Multi-marker analysis of circulating tumor cells in localized intermediate/high-risk and metastatic prostate cancer.

Circulating tumor cells (CTCs) are an established prognostic marker in metastatic prostate cancer (PrC) but have received little attention in localized high-risk disease. Peripheral blood was obtained from patients with early intermediate and high-risk PrC (n = 15) at baseline, after radiotherapy, and during follow-up, as well as from metastatic PrC patients (n = 23). CTCs were enriched using the microfluidic Parsortix® technology. CTC-related marker were quantified with qPCR and RNA in-situ hybridization (ISH). Positivity and associations to clinical parameters were assessed using McNemar test, Fisher Exact test or log-rank test. The overall positivity was high in both cohorts (87.0% metastatic vs. 66.7% early at baseline). A high concordance of qPCR and RNA ISH was achieved. In metastatic PrC, PSA and PSMA were prognostic for shorter overall survival. In early PrC patients, an increase of positive transcripts per blood sample was observed from before to after radiation therapy, while a decrease of positive markers was observed during follow-up. CTC analysis using the investigated qPCR marker panel serves as tool for achieving high detection rates of PrC patient samples even in localized disease. RNA ISH offers the advantage of confirming these markers at the single cell level. Employing the clinically relevant marker PSMA, our CTC approach can be used for diagnostic purposes to screen patients profiting from PSMA-directed PET-CT or PSMA-targeted therapy.

Prognostic significance of circulating tumor cells and tumor related transcripts in small cell lung cancer: A step further to clinical implementation.

Small-cell lung cancer (SCLC) is a fatal disease with limited treatment options. Circulating tumor cells (CTCs) in liquid biopsy samples may serve as predictive and prognostic biomarkers; but the analysis of CTCs is still challenging. By using microfluidic or density gradient CTC enrichment in combination with immunofluorescent (IF) staining or qPCR of CTC-related transcripts, we achieved a 60.8% to 88.0% positivity in SCLC blood samples. Epithelial and neuroendocrine transcripts including the druggable target DLL3 were associated with shorter overall survival (OS), indicating the clinical value of these markers in terms of differential diagnosis and treatment decisions. High CTC counts and the presence of CTC duplets detected by IF staining were prognostic for OS, and thus may serve as indicators of disease progression or therapy failure. In patient samples with high CTC load detected by IF staining, a concordance of the transcripts positivity in circulating free plasma RNA and CTCs was observed. Our data emphasize the role of CTCs and CTC-related transcripts and underline the clinical value of liquid biopsy analysis in SCLC.

Amyloid-like p53 as prognostic biomarker in serous ovarian cancer-a study of the OVCAD consortium.

TP53 is the most commonly mutated gene in cancer and has been shown to form amyloid-like aggregates, similar to key proteins in neurodegenerative diseases. Nonetheless, the clinical implications of p53 aggregation remain unclear. Here, we investigated the presence and clinical relevance of p53 aggregates in serous ovarian cancer (OC). Using the p53-Seprion-ELISA, p53 aggregates were detected in 46 out of 81 patients, with a detection rate of 84.3% in patients with missense mutations. High p53 aggregation was associated with prolonged progression-free survival. We found associations of overall survival with p53 aggregates, but they did not reach statistical significance. Interestingly, p53 aggregation was significantly associated with elevated levels of p53 autoantibodies and increased apoptosis, suggesting that high levels of p53 aggregates may trigger an immune response and/or exert a cytotoxic effect. To conclude, for the first time, we demonstrated that p53 aggregates are an independent prognostic marker in serous OC. P53-targeted therapies based on the amount of these aggregates may improve the patient's prognosis.

Effect of short-term storage of blood samples on gene expression in lung cancer patients.

