport of nanoparticles into the brain, taking a parallel approach to investigate the impact on both antibody-functionalized gold nanoparticles (AuNPs) and cargo-loaded liposomes. We report that among three different low-range mean ligand densities (0.15, 0.3, and 0.6 ∗ 10 3 antibodies/μm 2 ), the highest density yielded the highest ability towards both targeting of the BCECs and subsequent transport across the BBB in vivo, and in vitro using primary cultures of the murine BBB. We also find that TfR-targeting on liposomes in the mouse may induce severe adverse effects after intravenous administration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Prognostic and predictive biomarkers of disease and treatment outcome are needed to ensure optimal treatment of patients with triple-negative breast cancer (TNBC). In a mass spectrometry-based global proteomic study of 44 formalin-fixed, paraffin-embedded (FFPE) primary TNBC tumors and 10 corresponding metastases, we found that Cytochrome P450 reductase (CYPOR) expression correlated with patient outcome. The correlation between CYPOR expression and outcome was further evaluated in a Danish cohort of 113 TNBC patients using immunohistochemistry and publicly available gene expression data from two cohorts of TNBC and basal-like breast cancer patients, respectively (N = 249 and N = 580). A significant correlation between high CYPOR gene expression and shorter recurrence-free survival (RFS), but not overall survival, was found in the cohort of 249 TNBC patients (p = 0.018, HR = 1.77, 95% CI 1.1-2.85), and this correlation was recapitulated in a cohort of 580 basal-like breast cancer patients (p = 0.018, HR = 1.4, 95% CI 1.06-1.86). High CYPOR protein expression was also associated with shorter RFS in the cohort of 113 TNBC patients (p = 0.017, HR = 2.73, 95% CI 1.20-6.19), particularly those who were lymph node tumor-negative (p = 0.029, HR = 5.22). Multivariate Cox regression analysis identified CYPOR as an independent prognostic factor for shorter RFS in TNBC patients (p = 0.032, HR = 2.19, 95% CI 1.07-4.47). Together, these data suggest high expression of CYPOR as an independent prognostic biomarker of shorter RFS, which could be used to identify patients who should receive more extensive adjuvant treatment and more aggressive surveillance., (© 2018 UICC.)

Background: Prognostic factors in breast cancer are often measured on a continuous scale, but categorized for clinical decision-making. The primary aim of this study was to evaluate if accounting for continuous non-linear effects of the three factors age at diagnosis, tumor size, and number of positive lymph nodes improves prognostication. These factors will most likely be included in the management of breast cancer patients also in the future, after an expected implementation of gene expression profiling for adjuvant treatment decision-making., Methods: Four thousand four hundred forty seven and 1132 women with primary breast cancer constituted the derivation and validation set, respectively. Potential non-linear effects on the log hazard of distant recurrences of the three factors were evaluated during 10 years of follow-up. Cox-models of successively increasing complexity: dichotomized predictors, predictors categorized into three or four groups, and predictors transformed using fractional polynomials (FPs) or restricted cubic splines (RCS), were used. Predictive performance was evaluated by Harrell's C-index., Results: Using FP-transformations, non-linear effects were detected for tumor size and number of positive lymph nodes in univariable analyses. For age, non-linear transformations did, however, not improve the model fit significantly compared to the linear identity transformation. As expected, the C-index increased with increasing model complexity for multivariable models including the three factors. By allowing more than one cut-point per factor, the C-index increased from 0.628 to 0.674. The additional gain, as measured by the C-index, when using FP- or RCS-transformations was modest (0.695 and 0.696, respectively). The corresponding C-indices for these four models in the validation set, based on the same transformations and parameter estimates from the derivation set, were 0.675, 0.700, 0.706, and 0.701., Conclusions: Categorization of each factor into three to four groups was found to improve prognostication compared to dichotomization. The additional gain by allowing continuous non-linear effects modeled by FPs or RCS was modest. However, the continuous nature of these transformations has the advantage of making it possible to form risk groups of any size.

Olefin cross-metathesis (CM) is a viable reaction for the modification of alkene-containing proteins. Although allyl sulfide or selenide side-chain motifs in proteins can critically enhance the rate of CM reactions, no efficient method for their site-selective genetic incorporation into proteins has been reported to date. Here, through the systematic evaluation of olefin-bearing unnatural amino acids for their metabolic incorporation, we have discovered S-allylhomocysteine (Ahc) as a genetically encodable Met analogue that is not only processed by translational cellular machinery but also a privileged CM substrate residue in proteins. In this way, Ahc was used for efficient Met codon reassignment in a Met-auxotrophic strain of E. coli (B834 (DE3)) as well as metabolic labeling of protein in human cells and was reactive toward CM in several representative proteins. This expands the use of CM in the toolkit for "tag-and-modify" functionalization of proteins.

