Measurable residual disease-guided treatment with azacitidine to prevent haematological relapse in patients with myelodysplastic syndrome and acute myeloid leukaemia (RELAZA2): an open-label, multicentre, phase 2 trial.

BACKGROUND: Monitoring of measurable residual disease (MRD) in patients with advanced myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML) who achieve a morphological complete remission can predict haematological relapse. In this prospective study, we aimed to determine whether MRD-guided pre-emptive treatment with azacitidine could prevent relapse in these patients. METHODS: The relapse prevention with azacitidine (RELAZA2) study is an open-label, multicentre, phase 2 trial done at nine university health centres in Germany. Patients aged 18 years or older with advanced MDS or AML, who had achieved a complete remission after conventional chemotherapy or allogeneic haemopoietic stem-cell transplantation, were prospectively screened for MRD during 24 months from baseline by either quantitative PCR for mutant NPM1, leukaemia-specific fusion genes (DEK-NUP214, RUNX1-RUNX1T1, CBFb-MYH11), or analysis of donor-chimaerism in flow cytometry-sorted CD34-positive cells in patients who received allogeneic haemopoietic stem-cell transplantation. MRD-positive patients in confirmed complete remission received azacitidine 75 mg/m2 per day subcutaneously on days 1-7 of a 29-day cycle for 24 cycles. After six cycles, MRD status was reassessed and patients with major responses (MRD negativity) were eligible for a treatment de-escalation. The primary endpoint was the proportion of patients who were relapse-free and alive 6 months after the start of pre-emptive treatment. Analyses were done per protocol. This trial is registered with ClincialTrials.gov, number NCT01462578, and finished recruitment on Aug 21, 2018. FINDINGS: Between Oct 10, 2011, and Aug 20, 2015, we screened 198 patients with advanced MDS (n=26) or AML (n=172), of whom 60 (30%) developed MRD during the 24-month screening period and 53 (88%) were eligible to start study treatment. 6 months after initiation of azacitidine, 31 (58%, 95% CI 44-72) of 53 patients were relapse-free and alive (p<0·0001; one-sided binomial test for null hypothesis pexp≤0·3). With a median follow-up of 13 months (IQR 8·5-22·8) after the start of MRD-guided treatment, relapse-free survival at 12 months was 46% (95% CI 32-59) in the 53 patients who were MRD-positive and received azacitidine. In MRD-negative patients, 12-month relapse-free survival was 88% (95% CI 82-94; hazard ratio 6·6 [95% CI 3·7-11·8], p<0·0001). The most common (grade 3-4) adverse event was neutropenia, occurring in 45 (85%) of 53 patients. One patient with neutropenia died because of an infection considered possibly related to study treatment. INTERPRETATION: Pre-emptive therapy with azacitidine can prevent or substantially delay haematological relapse in MRD-positive patients with MDS or AML who are at high risk of relapse. Our study also suggests that continuous MRD negativity during regular MRD monitoring might be prognostic for patient outcomes. FUNDING: Celgene Pharma, José Carreras Leukaemia Foundation, National Center for Tumor Diseases (NCT), and German Cancer Consortium (DKTK) Foundation.

Agonists and antagonists of GnRH-I and -II reduce metastasis formation by triple-negative human breast cancer cells in vivo.

Metastasis to bone is a frequent problem of advanced breast cancer. Particularly breast cancers, which do not express estrogen and progesterone receptors and which have no overexpression/amplification of the HER2-neu gene, so called triple-negative breast cancers, are considered as very aggressive and possess a bad prognosis. About 60% of all human breast cancers and about 74% of triple-negative breast cancers express receptors for gonadotropin-releasing hormone (GnRH), which might be used as a therapeutic target. Recently, we could show that bone-directed invasion of human breast cancer cells in vitro is time- and dose-dependently reduced by GnRH analogs. In the present study, we have analyzed whether GnRH analogs are able to reduce metastases of triple-negative breast cancers in vivo. In addition, we have evaluated the effects of GnRH analogs on tumor growth. To quantify formation of metastasis by triple-negative MDA-MB-435 and MDA-MB-231 human breast cancers, we used a real-time PCR method based on detection of human-specific alu sequences measuring accurately the amount of human tumor DNA in athymic mouse organs. To analyze tumor growth, the volumes of breast cancer xenotransplants into nude mice were measured. We could demonstrate that GnRH analogs significantly reduced metastasis formation by triple-negative breast cancer in vivo. In addition, we could show that GnRH analogs significantly inhibited the growth of breast cancer into nude mice. Side effects were not detectable. In conclusion, GnRH analogs seem to be suitable drugs for an efficacious therapy for triple-negative, GnRH receptor-positive human breast cancers to prevent metastasis formation.

