Advancing Biomarker Development Through Convergent Engagement: Summary Report of the 2nd International Danube Symposium on Biomarker Development, Molecular Imaging and Applied Diagnostics; March 14-16, 2018; Vienna, Austria.

Here, we report on the outcome of the 2nd International Danube Symposium on advanced biomarker development that was held in Vienna, Austria, in early 2018. During the meeting, cross-speciality participants assessed critical aspects of non-invasive, quantitative biomarker development in view of the need to expand our understanding of disease mechanisms and the definition of appropriate strategies both for molecular diagnostics and personalised therapies. More specifically, panelists addressed the main topics, including the current status of disease characterisation by means of non-invasive imaging, histopathology and liquid biopsies as well as strategies of gaining new understanding of disease formation, modulation and plasticity to large-scale molecular imaging as well as integrative multi-platform approaches. Highlights of the 2018 meeting included dedicated sessions on non-invasive disease characterisation, development of disease and therapeutic tailored biomarkers, standardisation and quality measures in biospecimens, new therapeutic approaches and socio-economic challenges of biomarker developments. The scientific programme was accompanied by a roundtable discussion on identification and implementation of sustainable strategies to address the educational needs in the rapidly evolving field of molecular diagnostics. The central theme that emanated from the 2nd Donau Symposium was the importance of the conceptualisation and implementation of a convergent approach towards a disease characterisation beyond lesion-counting "lumpology" for a cost-effective and patient-centric diagnosis, therapy planning, guidance and monitoring. This involves a judicious choice of diagnostic means, the adoption of clinical decision support systems and, above all, a new way of communication involving all stakeholders across modalities and specialities. Moreover, complex diseases require a comprehensive diagnosis by converging parameters from different disciplines, which will finally yield to a precise therapeutic guidance and outcome prediction. While it is attractive to focus on technical advances alone, it is important to develop a patient-centric approach, thus asking "What can we do with our expertise to help patients?"

Human papillomavirus (HPV) and somatic EGFR mutations are essential, mutually exclusive oncogenic mechanisms for inverted sinonasal papillomas and associated sinonasal squamous cell carcinomas.

Background: Inverted sinonasal (Schneiderian) papilloma (ISP) is a locally aggressive neoplasm often associated with sinonasal squamous cell carcinoma (SNSCC). While the etiology of ISP is not well understood, human papillomavirus (HPV) has been detected in a subset of cases. Our group recently identified activating somatic EGFR mutations in the majority of ISP and ISP-associated SNSCC. However, the relationship between EGFR mutations and HPV infection has not been explored. Patients and methods: We evaluated 58 ISP and 22 ISP-associated SNSCC (including 13 patients with matched ISP/SNSCC samples), as well as 14 SNSCC without clinical or pathologic evidence of an associated ISP. Formalin-fixed, paraffin-embedded samples were evaluated for EGFR mutations using Sanger sequencing and for HPV infection using GP5+/GP6+ PCR. HPV subtyping based on the L1 sequence was done for HPV positive cases including temporally distinct tumors for four patients. Clinicopathologic data including progression free survival was also analyzed. Results: All ISP and ISP-associated SNSCC demonstrated either an EGFR mutation or HPV infection. HPV and EGFR mutation were mutually exclusive in all cases of ISP-associated SNSCC and all but one ISP; this case was only weakly HPV positive, and analysis of a prior temporally distinct ISP specimen from this patient failed to show HPV infection, suggesting transient infection/incidental colonization. HPV subtypes in ISP and ISP-associated SNSCC were predominantly low-risk, in contrast with SNSCC without ISP association, which showed frequent high-risk HPV. All paired ISP and associated SNSCC samples demonstrated concordant HPV status and EGFR genotypes. ISP progression to SNSCC was significantly associated with the presence of HPV infection and the absence of an EGFR mutation (log-rank = 9.620, P = 0.002). Conclusions: Collectively our data show that EGFR mutations and HPV infection represent essential, alternative oncogenic mechanisms in ISP and ISP-associated SNSCC.

Subjective well-being among young people in five Eastern European countries.