Background: Endocrine resistance in estrogen receptor-positive (ER+) breast cancer is a major clinical problem and is associated with accelerated cancer cell growth, increased motility and acquisition of mesenchymal characteristics. However, the specific molecules and pathways involved in these altered features remain to be detailed, and may be promising therapeutic targets to overcome endocrine resistance., Methods: In the present study, we evaluated altered expression of epithelial-mesenchymal transition (EMT) regulators in ER+ breast cancer cell models of tamoxifen or fulvestrant resistance, by gene expression profiling. We investigated the specific role of increased SNAI2 expression in fulvestrant-resistant cells by gene knockdown and treatment with a SNAIL-p53 binding inhibitor, and evaluated the effect on cell growth, migration and expression of EMT markers. Furthermore, we evaluated SNAI2 expression by immunohistochemical analysis in metastatic samples from two cohorts of patients with breast cancer treated with endocrine therapy in the advanced setting., Results: SNAI2 was found to be significantly upregulated in all endocrine-resistant cells compared to parental cell lines, while no changes were observed in the expression of other EMT-associated transcription factors. SNAI2 knockdown with specific small interfering RNA (siRNA) converted the mesenchymal-like fulvestrant-resistant cells into an epithelial-like phenotype and reduced cell motility. Furthermore, inhibition of SNAI2 with specific siRNA or a SNAIL-p53 binding inhibitor reduced growth of cells resistant to fulvestrant treatment. Clinical evaluation of SNAI2 expression in two independent cohorts of patients with ER+ metastatic breast cancer treated with endocrine therapy in the advanced setting (N = 86 and N = 67) showed that high SNAI2 expression in the metastasis correlated significantly with shorter progression-free survival on endocrine treatment (p = 0.0003 and p = 0.004)., Conclusions: Our results suggest that SNAI2 is a key regulator of the aggressive phenotype observed in endocrine-resistant breast cancer cells, an independent prognostic biomarker in ER+ advanced breast cancer treated with endocrine therapy, and may be a promising therapeutic target in combination with endocrine therapies in ER+ metastatic breast cancer exhibiting high SNAI2 levels.

Rationale: The ability to treat invalidating neurological diseases is impeded by the presence of the blood-brain barrier (BBB), which inhibits the transport of most blood-borne substances into the brain parenchyma. Targeting the transferrin receptor (TfR) on the surface of brain capillaries has been a popular strategy to give a preferential accumulation of drugs or nanomedicines, but several aspects of this targeting strategy remain elusive. Here we report that TfR-targeted gold nanoparticles (AuNPs) can accumulate in brain capillaries and further transport across the BBB to enter the brain parenchyma. Methods: We characterized our targeting strategy both in vitro using primary models of the BBB and in vivo using quantitative measurements of gold accumulation by inductively-coupled plasma-mass spectrometry together with morphological assessments using light microscopy after silver enhancement and transmission electron microscopy with energy-dispersive X-ray spectroscopy. Results: We find that the uptake capacity is significantly modulated by the affinity and valency of the AuNP-conjugated antibodies. Specifically, antibodies with high and low affinities mediate a low and intermediate uptake of AuNPs into the brain, respectively, whereas a monovalent (bi-specific) antibody improves the uptake capacity remarkably. Conclusion: Our findings indicate that monovalent ligands may be beneficial for obtaining transcytosis of TfR-targeted nanomedicines across the BBB, which is relevant for future design of nanomedicines for brain drug delivery., Competing Interests: Competing interests: All authors have declared no conflicting interest in relation to the theme and contents of the manuscript. The anti-TfRA, anti-TfRD, and anti-TfRA/BACE1 antibodies were obtained from Genentech, Inc. via a Material Transfer Agreement (MTA A15783). Genentech, Inc. had no influence on study design, experimental procedures, data analysis, or preparation of the manuscript.

