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Epithelial–mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific microenvironment for tumor cells, we explored a potential link between EMT and coagulation that may provide EMT-positive CTCs with enhanced colonizing properties. Here we report that EMT induces tissue factor (TF), a major cell-associated initiator of coagulation and related procoagulant properties in the blood. TF blockade by antibody or shRNA diminished the procoagulant activity of EMT-positive cells, confirming a functional role for TF in these processes. Silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity, further strengthening the link between EMT and coagulation. Accordingly, EMT-positive cells exhibited a higher persistance/survival in the lungs of mice colonized after intravenous injection, a feature diminished by TF or ZEB1 silencing. In tumor cells with limited metastatic capability, enforcing expression of the EMT transcription factor Snail increased TF, coagulant properties, and early metastasis. Clinically, we identified a subpopulation of CTC expressing vimentin and TF in the blood of metastatic breast cancer patients consistent with our observations. Overall, our findings define a novel EMT–TF regulatory axis that triggers local activation of coagulation pathways to support metastatic colonization of EMT-positive CTCs. Cancer Res; 76(14); 4270–82. ©2016 AACR.

Supplementary Materials and Methods

Supplementary Table 1: list of siRNA used. Supplementary Table 2: list of primers used. Supplementary Table 3: list of antibodies used in different applications. Supplementary Table 4: list of patients and donors information

Supplementary Figure Legends

Background The spread of the COVID‐19 pandemic has led to a rapid reorganization in all human and hospital activities, with impact on cancer patients. Aim An analysis of cancer patients fears, and awareness of COVID‐19 has been done in this study. Methods and results We analyzed cancer patients' reactions to the pandemic and their perception of oncological care reorganization, through a 12‐item survey, proposed at the peak of pandemic and 3 months later. Overall, 237 patients were included in the study. During the peak of pandemic 34.6% of patients were more worried about COVID‐19 than cancer versus 26.4% in the post‐acute phase (p = .013). Although 49.8% of patients in the acute phase and 42.3% in the post‐acute phase considered their risk of death if infected ≥50%, and more than 70% of patients thought to be at higher risk of complications, the majority of them did not consider the possibility to stop or delay their treatment. Patients were more interested in following news about COVID‐19 than cancer and they complied with all preventive measures in more than 90% of the cases. Conclusions Although cancer patients worried about COVID‐19 and evaluated the risk of complication or death due to COVID‐19 as extremely high, they were still asking for the best oncological treatment.

Background: No specific safety data concerning systemic oncological treatments were available at time of COVID-19 outbreak in Belgium. In our hospital we decided to maintain adjuvant and early line treatments for metastatic disease in patients under 65 and without specific comorbidities and to apply a shared decision approach in other patients while following closely the safety of these treatments. Method(s): Real time safety monitoring was proposed to all patients treated for solid tumours in our day-care unit starting March 1st, 2020. After signing informed consent patients were asked questions concerning protective measures at home, signs of SARS-CoV-2 infection and hospitalisation. Patients' charts were reviewed for outcome, including death, after suspected or proven SARS-CoV-2 infection. Minimum follow-up was 5 weeks after day care unit attendance. Result(s): 387 patients were included in our registry between March 1st and March 31st, 2020. Median age was 64 years-old (range 27-90). Most patients suffered from lung (n=96), breast (n=93), gastrointestinal (n=87), gynaecological (n=38) or urological (n=33) cancers. 131 patients received (neo)adjuvant treatments, 256 patients were treated for metastatic disease. Patients received chemotherapy (n=170), immunotherapy (n=103), targeted therapy (n=68) or other combinations (n=46). Although Belgium had one of the highest infection rates in the world, safety data concerning risk of SARS-CoV-2 infection and outcomes were rather reassuring. A total of 11 patients had either suspected (n=5, 1.3%) or proven (n=6, 1.6%) SARS-CoV-2 infection. Only one 74 years old patient died of COVID-19, another 51 years old patient died of progressive disease but presented also suspicion of SARS-CoV-2 infection at the time of death. Conclusion(s): Analysis of our data for patients treated in March 2020 in the day-care unit are reassuring and suggest higher risk related to under-treatment compared to risk related to continuation of systemic therapy at time of COVID-19 outbreak. Patients' follow-up will be updated and additional analyses and data in particular for April 2020, when the infection rate was still extremely high in Belgium, will be presented. Legal entity responsible for the study: The authors. Funding(s): Fondation Leon Fredericq. Disclosure: A. Rorive: Travel/Accommodation/Expenses: BMS;MSD. B. Sautois: Advisory/Consultancy, Travel/Accommodation/Expenses: Janssen;Advisory/Consultancy: Clovis;Sanofi;Astellas. J. Collignon: Advisory/Consultancy, Travel/Accommodation/Expenses: Roche;Amgen;Pfizer;Advisory/Consultancy: Servier;Bayer;Merck;Lilly;Sanofi;Sirtex;Celgene;Ipsen;Novartis. P. Freres: Advisory/Consultancy: Ipsen;Merck;BMS. C. Gennigens: Advisory/Consultancy, Research grant/Funding (institution): Astra-Zeneca;Advisory/Consultancy: BMS;GSK;Lilly;MSD;Advisory/Consultancy, Travel/Accommodation/Expenses: Ipsen;Pfizer;Pharmamar;Roche. G. Jerusalem: Advisory/Consultancy, Research grant/Funding (institution), Travel/Accommodation/Expenses: Novartis;Roche;Pfizer;Speaker Bureau/Expert testimony, Travel/Accommodation/Expenses: Lilly;Advisory/Consultancy, Research grant/Funding (institution): Amgen;Advisory/Consultancy, Travel/Accommodation/Expenses: BMS;AstraZeneca;Daiichi Sankyo;Advisory/Consultancy: Abbvie;Travel/Accommodation/Expenses: Medimmune;MerckKGaA. All other authors have declared no conflicts of interest.Copyright © 2020

