Correction: Long-term Follow-up and Safety of Patients after an Upfront Therapy with Letrozole for Early Breast Cancer in Routine Clinical Care - The PreFace Study.

[This corrects the article DOI: 10.1055/a-2238-3153.].

A content-based review of mobile health applications for breast cancer prevention and education: Characteristics, quality and functionality analysis.

Objective: Mobile Health apps could be a feasible and effective tool to raise awareness for breast cancer prevention and to support women to change their behaviour to a healthier lifestyle. The aim of this study was to analyse the characteristics and quality of apps designed for breast cancer prevention and education. Methods: We conducted a systematic search for apps covering breast cancer prevention topics in the Google Play and Apple App Store accessible from Germany using search terms either in German or in English. Only apps with a last update after June 2020 were included. The apps identified were downloaded and evaluated by two independent researchers. App quality was analysed using the Mobile Application Rating Scale (MARS). Associations of app characteristics and MARS rating were analysed. Results: We identified 19 apps available in the Google Play Store and seven apps available in the Apple App Store that met all inclusion criteria. The mean MARS score was 3.07 and 3.50, respectively. Functionality was the highest-scoring domain. Operating system, developer (healthcare), download rates and time since the last update were significantly associated with overall MARS score. In addition, the presence of the following app functions significantly influenced MARS rating: breast self-examination tutorial, reminder for self-examination, documentation feature and education about breast cancer risk factors. Conclusions: Although most of the apps offer important features for breast cancer prevention, none of the analysed apps combined all functions. The absence of healthcare professionals' expertise in developing apps negatively affects the overall quality.

Long-term Follow-up and Safety of Patients after an Upfront Therapy with Letrozole for Early Breast Cancer in Routine Clinical Care - The PreFace Study.

Introduction: Adjuvant treatment of patients with early-stage breast cancer (BC) should include an aromatase inhibitor (AI). Especially patients with a high recurrence risk might benefit from an upfront therapy with an AI for a minimum of five years. Nevertheless, not much is known about the patient selection for this population in clinical practice. Therefore, this study analyzed the prognosis and patient characteristics of postmenopausal patients selected for a five-year upfront letrozole therapy. Patients and Methods: From 2009 to 2011, 3529 patients were enrolled into the adjuvant phase IV PreFace clinical trial (NCT01908556). Postmenopausal hormone receptor-positive BC patients, for whom an upfront five-year therapy with letrozole (2.5 mg/day) was indicated, were eligible. Disease-free survival (DFS), overall survival (OS) and safety in relation to patient and tumor characteristics were assessed. Results: 3297 patients started letrozole therapy. The majority of patients (n = 1639, 57%) completed the five-year treatment. 34.5% of patients continued with endocrine therapy after the mandated five-year endocrine treatment. Five-year DFS rates were 89% (95% CI: 88-90%) and five-year OS rates were 95% (95% CI: 94-96%). In subgroup analyses, DFS rates were 83%, 84% and 78% for patients with node-positive disease, G3 tumor grading, and pT3 tumors respectively. The main adverse events (any grade) were pain and hot flushes (66.8% and 18.3% of patients). Conclusions: The risk profile of postmenopausal BC patients selected for a five-year upfront letrozole therapy showed a moderate recurrence and death risk. However, in subgroups with unfavorable risk factors, prognosis warrants an improvement, which might be achieved with novel targeted therapies.

Unbiased high-dimensional flow cytometry identified NK and DC immune cell signature in Luminal A-type and triple negative breast cancer.

Breast cancer is the most common malignancy in women worldwide and a highly heterogeneous disease. Four different subtypes are described that differ in the expression of hormone receptors as well as the growth factor receptor HER2. Treatment modalities and survival rate depend on the subtype of breast cancer. However, it is still not clear which patients benefit from immunotherapeutic approaches such as checkpoint blockade. Thus, we aimed to decipher the immune cell signature of the different breast cancer subtypes based on high-dimensional flow cytometry followed by unbiased approaches. Here, we show that the frequency of NK cells is reduced in Luminal A and B as well as triple negative breast cancer and that the phenotype of residual NK cells is changed toward regulatory CD11b-CD16- NK cells. Further, we found higher frequencies of PD-1+ CD4+ and CD8+ T cells in triple negative breast cancer. Moreover, while Luminal A-type breast cancer was enriched for CD14+ cDC2 (named type 3 DC (DC3)), CD14- cDC2 (named DC2) were more frequent in triple negative breast cancer. In contrast, HER2-enriched breast cancer did not show major alterations in the composition of the immune cell compartment in the tumor microenvironment. These findings suggest that patients with Luminal A- and B-type as well as triple negative breast cancer might benefit from immunotherapeutic approaches targeting NK cells.

