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Background and objectives With disease-modifying treatment strategies on the horizon, stratification of individual patients at the earliest stages of Parkinson's disease (PD) is key-ideally already at clinical disease onset. Blood levels of neurofilament light chain (NfL) provide an easily accessible fluid biomarker that might allow capturing the conversion from prodromal to manifest PD. Methods We assessed longitudinal serum NfL levels in subjects converting from prodromal to manifest sporadic PD (converters), at-risk subjects, and matched controls (72 participants with ≈4 visits), using single-molecule array (Simoa) technique. Results While NfL levels were not increased at the prodromal stage, subjects converting to the manifest motor stage showed a significant intraindividual acceleration of the age-dependent increase of NfL levels. Conclusions The temporal dynamics of intraindividual NfL blood levels might mark the conversion to clinically manifest PD, providing a potential stratification biomarker for individual disease onset in the advent of precision medicine for PD. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Therapeutic success in treating patients with systemic lupus erythematosus (SLE) is limited by the multivariate disease etiology, multi-organ presentation, systemic involvement, and complex immunopathogenesis. Agents targeting B-cell differentiation and survival are not efficacious for all patients, indicating a need to target other inflammatory mediators. One such target is the type I interferon pathway. Type I interferons upregulate interferon gene signatures and mediate critical antiviral responses. Dysregulated type I interferon signaling is detectable in many patients with SLE and other autoimmune diseases, and the extent of this dysregulation is associated with disease severity, making type I interferons therapeutically tangible targets. The recent approval of the type I interferon-blocking antibody, anifrolumab, by the US Food and Drug Administration for the treatment of patients with SLE demonstrates the value of targeting this pathway. Nevertheless, the interferon pathway has pleiotropic biology, with multiple cellular targets and signaling components that are incompletely understood. Deconvoluting the complexity of the type I interferon pathway and its intersection with lupus disease pathology will be valuable for further development of targeted SLE therapeutics. This review summarizes the immune mediators of the interferon pathway, its association with disease pathogenesis, and therapeutic modalities targeting the dysregulated interferon pathway.

The 14 authors of the first review article on hidradenitis suppurativa (HS) pathogenesis published 2008 in EXPERIMENTAL DERMATOLOGY cumulating from the 1st International Hidradenitis Suppurativa Research Symposium held March 30-April 2, 2006 in Dessau, Germany with 33 participants were prophetic when they wrote "Hopefully, this heralds a welcome new tradition: to get to the molecular heart of HS pathogenesis, which can only be achieved by a renaissance of solid basic HS research, as the key to developing more effective HS therapy." (Kurzen et al. What causes hidradenitis suppurativa? Exp Dermatol 2008;17:455). Fifteen years later, there is no doubt that the desired renaissance of solid basic HS research is progressing with rapid steps and that HS has developed deep roots among inflammatory diseases in Dermatology and beyond, recognized as "the only inflammatory skin disease than can be healed". This anniversary article of 43 research-performing authors from all around the globe in the official journal of the European Hidradenitis Suppurativa Foundation e.V. (EHSF e.V.) and the Hidradenitis Suppurativa Foundation, Inc (HSF USA) summarizes the evidence of the intense HS clinical and experimental research during the last 15 years in all aspects of the disease and provides information of the developments to come in the near future., info:eu-repo/semantics/published