BACKGROUND: Subjective well-being incorporates elements of life satisfaction, happiness and optimism. It is increasingly relevant in the assessment of population health and economic development. There are strong continuities in well-being from youth into later life. Despite its significance, few global surveys capture subjective well-being. This paper describes patterns of well-being among young people in five Eastern European countries [Belarus, Bosnia and Herzegovina (BiH), the Former Yugoslav Republic of Macedonia, Serbia and Ukraine] and investigates association between demographic factors and well-being. METHODS: Nationally representative household surveys, including large Roma population samples, were conducted as part of UNICEF's Multiple Indicator Cluster Survey programme. Young people aged 15-24 years (N = 11 944) indicated their satisfaction with life, happiness and expectations about the future. Multilevel logistic regressions were conducted to determine the impact of individual-level predictors while accounting for country- and cluster-level variability. RESULTS: Around 40% of young people considered themselves very happy or very satisfied with their life overall. Three quarters reported optimism. Yet well-being varied greatly between countries, with youth in BiH and Ukraine reporting lowest levels of well-being. Current marriage, increasing wealth, higher education, rural residence and not having children were associated with greater well-being. CONCLUSIONS: Patterns of well-being in youth vary substantially between countries and are only partly accounted for by standard demographic characteristics. Despite higher rates of adolescent marriage and childbearing, and lower levels of educational attainment and employment, Roma youth had similar levels of well-being to the general population.

The utility of the radionuclide probe in parathyroidectomy for primary hyperparathyroidism.

INTRODUCTION Parathyroidectomy is the definitive treatment for primary hyperparathyroidism but the intraoperative identification of adenomas is challenging. The aim of this study was to evaluate the utility of a radionuclide probe (RNP) in addition to intraoperative parathyroid hormone ( IOPTH) measurement as an intraoperative diagnostic adjunct in patients undergoing parathyroidectomy for primary hyperparathyroidism. METHODS This was a retrospective cohort study of patients treated between 2004 and 2015 in a university affiliated teaching hospital. Patients were grouped into those with RNP use (RNP+) and those without (RNP-). The primary outcome measure was rate of operative failure, which included false positives. The diagnostic sensitivity and positive predictive value of both RNP and IOPTH were also evaluated. RESULTS A total of 298 patients were included in the study, 127 (42.6%) being in the RNP+ group and 171 (57.4%) in the RNP- group. The false positive rate for the RNP+ patients was 1.6% compared with 9.4% for RNP- patients (p=0.006, hazard ratio [HR]: 6.45). The rates of operative failure were 6.3% and 11.7% respectively (p=0.159, HR: 1.97). RNP use had a sensitivity of 92.0% and a positive predictive value of 98.3% compared with 78.6% and 95.2% respectively for IOPTH monitoring. CONCLUSIONS RNP use is associated with fewer false positives and reduced operative failure than IOPTH measurement. It also has a higher sensitivity and positive predictive value. RNP use is recommended in centres that have the required facilities.

Anorectal swabs as a marker of male-to-male sexual exposure in STI surveillance systems.

Identification of priority populations such as men who have sex with men (MSM) is important in surveillance systems to monitor trends of sexually transmitted infections (STIs). We explored using routinely collected non-behavioural data as a means to establish MSM status in surveillance by assessing anorectal swab as a marker of male-to-male sexual exposure. We used chlamydia testing data from a sexual health clinic, 2007-2012. Men reporting any male sexual partner(s) in the previous 12 months were considered MSM. The dataset was split into development and validation samples to develop a univariate predictive model and assess the model fit. The dataset included 30 358 individual men and 48 554 episodes of STI testing; 45% were among reported MSM and an anorectal swab was performed in 40% of testing episodes. Anorectal swabbing had good diagnostic performance as a marker for MSM status (sensitivity = 87%, specificity = 99%, positive predictive value = 98·6%, negative predictive value = 90·3%). The model showed good fit against the internal validation sample (area under the curve = 0·93). Anorectal swabs are a valid marker of MSM behaviour in surveillance data from sexual health clinics, and they are likely to be particularly useful for monitoring STI trends among MSM with higher risk behaviour.

NPM-ALK phosphorylates WASp Y102 and contributes to oncogenesis of anaplastic large cell lymphoma.