RANK and RANKL Expression in Tumors of Patients with Early Breast Cancer.

Introduction: The receptor activator of nuclear factor-κB (RANK) pathway was associated with the pathogenesis of breast cancer. Several studies attempted to link the RANK/RANKL pathway to prognosis; however, with inconsistent outcomes. We aimed to further contribute to the knowledge about RANK/RANKL as prognostic factors in breast cancer. Within this study, protein expression of RANK and its ligand, RANKL, in the tumor tissue was analyzed in association with disease-free survival (DFS) and overall survival (OS) in a study cohort of patients with early breast cancer. Patients and Methods: 607 samples of female primary and early breast cancer patients from the Bavarian Breast Cancer Cases and Controls Study were analyzed to correlate the RANK and RANKL expression with DFS and OS. Therefore, expression was quantified using immunohistochemical staining of a tissue microarray. H-scores were determined with the cut-off value of 8.5 for RANK and 0 for RANKL expression, respectively. Results: RANK and RANKL immunohistochemistry were assessed by H-score. Both biomarkers did not correlate (ρ = -0.04). According to molecular subtypes, triple-negative tumors and HER2-positive tumors showed a higher number of RANK-positive tumors (H-score ≥ 8.5), however, no subtype-specific expression of RANKL could be detected. Higher RANKL expression tended to correlate with a better prognosis. However, RANK and RANKL expression could not be identified as statistically significant prognostic factors within the study cohort. Conclusions: Tumor-specific RANK and RANKL expressions are not applicable as prognostic factors for DFS and OS, but might be associated with subtype-specific breast cancer progression.

WebPPG: Feasibility and Usability of Self-Performed, Browser-Based Smartphone Photoplethysmography.

Smartphones enable and facilitate biomedical studies as they allow the recording of various biomedical signals, including photoplethysmograms (PPG). However, user engagement rates in mobile health studies are reduced when an application (app) needs to be installed. This could be alleviated by using installation-free web apps. We evaluate the feasibility of browser-based PPG recording, conducting the first usability study on smartphone-based PPG. We present an at-home study using a web app and library for PPG recording using the rear camera and flash. The underlying library is freely made available to researchers. 25 Android users participated, using their own smartphones. The study consisted of a demographic and anamnestic questionnaire, the signal recording itself (60 s), and a consecutive usability questionnaire. After filtering, heart rate was extracted (14/17 successful), signal-to-noise ratios assessed (0.64 ± 0.50 dB, mean ± standard deviation), and quality was visually inspected (12/17 usable for diagnosis). Recording was not supported in 9 cases. This was due to the browser's insufficient support for the flash light API. The app received a System Usability Scale score of 82 ± 9, which is above the 90th percentile. Overall, browser flash light support is the main limiting factor for broad device support. Thus, browser-based PPG is not yet widely applicable, although most participants feel comfortable with the recording itself. The utilization of the user-facing camera might represent a more promising approach. This study contributes to the development of low-barrier, user-friendly, installation-free smartphone signal acquisition. This enables profound, comprehensive data collection for research and clinical practice.Clinical relevance- WebPPG offers low-barrier remote diagnostic capabilities without the need for app installation.

Usability and Perception of a Wearable-Integrated Digital Maternity Record App in Germany: User Study.