A majority of estrogen receptor positive (ER+) breast cancers are growth stimulated by estrogens. The ability to inhibit the ER signaling pathway is therefore of critical importance in the current treatment of ER+ breast cancers. It has been reported that 1α,25-dihydroxyvitamin D 3 down-regulates the expression of the CYP19A1 gene, encoding the aromatase enzyme that catalyzes the synthesis of estradiol. Furthermore, 1α,25-dihydroxyvitamin D 3 has also been reported to down-regulate the expression of estrogen receptor α (ERα), the main mediator of ER signaling. This study reports a novel transcription factor critical to 1α,25-dihydroxyvitamin D 3 -mediated regulation of estrogenic signaling in MCF-7 breast cancer cells. We have investigated the molecular mechanisms for the 1α,25-dihydroxyvitamin D 3 -mediated down-regulation of CYP19A1 and ERα gene expression in human MCF-7 breast cancer cells and found that Williams syndrome transcription factor (WSTF) plays a key role by binding to the promoters of CYP19A1 and ERα. Although sometimes reported as an inhibitor of gene expression, we found that WSTF acts as an activator of the promoter activity of both CYP19A1 and ERα. Silencing of WSTF by siRNA transfection resulted in decreased aromatase-dependent cell growth as well as decreased ER signaling in the cells. When cells were treated with 1α,25-dihydroxyvitamin D 3 , WSTF was dissociated from the promoters and the promoter activities of CYP19A1 and ERα were decreased. We have measured the expression of WSTF in ER-positive tumor-samples from breast cancer patients and found that WSTF is expressed in the majority of the investigated samples and that the expression is higher in cancer tissue than in normal tissue. However, we were not able to show any significant association between the WSTF expression in the tumor and the disease free and overall survival in this patient group who have received adjuvant tamoxifen treatment, nor between the WSTF expression and the expression of ERα, progesterone receptor or HER2. The major conclusions of this study are that WSTF acts as an activator of ER signaling in MCF-7 breast cancer cells, that this action can be inhibited by 1α,25-dihydroxyvitamin D 3 , and that the expression of WSTF is higher in breast cancer tissue than in normal tissue. WSTF may by a new target for treatment of estrogen-dependent breast cancer cell growth., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Current prognostic markers allocate the majority of lymph node (LN) negative and estrogen receptor (ER) positive breast cancer patients into the high-risk group. Accordingly, most patients receive systemic treatments although approximately 40% of these patients may have been cured by surgery and radiotherapy alone. Two studies identified seven prognostic microRNAs in systemically untreated, LN negative and ER positive breast cancer patients which may allow more precise patient classification. However, six of the seven microRNAs were analyzed in both studies but only found to be prognostic in one study. To validate their prognostic potential, we analyzed microRNA expression in an independent cohort ( n = 110) using a pair-matched study design minimizing dependence of classical markers. The expression of hsa-miR-548c-5p was significantly associated with abridged disease-free survival (hazard ratio [HR]:1.96, p = 0.027). Contradicting published results, high hsa-miR-516-3p expression was associated with favorable outcome (HR:0.29, p = 0.0068). The association is probably time-dependent indicating later relapse. Additionally, re-analysis of previously published expression data of two matching cohorts ( n = 100, n = 255) supports an association of hsa-miR-128-3p with shortened disease-free survival (HR:2.48, p = 0.0033) and an upregulation of miR-7-5p ( p = 0.0038; p = 0.039) and miR-210-3p ( p = 0.031) in primary tumors of patients who experienced metastases. Further analysis may verify the prognostic potential of these microRNAs., Competing Interests: CONFLICTS OF INTEREST The authors declare that no competing interests exist.

Cancer results from alterations at essential genomic sites and is characterized by uncontrolled cell proliferation, invasion and metastasis. Identification of driver genes of metastatic progression is essential, as metastases, not primary tumors, are fatal. To gain insight into the mutational concordance between different steps of malignant progression we performed exome sequencing and validation with targeted deep sequencing of successive steps of malignant progression from pre-invasive stages to asynchronous distant metastases in six breast cancer patients. Using the ratio of non-synonymous to synonymous mutations, a surprisingly large number of cancer driver genes, ranging between 3 and 145, were estimated to confer a selective advantage in the studied primary tumors. We report a substantial amount of metastasis specific mutations and a number of novel putative metastasis driver genes. Most notable are the DCC, ABCA13, TIAM2, CREBBP, BCL6B and ZNF185 genes, mainly mutated exclusively in metastases and highly likely driver genes of metastatic progression. We find different genes and pathways to be affected at different steps of malignant progression. The Adherens junction pathway is affected in four of the six studied patients and this pathway most likely plays a vital role in the metastatic process.

Background: In Denmark, national roll-out of a population-based, screening mammography program took place in 2007-2010. We report on outcome of the first four biennial invitation rounds., Methods: Data on screening outcome were retrieved from the 2015 and 2016 national screening quality reports. We calculated coverage by examination; participation after invitation; detection-, interval cancer- and false-positive rates; cancer characteristics; sensitivity and specificity, for Denmark and for the five regions., Results: At the national level coverage by examination remained at 75-77%; lower in the Capital Region than in the rest of Denmrk. Detection rate was slightly below 1% at first screen, 0.6% at subsequent screens, and one region had some fluctuation over time. Ductal carcinoma in situ (DCIS) constituted 13-14% of screen-detected cancers. In subsequent rounds, 80% of screen-detected invasive cancers were node negative and 40% ≤10 mm. False-positive rate was around 2%; higher for North Denmark Region than for the rest of Denmark. Three out of 10 breast cancers in screened women were diagnosed as interval cancers., Conclusions: High coverage by examination and low interval cancer rate are required for screening to decrease breast cancer mortality. Two pioneer local screening programs starting in the 1990s were followed by a decrease in breast cancer mortality of 22-25%. Coverage by examination and interval cancer rate of the national program were on the favorable side of values from the pioneer programs. It appears that the implementation of a national screening program in Denmark has been successful, though regional variations need further evaluation to assure optimization of the program.

A main controversy in cancer research is whether metastatic abilities are present in the most advanced clone of the primary tumor or result from independently acquired aberrations in early disseminated cancer cells as suggested by the linear and the parallel progression models, respectively. The genetic concordance between different steps of malignant progression is mostly unexplored as very few studies have included cancer samples separated by both space and time. We applied whole exome sequencing and targeted deep sequencing to 26 successive samples from six patients with metastatic estrogen receptor (ER)-positive breast cancer. Our data provide support for both linear and parallel progression towards metastasis. We report for the first time evidence of metastasis-to-metastasis seeding in breast cancer. Our results point to three distinct routes of metastasis emergence. This may have profound clinical implications and provides substantial novel molecular insights into the timing and mutational evolution of breast cancer metastasis.

