Prognostic impact of selection criteria of current adjuvant endocrine therapy trials NATALEE and monarchE in postmenopausal HRpos/HER2neg breast cancer patients treated with upfront letrozole.

BACKGROUND: The monarchE and NATALEE trials demonstrated the benefit of CDK4/6 inhibitor (CDK4/6i) therapy in adjuvant breast cancer (BC) treatment. Patient selection, based on clinical characteristics, delineated those at high (monarchE) and high/intermediate recurrence risk (NATALEE). This study employed a historical patient cohort to describe the proportion and prognosis of patients eligible for adjuvant CDK4/6i trials. METHODS: Between 2009 and 2011, 3529 patients were enrolled in the adjuvant PreFace clinical trial (NCT01908556). Eligibility criteria included postmenopausal patients with hormone receptor-positive (HRpos) BC for whom a five-year upfront therapy with letrozole was indicated. Patients were categorized into prognostic groups according to monarchE and NATALEE inclusion criteria, and their invasive disease-free survival (iDFS) and overall survival (OS) were assessed. RESULTS: Among 2891 HRpos patients, 384 (13.3 %) met the primary monarchE inclusion criteria. The majority (n = 261) qualified due to having ≥ 4 positive lymph nodes. For NATALEE, 915 out of 2886 patients (31.7 %) met the eligibility criteria, with 126 patients (13.7 %) being node-negative. Patients from monarchE with ≥ 4 positive lymph nodes and NATALEE with stage III BC exhibited the poorest prognosis (3-year iDFS rate 0.87). Patients ineligible for the trials demonstrated prognoses similar to the most favorable patient groups within the eligibility criteria. CONCLUSION: Patient populations eligible for monarchE and NATALEE trials differed. Nearly a third of the postmenopausal HRpos population, previously under upfront letrozole treatment, met the NATALEE prognostic eligibility criteria. As certain eligible groups had a prognosis similar to non-eligible patients, it might be interesting to explore additional patient groups for CDK4/6i therapy.

Susceptibility gene mutations in germline and tumors of patients with HER2-negative advanced breast cancer.

Germline mutations in BRCA1 and BRCA2 (gBRCA1/2) are required for a PARP inhibitor therapy in patients with HER2-negative (HER2-) advanced breast cancer (aBC). However, little is known about the prognostic impact of gBRCA1/2 mutations in aBC patients treated with chemotherapy. This study aimed to investigate the frequencies and prognosis of germline and somatic BRCA1/2 mutations in HER2- aBC patients receiving the first chemotherapy in the advanced setting. Patients receiving their first chemotherapy for HER2- aBC were retrospectively selected from the prospective PRAEGNANT registry (NCT02338167). Genotyping of 26 cancer predisposition genes was performed with germline DNA of 471 patients and somatic tumor DNA of 94 patients. Mutation frequencies, progression-free and overall survival (PFS, OS) according to germline mutation status were assessed. gBRCA1/2 mutations were present in 23 patients (4.9%), and 33 patients (7.0%) had mutations in other cancer risk genes. Patients with a gBRCA1/2 mutation had a better OS compared to non-mutation carriers (HR: 0.38; 95%CI: 0.17-0.86). PFS comparison was not statistically significant. Mutations in other risk genes did not affect prognosis. Two somatic BRCA2 mutations were found in 94 patients without gBRCA1/2 mutations. Most frequently somatic mutated genes were TP53 (44.7%), CDH1 (10.6%) and PTEN (6.4%). In conclusion, aBC patients with gBRCA1/2 mutations had a more favorable prognosis under chemotherapy compared to non-mutation carriers. The mutation frequency of ~5% with gBRCA1/2 mutations together with improved outcome indicates that germline genotyping of all metastatic patients for whom a PARP inhibitor therapy is indicated should be considered.

Correlation of RANK and RANKL with mammographic density in primary breast cancer patients.

PURPOSE: The receptor activator of nuclear factor kappa B (RANK) and its ligand (RANKL) have been shown to promote proliferation of the breast and breast carcinogenesis. The objective of this analysis was to investigate whether tumor-specific RANK and RANKL expression in patients with primary breast cancer is associated with high percentage mammographic density (PMD), which is a known breast cancer risk factor. METHODS: Immunohistochemical staining of RANK and RANKL was performed in tissue microarrays (TMAs) from primary breast cancer samples of the Bavarian Breast Cancer Cases and Controls (BBCC) study. For RANK and RANKL expression, histochemical scores (H scores) with a cut-off value of > 0 vs 0 were established. PMD was measured in the contralateral, non-diseased breast. Linear regression models with PMD as outcome were calculated using common predictors of PMD (age at breast cancer diagnosis, body mass index (BMI) and parity) and RANK and RANKL H scores. Additionally, Spearman rank correlations (ρ) between PMD and RANK and RANKL H score were performed. RESULTS: In the final cohort of 412 patients, breast cancer-specific RANK and RANKL expression was not associated with PMD (P = 0.68). There was no correlation between PMD and RANK H score (Spearman's ρ = 0.01, P = 0.87) or RANKL H score (Spearman's ρ = 0.04, P = 0.41). RANK expression was highest in triple-negative tumors, followed by HER2-positive, luminal B-like and luminal A-like tumors, while no subtype-specific expression of RANKL was found. CONCLUSION: Results do not provide evidence for an association of RANK and RANKL expression in primary breast cancer with PMD.