Background: Although digital maternity records (DMRs) have been evaluated in the past, no previous work investigated usability or acceptance through an observational usability study. Objective: The primary objective was to assess the usability and perception of a DMR smartphone app for pregnant women. The secondary objective was to assess personal preferences and habits related to online information searching, wearable data presentation and interpretation, at-home examination, and sharing data for research purposes during pregnancy. Methods: A DMR smartphone app was developed. Key features such as wearable device integration, study functionalities (eg, questionnaires), and common pregnancy app functionalities (eg, mood tracker) were included. Women who had previously given birth were invited to participate. Participants completed 10 tasks while asked to think aloud. Sessions were conducted via Zoom. Video, audio, and the shared screen were recorded for analysis. Task completion times, task success, errors, and self-reported (free text) feedback were evaluated. Usability was measured through the System Usability Scale (SUS) and User Experience Questionnaire (UEQ). Semistructured interviews were conducted to explore the secondary objective. Results: A total of 11 participants (mean age 34.6, SD 2.2 years) were included in the study. A mean SUS score of 79.09 (SD 18.38) was achieved. The app was rated "above average" in 4 of 6 UEQ categories. Sixteen unique features were requested. We found that 5 of 11 participants would only use wearables during pregnancy if requested to by their physician, while 10 of 11 stated they would share their data for research purposes. Conclusions: Pregnant women rely on their medical caregivers for advice, including on the use of mobile and ubiquitous health technology. Clear benefits must be communicated if issuing wearable devices to pregnant women. Participants that experienced pregnancy complications in the past were overall more open toward the use of wearable devices in pregnancy. Pregnant women have different opinions regarding access to, interpretation of, and reactions to alerts based on wearable data. Future work should investigate personalized concepts covering these aspects.

Ex Vivo Chromosomal Radiosensitivity Testing in Patients with Pathological Germline Variants in Breast Cancer High-Susceptibility Genes BReast CAncer 1 and BReast CAncer 2.

BACKGROUND: Individual radiosensitivity is an important factor in the occurrence of undesirable consequences of radiotherapy. The potential for increased radiosensitivity has been linked to highly penetrant heterozygous mutations in DNA repair genes such as BRCA1 and BRCA2. By studying the chromosomal radiosensitivity of BRCA1/2 mutation carriers compared to the general population, we study whether increased chromosomal radiation sensitivity is observed in patients with BRCA1/2 variants. METHODS: Three-color-fluorescence in situ hybridization was performed on ex vivo-irradiated peripheral blood lymphocytes from 64 female patients with a heterozygous germline BRCA1 or BRCA2 mutation. Aberrations in chromosomes #1, #2 and #4 were analyzed. Mean breaks per metaphase (B/M) served as the parameter for chromosomal radiosensitivity. The results were compared with chromosomal radiosensitivity in a cohort of generally healthy individuals and patients with rectal cancer or breast cancer. RESULTS: Patients with BRCA1/2 mutations (n = 64; B/M 0.47) overall showed a significantly higher chromosomal radiosensitivity than general healthy individuals (n = 211; B/M 0.41) and patients with rectal cancer (n = 379; B/M 0.44) and breast cancer (n = 147; B/M 0.45) without proven germline mutations. Chromosomal radiosensitivity varied depending on the locus of the BRCA1/2 mutation. CONCLUSIONS: BRCA1/2 mutations result in slightly increased chromosomal sensitivity to radiation. A few individual patients have a marked increase in radiation sensitivity. Therefore, these patients are at a higher risk for adverse therapeutic consequences.

Feasibility and Acceptance of Self-Guided Mobile Ultrasound among Pregnant Women in Routine Prenatal Care.

BACKGROUND AND OBJECTIVES: Mobile and remote ultrasound devices are becoming increasingly available. The benefits and possible risks of self-guided ultrasound examinations conducted by pregnant women at home have not yet been well explored. This study investigated aspects of feasibility and acceptance, as well as the success rates of such examinations. METHODS: In this prospective, single-center, interventional study, forty-six women with singleton pregnancies between 17 + 0 and 29 + 6 weeks of gestation were included in two cohorts, using two different mobile ultrasound systems. The participants examined the fetal heartbeat, fetal profile and amniotic fluid. Aspects of feasibility and acceptance were evaluated using a questionnaire. Success rates in relation to image and video quality were evaluated by healthcare professionals. RESULTS: Two thirds of the women were able to imagine performing the self-guided examination at home, but 87.0% would prefer live support by a professional. Concerns about their own safety and that of the child were expressed by 23.9% of the women. Success rates for locating the target structure were 52.2% for videos of the fetal heartbeat, 52.2% for videos of the amniotic fluid in all four quadrants and 17.9% for videos of the fetal profile. CONCLUSION: These results show wide acceptance of self-examination using mobile systems for fetal ultrasonography during pregnancy. Image quality was adequate for assessing the amniotic fluid and fetal heartbeat in most participants. Further studies are needed to determine whether ultrasound self-examinations can be implemented in prenatal care and how this would affect the fetomaternal outcome.