Kisspeptin-10 inhibits bone-directed migration of GPR54-positive breast cancer cells: Evidence for a dose-window effect.

OBJECTIVES: The KiSS-1 gene product is absent or expressed at low level in metastatic breast cancer compared with their nonmetastatic counterparts. A deca-peptide derived from the KiSS-1 gene product, designated kisspeptin-10 (Kp-10), activates a receptor coupled to Gαq subunits (GPR54 or KiSS-1R). In this study we have analyzed whether Kp-10 treatment affects bone-directed migration of GPR54-positive breast cancer cells. METHODS: GPR54 expression was analyzed using immune cytochemistry. Bone-directed breast cancer cell invasion was measured by assessment of the breast cancer cell migration rate through an artificial basement membrane. Chemokine receptor CXCR4 and stromal cell-derived factor-1 (SDF-1) mRNA expression was quantified using semi-quantitative RT-PCR. CXCR4 protein expression and SDF-1 protein secretion were measured using the western blot technique. RESULTS: Breast cancer cell invasion was increased when cocultured with MG63 osteoblast-like cells. Treatment with KP-10 reduced the ability to invade a reconstituted basement membrane and to migrate in response to the cellular stimulus. This effect was significant in a dose-window of 10⁻9 M to 10⁻¹¹ M. Searching for the molecular mechanisms we found that KP-10 treatment significantly reduces expression of the chemokine receptor CXCR4 by the breast cancer cells. In addition, expression and secretion of its ligand SDF-1 by the MG63 cells were significantly reduced. Furthermore, SDF-1-induced CXCR4 signaling was down-regulated. CONCLUSIONS: These data represent the first report that KP-10 inhibits bone-directed migration of GPR54-positive breast cancer cells. In addition, we found evidence for a KP-10 dose-window effect. Furthermore, the SDF-1/CXCR4 system seems to be involved in the anti-migratory action of KP-10.

[What speech therapists, occupational therapists and physical therapist need to know to become evidence-based practitioners: A cross-sectional study]. / Lernbedarf von PraktikerInnen der Logopädie, Ergotherapie und Physiotherapie für evidenzbasiertes Arbeiten - Querschnittsstudie.

BACKGROUND: Today we are faced with changes in society and healthcare needs resulting from demographic transition. Among many other developments, the increasing complexity of healthcare creates new requirements for health professionals. To meet these requirements, such as evidence-based practice, professionally experienced therapists need additional training courses to acquire scientific competencies in addition to their professional competence. Certification courses for practitioners in the fields of speech therapy, occupational therapy and physical therapy offer a way to achieve these scientific competencies. AIM: The aim of this study was to gather empirical data that help to develop scientific contents for additional training courses on a higher education level. These contents must fit the learning needs of the target group with regard to evidence-based practice. METHODS: In a multi-method approach a questionnaire was created consisting of four parts. These contained closed questions on self-assessed learning needs for competencies in evidence-based practice with and without links to therapeutic practice, open questions about the design of a certification course as well as socio-demographic questions. The results were shown using frequencies and correlations between learning, work experience and level of education. Contextual correlations were calculated using Kendall's tau correlation of Cramer's V, a contingency coefficient based on Chi square. Answers to the open questions were assigned to subcategories. RESULTS: The answers of 70 therapists (11,5 % speech therapist, 17,1 % occupational therapists, 70 % physical therapists, 1,4 % unspecified) were evaluated. The results demonstrate that there are high learning needs (> 80 %) of the total sample regarding content of assessments and test procedures, evidence-based practice as well as clinical decision-making and interprofessional cooperation. The therapists see a high demand for additional skills combined with scientific knowledge and their own professional experience (92,8 %). Almost 90 % identified high learning requirements for the skill of critically reflecting on the treatments they provided. Depending on prior professional experience, learning needs differed for evidence-based practice with and without links to therapeutic practice. As to the design of certification courses, the participants preferred low costs, a flexible time-structure as well as teaching methods making the input more vivid and comprehensible. DISCUSSION: Scientific qualification of healthcare practitioners must address the learning requirements for evidence-based practice. For this purpose, teaching of scientific work skills such as scientific writing, evidence-based practice and quantitative and qualitative methods is recommended. The fact that the questionnaire has not been validated may limit the validity of the results. Due to the indirect distribution of the questionnaires, the survey may have been liable to non-response bias.