Colorectal cancer is an important cancer worldwide, whose etiology is not fully understood. Known causes include several genetic factors, overweight/obesity, tobacco smoking, and heavy alcohol drinking. An etiologic role of diet is highly plausible but the evidence for specific factors, with the possible exception of processed meat, is not conclusive. Workers exposed to asbestos have been found at increased risk of colorectal cancer in several studies, but the evidence is not sufficiently strong to conclude in favor of a causal association. No other occupational factors have been linked to colorectal cancer. Liver cancer is a common cancer in many regions of the world and is the second cause of cancer-specific mortality. About 75% of liver cancers are hepatocellular carcinoma (HCC), the second most frequent type being cholangiocarcinoma (CCA). HCC develops in the context of a web of interactions between viral (HBV, HCV), environmental (alcohol, aflatoxin), and metabolic (fatty liver disease, obesity) factors. Genetic predisposition accounts for only a small fraction of the global burden of HCC. The only established occupational cause of liver cancer is vinyl chloride, which causes a rare type of neoplasm, angiosarcoma, and has also been associated with HCC. Detection and diagnosis of HCC is complicated by its occurrence in a background of chronic liver disease characterized by inflammation and cycles of hepatocyte proliferation and destruction. Markers used in clinical practice include serological and molecular markers of viral hepatitis, enzymatic tests for liver function and injury, and a growing list of plasma-based tumor markers, the current gold standard being alpha-fetoprotein (AFP). Recent research has identified molecular changes in transcriptome, microRNAome, epigenome and, significantly, plasma proteome, that pave the way to the development of a new generation of biomarkers for early detection of HCC in different etiological contexts.

Background: A more reliable classification of skin inflammation and severity of active disease results from ultrasound sonography and the new hidradenitis suppurativa/acne inversa (HS) classification system IHS4. However, an objective assessment of skin inflammation in a continuous mode is still the ultimate goal. Long-wave medical infrared thermography (MIT) may offer a blood flow and temperature differential assessment in inflammatory conditions. Objective: To evaluate the application of MIT in HS. Methods: Standardized photography of the areas involved or been candidates for HS involvement was performed and MIT pictures were taken simultaneously and superimposed on the photographs of 18 patients (11 female, 7 male, median age 38.75 years [95% confidence interval 28.5 and 51 years], Hurley score I 5.6%, Hurley score II 38.9%, and Hurley score III 55.5%). A modification of the Otsu’s method facilitated the automatic lesion segmentation from the background, depicting the inflammation area. Moreover, MIT was administered in real-time mode during radical HS surgery. Results: A 1°C temperature difference from a corresponding symmetric body region was indicative of inflammation. MIT figures detected a gradual increase of skin temperature from 33.0°C in healthy skin on average to 35.0–36.6°C at the center of inflamma tory lesions in the axilla and to 35.4–36.9°C at the center of inflammation in the groin area. Real-time MIT assessment enabled the definition of the margins and depth of the surgical intervention during the procedure. Conclusion: MIT is a promising tool for the detection of inflammation severity in HS lesions and can be used as a clinical biomarker in evaluation studies of medical and surgical HS treatment.

Identification of improved translatable biomarkers of nephrotoxicity is an unmet safety biomarker need. Fatty-acid-binding protein 4 (FABP4) was previously found to be associated with clinical renal dysfunction and was proposed as a biomarker of glomerular damage. The aim of this study was to evaluate FABP4 as a potential preclinical biomarker of drug-induced kidney injury (DIKI). Han-Wistar rats were dosed with cisplatin [2.5 mg/kg, single, intraperitoneally (i.p.)], puromycin (10 mg/kg, daily, i.p.) or N-phenylanthranylic acid [NPAA, 500 mg/kg, daily, per os (p.o.)] over a 28-day period to induce proximal tubule, glomerular or collecting duct injury, respectively. An increase in urinary FABP4 levels was observed on days 1 and 3 after NPAA treatment and on days 14, 21, and 28 after puromycin treatment, whereas cisplatin treatment had no effect. No significant changes were reported for plasma levels of FABP4 after any treatment. Interestingly, immunohistochemical analysis showed a marked decrease in FABP4 expression in the loop of Henle on day 7 after NPAA treatment and a complete loss of FABP4 expression on day 14 after puromycin treatment. The magnitude of increase in FABP4 urinary levels in response to NPAA and puromycin was higher than for established preclinical biomarkers serum creatinine, clusterin, or cystatin C. Our results suggest that FABP4 has the potential for preclinical application as a biomarker of DIKI.