Non-coding RNAs (ncRNA) represent 1/5 of the mammalian transcript number, and 90% of the genome length is transcribed. Many ncRNAs play a role in cancer. Among them, non-coding natural antisense transcripts (ncNAT) are RNA sequences that are complementary and overlapping to those of either protein-coding (PCT) or non-coding transcripts. Several ncNATs were described as regulating protein coding gene expression on the same loci, and they are expected to act more frequently in cis compared to other ncRNAs that commonly function in trans. In this work, 22 breast cancers expressing estrogen receptors and their paired adjacent non-malignant tissues were analyzed by strand-specific RNA sequencing. To highlight ncNATs potentially playing a role in protein coding gene regulations that occur in breast cancer, three different data analysis methods were used: differential expression analysis of ncNATs between tumor and non-malignant tissues, differential correlation analysis of paired ncNAT/PCT between tumor and non-malignant tissues, and ncNAT/PCT read count ratio variation between tumor and non-malignant tissues. Each of these methods yielded lists of ncNAT/PCT pairs that were enriched in survival-associated genes. This work highlights ncNAT lists that display potential to affect the expression of protein-coding genes involved in breast cancer pathology.

Background: Triple-negative breast cancers (TNBC) are heterogeneous cancers with poor prognosis. We aimed to determine the clinical relevance of membrane type-4 matrix metalloproteinase (MT4-MMP), a membrane type matrix metalloproteinase that interacts with epidermal growth factor receptor (EGFR) overexpressed in >50% of TNBC. Methods: We conducted a retrospective immunohistochemical analysis on human TNBC samples (n=81) and validated our findings in in vitro and in vivo assays. Results: Membrane type-4 matrix metalloproteinase and EGFR are produced in 72.5% of TNBC samples, whereas those proteins are faintly produced by healthy tissues. Unexpectedly, tumour relapse after chemotherapy was reduced in samples highly positive for MT4-MMP. Mechanistically, this is ascribed to a higher sensitivity of MT4-MMP-producing cells to alkylating or intercalating chemotherapeutic agents, as assessed in vitro. In sharp contrast, MT4-MMP expression did not affect tumour cell sensitivity to paclitaxel that interferes with protease trafficking. Importantly, MT4-MMP expression sensitised cancer cells to erlotinib, a tyrosine kinase EGFR inhibitor. In a pre-clinical model, the growth of MT4-MMP overexpressing xenografts, but not of control ones, was reduced by epirubicin or erlotinib. The combination of suboptimal drug doses blocked drastically the growth of MT4-MMP-producing tumours. Conclusions: We demonstrate that MT4-MMP defines a sub-population of TNBC sensitive to a combination of DNA-targeting chemotherapeutic agents and anti-EGFR drugs.