The Chorioallantoic Membrane Xenograft Assay as a Reliable Model for Investigating the Biology of Breast Cancer.

The chorioallantoic membrane (CAM) assay is an alternative in vivo model that allows for minimally invasive research of cancer biology. Using the CAM assay, we investigated phenotypical and functional characteristics (tumor grade, mitosis rate, tumor budding, hormone receptor (HR) and HER2 status, Ki-67 proliferation index) of two breast cancer cell lines, MCF-7 and MDA-MB-231, which resemble the HR+ (luminal) and triple-negative breast cancer (TNBC) subgroups, respectively. Moreover, the CAM results were directly compared with murine MCF-7- and MDA-MB-231-derived xenografts and human patient TNBC tissue. Known phenotypical and biological features of the aggressive triple-negative breast cancer cell line (MDA-MB-231) were confirmed in the CAM assay, and mouse xenografts. Furthermore, the histomorphological and immunohistochemical variables assessed in the CAM model were similar to those in human patient tumor tissue. Given the confirmation of the classical biological and growth properties of breast cancer cell lines in the CAM model, we suggest this in vivo model to be a reliable alternative test system for breast cancer research to reduce murine animal experiments.

Evaluation of automated techniques for extraction of circulating cell-free DNA for implementation in standardized high-throughput workflows.

Analysis of circulating cell-free DNA (ccfDNA) is a suitable tool for detecting somatic mutations for the purpose of making decisions on treatment, monitoring treatment response, and predicting survival. High-throughput techniques for ccfDNA extraction are essential to implementing ccfDNA testing in the clinical setting. We set out to compare two automated techniques with regard to hands-on time, ccfDNA output and integrity, and circulating mitochondrial DNA (mtDNA). CcfDNA was isolated using the EZ1&2 ccfDNA field test kit (EZ2 kit, QIAGEN) and the Maxwell RSC ccfDNA plasma kit (Maxwell kit, Promega). DNA was extracted from plasma of 30 breast cancer patients enrolled in the iMODE-B (#325_19B; 12.10.2020) study. Real-time PCR, fluorescence-based detection and automated electrophoresis were used to assess ccfDNA concentrations. The ccfDNA yield was significantly higher when extracted with the EZ2 kit. The EZ2 kit enabled the isolation of a higher proportion of short fragments and a lower proportion of long fragments, resulting in lower DNA integrity. Significantly lower mtDNA quantities were detected in the Maxwell eluate than in the EZ2 eluate. Thus, decisions on which extraction method to use should proceed on the basis of the required input for downstream applications, the anticipated fragment size and minimum hands-on time.

Return of individual genomic research results within the PRAEGNANT multicenter registry study.

PURPOSE: The PRAEGNANT study is a registry study for metastatic breast cancer patients, focusing on biomarker detection. Recently, within this study, genetic alterations in 37 breast cancer predisposition genes were analyzed and genetic findings were detected for 396 participants. The aim of this project was to return genetic results to the physicians and to analyze actions taken (e.g., disclosure of results to patients, validation of results, clinical impact, and impact on the patient's quality of life) using a questionnaire. METHODS: 235 questionnaires were sent out to the study centers, with each questionnaire representing one patient with a genetic finding. The questionnaire consisted of twelve questions in the German language, referring to the disclosure of results, validation of test results, and their impact on treatment decisions and on the patient's quality of life. RESULTS: 135 (57.5%) questionnaires were completed. Of these, 46 (34.1%) stated that results were returned to the patients. In 80.0% (N = 36) of cases where results were returned, the patient had not been aware of the finding previously. For 27 patients (64.3%), genetic findings had not been validated beforehand. All validation procedures (N = 15) were covered by the patients' health insurance. For 11 (25.0%) patients, physicians reported that the research results influenced current or future decision-making on treatment, and for 37.8% (N = 17) the results influenced whether family members will be genetically tested. CONCLUSION: This study provides novel insights into the return of research results and into clinical and personal benefits of disclosure of genetic findings within a German registry.