Mechanisms by which NPM-ALK signaling regulates cell migration, invasion and contributes to the oncogenesis of anaplastic large cell lymphoma (ALCL) are not completely understood. In an attempt to identify novel actin signaling pathways regulated by NPM-ALK, a comprehensive phosphoproteome analysis of ALCL cell lines was performed in the presence or absence of NPM-ALK activity. Numerous phosphoproteins involved in actin dynamics including Wiskott-Aldrich syndrome protein (WASp) were regulated by NPM-ALK. Network analysis revealed that WASp is a central component of the NPM-ALK-dependent actin signaling pathway. Here we show that NPM-ALK phosphorylates WASp at its known activation site (Y290) as well as at a novel residue (Y102). Phosphorylation of WASp at Y102 negatively regulates its interaction with Wiskott-Aldrich interacting protein and decreases its protein stability. Phosphorylation of WASp at Y102 enhances anchorage-independent growth and tumor growth in an in vivo xenograft model and enhances invasive properties of ALCL. We show that knock-down of WASp or expression of Y102F mutant of WASp decreases colony formation and in vivo tumor growth. Our results show that WASp is a novel substrate of ALK and has a critical role in regulating invasiveness and oncogenesis of ALCL.

Effects of Melatonin on Colchicine-Treated PLBs of Dendrobium sonia-28 Orchid.

Dendrobium hybrid orchid is popular in orchid commercial industry due to its short life cycle and ability to produce various types of flower colours. This study was conducted to identify the morphological, biochemical and scanning electron microscopy (SEM) analysis in the Dendrobium sonia-28 orchid plants. In this study, 0.05 and 0.075 % of colchicine-treated Dendrobium sonia-28 (4-week-old culture) protocorm-like bodies (PLBs) were treated in different concentrations of melatonin (MEL) posttreatments (0, 0.05, 0.1, 0.5, 1, 5 and 10 µM). Morphological parameters such as number of shoots, growth index and number of PLBs were determined. In the 0.05 and 0.075 % of colchicine-treated PLBs which were posttreated with 0.05 µM MEL resulted in the highest value of the morphological parameters tested based on the number of shoots (84.5 and 96.67), growth index (16.94 and 12.15) and number of PLBs (126.5 and 162.33), respectively. SEM analysis of the 0.05 µM MEL posttreatment on both the colchicine-treated regenerated PLBs showed irregular cell lineages, and some damages occurred on the stomata. This condition might be due to the effect of plasmolyzing occurred in the cell causing irregular cell lineages.

Hemilaminectomy for large spinal extradural meningeal cysts: A case report and review of surgical techniques.

Spinal extradural meningeal cysts (SEMC) are uncommon causes of back pain. The literature contains only case reports of this pathology, and treatment remains controversial due to its rarity. We present a case of SEMC and describe an approach via hemilaminectomy, with the choice of side guided by radiological imaging, followed by complete excision of the cyst and repair of the underlying dural defect.

Performance of signal-to-noise ratio estimation for scanning electron microscope using autocorrelation Levinson-Durbin recursion model.

A new technique to quantify signal-to-noise ratio (SNR) value of the scanning electron microscope (SEM) images is proposed. This technique is known as autocorrelation Levinson-Durbin recursion (ACLDR) model. To test the performance of this technique, the SEM image is corrupted with noise. The autocorrelation function of the original image and the noisy image are formed. The signal spectrum based on the autocorrelation function of image is formed. ACLDR is then used as an SNR estimator to quantify the signal spectrum of noisy image. The SNR values of the original image and the quantified image are calculated. The ACLDR is then compared with the three existing techniques, which are nearest neighbourhood, first-order linear interpolation and nearest neighbourhood combined with first-order linear interpolation. It is shown that ACLDR model is able to achieve higher accuracy in SNR estimation.

Oncogenic Y641 mutations in EZH2 prevent Jak2/ß-TrCP-mediated degradation.

EZH2 (enhancer of zeste homolog 2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2), which trimethylates histone H3 (H3K27) to mediate gene repression. Somatic mutations, overexpression and hyperactivation of EZH2 have been implicated in the pathogenesis of several forms of cancer. In particular, recurrent gain-of-function mutations targeting EZH2 Y641 occur most frequently in follicular lymphoma and aggressive diffuse large B-cell lymphoma and are associated with H3K27me3 hyperactivation, which contributes to lymphoma pathogenesis. However, the post-translational mechanisms of EZH2 regulation are not completely understood. Here we show that EZH2 is a novel interactor and substrate of the SCF E3 ubiquitin ligase ß-TrCP (FBXW1). ß-TrCP ubiquitinates EZH2 and Jak2-mediated phosphorylation on Y641 directs ß-TrCP-mediated EZH2 degradation. RNA interference-mediated silencing of ß-TrCP or inhibition of Jak2 results in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the EZH2(Y641) mutants recurrently implicated in lymphoma pathogenesis are unable to bind ß-TrCP. Further, endogenous EZH2(Y641) mutants in lymphoma cells exhibit increased EZH2 stability and H3K27me3 hyperactivity. Our studies demonstrate that ß-TrCP has an important role in controlling H3K27 trimethylation activity and lymphoma pathogenesis by targeting EZH2 for degradation.