OBJECTIVES: The stability of gene transcripts associated with the presence of circulating tumor cells (CTCs) has been predominantly studied in cultured cancer cell lines added to blood samples under artificial conditions. In the present study the effect of storage on CTC-related transcripts was assessed in blood samples taken from patients with non-small lung cancer (n=58). METHODS: The blood samples were split in two equal parts to compare the gene expression with and without storage for 24 h at ambient temperature without preservative added. After enrichment using the microfluidic Parsortix® technology, the expression levels of selected genes were assessed using quantitative PCR following a gene-specific pre-amplification. The prognostic relevance of each gene in fresh and stored blood samples was evaluated using the R-package Survminer. RESULTS: Some genes were either not affected (TWIST1, CDH5, CK19) or upregulated upon storage (NANOG, MET, UCHL1) but still associated with poor prognosis. In contrast, ERBB3, PTHLH, EpCAM, and TERT were no longer associated with the overall survival of the patients. CONCLUSIONS: The study demonstrates the surprising stability of CTC-related transcripts, which makes overnight shipping of native blood samples possible. Careful verification is required when using model systems - such as normal blood spiked with tumor cells - or other CTC-related markers, as individual transcripts may respond differently to storage.

Comparison of microfluidic platforms for the enrichment of circulating tumor cells in breast cancer patients.

PURPOSE: Circulating tumor cells (CTCs) hold promise to be a non-invasive measurable biomarker in all cancer stages. Because the analysis of CTCs is still a technical challenge, we compared different types of microfluidic enrichment protocols to isolate these rare cells from the blood. METHODS: Blood samples from patients with early and metastatic breast cancer (BC) were processed using the microfluidic Parsortix® technology employing (i) a single-step cell separation using the standard GEN3D6.5 microfluidic cassette, (ii) a two-step separation with an upfront pre-enrichment, and (iii) a two-step separation with a different type of cassette. In the enriched cells, the gene expression levels of CTC-related transcripts were assessed using quantitative real-time PCR (qPCR) by Taqman® and Lightcycler (LC) technology. RESULTS: 23/60 (38.3%) BC samples were assigned as positive due to the presence of at least one gene marker beyond the threshold level. The prevalence of epithelial markers was significantly higher in metastatic compared to early BC (EpCAM: 31.3% vs. 7.3%; CK19: 21.1% vs. 2.4%). A high level of concordance was observed between CK19 assessed by Taqman® and LC technology, and for detection of the BC-specific gene SCGB2A2. An upfront pre-enrichment resulted in lower leukocyte contamination, at the cost of fewer tumor cells captured. CONCLUSION: The Parsortix® system offers both reasonable recovery of tumor cells and depletion of contaminating leukocytes when the single-step separation using the GEN3D6.5 cassette is employed. Careful selection of suitable markers and cut-off thresholds is an essential point for the subsequent molecular analysis of the enriched cells.

Cancer Stem Cell-Like Circulating Tumor Cells Are Prognostic in Non-Small Cell Lung Cancer.

Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), less than 10% of patients survive the first five years when the disease has already spread at primary diagnosis. METHODS: Blood samples were taken from 118 NSCLC patients at primary diagnosis or at progression of the disease before the start of a new treatment line and enriched for circulating tumor cells (CTCs) by microfluidic Parsortix™ (Angle plc, Guildford GU2 7AF, UK) technology. The gene expression of epithelial cancer stem cell (CSC), epithelial to mesenchymal (EMT), and lung-related markers was assessed by qPCR, and the association of each marker with overall survival (OS) was evaluated using log-rank tests. RESULTS: EpCAM was the most prevalent transcript, with 53.7% positive samples at primary diagnosis and 25.6% at recurrence. EpCAM and CK19, as well as NANOG, PROM1, TERT, CDH5, FAM83A, and PTHLH transcripts, were associated with worse OS. However, only the CSC-specific NANOG and PROM1 were related to the outcome both at primary diagnosis (NANOG: HR 3.21, 95%CI 1.02-10.14, p = 0.016; PROM1: HR 4.23, 95% CI 0.65-27.56, p = 0.007) and disease progression (NANOG: HR 4.17, 95%CI 0.72-24.14, p = 0.025; PROM1: HR 4.77, 95% CI 0.29-78.94, p = 0.032). CONCLUSIONS: The present study further underlines the relevance of the molecular characterization of CTCs. Our multi-marker analysis highlighted the prognostic value of cancer stem cell-related transcripts at primary diagnosis and disease progression.

Polymer-Ligand-Based ELISA for Robust, High-Throughput, Quantitative Detection of p53 Aggregates.