Purpose: Resistance to endocrine therapy in estrogen receptor-positive (ER + ) breast cancer remains a major clinical problem. Recently, the CDK4/6 inhibitor palbociclib combined with letrozole or fulvestrant was approved for treatment of ER + advanced breast cancer. However, the role of CDK4/6 in endocrine resistance and their potential as predictive biomarkers of endocrine treatment response remains undefined., Experimental Design: We investigated the specific role of increased CDK6 expression in fulvestrant-resistant cells by gene knockdown and treatment with palbociclib, and evaluated the effect in cell proliferation, apoptosis, and kinase activity. Furthermore, we evaluated CDK6 expression in metastatic samples from breast cancer patients treated or not with fulvestrant., Results: We found increased expression of CDK6 in two fulvestrant-resistant cell models versus sensitive cells. Reduction of CDK6 expression impaired fulvestrant-resistant cell growth and induced apoptosis. Treatment with palbociclib resensitized fulvestrant-resistant cells to fulvestrant through alteration of retinoblastoma protein phosphorylation. High CDK6 levels in metastatic samples from two independent cohorts of breast cancer patients treated with fulvestrant (N = 45 and 46) correlated significantly with shorter progression-free survival (PFS) on fulvestrant treatment (P = 0.0006 and 0.018), whereas no association was observed in patients receiving other first- or second-/third-line endocrine treatments (N = 68, P = 0.135 and 0.511, respectively)., Conclusions: Our results indicate that upregulation of CDK6 may be an important mechanism in overcoming fulvestrant-mediated growth inhibition in breast cancer cells. Patients with advanced ER + breast cancer exhibiting high CDK6 expression in the metastatic lesions show shorter PFS upon fulvestrant treatment and thus may benefit from the addition of CDK4/6 inhibitors in their therapeutic regimens. Clin Cancer Res; 22(22); 5514-26. ©2016 AACR., (©2016 American Association for Cancer Research.)

Our on-going work to develop an intracortical visual prosthesis has motivated the design, fabrication, and testing of a Wireless Floating Microelectrode Array (WFMA) stimulator. This implantable device can be used for an electrical stimulation interface in the peripheral and the central nervous system. Previously, its use in a sciatic nerve rodent model was described. Here implantation of two WFMAs in motor cortex of two NHP (macaque - two devices/animal) is presented. Preliminary functional tests show the implanted devices to be fully functional with stimulation-induced motor movements obtained. Functional testing is on-going.

Functionalization of nanoparticles is a key element for improving specificity of drug delivery systems toward diseased tissue or cells. In the current study we report a highly efficient and chemoselective method for post-functionalization of liposomes with biomacromolecules, which equally well can be used for functionalization of other nanoparticles or solid surfaces. The method exploits a synergistic effect of having both affinity and covalent anchoring tags on the surface of the liposome. This was achieved by synthesizing a peptide linker system that uses Cu-free strain-promoted click chemistry in combination with histidine affinity tags. The investigation of post-functionalization of PEGylated liposomes was performed with a cyclic RGDfE peptide. By exploring both affinity and covalent tags a 98 ± 2.0% coupling efficiency was achieved, even a diluted system showed a coupling efficiency of 87 ± 0.2%. The reaction kinetics and overall yield were quantified by HPLC. The results presented here open new possibilities for constructing complex nanostructures and functionalized surfaces.

Triple-negative breast cancer (TNBC) represents a heterogeneous subgroup with generally poor outcome and lack of an effective targeted therapy. Prognostic or predictive biomarkers to guide treatment decisions for this group of patients are needed. To evaluate the potential of S100A14 protein as a novel biomarker in TNBC, the protein expression of S100A14 was correlated with clinical outcomes in a Pilot Sample set and a Danish cohort of predominantly TNBC patients. Kaplan-Meier analysis identified a prognostic impact of S100A14 on disease-free survival and overall survival, showing that tumors with high S100A14 protein expression levels were significantly correlated with poor outcome in TNBC patients (p = 0.017; p = 0.038), particularly those in the basal-like subgroup (p = 0.006; p = 0.037). Importantly, TNBC patients with high S100A14 expression, but tumor-negative axillary lymph nodes (N-), had equally poor outcomes as those with tumor-positive axillary lymph nodes (N+), while TNBC/N- patients with low S100A14 expression had a significantly better disease free survival (p = 0.013). Multivariate analysis revealed that S100A14 is an independent prognostic factor for TNBC patients (p = 0.024; p = 0.05). At the cellular level, S100A14 was found to be expressed in epithelial-like, but not in mesenchymal-like, TNBC cells in vitro. S100A14 is an independent prognostic factor in TNBC and a novel potential therapeutic target in TNBC., (© 2015 UICC.)