The authors asked breast cancer (BC) patients to participate in 1 of 3 mind-body interventions (cognitive-behavioral therapy (CBT), yoga, or self-hypnosis) to explore their feasibility, ease of compliance, and impact on the participants' distress, quality of life (QoL), sleep, and mental adjustment. Ninety-nine patients completed an intervention (CBT: n = 10; yoga: n = 21; and self-hypnosis: n = 68). Results showed high feasibility and high compliance. After the interventions, there was no significant effect in the CBT group but significant positive effects on distress in the yoga and self-hypnosis groups, and, also, on QoL, sleep, and mental adjustment in the self-hypnosis group. In conclusion, mind-body interventions can decrease distress in BC patients, but RCTs are needed to confirm these findings.

Epithelial–mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific microenvironment for tumor cells, we explored a potential link between EMT and coagulation that may provide EMT-positive CTCs with enhanced colonizing properties. Here we report that EMT induces tissue factor (TF), a major cell-associated initiator of coagulation and related procoagulant properties in the blood. TF blockade by antibody or shRNA diminished the procoagulant activity of EMT-positive cells, confirming a functional role for TF in these processes. Silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity, further strengthening the link between EMT and coagulation. Accordingly, EMT-positive cells exhibited a higher persistance/survival in the lungs of mice colonized after intravenous injection, a feature diminished by TF or ZEB1 silencing. In tumor cells with limited metastatic capability, enforcing expression of the EMT transcription factor Snail increased TF, coagulant properties, and early metastasis. Clinically, we identified a subpopulation of CTC expressing vimentin and TF in the blood of metastatic breast cancer patients consistent with our observations. Overall, our findings define a novel EMT–TF regulatory axis that triggers local activation of coagulation pathways to support metastatic colonization of EMT-positive CTCs. Cancer Res; 76(14); 4270–82. ©2016 AACR.

The complement system is a crucial part of innate and adaptive immunity which exerts a significant evolutionary pressure on pathogens. It has selected for those pathogens, mainly micro-organisms but also parasites, that have evolved countermeasures. The characterization of how pathogens evade complement attack is a rapidly developing field of current research. In recent years, multiple complement evasion strategies have been characterized. In this review, we focus on complement escape mechanisms expressed by hematophagous parasites, a heterogeneous group of metazoan parasites that share the property of ingesting the whole blood of their host. Complement inhibition is crucial for parasite survival within the host tissue or to facilitate blood feeding. Finally, complement inhibition by hematophagous parasites may also contribute to their success as pathogen vectors.

The alternative pathway of complement is an important innate defence against pathogens including ticks. This component of the immune system has selected for pathogens that have evolved countermeasures. Recently, a salivary protein able to inhibit the alternative pathway was cloned from the American tick Ixodes scapularis (Valenzuela et al., 2000; J. Biol. Chem. 275, 18717–18723). Here, we isolated two different sequences, similar to Isac, from the transcriptome of I. ricinus salivary glands. Expression of these sequences revealed that they both encode secreted proteins able to inhibit the complement alternative pathway. These proteins, called I. ricinus anticomplement (IRAC) protein I and II, are coexpressed constitutively in I. ricinus salivary glands and are upregulated during blood feeding. Also, we demonstrated that they are the products of different genes and not of alleles of the same locus. Finally, phylogenetic analyses demonstrate that ticks belonging to the Ixodes ricinus complex encode a family of relatively small anticomplement molecules undergoing diversification by positive Darwinian selection.

Circulating microRNAs (miRNAs) are increasingly recognized as powerful biomarkers in several pathologies, including breast cancer. Here, their plasmatic levels were measured to be used as an alternative screening procedure to mammography for breast cancer diagnosis. A plasma miRNA profile was determined by RT-qPCR in a cohort of 378 women. A diagnostic model was designed based on the expression of 8 miRNAs measured first in a profiling cohort composed of 41 primary breast cancers and 45 controls, and further validated in diverse cohorts composed of 108 primary breast cancers, 88 controls, 35 breast cancers in remission, 31 metastatic breast cancers and 30 gynecologic tumors. A receiver operating characteristic curve derived from the 8-miRNA random forest based diagnostic tool exhibited an area under the curve of 0.81. The accuracy of the diagnostic tool remained unchanged considering age and tumor stage. The miRNA signature correctly identified patients with metastatic breast cancer. The use of the classification model on cohorts of patients with breast cancers in remission and with gynecologic cancers yielded prediction distributions similar to that of the control group. Using a multivariate supervised learning method and a set of 8 circulating miRNAs, we designed an accurate, minimally invasive screening tool for breast cancer.