Fbxo45-mediated degradation of the tumor-suppressor Par-4 regulates cancer cell survival.

Prostate apoptosis response protein 4 (Par-4) also known as PRKC apoptosis WT1 regulator is a tumor suppressor that selectively induces apoptosis in cancer cells. However, its post-translational regulation by ubiquitin-mediated proteolysis and the cellular machinery that is responsible for its proteasomal degradation are unknown. Using immunopurification and an unbiased mass spectrometry-based approach, we show that Par-4 interacts with the SPRY-domain containing E3 ubiquitin ligase Fbxo45 through a short consensus sequence motif. Fbxo45 interacts with Par-4 in the cytoplasm and mediates its ubiquitylation and proteasomal degradation. Fbxo45 silencing results in stabilization of Par-4 with increased apoptosis. Importantly, a Par-4 mutant that is unable to bind Fbxo45 is stabilized and further enhances staurosporine-induced apoptosis. Co-expression of Fbxo45 with Par-4 protects cancer cells against Par-4-induced apoptosis. Our studies reveal that Fbxo45 is the substrate-receptor subunit of a functional E3 ligase for Par-4 that has a critical role in cancer cell survival.

The role of front-line anthracycline-containing chemotherapy regimens in peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) are a heterogenous group of aggressive non-Hodgkin's lymphomas that are incurable in the majority of patients with current therapies. Outcomes associated with anthracycline-based therapies are suboptimal, but remain the standard of care for most patients, even though the benefits of this approach remain uncertain. This study retrospectively examined outcomes in a cohort of North American PTCL patients treated with both anthracycline- and nonanthracycline-containing regimens. The incorporation of anthracycline-containing regimens was associated with improved progression-free survival (PFS) and overall survival (OS). Patients treated with nonanthracycline-containing regimens were more likely to have high-risk features and were less likely to undergo high-dose therapy and stem cell transplantation. However, anthracycline use remained an independent predictor of improved PFS and OS when adjusting for these confounding variables. Anthracycline-based regimens and consolidation with high-dose therapy and autologous stem cell transplantation in appropriately selected patients remains a viable option for patients unable to participate in a clinical trial. Long-term disease-free survival is not optimal, highlighting the need for an improved understanding of disease pathogenesis, and the development of novel therapeutic strategies.

Rapid genotyping of Plasmodium vivax Pvs25 and Pv38 genes by using mismatch specific endonuclease.

Mismatch specific endonuclease (MSE) method was used to detect natural polymorphisms in Pvs25 and Pv38 genes of Plasmodium vivax. Eighty seven patients with P. vivax were recruited in the Republic of Korea (ROK). Pvs25 and Pv38 genes were amplified by polymerase chain reaction (PCR), and the PCR amplicons were mixed with reference DNA sequences. Following the denaturation and gradual annealing, the product mixtures were cleaved by the MSE. Heteroduplex types were readily detected by gel electrophoresis, where extra bands with shorter sizes would appear from the cleavage. After MSE cleavage of 657- bp product from Pvs25 mixtures, three genotypes were detected, while Pv38 mixtures with 1220-bp products presented two genotypes in ROK isolates. After the MSE cleavage, the mismatched samples of Pvs25 and Pv38 were completely sequenced, and the results were in complete agreement with the MSE analyses. In conclusion, genotyping of Pvs25 and Pv38 with MSE cleavage could be a potential method for the high-throughput screening of the large field samples.

Rapid genotyping of Plasmodium vivax Pvs25 and Pv38 genes by using mismatch specific endonuclease

Mismatch specific endonuclease (MSE) method was used to detect natural polymorphisms in Pvs25 and Pv38 genes of Plasmodium vivax. Eighty seven patients with P. vivax were recruited in the Republic of Korea (ROK). Pvs25 and Pv38 genes were amplified by polymerase chain reaction (PCR), and the PCR amplicons were mixed with reference DNA sequences. Following the denaturation and gradual annealing, the product mixtures were cleaved by the MSE. Heteroduplex types were readily detected by gel electrophoresis, where extra bands with shorter sizes would appear from the cleavage. After MSE cleavage of 657- bp product from Pvs25 mixtures, three genotypes were detected, while Pv38 mixtures with 1220-bp products presented two genotypes in ROK isolates. After the MSE cleavage, the mismatched samples of Pvs25 and Pv38 were completely sequenced, and the results were in complete agreement with the MSE analyses. In conclusion, genotyping of Pvs25 and Pv38 with MSE cleavage could be a potential method for the high-throughput screening of the large field samples.

Generation of potent cytotoxic T lymphocytes against castration-resistant prostate cancer cells by dendritic cells loaded with dying allogeneic prostate cancer cells.

To induce a potent cytotoxic T lymphocyte (CTL) response in dendritic cell (DC)-based immunotherapy against prostate cancer, various tumour antigens should be loaded onto DCs. The aim of this study was to establish a method of immunotherapy for castration-resistant prostate cancer (CRPC) using prostate cancer-specific CTLs generated in vitro by DCs. Monocyte-derived DCs from patients with CRPC were induced to mature using a standard cytokine cocktail (in IL-1ß, TNF-α, IL-6 and PGE(2) : standard DCs, sDCs) or using an α-type 1-polarized DC (αDC1) cocktail (in IL-1ß, TNF-α, IFN-α, IFN-γ and polyinosinic:polycytidylic acid) and loaded with the UVB-irradiated CRPC cell line PC-3. Antigen-loaded DCs were evaluated by morphological and functional assays. The αDC1s significantly increased the expression of several molecules related to DC maturation, regardless of whether the αDC1s were loaded with tumour antigens or not, compared to sDCs. The αDC1s showed a higher production of interleukin-12 both during maturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumour antigens, as compared to standard DCs (sDCs). Prostate cancer-specific CTLs against autologous CRPC cells were successfully induced by αDC1s loaded with dying PC-3 cells. Autologous αDC1s loaded with an allogeneic CRPC cell line can generate greater CRPC-specific CTL responses as compared to sDCs and may provide a novel source of DC-based vaccines that can be used for the development of immunotherapy in patients with CRPC.

Quantifying errors in the estimation of tuberculosis mortality in a population of South African miners.

BACKGROUND: All-cause mortality, based on national tuberculosis programme (NTP) register deaths, may under- or overestimate tuberculosis (TB) specific mortality in the population. OBJECTIVE: To assess the factors influencing this measurement in a single large population with high TB prevalence and mortality. METHODS: Routinely collected data on TB cases and treatment outcomes were linked to population data from a cohort of South African miners from 1995 to 2008. Vital status and cause of death were determined from multiple sources, including the TB programme, death register and autopsy. RESULTS: The TB mortality rate, based on 430 deaths on the TB register, was 192/100,000 person-years (py). Many of these deaths (57%) were not caused by TB, and 483 TB deaths were identified outside the programme. Overall, there were 674 TB-specific deaths; the TB-specific mortality rate was 302/100,000 py. These deaths included 191 (28%) on the TB register, 23 (3%) among defaulters/transfers, 153 (23%) after anti-tuberculosis treatment and 307 (46%) in men who had never been on the programme. CONCLUSIONS: This study highlights methodological issues in estimating TB mortality. In this population, a method using the product of TB incidence and case fatality consistently underestimated TB mortality. Accurate estimates of TB-specific mortality are crucial for the proper evaluation of TB control programmes.

NPM-ALK signals through glycogen synthase kinase 3ß to promote oncogenesis.

Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70-80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric tyrosine kinase, which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3ß as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3ß. Expression of NPM-ALK in 293T cells led to an increase of pS(9)-GSK3ß (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3ß levels. Phosphorylation of pS(9)-GSK3ß by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3ß substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3ß. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3ß inhibition. Furthermore, stable knockdown of GSK3ß conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS(9)-GSK3ß and CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies, and showed a significant correlation (P<0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS(9)-GSK3ß and CDC25A expression (P<0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S(9)-GSK3ß by PI3K/AKT. The subsequent inhibition of GSK3ß activity results in accumulation of CDC25A and Mcl-1, which confers the advantage of growth and protection from apoptosis. These findings provide support for the role of GSK3ß as a mediator of NPM-ALK oncogenesis.