Expression of osteoprotegerin and receptor activator of nuclear factor-kappaB ligand (RANKL) in HCC70 breast cancer cells and effects of treatment with gonadotropin-releasing hormone on RANKL expression.

BACKGROUND: The majority of human breast cancers and in addition most breast-cancer cell lines express gonadotropin-releasing hormone (GnRH) receptors. Their proliferation and in addition their bone-directed invasion is time- and dose-dependently reduced by GnRH. Osteolytic metastases are characteristic for breast cancer-derived metastasis. Since the osteolytic activity depends on the receptor activator of nuclear factor-kappaB (NFkappaB) ligand (RANKL)/osteoprotegerin (OPG) ratio, we analyzed RANKL and OPG expression in different breast-cancer cell lines. METHODS: Different human breast-cancer cell lines were tested for expression of GnRH receptor, OPG and RANKL. Using a co-culture system of breast-cancer cell lines and human primary osteoblasts (hOB), we analyzed the expression of OPG and RANKL in the GnRH receptor-positive breast-cancer cell line HCC70 co-cultured with or without hOB. In addition, we assessed the effects of GnRH analog treatment on OPG and RANKL mRNA and protein levels. RESULTS: All tested breast-cancer cell lines were GnRH receptor-positive. The majority of these cell lines expressed OPG but not RANKL. The HCC70 breast-cancer cell line derived from an invasive ductal carcinoma with metastases was positive for both OPG and RANKL. The expression of RANKL by HCC70 cells was increased when co-cultured with hOB. Treatment with GnRH analogs reduced the expression of RANKL by HCC70 cells co-cultured with hOB. No effects were observed on breast cancer OPG expression. CONCLUSIONS: These data show that the majority of human breast-cancer cell lines express OPG but not RANKL. The HCC70 breast-cancer cell line is RANKL-positive. Co-culture of HCC70 breast cancer cells with hOB increases RANKL expression. Activation of tumor GnRH receptors reduces RANKL expression. These experiments demonstrate that HCC70 breast cancer cells are able to activate osteoclasts directly via RANKL. The interaction between HCC70 breast cancer cells and osteoblasts induces osteoclastogenesis through an increase of RANKL expression. GnRH seems to play an important role by modulating the RANKL expression in HCC70 breast cancer cells.

Invasion and increased expression of S100A4 and CYR61 in mesenchymal transformed breast cancer cells is downregulated by GnRH.

S100 calcium binding protein A4 (S100A4) and cysteine-rich angiogenic inducer 61 (CYR61) play important roles in epithelial-mesenchymal-transition (EMT), invasion and metastasis by promoting cancer cell motility. Recently we were able to show that invasion of GnRH receptor-positive breast cancer cells is time- and dose-dependently reduced by GnRH analogs. We have now analyzed whether GnRH treatment affects S100A4 and CYR61 in mesenchymal transformed breast cancer cells. S100A4 and CYR61 expression was analyzed using RT-PCR. Invasion was quantified by assessment of breast cancer cell migration rate through an artificial basement membrane. The role of S100A4 and CYR61 in invasion of breast cancer cells was analyzed by neutralizing their biological activity. Expression of S100A4, CYR61 and GnRH receptor in human breast cancers, normal and other non-malignant breast tissues was analyzed by immuno-histochemistry. Invasion and expression of S100A4 and CYR61 in MDA-MB-231 breast cancer cells were significant higher as compared with MCF-7 breast cancer cells. Invasion and expression of S100A4 and CYR61 were significantly increased in mesenchymal transformed MCF-7 cells (MCF-7-EMT). The increased invasion of MCF-7-EMT cells could be reduced by anti-S100A4 and anti-CYR61 antibodies. In addition, invasion of MDA-MB-231 cells was decreased by anti-S100A4 and anti-CYR61 antibodies. Treatment of MCF-7-EMT and MDA-MB-231 cells with GnRH agonist Triptorelin resulted in a significant decrease of invasion and expression of S100A4 and CYR61. Both, S100A4 and CYR61 were found highly expressed in biopsy specimens of breast hyperplasia and malignant breast cancers. GnRH receptor expression was detectable in approximately 71% of malignant breast cancers. Our findings suggest that S100A4 and CYR61 play major roles in breast cancer invasion. Both, invasion and expression of S100A4 and CYR61 can be inhibited by GnRH treatment.