CYPOR is a novel and independent prognostic biomarker of recurrence-free survival in triple-negative breast cancer patients.

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.

Genetic Incorporation of Olefin Cross-Metathesis Reaction Tags for Protein Modification.

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.

SNAI2 upregulation is associated with an aggressive phenotype in fulvestrant-resistant breast cancer cells and is an indicator of poor response to endocrine therapy in estrogen receptor-positive metastatic breast cancer.

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.

Non-linear transformations of age at diagnosis, tumor size, and number of positive lymph nodes in prediction of clinical outcome in breast cancer.

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.

Outcome of breast cancer screening in Denmark.

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.

Affinity Induced Surface Functionalization of Liposomes Using Cu-Free Click Chemistry.

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.

Long non-coding RNA expression profiles predict metastasis in lymph node-negative breast cancer independently of traditional prognostic markers.

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.

S100A14 is a novel independent prognostic biomarker in the triple-negative breast cancer subtype.

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.

Aurora kinase B is important for antiestrogen resistant cell growth and a potential biomarker for tamoxifen resistant breast cancer.

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.

Deciphering the monocyte-targeting mechanisms of PEGylated cationic liposomes by investigating the biomolecular corona.

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.

Ensemble-based classification using microRNA expression identifies a breast cancer patient subgroup with an ultralow long-term risk of metastases.

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.

HER2-targeted, enzyme-activated liposomes show superior in vivo efficacy in an ovarian cancer model.

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.

Long non-coding RNA HOTAIR is an independent prognostic marker of metastasis in estrogen receptor-positive primary breast cancer.

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.

Mechanisms of selective monocyte targeting by liposomes functionalized with a cationic, arginine-rich lipopeptide.

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.

Enhancing Adoptive Cell Therapy by T Cell Loading of SHP2 Inhibitor Nanocrystals before Infusion.

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.

Co-targeting CDK2 and CDK4/6 overcomes resistance to aromatase and CDK4/6 inhibitors in ER+ breast cancer.

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.

Mammography screening in Denmark.

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.

Low risk of recurrence in breast cancer with negative sentinel node.

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.

Comparison of the Metastasis Predictive Potential of mRNA and Long Non-Coding RNA Profiling in Systemically Untreated Breast Cancer.

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.

Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer.

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.