Smartphone-Based Colorimetric Analysis of Urine Test Strips for At-Home Prenatal Care.

OBJECTIVE: Clinical urine tests are a key component of prenatal care. As of now, urine test strips are evaluated through a time consuming, often error-prone and operator-dependent visual color comparison of test strips and reference cards by medical staff. METHODS AND PROCEDURES: This work presents an automated pipeline for urinalysis with urine test strips using smartphone camera images in home environments, combining several image processing and color combination techniques. Our approach is applicable to off-the-shelf test strips in home conditions with no additional hardware required. For development and evaluation of our pipeline we collected image data from two sources: i) A user study (26 participants, 150 images) and ii) a lab study (135 images). RESULTS: We trained a region-based convolutional neural network that is able to detect the urine test strip location and orientation in images with a wide variety of light conditions, backgrounds and perspectives with an accuracy of 85.5%. The reference card can be robustly detected through a feature matching approach in 98.6% of the images. Color comparison by Hue channel (0.81 F1-Score), Matching factor (0.80 F1-Score) and Euclidean distance (0.70 F1-Score) were evaluated to determine the urinalysis results. CONCLUSION: We show that an automated smartphone-based colorimetric analysis of urine test strips in a home environment is feasible. It facilitates examinations and provides the possibility to shift care into an at-home environment. CLINICAL IMPACT: The findings demonstrate that routine urine examinations can be transferred into the home environment using a smartphone. Simultaneously, human error is avoided, accuracy is increased and medical staff is relieved.

Identification of Two Genetic Loci Associated with Leukopenia after Chemotherapy in Patients with Breast Cancer.

PURPOSE: To identify molecular predictors of grade 3/4 neutropenic or leukopenic events (NLE) after chemotherapy using a genome-wide association study (GWAS). EXPERIMENTAL DESIGN: A GWAS was performed on patients in the phase III chemotherapy study SUCCESS-A (n = 3,322). Genotyping was done using the Illumina HumanOmniExpress-12v1 array. Findings were functionally validated with cell culture models and the genotypes and gene expression of possible causative genes were correlated with clinical treatment response and prognostic outcomes. RESULTS: One locus on chromosome 16 (rs4784750; NLRC5; P = 1.56E-8) and another locus on chromosome 13 (rs16972207; TNFSF13B; P = 3.42E-8) were identified at a genome-wide significance level. Functional validation revealed that expression of these two genes is altered by genotype-dependent and chemotherapy-dependent activity of two transcription factors. Genotypes also showed an association with disease-free survival in patients with an NLE. CONCLUSIONS: Two loci in NLRC5 and TNFSF13B are associated with NLEs. The involvement of the MHC I regulator NLRC5 implies the possible involvement of immuno-oncological pathways.

MUC1 (CA27.29) before and after Chemotherapy and Prognosis in High-Risk Early Breast Cancer Patients.

Soluble MUC1 has been discussed as a biomarker for predicting prognosis, treatment efficacy, and monitoring disease activity in breast cancer (BC) patients. Most studies in adjuvant settings have used preoperative assessment. This study, part of the SUCCESS-A trial (NCT02181101), assessed the prognostic value of soluble MUC1 before and after standard adjuvant chemotherapy. Patients with high-risk BC were treated within the SUCCESS-A trial with either three cycles of 5-fluorouracil, epirubicin, and cyclophosphamide followed by three cycles of docetaxel or three cycles of FEC followed by three cycles of docetaxel and gemcitabine. Cox regression analyses were performed to investigate the prognostic value of CA27.29 before and after chemotherapy relative to disease-free survival (DFS), along with established BC prognostic factors such as age, body mass index, tumor size, nodal status, estrogen receptor, progesterone receptor, HER2 status, and grading. Pre-chemotherapy and post-chemotherapy CA27.29 assessments were available for 2687 patients of 3754 randomized patients. Pre-chemotherapy CA27.29 assessment was associated with DFS in addition to established prognostic factors. It had no prognostic value in node-negative patients, but there was a clear association in node-positive patients. Post-chemotherapy CA27.29 assessment did not add any prognostic value, either on its own or in addition to pre-chemotherapy CA27.29 assessment.