To gain insight into miRNA regulation in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using global miRNA profiling, 28 miRNAs were found to exhibit significantly altered expression between isogenic metastasizing and non-metastasizing cancer cells, with miR-155 being the most differentially expressed. Highly metastatic mesenchymal-like CL16 cancer cells showed very low miR-155 expression, and miR-155 overexpression in these cells lead to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. Our experiments addressing the underlying mechanism of the altered tumor burden revealed that miR-155-overexpressing CL16 cells were less invasive than CL16 control cells in vitro, while miR-155 overexpression had no effect on cancer cell proliferation or apoptosis in established lung tumors. To identify proteins regulated by miR-155 and thus delineate its function in our cell model, we compared the proteome of xenograft tumors derived from miR-155-overexpressing CL16 cells and CL16 control cells using mass spectrometry-based proteomics. >4,000 proteins were identified, of which 92 were consistently differentially expressed. Network analysis revealed that the altered proteins were associated with cellular functions such as movement, growth and survival as well as cell-to-cell signaling and interaction. Downregulation of the three metastasis-associated proteins ALDH1A1, PIR and PDCD4 in miR-155-overexpressing tumors was validated by immunohistochemistry. Our results demonstrate that miR-155 inhibits the ability of cancer cells to extravasate and/or colonize at distant organs and brings additional insight into the complexity of miR-155 regulation in metastatic seeding.

Introduction: Patients with clinically and pathologically similar breast tumors often have very different outcomes and treatment responses. Current prognostic markers allocate the majority of breast cancer patients to the high-risk group, yielding high sensitivities in expense of specificities below 20%, leading to considerable overtreatment, especially in lymph node-negative patients. Seventy percent would be cured by surgery and radiotherapy alone in this group. Thus, precise and early indicators of metastasis are highly desirable to reduce overtreatment. Previous prognostic RNA-profiling studies have only focused on the protein-coding part of the genome, however the human genome contains thousands of long non-coding RNAs (lncRNAs) and this unexplored field possesses large potential for identification of novel prognostic markers., Methods: We evaluated lncRNA microarray data from 164 primary breast tumors from adjuvant naïve patients with a mean follow-up of 18 years. Eighty two patients who developed detectable distant metastasis were compared to 82 patients where no metastases were diagnosed. For validation, we determined the prognostic value of the lncRNA profiles by comparing the ability of the profiles to predict metastasis in two additional, previously-published, cohorts., Results: We showed that lncRNA profiles could distinguish metastatic patients from non-metastatic patients with sensitivities above 90% and specificities of 64-65%. Furthermore; classifications were independent of traditional prognostic markers and time to metastasis., Conclusions: To our knowledge, this is the first study investigating the prognostic potential of lncRNA profiles. Our study suggest that lncRNA profiles provide additional prognostic information and may contribute to the identification of early breast cancer patients eligible for adjuvant therapy, as well as early breast cancer patients that could avoid unnecessary systemic adjuvant therapy. This study emphasizes the potential role of lncRNAs in breast cancer prognosis.

Background: Resistance to antiestrogen therapy is a major clinical challenge in the treatment of estrogen receptor α (ER)-positive breast cancer. The aim of the study was to explore the growth promoting pathways of antiestrogen resistant breast cancer cells to identify biomarkers and novel treatment targets., Methods: Antiestrogen sensitive and resistant T47D breast cancer cell lines were used as model systems. Parental and fulvestrant resistant cell lines were subjected to a kinase inhibitor library. Kinase inhibitors preferentially targeting growth of fulvestrant resistant cells were identified and the growth inhibitory effect verified by dose-response cell growth experiments. Protein expression and phosphorylation were investigated by western blot analysis. Cell cycle phase distribution and cell death were analyzed by flow cytometry. To evaluate Aurora kinase B as a biomarker for endocrine resistance, immunohistochemistry was performed on archival primary tumor tissue from breast cancer patients who have received adjuvant endocrine treatment with tamoxifen., Results: The selective Aurora kinase B inhibitor barasertib was identified to preferentially inhibit growth of fulvestrant resistant T47D breast cancer cell lines. Compared with parental cells, phosphorylation of Aurora kinase B was higher in the fulvestrant resistant T47D cells. Barasertib induced degradation of Aurora kinase B, caused mitotic errors, and induced apoptotic cell death as measured by accumulation of SubG1 cells and PARP cleavage in the fulvestrant resistant cells. Barasertib also exerted preferential growth inhibition of tamoxifen resistant T47D cell lines. Finally, high percentage of Aurora kinase B positive tumor cells was significantly associated with reduced disease-free and overall survival in 261 ER-positive breast cancer patients, who have received tamoxifen as first-line adjuvant endocrine treatment., Conclusions: Our results indicate that Aurora kinase B is a driving factor for growth of antiestrogen resistant T47D breast cancer cell lines, and a biomarker for reduced benefit of tamoxifen treatment. Thus, inhibition of Aurora kinase B, e.g. with the highly selective kinase inhibitor barasertib, could be a candidate new treatment for breast cancer patients with acquired resistance to antiestrogens.