Attrition in the First Three Therapy Lines in Patients with Advanced Breast Cancer in the German Real-World PRAEGNANT Registry.

Background With more effective therapies for patients with advanced breast cancer (aBC), therapy sequences are becoming increasingly important. However, some patients might drop out of the treatment sequence due to deterioration of their life status. Since little is known about attrition in the real-world setting, this study assessed attrition in the first three therapy lines using a real-world registry. Methods Patients with information available on the first three therapy lines were selected from the German PRAEGNANT registry (NCT02338167). Attrition was determined for each therapy line using competing risk analyses, with the start of the next therapy line or death as endpoints. Additionally, a simple attrition rate was calculated based on the proportion of patients who completed therapy but did not start the next therapy line. Results Competitive risk analyses were performed on 3988 1st line, 2651 2nd line and 1866 3rd line patients. The probabilities of not starting the next therapy line within 5 years after initiation of 1st, 2nd and 3rd line therapy were 30%, 24% and 24% respectively. Patients with HER2-positive disease had the highest risk for attrition, while patients with HRpos/HER2neg disease had the lowest risk. Attrition rates remained similar across molecular subgroups in the different therapy lines. Conclusion Attrition affects a large proportion of patients with aBC, which should be considered when planning novel therapy concepts that specifically address the sequencing of therapies. Taking attrition into account could help understand treatment effects resulting from sequential therapies and might help develop treatment strategies that specifically aim at maintaining quality of life.

CDK4/6 Inhibition - Therapy Sequences and the Quest to Find the Best Biomarkers - an Overview of Current Programs.

In recent years, new targeted therapies have been developed to treat patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) breast cancer. Some of these therapies have not just become the new therapy standard but also led to significantly longer overall survival rates. The cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) have become the therapeutic standard for first-line therapy. Around 70 - 80% of patients are treated with a CDK4/6i. In recent years, a number of biomarkers associated with progression, clonal selection or evolution have been reported for CDK4/6i and their endocrine combination partners. Understanding the mechanisms behind treatment efficacy and resistance is important. A better understanding could contribute to planning the most effective therapeutic sequences and utilizing basic molecular information to overcome endocrine resistance. One study with large numbers of patients which aims to elucidate these mechanisms is the Comprehensive Analysis of sPatial, TempORal and molecular patterns of ribociclib efficacy and resistance in advanced Breast Cancer patients (CAPTOR BC) trial. This overview summarizes the latest clinical research on resistance to endocrine therapies, focusing on CDK4/6 inhibitors and discussing current study concepts.

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.

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.

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.

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.

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.

Influence of labor on direct and indirect determinants of placental 11beta-hydroxysteroid dehydrogenase activity.

PURPOSE: Labor is a complex process involving multiple para-, auto- and endocrine cascades. The interaction of cortisol, corticotropin-releasing hormone (CRH) and progesterone is essential. The action of cortisol on the human feto-placental unit is regulated by 11beta-hydroxysteroid dehydrogenase type 2 (11ß-HSD2/HSD11B2) that converts cortisol into inactive cortisone. The majority of studies on the assessment of placental 11ß-HSD2 function determined indirect activity parameters. It remains elusive if indirect measurements correlate with enzymatic function and if these parameters are affected by potential confounders (e.g., mode of delivery). Thus, we compared determinants of indirect 11ß-HSD2 tissue activity with its direct enzymatic turnover rate in placental samples from spontaneous births and cesarean (C)-sections. METHODS: Using LC-MS/MS, we determined CRH, cortisol, cortisone, progesterone and 17-hydroxy(OH)-progesterone in human term placentas (spontaneous birth vs. C-section, n = 5 each) and measured the enzymatic glucocorticoid conversion rates in placental microsomes. Expression of HSD11B1, 2 and CRH was determined via qRT-PCR in the same samples. RESULTS: Cortisol-cortisone ratio correlated with direct microsomal enzymatic turnover. While this observation seemed independent of sampling site, a strong influence of mode of delivery on tissue steroids was observed. The mRNA expression of HSD11B2 correlated with indirect and direct cortisol turnover rates in C-section placentas only. In contrast to C-sections, CRH, cortisol and cortisone levels were significantly increased in placental samples following spontaneous birth. CONCLUSION: Labor involves a series of complex hormonal processes including activation of placental CRH and glucocorticoid metabolism. This has to be taken into account when selecting human cohorts for comparative analysis of placental steroids.