Heart Rate Measurement Accuracy of Fitbit Charge 4 and Samsung Galaxy Watch Active2: Device Evaluation Study.

BACKGROUND: Fitness trackers and smart watches are frequently used to collect data in longitudinal medical studies. They allow continuous recording in real-life settings, potentially revealing previously uncaptured variabilities of biophysiological parameters and diseases. Adequate device accuracy is a prerequisite for meaningful research. OBJECTIVE: This study aims to assess the heart rate recording accuracy in two previously unvalidated devices: Fitbit Charge 4 and Samsung Galaxy Watch Active2. METHODS: Participants performed a study protocol comprising 5 resting and sedentary, 2 low-intensity, and 3 high-intensity exercise phases, lasting an average of 19 minutes 27 seconds. Participants wore two wearables simultaneously during all activities: Fitbit Charge 4 and Samsung Galaxy Watch Active2. Reference heart rate data were recorded using a medically certified Holter electrocardiogram. The data of the reference and evaluated devices were synchronized and compared at 1-second intervals. The mean, mean absolute error, mean absolute percentage error, Lin concordance correlation coefficient, Pearson correlation coefficient, and Bland-Altman plots were analyzed. RESULTS: A total of 23 healthy adults (mean age 24.2, SD 4.6 years) participated in our study. Overall, and across all activities, the Fitbit Charge 4 slightly underestimated the heart rate, whereas the Samsung Galaxy Watch Active2 overestimated it (-1.66 beats per minute [bpm]/3.84 bpm). The Fitbit Charge 4 achieved a lower mean absolute error during resting and sedentary activities (seated rest: 7.8 vs 9.4; typing: 8.1 vs 11.6; laying down [left]: 7.2 vs 9.4; laying down [back]: 6.0 vs 8.6; and walking slowly: 6.8 vs 7.7 bpm), whereas the Samsung Galaxy Watch Active2 performed better during and after low- and high-intensity activities (standing up: 12.3 vs 9.0; walking fast: 6.1 vs 5.8; stairs: 8.8 vs 6.9; squats: 15.7 vs 6.1; resting: 9.6 vs 5.6 bpm). CONCLUSIONS: Device accuracy varied with activity. Overall, both devices achieved a mean absolute percentage error of just <10%. Thus, they were considered to produce valid results based on the limits established by previous work in the field. Neither device reached sufficient accuracy during seated rest or keyboard typing. Thus, both devices may be eligible for use in respective studies; however, researchers should consider their individual study requirements.

Variable Expression of the Disialoganglioside GD2 in Breast Cancer Molecular Subtypes.

The disialoganglioside GD2 is a tumor-associated antigen that may allow for the application of targeted immunotherapies (anti-GD2 antibodies, GD2 CAR T cells) in patients with neuroblastoma and other solid tumors. We retrospectively investigated GD2 expression in a breast cancer cohort, using immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays (TMAs), and its impact on survival. GD2 expression on IHC (n = 568) and IF (n = 503) was investigated in relation to subtypes and patient outcome. Overall, 50.2% of the 568 IHC-assessed samples and 69.8% of the 503 IF-assessed samples were GD2-positive. The highest proportion of GD2-positive tumors was observed in luminal tumors. Significantly fewer GD2-positive cases were detected in triple-negative breast cancer (TNBC) compared with other subtypes. The proportion of GD2-expressing tumors were significantly lower in HER2-positive breast cancer in comparison with luminal tumors on IF staining (but not IHC). GD2 expression of IHC or IF was not significantly associated with disease-free or overall survival, in either the overall cohort or in individual subtypes. However, GD2 expression can be seen in more than 50% of breast cancer cases, with the highest frequency in hormone receptor-positive tumors. With this high expression frequency, patients with GD2-positive advanced breast cancer of all subtypes may benefit from GD2-targeting immunotherapies, which are currently subject to clinical testing.

Comparison of methods for isolation and quantification of circulating cell-free DNA from patients with endometriosis.

