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Single circulating tumor cell (sCTC) analysis enables the determination of predominant CTC phenotypes and genotypes. We previously demonstrated the feasibility of sCTC detection and genomic characterization in high‐grade serous ovarian cancer (HGSOC) by combining immune‐magnetic enrichment and image‐based sorting, followed by whole‐genome amplification (WGA) and next‐generation sequencing‐based copy number alteration analysis (CNA). Here we aimed to improve our workflow by incorporating HGSOC‐specific markers, folate receptor alpha (FRα), and markers to identify epithelial (cytokeratin) and mesenchymal (vimentin) phenotypes for the phenotypic as well as genotypic analysis of sCTCs over the course of treatment in 42 HGSOC patients. We detected a significant reduction of FRα‐positive cells (P = 0.0205) and an expansion of cells with a high nuclear staining and no target antigen expression (P = 0.002). Before treatment, sCTCs showed an enrichment in CNAs of Chromosomes 2, 7, and 12, while CNA dynamics of sCTCs suggested a potential selection of distinct CNAs specific to the homologous recombination pathway. sCTCs revealed persistent CNAs in the CDK4 and emerging ones in the ALK oncogene. Notably, primary tumors revealed considerable fractions of shared genomic aberrations. Single circulating tumor cells (sCTCs) from high‐grade serous ovarian cancer patients were enriched, imaged, and genomically profiled using WGA and NGS at different time points during treatment. sCTCs revealed enrichment of alterations in Chromosomes 2, 7, and 12 as well as persistent or emerging oncogenic CNAs, supporting sCTC identity. Notably, primary tumors revealed considerable fractions of shared genomic aberrations.

MicroRNAs (miRNAs: short non-coding RNAs) are emerging as a class of potential novel tumor markers, as their dysregulation is being increasingly reported in various types of cancers. In the present study, we investigated the transcription status of miRNA-148a (miR-148a) in human pancreatic ductal adenocarcinoma (PDAC) and its role in the regulation of the dual specificity protein phosphatase CDC25B. We observed that miR-148a exhibited a significant 4-fold down-regulation in PDAC as opposed to normal pancreatic ductal cells. In addition, we observed that stable lentiviral-mediated overexpression of miR-148a in the pancreatic cancer cell line IMIM-PC2, inhibited tumor cell growth and colony formation. Furthermore, CDC25B was identified as a potential target of miR-148a by in silico analysis using PicTar, Targetscan and miRanda in conjunction with gene ontology analysis. The proposed interaction between miR-148a and the 3′ untranslated region (UTR) of CDC25B was verified by in-vitro luciferase assays. We demonstrate that the activity of a luciferase reporter containing the 3′UTR of CDC25B was repressed in the presence of miR-148a mimics, confirming that miR-148a targets the 3′UTR of CDC25B. Finally, CDC25B was down-regulated at the protein level in miR-148a overexpressing IMIM-PC2-cells, and in transiently transfected pancreatic cell lines (as detected by Western blot analysis), as well as in patient tumor samples (as detected by immunohistochemistry). In summary, we identified CDC25B as a novel miR-148a target which may confer a proliferative advantage in PDAC.

Purpose Endocrine therapies, including tamoxifen or aromatase inhibitors, are indispensable for the treatment of patients with estrogen receptor (ER)- and/or progesterone-positive breast cancer. Whereas tamoxifen displays partial ER agonistic effects in bone, aromatase inhibitors increase bone resorption and fracture risk. The Wnt inhibitors dickkopf-1 (DKK-1) and sclerostin negatively impact bone formation and are considered targets for the treatment of bone disorders. However, the effect of endocrine therapies on serum DKK-1 and sclerostin levels in patients with primary breast cancer remains elusive. Methods Serum DKK-1 and sclerostin levels were measured at primary diagnosis as well as 3–5 days and 12 months after surgery in a cohort of 45 pre- and postmenopausal women with primary estrogen receptor-positive breast cancer treated with adjuvant tamoxifen or aromatase inhibitors. Results Mean baseline levels ±SD for DKK-1 and sclerostin were 29.7 ± 14.6 and 27.1 ± 16.2 pmol/l, respectively. A significant negative correlation of DKK-1 levels and age was observed ( r  = −0.32; p  < 0.05), but not for sclerostin. Of note, DKK-1 levels were significantly lower in peri- and postmenopausal women compared to premenopausal patients (−47%; p  < 0.05). In tamoxifen-treated patients, DKK-1 levels were reduced by 35% ( p  < 0.01) one year after surgery but remained unaltered in patients treated with aromatase inhibitors. No significant changes were observed for sclerostin. Conclusion DKK-1 serum levels were reduced in breast cancer patients receiving an adjuvant therapy with tamoxifen, possibly contributing to its bone-protective properties.