Evolution of the breast cancer genome from pre-invasive stages to asynchronous metastasis is complex and mostly unexplored, but highly demanded as it may provide novel markers for and mechanistic insights in cancer progression. The increasing use of personalized therapy of breast cancer necessitates knowledge of the degree of genomic concordance between different steps of malignant progression as primary tumors often are used as surrogates of systemic disease. Based on exome sequencing we performed copy number profiling and point mutation detection on successive steps of breast cancer progression from one breast cancer patient, including two different regions of Ductal Carcinoma In Situ (DCIS), primary tumor and an asynchronous metastasis. We identify a remarkable landscape of somatic mutations, retained throughout breast cancer progression and with new mutational events emerging at each step. Our data, contrary to the proposed model of early dissemination of metastatic cells and parallel progression of primary tumors and metastases, provide evidence of linear progression of breast cancer with relatively late dissemination from the primary tumor. The genomic discordance between the different stages of tumor evolution in this patient emphasizes the importance of molecular profiling of metastatic tissue directing molecularly targeted therapy at recurrence.

The underlying mechanisms leading to antiestrogen resistance in estrogen-receptor α (ER)-positive breast cancer is still poorly understood. The aim of this study was therefore to identify biomarkers and novel treatments for antiestrogen resistant breast cancer. We performed a kinase inhibitor screen on antiestrogen responsive T47D breast cancer cells and T47D-derived tamoxifen and fulvestrant resistant cell lines. We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells. Furthermore western blot analysis showed increased expression and phosphorylation of Src in the resistant cells and that dasatinib inhibited phosphorylation of Src and also signaling via Akt and Erk in all cell lines. Immunoprecipitation revealed Src: ER complexes only in the parental T47D cells. In fulvestrant resistant cells, Src formed complexes with the Human Epidermal growth factor Receptor (HER)1 and HER2. Neither HER receptors nor ER were co-precipitated with Src in the tamoxifen resistant cell lines. Compared to treatment with dasatinib alone, combined treatment with dasatinib and fulvestrant had a stronger inhibitory effect on tamoxifen resistant cell growth, whereas dasatinib in combination with tamoxifen had no additive inhibitory effect on fulvestrant resistant growth. When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival. In conclusion, Src was identified as target for treatment of antiestrogen resistant T47D breast cancer cells. For tamoxifen resistant T47D cells, combined treatment with dasatinib and fulvestrant was superior to treatment with dasatinib alone. Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen.

Background: In more than 70% of families with a strong history of breast and ovarian cancers, pathogenic mutation in BRCA1 or BRCA2 cannot be identified, even though hereditary factors are expected to be involved. It has been proposed that tumors with similar molecular phenotypes also share similar underlying pathophysiological mechanisms. In the current study, the aim was to investigate if global RNA profiling can be used to identify functional subgroups within breast tumors from families tested negative for BRCA1/2 germline mutations and how these subgroupings relate to different breast cancer patients within the same family., Methods: In the current study we analyzed a collection of 70 frozen breast tumor biopsies from a total of 58 families by global RNA profiling and promoter methylation analysis., Results: We show that distinct functional subgroupings, similar to the intrinsic molecular breast cancer subtypes, exist among non-BRCA1/2 breast cancers. The distribution of subtypes was markedly different from the distribution found among BRCA1/2 mutation carriers. From 11 breast cancer families, breast tumor biopsies from more than one affected family member were included in the study. Notably, in 8 of these families we found that patients from the same family shared the same tumor subtype, showing a tendency of familial aggregation of tumor subtypes (p-value = 1.7e-3). Using our previously developed BRCA1/2-signatures, we identified 7 non-BRCA1/2 tumors with a BRCA1-like molecular phenotype and provide evidence for epigenetic inactivation of BRCA1 in three of the tumors. In addition, 7 BRCA2-like tumors were found., Conclusions: Our finding indicates involvement of hereditary factors in non-BRCA1/2 breast cancer families in which family members may carry genetic susceptibility not just to breast cancer but to a particular subtype of breast cancer. This is the first study to provide a biological link between breast cancers from family members of high-risk non-BRCA1/2 families in a systematic manner, suggesting that future genetic analysis may benefit from subgrouping families into molecularly homogeneous subtypes in order to search for new high penetrance susceptibility genes.

In order to track the on-going changes and ultimate reliability of neural recording and stimulation arrays, it is beneficial to regularly characterize electrode arrays within the use environment. Microelectrodes used for neural stimulation or recording research can have different behaviors in-vivo vs. in- vitro, and once implanted the success of the experiment often hinges upon knowing the stability, changes, or deterioration of the electrodes. This paper describes a new instrument that is capable of batch characterizing 16 electrodes using cyclic voltammetry, electrochemical impedance spectroscopy and charge injection measurements. The latest web based technology was applied to the software design, which greatly facilitates electrode data sharing among researchers.