Drug-induced kidney injury (DIKI) remains a significant concern during drug development. Whereas FDA-endorsed urinary protein biomarkers encounter limitations including the lack of translatability, there is a considerable interest surrounding the application of microRNAs (miRNAs) in the renal biomarker space. Current knowledge about the value of these novel biomarkers for subacute preclinical rodent studies is still sparse. In this work, Wistar rats were treated with three nephrotoxic compounds-cisplatin (CIS, proximal tubule, 2.5 mg/kg, intraperitoneal [i.p.]), puromycin (PUR, glomerulus, 20/10 mg/kg, i.p.) and N-phenylanthranylic acid (NPAA, collecting ducts, 500 mg/kg, per os)-for up to 28 days to evaluate the performance of a panel of 68 urinary miRNAs as potential nephron segment-specific biomarkers. Out of these 68 kidney injury associated-miRNAs, our selection strategy ultimately revealed rno-miR-34c-5p significantly dysregulated after CIS single administration, and rno-miR-335 and rno-miR-155-5p significantly dysregulated after PUR treatment. In contrast, NPAA daily administration strongly altered the expression profile of 28 miRNAs, with rno-miR-210-3p displaying the most robust changes. A thorough evaluation showed that these miRNA candidates could complement urinary protein biomarkers to detect CIS- or PUR-induced kidney injury in a subacute setting, with a mechanistic (based on rno-miR-34c-5p) and/or a kidney injury detection potential. Our results also provide the first evidence that urinary miRNAs could enhance the detection of collecting duct damage. Overall, these data improve our understanding of the utility of urinary miRNAs as DIKI biomarkers in a subacute DIKI preclinical setting and support the value of using urinary biomarker panels comprising proteins and miRNAs.

Background: Despite existing guidelines and methods for standardized clinical photography in dermatology and plastic surgery, human skin exhibits exquisite site-specific morphologies and functions, and each body region can exhibit an individual pathologic phenotype. The aim of this work was to develop a standardized, representative and reproducible documentation of the multilocular hidradenitis suppurativa/acne inversa (HS) lesions, a disease mostly occurring in skin folds. Method: Optimal body positions for medical photography of candidate areas for HS involvement were evaluated. Pictures of volunteers were taken, and indicative scientific graphics were designed. Results: Due to the variability of HS lesions and the fact of their localization in skin folds, a standardized, reproducible photographic documentation of HS candidate skin areas (50 cm from the skin surface) is proposed. The photographic series includes: (1) right armpit, (2) left armpit, (3) right groin, (4) left groin, (5) genital area, (6) anal area and anal fold, (7) right buttock, (8) left buttock, (9) chest area, (10) mons pubis, (11) right submammary area (females), (12) left submammary area (females). The protocol is accompanied with indicative scientific graphics for photography-proper positioning of the relevant body parts for documentation of potentially flat skin areas. In addition, former proposals for technical standardization of photography in dermatology, regarding instrumentation, environmental lighting and background colour, have to be respected. Conclusion: Standardized photography of candidate skin areas for HS involvement will allow longitudinal intraindividual and interindividual evaluation of the disease course as well as prospective and retrospective comparative studies.

Drug-induced cardiac injury (DICI) detection remains a major safety issue in drug development. While circulating microRNAs (miRs) have emerged as promising translational biomarkers, novel early detection biomarkers of cardiotoxicity are needed. This work aims at evaluating whether a panel of putative cardiac injury plasma miRs could serve as early DICI biomarkers in a 4-day rat preclinical model. Out of a panel of 68 selected targets, we identified plasma miR-208a-3p as being significantly upregulated after single administration with either isoproterenol (ISO) or allylamine (AAM). This provides the first evidence of miR-208a-3p detection after AAM administration. Moreover, similarly to cardiac troponins (cTn), plasma miR-208a-3p expression profile appears to be compound-specific with most significant early changes occurring in ISO-treated rats. Overall, miR-208a-3p performance in detecting the severity of myocardial injury, as well as the magnitude of miR-208a-3p increase after ISO or AAM administration, were comparable to that of cTn. Our results highlight the importance of assessing the whole time-dependent profiles of miR expression. Hence, time course evaluation revealed plasma miR candidates whose expression was not stable across the duration of the study in the vehicle group, restricting their utility as cardiac injury-specific biomarkers. In light of these findings, miR-208a-3p has a potential to complement the existing biomarkers of cardiac injury specifically in the context of evaluating toxicity in a time-dependant manner. Assessment of miR-208a-3p in other DICI settings would strengthen its robustness as an early detection biomarker leading to a warranted extensive and rigorous validation.