Background We recently showed that LOH proximal to M6P/IGF2R locus ( D6S1581 ) in primary ovarian tumors is predictive for the presence of disseminated tumor cells (DTC) in the bone marrow (BM). For therapy-monitoring, it would be highly desirable to establish a blood-based biomarker. Therefore, we quantified circulating DNA (cirDNA) in sera of 63 ovarian cancer patients before surgery and after chemotherapy, measured incidence of LOH at four cancer-relevant chromosomal loci, correlated LOH with tumor cell spread to the BM and evaluated prognostic significance of LOH. Methods cirDNA was fractionated into high- and low molecular-weight fraction (HMWF, LMWF) for LOH-profiling, utilizing PCR-based fluorescence microsatellite analysis. BM aspirates were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Results cirDNA levels in the HMWF before surgery were predictive for residual tumor load (p = 0.017). After chemotherapy, we observed a significant decline of cirDNA in the LMWF (p = 0.0001) but not in the HMWF. LOH was prevalently detected in the LMWF with an overall frequency of 67%, only moderately ablating after chemotherapy (45%). Before surgery, LOH in the LMWF at marker D10S1765 and D13S218 significantly correlated with tumor grading and FIGO stage (p = 0.033, p = 0.004, respectively). In both combined fractions, LOH at D6S1581 additionally associated with overall survival (OS) (p = 0.030). Moreover, solely LOH at D10S1765 in LMWF after therapy correlated with DTC in BM after therapy (p = 0.017). Conclusion We demonstrate the applicability and necessity of DNA-fractionation prior to analyzing circulating LOH and identify LOH at D10S1765 and D6S1581 as novel blood-based biomarkers for ovarian cancer, being relevant for therapy-monitoring.

Background Ovarian cancer remains the most fatal gynecological malignancy. Current therapeutic options are limited due to late diagnosis in the majority of the cases, metastatic spread to the peritoneal cavity and the onset of chemo-resistance. Thus, novel therapeutic approaches are required. Statins and amino-bisphosphonates are inhibitors of the mevalonate pathway, which is a fundamental pathway of cellular metabolism, essential for cholesterol production and posttranslational protein farnesylation and geranylgeranylation. While this pathway has emerged as a promising treatment target in several human malignancies, its potential as a therapeutic approach in ovarian cancer is still not fully understood. Methods Human ovarian cancer cell lines (IGROV-1, A2780, A2780cis) were treated with increasing concentrations (0.5-100 μM) of statins (simvastatin, atorvastatin, rosuvastatin) and zoledronic acid. Effects on cell vitality and apoptosis were assessed using Cell Titer Blue®, Caspase 3/7 Glo®, clonogenic assays as well as cleaved poly (ADP-ribose) polymerase (cPARP) detection. The inhibition of the mevalonate pathway was confirmed using Western Blot of unprenylated Ras and Rap1a proteins. Quantitative real-time PCR and ELISA were used to analyze modulations on several key regulators of ovarian cancer tumorigenesis. Results The treatment of IGROV-1 and A2780 cells with statins and zoledronic acid reduced vitality (by up to 80%; p  < 0.001) and induced apoptosis by up to 8-folds ( p  < 0.001) in a dose-dependent fashion. Rescue experiments using farnesyl pyrophosphate or geranylgeranyl pyrophosphate evidenced that blocked geranylgeranylation is the major underlying mechanism of the pro-apoptotic effects. Gene expression of the tumor-promoting cytokines and mediators, such as transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF), interleukin (IL)-8, and IL-6 were significantly suppressed by statins and zoledronic acid by up to 90% ( p  < 0.001). For all readouts, simvastatin was most potent of all agents used. Cisplatin-resistant A2780cis cells showed a relative resistance to statins and zoledronic acid. However, similar to the effects in A2780 cells, simvastatin and zoledronic acid significantly induced caspase 3/7 activation (6-folds; p  < 0.001). Conclusion Our in vitro findings point to promising anti-tumor effects of statins and zoledronic acid in ovarian cancer and warrant additional validation in preclinical and clinical settings.