Expression of HOX transcript antisense intergenic RNA (HOTAIR)--a long non-coding RNA--has been examined in a variety of human cancers, and overexpression of HOTAIR is correlated with poor survival among breast, colon, and liver cancer patients. In this retrospective study, we examine HOTAIR expression in 164 primary breast tumors, from patients who do not receive adjuvant treatment, in a design that is paired with respect to the traditional prognostic markers. We show that HOTAIR expression differs between patients with or without a metastatic endpoint, respectively. Survival analysis shows that high HOTAIR expression in primary tumors is significantly associated with worse prognosis independent of prognostic markers (P = 0.012, hazard ratio (HR) 1.747). This association is even stronger when looking only at estrogen receptor (ER)-positive tumor samples (P = 0.0086, HR 1.985). In ER-negative tumor samples, we are not able to detect a prognostic value of HOTAIR expression, probably due to the limited sample size. These results are successfully validated in an independent dataset with similar associations (P = 0.018, HR 1.825). In conclusion, our findings suggest that HOTAIR expression may serve as an independent biomarker for the prediction of the risk of metastasis in ER-positive breast cancer patients.

Background: The aim was to confirm a previously defined prognostic index, combining a proliferation marker, histological grade, and estrogen receptor (ER) in different subsets of primary N0/N1 chemo-naïve breast cancer patients., Methodsdesign: In the present study, including 1,854 patients, Ki67 was used in the index (KiGE), since it is the generally accepted proliferation marker in clinical routine. The low KiGE-group was defined as histological grade 1 patients and grade 2 patients which were ER-positive and had low Ki67 expression. All other patients made up the high KiGE-group. The KiGE-index separated patients into two groups with different prognosis. In multivariate analysis, KiGE was significantly associated with disease-free survival, when adjusted for age at diagnosis, tumor size and adjuvant endocrine treatment (hazard ratio: 3.5, 95% confidence interval: 2.6-4.7, P<0.0001)., Discussion: We have confirmed a prognostic index based on a proliferation marker (Ki67), histological grade, and ER for identification of a low-risk group of patients with N0/N1 primary breast cancer. For this low-risk group constituting 57% of the patients, with a five-year distant disease-free survival of 92%, adjuvant chemotherapy will have limited effect and may be avoided.

Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority of BRCA1 tumors were basal-like while BRCA2 tumors were mainly luminal B. Using RNA profiles, we were able to distinguish BRCA1 tumors from sporadic tumors among basal-like tumors with 83% accuracy and BRCA2 from sporadic tumors among luminal B tumors with 89% accuracy. Furthermore, subtype-specific BRCA1/2 gene signatures were successfully validated in two independent data sets with high accuracies. Although additional validation studies are required, indication of BRCA1/2 involvement ("BRCAness") by RNA profiling could potentially be valuable as a tool for distinguishing pathogenic mutations from benign variants, for identification of undetected mutation carriers, and for selecting patients sensitive to new therapeutics such as PARP inhibitors.

The task of chronic monitoring and characterizing a large number of microelectrodes can be tedious and error prone, especially if needed to be done in vivo. This paper presents a lab instrument that automates the measurement and data processing, allowing for large numbers of electrodes to be characterized within a short time period. A version 1.0 of the Electrode Analyser System (EAS 1.0) has already been used in various neural engineering laboratories, as well by one electrode array manufacturer. The goal of the current work is to implement the EAS 2.0 system that provides improved performance beyond that of the 1.0 system, as well as reducing size and cost.

The Danish Quality Database of Mammography Screening (DKMS) was established in 2007, when screening was implemented on a nationwide basis and offered biennially to all Danish women aged 50-69 years. The primary aims of the database are to monitor and evaluate the quality of the screening program and - after years of follow-up - to evaluate the effect of nationwide screening on breast cancer-specific mortality. Here, we describe the database and present results for quality assurance from the first round of national screening. The steering committee for the DKMS defined eleven organizational and clinical quality indicators and standards to monitor the Danish breast cancer screening program. We calculated the relevant proportions and ratios with 95% confidence intervals for each quality indicator. All indicators were assessed on a national and regional level. Of 670,039 women invited for mammography, 518,823 (77.4%) participated. Seventy-one percent of the women received the result of their mammography examination within 10 days of screening, and 3% of the participants were recalled for further investigation. Among all detected cancers, 86% were invasive cancers, and the proportion of women with node negative cancer was 67%. There were 36% women with small cancers, and the ratio of surgery for benign lesions to malignant lesions was 1:6.3. A total of 80% of women with invasive cancers were treated with breast conserving therapy. Screening interval and interval cancers were not relevant in the first round, and data regarding radiation dose were not available at the time of evaluation. Overall, the quality indicators showed satisfactory quality in the first round of national breast cancer screening in Denmark. The DKMS is a potentially valuable tool for improving quality and conducting research in the field of breast cancer screening.