Aim: To set up a targeted methylation analysis using semiconductor sequencing and evaluate the potential for studying methylation in circulating cell-free DNA (cfDNA). Materials & Methods: Methylation of VIM, FBLN1, LTBP2, HINT2, h19 and IGF2 was analyzed in plasma cfDNA and white blood cell DNA obtained from eight hepatocellular carcinoma patients and eight controls using Ion Torrent™ PGM sequencer. Results: h19 and IGF2 showed consistent methylation levels and methylation was detected for VIM and FBLN1, whereas LTBP2 and HINT2 did not show methylation for target regions. VIM gene promoter methylation was higher in HCC cfDNA than in cfDNA of controls or white blood cell DNA. Conclusion: Semiconductor sequencing is a suitable method for analyzing methylation profiles in cfDNA. Furthermore, differences in cfDNA methylation can be detected between controls and hepatocellular carcinoma cases, even though due to the small sample set these results need further validation.

// Marcia Cristina T. dos Santos 1 , Miguel Arturo Barreto-Sanz 2 , Bruna Renata S. Correia 3 , Rosie Bell 4 , Catherine Widnall 5 , Luis Tosar Perez 6 , Caroline Berteau 5 , Claudia Schulte 7 , Dieter Scheller 8 , Daniela Berg 7, 9 , Walter Maetzler 7, 9 , Pedro A. F. Galante 3 and Andre Nogueira da Costa 1 1 Experimental Medicine and Diagnostics, Global Exploratory Development, UCB Biopharma SPRL, Braine-l'Alleud, Belgium 2 SimplicityBio SA, Monthey, Switzerland 3 Hospital Sirio Libanes, Sao Paulo, Brazil 4 Centre for Misfolding Diseases, University of Cambridge, Cambridge, UK 5 Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK 6 Bioanalytical Sciences, Non Clinical Development, UCB Biopharma SPRL, Belgium 7 Hertie Institute for Clinical Brain Research, Department of Neurodegeneration, University of Tuebingen and German Center for Neurodegenerative Diseases, Tuebingen, Germany 8 Consultancy Neuropharm, Neukirchener, Neuss, Germany 9 Department of Neurology, Christian-Albrechts-University Kiel, Kiel, Germany Correspondence to: Andre Nogueira da Costa, email: andre.dacosta@ucb.com Keywords: exosomal miRNA; early stage PD diagnosis; CSF; machine learning Received: January 06, 2018 Accepted: February 25, 2018 Published: April 03, 2018 ABSTRACT Parkinson’s Disease is the second most common neurodegenerative disorder, affecting 1–2% of the elderly population. Its diagnosis is still based on the identification of motor symptoms when a considerable number of dopaminergic neurons are already lost. The development of translatable biomarkers for accurate diagnosis at the earliest stages of PD is of extreme interest. Several microRNAs have been associated with PD pathophysiology. Consequently, microRNAs are emerging as potential biomarkers, especially due to their presence in Cerebrospinal Fluid and peripheral circulation. This study employed small RNA sequencing, protein binding ligand assays and machine learning in a cross-sectional cohort comprising 40 early stage PD patients and 40 well-matched controls. We identified a panel comprising 5 microRNAs (Let-7f-5p, miR-27a-3p, miR-125a-5p, miR-151a-3p and miR-423-5p), with 90% sensitivity, 80% specificity and 82% area under the curve (AUC) for the differentiation of the cohorts. Moreover, we combined miRNA profiles with hallmark-proteins of PD and identified a panel (miR-10b-5p, miR-22-3p, miR-151a-3p and α-synuclein) reaching 97% sensitivity, 90% specificity and 96% AUC. We performed a gene ontology analysis for the genes targeted by the microRNAs present in each panel and showed the likely association of the models with pathways involved in PD pathogenesis.