Metastasis‐associated in colon cancer 1 (MACC1) and S100 calcium‐binding protein A4 (S100A4) are prominent inducers of tumor progression and metastasis. For the first time, we systematically tracked circulating serum levels of MACC1 and S100A4 transcripts in the course of surgery and chemotherapy and analyzed their clinical relevance for ovarian cancer. MACC1 and S100A4 transcripts were quantified in a total of 318 serum samples from 79 ovarian cancer patients by RT‐qPCR and digital droplet PCR, respectively. MACC1 and S100A4 transcripts were significantly elevated in serum of ovarian cancer patients, compared to healthy controls (P = 0.024; P < 0.001). At primary diagnosis, high levels of MACC1 or S100A4 correlated with advanced FIGO stage (P = 0.042; P = 0.008), predicted suboptimal debulking surgery and indicated shorter progression‐free survival (PFS; P = 0.003; P = 0.001) and overall survival (OS; P = 0.001; P = 0.002). This is the first study in ovarian cancer to propose circulating MACC1 and S100A4 transcripts as potential liquid biopsy markers. We analyzed the clinical relevance of circulating MACC1 and S100A4 transcripts in ovarian cancer patients. We report that high serum levels of both transcripts at primary diagnosis (but not in the course of surgery and chemotherapy) are associated with advanced disease and indicate poor survival. Our results suggest blood‐based MACC1 and S100A4 transcripts as innovative liquid biopsy markers for ovarian cancer.

Background: Atu027 is a liposomally formulated short interfering RNA with anti-metastatic activity, which silences the expression of protein kinase N3 (PKN3) in the vascular endothelium. This trial was designed to assess the safety, pharmacokinetics and efficacy of Atu027 in combination with gemcitabine in advanced pancreatic carcinoma (APC). Methods: In total, 23 patients (pts) with inoperable APC were randomly assigned to gemcitabine combined with two different Atu027 schedules (0.235 mg/kg once weekly vs. 0.235 mg/kg twice weekly). ClinicalTrials.gov Identifier: NCT01808638. Results: The treatment was well-tolerated. There were Grade 3 adverse events (AEs) in 9/11 pts (arm 1) and 11/12 pts (arm 2), while Grade 4 AEs were reported for two pts in each arm. The AEs were mainly laboratory abnormalities without clinical significance. The median progression-free survival reached statistical significance in patients who had metastatic disease (1.6 vs. 2.9 months, p = 0.025). Disease control during treatment was achieved in 4/11 pts (arm 1) and in 7/12 pts (arm 2). Pts in arm 1 experienced stable global health status while pts in arm 2 reported improvement. Conclusions: Combining Atu027 with gemcitabine is safe and well tolerated. In pts with metastatic APC, twice-weekly Atu027 is associated with significantly improved outcomes. Our clinical results support the significant involvement of the vascular endothelium in the spread of cancer, and thus the further investigation of its target role.

We assess the disputed phase relations between forcing and climatic response in the early Pleistocene with a spliced Gelasian (∼2.6–1.8 Ma) multi-proxy record from the southern North Sea basin. The cored sections couple climate evolution on both land and sea during the intensification of Northern Hemisphere glaciation (NHG) in NW Europe, providing the first well-constrained stratigraphic sequence of the classic terrestrial Praetiglian stage. Terrestrial signals were derived from the Eridanos paleoriver, a major fluvial system that contributed a large amount of freshwater to the northeast Atlantic. Due to its latitudinal position, the Eridanos catchment was likely affected by early Pleistocene NHG, leading to intermittent shutdown and reactivation of river flow and sediment transport. Here we apply organic geochemistry, palynology, carbonate isotope geochemistry, and seismostratigraphy to document both vegetation changes in the Eridanos catchment and regional surface water conditions and relate them to early Pleistocene glacial–interglacial cycles and relative sea level changes. Paleomagnetic and palynological data provide a solid integrated timeframe that ties the obliquity cycles, expressed in the borehole geophysical logs, to Marine Isotope Stages (MIS) 103 to 92, independently confirmed by a local benthic oxygen isotope record. Marine and terrestrial palynological and organic geochemical records provide high-resolution reconstructions of relative terrestrial and sea surface temperature (TT and SST), vegetation, relative sea level, and coastal influence. During the prominent cold stages MIS 98 and 96, as well as 94, the record indicates increased non-arboreal vegetation, low SST and TT, and low relative sea level. During the warm stages MIS 99, 97, and 95 we infer increased stratification of the water column together with a higher percentage of arboreal vegetation, high SST, and relative sea level maxima. The early Pleistocene distinct warm–cold alterations are synchronous between land and sea, but lead the relative sea level change by 3000–8000 years. The record provides evidence for a dominantly Northern Hemisphere-driven cooling that leads the glacial buildup and varies on the obliquity timescale. Southward migration of Arctic surface water masses during glacials, indicated by cool-water dinoflagellate cyst assemblages, is furthermore relevant for the discussion on the relation between the intensity of the Atlantic meridional overturning circulation and ice sheet growth.