Recently, vacuum-based preservation of surgical specimens has been proposed as a safe alternative to formalin fixation at the surgical theater. The method seems feasible from a practical point of view, but no systematic study has examined the effect of vacuum sealing alone with respect to tissue preservation. In this study, we therefore subjected tissue samples from 5 different organs to treatments with and without vacuum sealing and cooling at 4°C to study the effect of vacuum sealing of surgical specimens with respect to tissue preservation and compare it with the effect of cooling. No preserving effect of vacuum sealing was observed with respect to cellular morphology, detection of immunohistochemical epitopes, or RNA integrity. In contrast, storage at 4°C was shown to preserve tissue to a higher degree than storage at room temperature for all included endpoints, independently of whether the tissue was subjected to vacuum sealing or not. We, therefore, conclude that vacuum sealing is not an alternative to cooling on ice.

Mammography screening is offered healthy women, and a high standard on professional and organizational level is mandatory not only in the screening programme but even in the diagnostic work-up and treatment. The main goal is to achieve a substantial reduction in disease specific mortality, but it is not possible to evaluate the effect on mortality until several years later, and continuously monitoring of the quality of all aspects of a screening programme is necessary. Based on other European guidelines, 11 quality indicators have been defined, and guidelines concerning organizational requirements for a Danish screening programme as well as recommendations for the radiographic and radiological work have been drawn up.

Introduction: The sentinel lymph node (SLN) procedure has emerged as a safe staging method with a low morbidity. The objective of the present study was to examine the recurrence rates including especially the axillary recurrence rate in SLN-negative patients after a long follow-up period., Material and Methods: A total of 344 breast cancer patients were referred to SLN biopsy at our department from January 2000 to May 2005. Lymphoscintigraphy with (99m)Tc-nanocolloid was followed by same-day radioprobe-guided surgery. Among the 344 patients, 181 were SLN-negative. The group of SLN-negative patients was followed with regard to recurrence in general and axillary recurrence in particular by reviewing their respective medical files from control visits., Results: The identification rate (IR) was 99% (340/344). Extra-axillary SLNs were detected in seven patients (4%). One patient had an axillary recurrence 39 months after the primary operation, corresponding to an axillary recurrence rate of 0.6% after a median follow-up of 60 months (range 7-93)., Conclusion: With a high IR and an axillary recurrence rate of 0.6% after five years of follow-up, our data suggest that the SLN procedure is a valid and accurate method for the staging of breast cancer patients.

Introduction: In the year 2000 a quality assurance programme for the preoperative breast diagnostics was introduced in Denmark. The programme was based on the "European guidelines for quality assurance in breast cancer screening and diagnosis" where - among other measures - five cytological diagnostic classes were introduced. The aim of this study was to evaluate the quality assurance programme in a screening population to determine whether fine needle aspiration cytology (FNAC) as first choice remains a useful tool in the preoperative diagnostics, or if needle core biopsy should be the first-choice treatment., Material and Methods: 767 women had FNAC performed from a total of 783 lesions at the Mammography Clinic, University Hospital Odense. All FNACs were compared with the final histology diagnosis. Nine statistical parameters were calculated according to the European guidelines., Results: A total of 66% of the 783 FNACs had a malignant cytology diagnosis, which in 99% of the cases turned out to be the correct diagnosis. Four lesions were false positives all of which represented benign proliferative breast diseases. The surgical procedures in these cases were either excisional biopsy or lumpectomy. The values of eight of the nine mutually dependent statistical parameters of quality scored within the recommended threshold values. Specificity was the only parameter that fell outside the recommended threshold values., Conclusion: Although specificity in our study is lower than recommended, we find that the use of FNAC as first-choice in triple diagnostics is a useful tool.

Purpose: In the low-risk group of breast cancer patients, a subgroup experiences metastatic recurrence of the disease. The aim of this study was to examine the performance of gene sets, developed mainly from high-risk tumors, in a group of low-malignant tumors., Experimental Design: Twenty-six tumors from low-risk patients and 34 low-malignant T2 tumors from patients with slightly higher risk have been examined by genome-wide gene expression analysis. Nine prognostic gene sets were tested in this data set., Results: A 32-gene profile (HUMAC32) that accurately predicts metastasis has previously been developed from this data set. In the present study, six of the eight other gene sets have prognostic power in the low-malignant patient group, whereas two have no prognostic value. Despite a relatively small overlap between gene sets, there is high concordance of classification of samples. This, together with analysis of functional gene groups, indicates that the same pathways may be represented by several of the gene sets. However, the results suggest that low-risk patients may be classified more accurately with gene signatures developed especially for this patient group., Conclusion: Several gene sets, mainly developed in high-risk cancers, predict metastasis from low-malignant cancer.

The golden standard in non-operative breast cancer diagnosis is the triple test, a combination of clinical evaluation, mammography/ultrasound and needle biopsy, either fine needle aspiration cytology (FNAC) or histological core biopsy. FNAC and core biopsy both have advantages and disadvantages but neither of them can act as a decisive diagnostic procedure on its own. The final diagnosis should always be a consensus between the three diagnostic modalities in the triple test. Quality assurance of the pathological diagnosis is a must. The number of uncertain diagnoses i.e. atypia or suspicion of malignancy should be kept at a minimum. These diagnostic categories call for additional diagnostic procedures and thereby cause a delay in reaching the final diagnosis leading to definitive treatment.