RESEARCH QUESTION: Which is the optimal extraction method for isolating and quantifying circulating cell-free DNA (ccfDNA) from patients with endometriosis? Endometriosis is a common benign disease, associated with pain, infertility and reduced quality of life. Endometriosis is also a known risk factor for various cancers. Robust biomarkers for early detection and prediction of prognosis, however, are lacking. CcfDNA is an easy to obtain biomarker associated with prognosis of cancer patients and enables non-invasive analysis of somatic mutations. Recently, elevated levels of ccfDNA were detected in patients with endometriosis. DESIGN: Two different ccfDNA extraction methods were compared: Maxwell RSC ccfDNA plasma kit (Maxwell) and QiAamp minElute ccfDNA mini kit (QIAamp). The ccfDNA and circulating mitochondrial DNA (mtDNA) quantities from 34 patients diagnosed with endometriosis were analysed. Fluorometric measurement and quantitative reverse transcription polymerase chain reaction (qRT-PCR) of short and long ALU and mtDNA fragments were used to quantiy ccfDNA. RESULTS: The yield of ccfDNA isolated with the Maxwell method was significantly higher compared with the QIAamp method (P < 0.0001). Integrity of ccfDNA was significantly higher in the QIAamp isolate (P < 0.0001). Recovered mtDNA was not significantly different between both extraction methods used. CONCLUSIONS: The choice of extraction method can significantly influence the ccfDNA output and integrity. Both methods, however, enabled isolation of sufficient ccfDNA for further downstream applications. With this approach, isolation of ccfDNA could enable the non-invasive detection and analysis of somatic mutation within endometriosis tissue.

Reproducibility of mRNA-Based Testing of ESR1, PGR, ERBB2, and MKI67 Expression in Invasive Breast Cancer-A Europe-Wide External Quality Assessment.

Estrogen receptor (ER), progesterone receptor (PgR), Ki-67, and HER2 immunohistochemistry (IHC) together with HER2 in situ hybridization (ISH) are utilized to classify invasive breast cancer (IBC) into predictive molecular subtypes. As IHC evaluation may be hampered by analytical errors, gene expression assays could offer a reliable alternative. In this first Europe-wide external quality assessment (EQA) study, we investigated performance of mRNA-based Xpert® Breast Cancer STRAT4 (CE-IVD) in five European laboratories. The cohort comprised ten pre-therapy IBC core biopsies diagnosed in the coordinating center (CC). STRAT4 binary (positive or negative) mRNA results of each marker (ESR1, PGR, ERBB2, MKI67) were compared with the gold standard IHC/ISH performed by the CC. Sensitivity, specificity, and accuracy of ESR1 and ERBB2 mRNA were 100% for all samples. In contrast, PGR expression was falsely negative for one case by two sites and MKI67 falsely negative for two cases (respectively by four and one sites). These cases had STRAT4 expression values close to assay cut-offs and immunohistochemically presented heterogeneous low positive PgR and heterogeneous Ki-67. Our EQA shows that STRAT4 mRNA assay may be a reproducible method to evaluate ER, PgR, HER2, and Ki-67 status. However, cases with expression values close to assay cut-offs should be carefully reviewed.

Procoagulant Extracellular Vesicles Alter Trophoblast Differentiation in Mice by a Thrombo-Inflammatory Mechanism.

Procoagulant extracellular vesicles (EV) and platelet activation have been associated with gestational vascular complications. EV-induced platelet-mediated placental inflammasome activation has been shown to cause preeclampsia-like symptoms in mice. However, the effect of EV-mediated placental thrombo-inflammation on trophoblast differentiation remains unknown. Here, we identify that the EV-induced thrombo-inflammatory pathway modulates trophoblast morphology and differentiation. EVs and platelets reduce syncytiotrophoblast differentiation while increasing giant trophoblast and spongiotrophoblast including the glycogen-rich cells. These effects are platelet-dependent and mediated by the NLRP3 inflammasome. In humans, inflammasome activation was negatively correlated with trophoblast differentiation marker GCM1 and positively correlated with blood pressure. These data identify a crucial role of EV-induced placental thrombo-inflammation on altering trophoblast differentiation and suggest platelet activation or inflammasome activation as a therapeutic target in order to achieve successful placentation.