Metastasis‐associated in colon cancer 1 ( MACC 1) and S100 calcium‐binding protein A4 (S100A4) are prominent inducers of tumor progression and metastasis. For the first time, we systematically tracked circulating serum levels of MACC 1 and S100A4 transcripts in the course of surgery and chemotherapy and analyzed their clinical relevance for ovarian cancer. MACC 1 and S100A4 transcripts were quantified in a total of 318 serum samples from 79 ovarian cancer patients by RT ‐ qPCR and digital droplet PCR , respectively. MACC 1 and S100A4 transcripts were significantly elevated in serum of ovarian cancer patients, compared to healthy controls ( P  = 0.024; P  < 0.001). At primary diagnosis, high levels of MACC 1 or S100A4 correlated with advanced FIGO stage ( P  = 0.042; P  = 0.008), predicted suboptimal debulking surgery and indicated shorter progression‐free survival ( PFS ; P  = 0.003; P  = 0.001) and overall survival ( OS ; P  = 0.001; P  = 0.002). This is the first study in ovarian cancer to propose circulating MACC 1 and S100A4 transcripts as potential liquid biopsy markers.

BCG vaccination, combined with adenoviral-delivered boosts, represents a reasonable strategy to augment, broaden and prolong immune protection against tuberculosis (TB). We tested BCG (SSI1331) (in 6 animals, delivered intradermally) and a recombinant (rBCG) AFRO-1 expressing perfringolysin (in 6 animals) followed by two boosts (delivered intramuscullary) with non-replicating adenovirus 35 (rAd35) expressing a fusion protein composed of Ag85A, Ag85B and TB10.4, for the capacity to induce antigen-specific cellular immune responses in rhesus macaques (Macaca mulatta). Control animals received diluent (3 animals). Cellular immune responses were analyzed longitudinally (12 blood draws for each animal) using intracellular cytokine staining (TNF-alpha, IL-2 and IFN-gamma), T cell proliferation was measured in CD4(+), CD8alpha/beta(+), and CD8alpha/alpha(+) T cell subsets and IFN-gamma production was tested in 7 day PBMC cultures (whole blood cell assay, WBA) using Ag85A, Ag85B, TB10.4 recombinant proteins, PPD or BCG as stimuli. Animals primed with AFRO-1 showed i) increased Ag85B-specific IFN-gamma production in the WBA assay (median >400 pg/ml for 6 animals) one week after the first boost with adenoviral-delivered TB-antigens as compared to animals primed with BCG (<200 pg/ml), ii) stronger T cell proliferation in the CD8alpha/alpha(+) T cell subset (proliferative index 17%) as compared to BCG-primed animals (proliferative index 5% in CD8alpha/alpha(+) T cells). Polyfunctional T cells, defined by IFN-gamma, TNF-alpha and IL-2 production were detected in 2/6 animals primed with AFRO-1 directed against Ag85A/b and TB10.4; 4/6 animals primed with BCG showed a Ag85A/b responses, yet only a single animal exhibited Ag85A/b and TB10.4 reactivity. AFRO-1 induces qualitatively and quantitatively different cellular immune responses as compared with BCG in rhesus macaques. Increased IFN-gamma-responses and antigen-specific T cell proliferation in the CD8alpha/alpha+ T cell subset represents a valuable marker for vaccine-take in BCG-based TB vaccine trials.