A growing number of diseases are being linked to protein misfolding and amyloid formation. Recently, p53 was also shown to associate into amyloid aggregates, raising the question of whether cancer development is associated with protein aggregation as well. However, a lack of suitable tools has hampered the evaluation of their clinical relevance. Herein, we report an enzyme-linked-immunosorbent-assay (ELISA) system based on a polyionic, high-molecular-weight ligand that specifically captures aggregated oligomers and amyloid proteins. We proved that naturally occurring tetramers of p53 are not bound, but high-molecular-weight aggregates are bound and subsequently detected. For the first time, this assay allows the quantitative detection of p53 aggregates from cell lysates, which was demonstrated using 22 ovarian-cancer cell lines as well as 7 patient-derived tumor tissues. The levels of p53 aggregates within the missense-mutated tissue samples varied more than 12-fold. This simple, robust method allows studying the abundance and clinical relevance of protein aggregates. This could help our understanding of the role of protein misfolding in cancer or even in predicting therapy responses to aggregation-targeting drugs.

Assessing adherence to multiple medications and in daily life among patients with multimorbidity.

OBJECTIVE: Chronic conditions often require multiple medication intake. However, past research has focused on assessing overall adherence or adherence to a single index medication only. This study explored adherence measures for multiple medication intake, and in daily life, among patients with multiple chronic conditions (i.e. multimorbidity). DESIGN: Eighty-four patients with multimorbidity and multiple-medication regimens completed three monthly panel questionnaires. A randomly assigned subsample additionally completed a 30-day daily diary. MAIN OUTCOME MEASURE: The Non-Adherence Report; a brief self-report measure of adherence to each prescribed medication (NAR-M), and in daily life. We further assessed the Medication Adherence Report Scale (MARS), and a subsample of participants were randomised to electronic adherence monitoring. RESULTS: The NAR-M indicated M = 94.7% adherence at Time 1 (SD = 9.3%). The NAR-M was significantly correlated with the MARS (rt1 = .52, rt2 = .57, and rt3 = .65; p < .001), and in tendency with electronically assessed adherence (rt2 = .45, rt3 = .46, p < .10). Variance components analysis indicated that between-person differences accounted for 10.2% of the variance in NAR-M adherence rates, whereas 22.9% were attributable to medication by person interactions. CONCLUSION: This study highlights the importance and feasibility of studying adherence to multiple medications differentially, and in daily life. Future studies may use these measures to investigate within-person and between-medication differences in adherence.

Chronic Pain: How Challenging Are DDIs in the Analgesic Treatment of Inpatients with Multiple Chronic Conditions?

BACKGROUND: Chronic pain is common in multimorbid patients. However, little is known about the implications of chronic pain and analgesic treatment on multimorbid patients. This study aimed to assess chronic pain therapy with regard to the interaction potential in a sample of inpatients with multiple chronic conditions. METHODS AND FINDINGS: We conducted a retrospective study with all multimorbid inpatients aged ≥18 years admitted to the Department of Internal Medicine of University Hospital Zurich in 2011 (n = 1,039 patients). Data were extracted from the electronic health records and reviewed. We identified 433 hospitalizations of patients with chronic pain and analyzed their combinations of chronic conditions (multimorbidity). We then classified all analgesic prescriptions according to the World Health Organization (WHO) analgesic ladder. Furthermore, we used a Swiss drug-drug interactions knowledge base to identify potential interactions between opioids and other drug classes, in particular coanalgesics and other concomitant drugs. Chronic pain was present in 38% of patients with multimorbidity. On average, patients with chronic pain were aged 65.7 years and had a mean number of 6.6 diagnoses. Hypertension was the most common chronic condition. Chronic back pain was the most common painful condition. Almost 90% of patients were exposed to polypharmacotherapy. Of the chronic pain patients, 71.1% received opioids for moderate to severe pain, 43.4% received coanalgesics. We identified 3,186 potential drug-drug interactions, with 17% classified between analgesics (without coanalgesics). CONCLUSIONS: Analgesic drugs-related DDIs, in particular opioids, in multimorbid patients are often complex and difficult to assess by using DDI knowledge bases alone. Drug-multimorbidity interactions are not sufficiently investigated and understood. Today, the scientific literature is scarce for chronic pain in combination with multiple coexisting medical conditions and medication regimens. Our work may provide useful information to enable further investigations in multimorbidity research within the scope of potential interactions and chronic pain.

Determination of the minimum fully protective dose of adenovirus-based DIVA vaccine against peste des petits ruminants virus challenge in East African goats.