Triple-negative breast cancers (TNBCs) are defined by the absence of estrogen and progesterone receptors and the absence of HER2 overexpression. These cancers represent a heterogeneous breast cancer subtype with a poor prognosis. Few systemic treatment options exist besides the use of chemotherapy (CT). The heterogeneity of the disease has limited the successful development of targeted therapy in unselected patient populations. Currently, there are no approved targeted therapies for TNBC. However, intense research is ongoing to identify specific targets and develop additional and better systemic treatment options. Standard adjuvant and neoadjuvant regimens include anthracyclines, cyclophosphamide, and taxanes. Platinum-based CT has been proposed as another CT option of interest in TNBC. We review the role of this therapy in general, and particularly in patients carrying BRCA germ-line mutations. Available data concerning the role of platinum-based CT in TNBC were acquired primarily in the neoadjuvant setting. The routine use of platinum-based CT is not yet recommended by available guidelines. Many studies have reported the molecular characterization of TNBCs. Several actionable targets have been identified. Novel therapeutic strategies are currently being tested in clinical trials based on promising results observed in preclinical studies. These targets include androgen receptor, EGFR, PARP, FGFR, and the angiogenic pathway. We review the recent data on experimental drugs in this field. We also discuss the recent data concerning immunologic checkpoint inhibitors.

Antigen expression and purification are laborious, time consuming and frequently difficult steps in the process of antibody production. In the present study, we developed a method avoiding these two steps. This method relies on the injection of histocompatible living cells stably expressing the antigen as a cell surface type II transmembrane fusion protein. A vector, nicknamed pCD1-CD134L, was constructed to express the antigen fused at the carboxyterminal end of the human CD134 ligand (CD134L) type II transmembrane protein on the surface of eucaryotic cells. This vector was shown to induce cell surface expression of epitopes from human c-Myc (soluble protein), uterogloblin-related protein 1 (secreted protein) and CD94 (type II transmembrane protein). Using this vector, we developed a method to produce antibodies without antigen production. The flowchart of this method is as follows: (i) cloning of the antigen in the pCD1-CD134L vector; (ii) production of a histocompatible cell line stably expressing the CD134L-antigen fusion protein; (iii) testing for cell surface expression of the fusion protein by targeting the CD134L carrier; and (iv) prime-boost immunisation with living cells expressing the fusion protein. This method was successfully used for production of polyclonal antibodies raised against Ixodes ricinus calreticulin (secreted protein) in mice and for production of monoclonal antibodies raised against an epitope of Vaccinia virus A56 (type I transmembrane protein) protein in rat. The present study is the first to demonstrate the use of a type II transmembrane protein as a carrier for cell surface display of antigens.

Several observations suggest that inhibition of the host complement alternative pathway by Ixodes tick saliva is crucial to achieve blood feeding. We recently described two paralogous anti-complement proteins called Ixodes ricinus anti-complement (IRAC) proteins I and II co-expressed in I. ricinus salivary glands. Phylogenetic analyses suggested that these sequences were diversifying by a process of positive Darwinian selection, possibly leading to molecules with different biological properties. In the present study, we tested the hypothesis that each paralogue may have different inhibitory activities against the complement of different natural host species, thereby contributing to broaden the host range of I. ricinus ticks. IRAC I and IRAC II were tested against the complement of eight I. ricinus natural host species (six mammals and two birds). The results demonstrate that IRAC I and IRAC II have broad and complementary inhibition activities against the complement of different host species. This report is the first description of paralogous anti-complement molecules encoded by a pathogen with broad and complementary inhibitory activities against the complement of different host species.

e11549 Background: Several retrospective studies and 3 prospective studies have shown discordance in receptor status between primary tumor and metastasis leading to a treatment modification in about one out of 7 patients. However, some clinicians report unpublished data suggesting that these findings are not observed in all centers. We did prospectively a biopsy at suspicion of first relapse in our center and evaluated the impact of this biopsy on the management of our patients. Methods: Thirty four patients underwent a biopsy between October 2011 and January 2013. Hormonal receptor status was determined by the same IHC methodology for primary and metastatic tissue. Primary tumors were reanalyzed if necessary with our current IHC technique. A change in ER or PgR status was considered clinically significant if quantitative staining changed from

International audience; Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane.