These results emphasize the potential of CTCs and EVs for therapy monitoring and the need for critical evaluation of the implementation of any liquid biopsy in clinical practice.

BackgroundCirculating tumor cells (CTC) are discussed to be an ideal surrogate marker for individualized treatment in metastatic breast cancer (MBC) since metastatic tissue is often difficult to obtain for repeated analysis. We established a nine gene qPCR panel to characterize the heterogeneous CTC population in MBC patients including epithelial CTC, their receptors (EPCAM, ERBB2, ERBB3, EGFR) CTC in Epithelial-Mesenchymal-Transition [(EMT); PIK3CA, AKT2), stem cell-like CTC (ALDH1) as well as resistant CTC (ERCC1, AURKA] to identify individual therapeutic targets.ResultsAt TP0, at least one marker was detected in 84%, at TP1 in 74% and at TP2 in 79% of the patients, respectively. The expression of ERBB2, ERBB3 and ERCC1 alone or in combination with AURKA was significantly associated with therapy failure. ERBB2 + CTC were only detected in patients not receiving ERBB2 targeted therapies which correlated with no response. Furthermore, patients responding at TP2 had a significantly prolonged overall-survival than patients never responding (p = 0.0090).Patients and Methods2 × 5 ml blood of 62 MBC patients was collected at the time of disease progression (TP0) and at two clinical staging time points (TP1 and TP2) after 8–12 weeks of chemo-, hormone or antibody therapy for the detection of CTC (AdnaTest EMT-2/StemCell Select™, QIAGEN Hannover GmbH, Germany). After pre-amplification, multiplex qPCR was performed. Establishment was performed using various cancer cell lines. PTPRC (Protein tyrosine phosphatase receptor type C) and GAPDH served as controls.ConclusionsMonitoring MBC patients using a multimarker qPCR panel for the characterization of CTC might help to treat patients accordingly in the future.

BackgroundIn search of potential early biomarkers for timely prediction of gestational diabetes mellitus (GDM), we focused on afamin, a vitamin E–binding protein in human plasma.. Afamin plays a role in anti-apoptotic cellular processes related to oxidative stress and is associated with insulin resistance and other features of metabolic syndrome. During uncomplicated pregnancy its serum concentrations increase linearly. The aim of this study was to investigate the suitability of afamin as early marker for predicting GDM.MethodsIn a first-trimester cohort from a prospective observational study of adverse pregnancy outcomes we secondarily analyzed afamin concentrations in 59 patients diagnosed with GDM and 51 controls. Additionally, afamin concentrations were cross-sectionally examined in a mid-trimester cohort of 105 women and compared with results from a simultaneously performed oral glucose tolerance test (OGTT). Subgroup analysis comparing patients treated with either insulin (iGDM) or dietary intervention (dGDM) was performed in both cohorts. Patients were recruited at the University Hospital Essen, Germany, between 2003 and 2016.ResultsResults were adjusted for body-mass-index (BMI) and gestational age. First and mid-trimester cohorts yielded significantly elevated afamin concentrations in patients with pathological OGTT compared to patients without GDM (first trimester cohort: mean, 113.4 mg/l; 95% CI, 106.4–120.5 mg/l and 87.2 mg/l; 95% CI, 79.7–94.7 mg/l; mid-trimester cohort: mean, 182.9 mg/l; 95% CI, 169.6–196.2 mg/l and 157.3 mg/l; 95% CI, 149.1–165.4 mg/l, respectively). In the first-trimester cohort, patients developing iGDM later in pregnancy presented with significantly higher afamin concentrations compared to patients developing dGDM and compared to patients without GDM. In the mid-trimester cohort, mean concentrations of afamin differed significantly between patients with dGDM compared to controls and between patients with iGDM and controls. Patients with iGDM showed only slightly higher afamin levels compared to patients with dGDM.ConclusionAfamin may serve as a new early biomarker for pathological glucose metabolism during pregnancy. Further research is needed to determine afamin’s concentrations during pregnancy, its predictive value for early detection of pregnancies at high risk to develop GDM and its diagnostic role during the second trimester.

could be a key marker in distinguishing R from NR, and was powerful in identifying CTCs.

Liquid biopsy analytes such as cell-free DNA (cfDNA) and circulating tumor cells (CTCs) exhibit great potential for personalized treatment. Since cfDNA and CTCs are considered to give additive information and blood specimens are limited, isolation of cfDNA and CTC in an “all from one tube” format is desired. We investigated whether cfDNA variant sequencing from CTC-depleted blood (CTC-depl. B; obtained after positive immunomagnetic isolation of CTCs (AdnaTest EMT-2/Stem Cell Select, QIAGEN)) impacts the results compared to cfDNA variant sequencing from matched whole blood (WB). Cell-free DNA was isolated using matched WB and CTC-depl. B from 17 hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2−) metastatic breast cancer patients (QIAamp MinElute ccfDNA Kit, QIAGEN). Cell-free DNA libraries were constructed (customized QIAseq Targeted DNA Panel for Illumina, QIAGEN) with integrated unique molecular indices. Sequencing (on the NextSeq 550 platform, Illumina) and data analysis (Ingenuity Variant Analysis) were performed. RNA expression in CTCs was analyzed by multimarker quantitative PCR. Cell-free DNA concentration and size distribution in the matched plasma samples were not significantly different. Seventy percent of all variants were identical in matched WB and CTC-depl. B, but 115/125 variants were exclusively found in WB/CTC-depl. B. The number of detected variants per patient and the number of exclusively detected variants per patient in only one cfDNA source did not differ between the two matched cfDNA sources. Even the characteristics of the exclusively detected cfDNA variants in either WB or CTC-depl. B were comparable. Thus, cfDNA variants from matched WB and CTC-depl. B exhibited no relevant differences, and parallel isolation of cfDNA and CTCs from only 10 mL of blood in an “all from one tube” format was feasible. Matched cfDNA mutational and CTC transcriptional analyses might empower a comprehensive liquid biopsy analysis to enhance the identification of actionable targets for individual therapy strategies.

Cell-free DNA (cfDNA) is described to mirror intratumoral heterogeneity and gives insight about clonal evolution for improved therapeutic decisions. We sequenced cfDNA of a hormone receptor-positive, HER2-negative metastatic breast cancer (MBC) cohort with a high coverage to examine the prevalence and relevance of any detected variant. cfDNA of 44 MBC patients was isolated, followed by library construction using a customized targeted DNA panel with integrated unique molecular indices analyzing AKT1,

IntroductIon Soluble forms of tumor necrosis factor (TNF) membrane receptors 1 and 2 (sTNFR1 and sTNFR2) are present in body fluids. Their higher concentrations are observed in a number of diseases, including inflammatory bowel diseases (IBDs). sTNFR1 and sTNFR2 are capable of binding TNF-α, acting as an inhibitor that competes with a membrane receptor. The results of the available studies on sTNFR1 and sTNFR2 concentrations in IBDs and their association with disease activity are ambiguous.objectIves The aim of the study was to assess sTNFR1 and sTNFR2 concentrations and their correlation with disease activity in patients with IBD.PAtIents And methods Plasma levels of TNF-α, sTNFR1, and sTNFR2 were measured in 55 consecutive patients with ulcerative colitis (UC), 50 subjects with Crohn's disease (CD), and 41 healthy controls. We assessed the associations of those markers with other inflammatory markers, disease activity and location, type of treatment, and complications.results Positive correlations were observed between CD activity and sTNFR1 and sTNFR2 levels (r = 0.42 for both, P <0.01) as well as between UC activity and sTNFR1 and sTNFR2 levels (r = 0.63, P <0.0001; r = 0.47, P <0.001; respectively). TNF-α levels correlated only with CD activity (r = 0.29, P <0.05). In patients with nonactive UC, higher sTNFR2 levels were observed compared with controls. In patients with CD, higher TNF-α and sTNFR2 levels were demonstrated in patients who developed complications.conclusIons sTNFR1 and sTNFR2 are more sensitive inflammatory markers than TNF-α in the assessment of disease activity in patients with CD and UC. Higher sTNFR2 levels are observed in patients with CD and complications.

Background B7‐H4, a checkpoint molecule of the B7 family, regulates a broad spectrum such as T‐cell activation, cytokine secretion, tumour progression, and invasion capacities. Our previous data revealed that soluble B7‐H4 (sB7‐H4) blood serum levels are elevated in women at high risk for the hypertensive pregnancy disorder preeclampsia (PE) in the first trimester, as well as in patients with confirmed early/late‐onset PE. Aim We here aim to investigate the expression pattern of B7‐H4 in placental tissues of PE and HELLP Syndrome versus control group. Methods B7‐H4 protein expression and localization were investigated by immunoblotting and co‐immunohistochemistry in placental chorionic villous and decidual basalis tissues. Results B7‐H4 protein was prominently expressed at the cell membrane, in the cytoplasm of the syncytiotrophoblast (STB) and interstitial extravillous trophoblast (EVT). B7‐H4 protein levels in placental chorionic villous tissue were significantly higher in women with early‐onset/late‐onset PE and HELLP, while it was decreased in decidual basalis tissues of early‐onset PE and HELLP compared with controls. Conclusion B7‐H4 was inversely expressed in placental chorionic villous and decidual basalis tissues of PE and HELLP patients. The increase in B7‐H4 in the STB in PE and HELLP may lead to excessive apical expression and release of soluble B7‐H4 in the maternal circulation. In contrast, the decrease in B7‐H4 in decidual basalis tissues could be related to the decrease in invasion ability of the EVT in PE. Thus, the current results strongly suggest that B7‐H4 is involved in the pathogenesis of PE and HELLP.