Peste des petits ruminants virus (PPRV) causes an economically important disease of sheep and goats, primarily in developing countries. It is becoming the object of intensive international control efforts. Current vaccines do not allow vaccinated and infected animals to be distinguished (no DIVA capability). We have previously shown that recombinant, replication-defective, adenovirus expressing the PPRV H glycoprotein (AdH) gives full protection against wild type PPRV challenge. We have now tested lower doses of the vaccine, as well as AdH in combination with a similar construct expressing the PPRV F glycoprotein (AdF). We show here that, in a local breed of goat in a country where PPR disease is common (Kenya), as little as 10(7) pfu of AdH gives significant protection against PPRV challenge, while a vaccine consisting of 10(8) pfu of each of AdH and AdF gives apparently sterile protection. These findings underline the utility of these constructs as DIVA vaccines for use in PPR control.

Different functions of the common P/V/W and V-specific domains of rinderpest virus V protein in blocking IFN signalling.

The V proteins of paramyxoviruses are composed of two evolutionarily distinct domains, the N-terminal 75 % being common to the viral P, V and W proteins, and not highly conserved between viruses, whilst the remaining 25 % consists of a cysteine-rich V-specific domain, which is conserved across almost all paramyxoviruses. There is evidence supporting a number of different functions of the V proteins of morbilliviruses in blocking the signalling pathways of type I and II IFNs, but it is not clear which domains of V are responsible for which activities and whether all these activities are required for effective blockade of IFN signalling. We have shown here that the two domains of rinderpest virus V protein have distinct functions: the N-terminal domain acted to bind STAT1, whilst the C-terminal V-specific domain interacted with the IFN receptor-associated kinases Jak1 and Tyk2. Effective blockade of IFN signalling required the intact V protein.

PP057. ENOSI4 and EPHX1 polymorphisms affect maternal susceptibility topreeclampsia - Analysis of five polymorphisms predisposing tocardiovascular disease in 279 caucasian and 241 african women.

OBJECTIVES: Clinical studies have documented a familiar tendency to develop preeclampsia and patients with impaired endothelial health are at increased risk. We studied genetic polymorphisms associated to cardiovascular disease and impaired endothelial function in women with and without preeclampsia. METHODS: 241 African and 279 Caucasian women were recruited for genetic testing of the polymorphisms epoxide hydrolase 1 (EPXH1), endothelial nitric oxide synthase (NOS3) on exon 7 and variable nucleotide tandem repeats in intron 4 (NOS3I4a/b), angiotensinogen and the estrogen receptor1 polymorphism in intron 1. RESULTS: Of 241 African women, 95 developed preeclampsia and out of the 279 Caucasian women 81 had preeclampsia. Carriers of the NOS3I4a/b polymorphism had a 1.7-fold increased risk to develop preeclampsia. There was no difference in distribution of the other tested polymorphism. Furthermore we could show a fourfold reduction to encounter severe course of preeclampsia (defined as occurrence of HELLP-syndrome or eclampsia) in carriers of the EPHX1 polymorphism encoding histidine. CONCLUSIONS: Our data reveal a highly significant association of the NOS3I4a/b polymorphism with increased risk to develop preeclampsia in pregnancy. The shown association of EPXH1 polymorphism with the severity of preeclampsia strengthen the concept, that individual susceptibility determines the capability of the maternal organism to deal with the pregnancy derived agents causing preeclampsia.

Inhibition of interferon induction and action by the nairovirus Nairobi sheep disease virus/Ganjam virus.

The Nairoviruses are an important group of tick-borne viruses that includes pathogens of man (Crimean Congo hemorrhagic fever virus) and livestock animals (Dugbe virus, Nairobi sheep disease virus (NSDV)). NSDV is found in large parts of East Africa and the Indian subcontinent (where it is known as Ganjam virus). We have investigated the ability of NSDV to antagonise the induction and actions of interferon. Both pathogenic and apathogenic isolates could actively inhibit the induction of type 1 interferon, and also blocked the signalling pathways of both type 1 and type 2 interferons. Using transient expression of viral proteins or sections of viral proteins, these activities all mapped to the ovarian tumour-like protease domain (OTU) found in the viral RNA polymerase. Virus infection, or expression of this OTU domain in transfected cells, led to a great reduction in the incorporation of ubiquitin or ISG15 protein into host cell proteins. Point mutations in the OTU that inhibited the protease activity also prevented it from antagonising interferon induction and action. Interestingly, a mutation at a peripheral site, which had little apparent effect on the ability of the OTU to inhibit ubiquitination and ISG15ylation, removed the ability of the OTU to block the induction of type 1 and the action of type 2 interferons, but had a lesser effect on the ability to block type 1 interferon action, suggesting that targets other than ubiquitin and ISG15 may be involved in the actions of the viral OTU.

KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffin-embedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay.

AIMS: To evaluate a reverse-hybridisation assay (strip assay) designed for the sensitive detection of 10 mutations in codons 12 and 13 of the KRAS gene. The strip assay relies on mutant-enriched PCR followed by reverse-hybridisation of biotinylated amplification products to oligonucleotide probes immobilised as an array of parallel lines on nitrocellulose test strips. METHODS: The strip assay was used to analyse genomic DNA isolated from 120 formalin-fixed paraffin-embedded (FFPE) ovarian tissue samples. The samples were analysed in parallel using a biochip-based protocol (biochip assay) covering the same mutation spectrum, and results were compared with respect to sensitivity, specificity and operational input. RESULTS: The strip assay identified 19 (16%) of 120 FFPE samples to carry a KRAS mutation; results were in agreement with those obtained by biochip hybridisation. Both assays had an analytical sensitivity of 1% when performed on FFPE-extracted DNA with approximately the same operational input needed for post-PCR processing. In contrast to the biochip assay, strip assay hybridisation may be automated to a large extent. CONCLUSIONS: The strip assay is an accurate and sensitive tool for the low to medium throughput detection of KRAS mutation in genomic DNA isolated from FFPE tissue.

Sensitive detection of KRAS mutations in archived formalin-fixed paraffin-embedded tissue using mutant-enriched PCR and reverse-hybridization.

Recently, evidence has emerged indicating that assessment of KRAS mutations before anti-epidermal growth factor receptor therapy improves outcome in patients with metastatic colorectal cancer (CRC). We report here a novel reverse-hybridization (RH) assay to screen for KRAS mutations in formalin-fixed paraffin-embedded colorectal tissue samples. We combined mutant-enriched PCR based on peptide nucleic acid clamping and RH of amplification products to nitrocellulose test strips that contained a parallel array of oligonucleotide probes targeting 10 frequent mutations in codons 12 and 13 of the KRAS gene. DNA mixing experiments, which included eight different tumor cell lines with known KRAS mutations, were performed to examine the sensitivity of mutation detection. All KRAS mutations present in tumor cell lines were unambiguously identified by the RH assay with 1% of each cell line DNA diluted in normal DNA. RH was then used to screen for KRAS mutations in 74 colorectal tumor and 4 normal control samples. Twenty-six (35%) of the 74 tumor samples showed KRAS mutations. No mutation was found in the four samples of normal colorectal tissue. DNA sequencing without previous mutant enrichment, however, failed to detect four (15%) out of 26 KRAS-positive formalin-fixed paraffin-embedded samples (FFPE). This finding suggests that even after microdissection, mutant sequences in a given DNA isolate can be rare and more sensitive methods are needed for mutation analysis.

MAPKAP kinase 3pK phosphorylates and regulates chromatin association of the polycomb group protein Bmi1.

Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14(ARF), which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process.

Stabilization of neurotensin analogues: effect on peptide catabolism, biodistribution and tumor binding.

Neurotensin (NT) receptors in pancreatic and other neuroendocrine tumors are promising targets for imaging and therapeutic purposes. Here, we report on the effect of distinct changes in the peptide chain on catabolism in vitro for five radiolabeled [99mTc] neurotensin analogues having high affinity for neurotensin receptors. Substitution of NT(1-7) by (NalphaHis)Ac--the Tc-binding moiety--combined with a reduced bond 8-9 (CH2NH), N-methylation of peptide bonds or replacement of Ile(12) by tertiary leucin (Tle) led to peptide stabilization of various degrees. Biodistribution studies in nude mice bearing HT29 xenografts showed higher tumor uptake with more stable peptides, yielding high tumor to blood ratios of up to 70.