The development of new medicines is a long and expensive process. Despite growing efforts in R&D over the last decades, attrition rate due to safety issues (especially cardiac and hepatic toxicity) remains a major challenge for the pharmaceutical industry. This may lead to market withdrawal or late stage halting of a drug development program. Consequently, early detection of toxicity issues is critical to avoid late-stage failures. To this end, development of predictive toxicology assays and models have become a strategic matter for drug makers. An integrated approach confronting knowledge-based data sources with in vitro and in vivo experimental data should be performed. A well-defined balance between in vivo and in vitro assays should guide the safety assessment process and include a rationale taking into account ethical considerations as well as associated resourcing involved with animal use. Innovation in de-risking strategies may support refinement of regulatory testing and contribute to (i) improve drug safety evaluation alleviating assessment of the risk-benefit ratio and (ii) promote the access to safe drugs for patients. In this review, promising innovative approaches aiming at facilitating early detection of toxicity during drug development are described.

Investigation of genomic changes in cardiotoxicity can provide novel biomarkers and insights into molecular mechanisms of drug-induced cardiac injury (DICI). The main objective of this study was to identify and characterize dysregulated microRNAs (miRNAs) in the heart associated with cardiotoxicity. Wistar rats were dosed once with either isoproterenol (1.5 mg/kg, i.p), allylamine (100 mg/kg, p.o.) or the respective vehicle controls. Heart tissue was collected at 24 h, 48 h and 72 h post-drug administration and used for histopathological assessment, miRNA profiling, immunohistochemical analysis and in situ hybridization. Multiplex analysis of 68 miRNAs in the heart revealed a significant upregulation of several miRNAs (miR-19a-3p, miR-142-3p, miR-155-5p, miR-208b-3p, miR-21-5p) after isoproterenol and one miRNA (miR-21-5p) after allylamine administration. Localization of miR-21-5p was specific to inflammatory cell infiltrates in the heart after both treatments. Immunohistochemical analysis of Stat3, a known miR-21-5p regulator, also confirmed its upregulation in cardiomyocytes and inflammatory cell infiltrates. The toxicity signatures based on miRNA networks, identified in vivo, can potentially be used as mechanistic biomarkers as well as to study cardiotoxicity in vitro in order to develop sensitive tools for early hazard identification and risk assessment.

Impulsive-compulsive disorders in Parkinson’s disease patients have been described as behavioural or substance addictions including pathological gambling or compulsive medication use of dopamine replacement therapy. A substantial gap remains in the understanding of these disorders. We previously demonstrated that the rewarding effect of the D2/D3 agonist pramipexole was enhanced after repeated exposure to L-dopa and alpha-synuclein mediated dopaminergic nigral loss with specific transcriptional signatures suggesting a key involvement of the glutamatergic pathway. Here, we further investigate the therapeutic potential of metabotropic glutamate receptor 5 antagonism in Parkinson’s disease/dopamine replacement therapy related bias of reward-mediated associative learning. We identified protein changes underlying the striatal remodelling associated with the pramipexole-induced conditioned place preference. Acquisition and expression of the pramipexole-induced conditioned place preference were abolished by the metabotropic glutamate receptor 5 antagonist 2-methyl-6-phenylethynyl (pyridine) (conditioned place preference scores obtained with pramipexole conditioning were reduced by 12.5% and 125.8% when 2-methyl-6-phenylethynyl (pyridine) was co-administrated with pramipexole or after the pramipexole conditioning, respectively). Up-regulation of the metabotropic glutamate receptor 5 was found in the dorsomedial-striatum and nucleus accumbens core. Activation of these two brain sub-regions was also highlighted through FosB immunohistochemistry. Convergent molecular and pharmacological data further suggests metabotropic glutamate receptor 5 as a promising therapeutic target for the management of Parkinson’s disease/dopamine replacement therapy related reward bias.

Chronic Hepatitis B (HB) is the main risk factor for chronic liver disease (CLD) and hepatocellular carcinoma (HCC) in many low-resource countries, where diagnosis is constrained by lack of clinical, histopathological and biomarker resources. We have used proteomics to detect plasma biomarkers that outperform α-Fetoprotein (AFP), the most widely used biomarker for HCC diagnosis in low-resource contexts. Deep-plasma proteome analysis was performed in HCC patients, patients with CLD and in HB-carrier controls from Thailand (South-East Asia) and The Gambia (West-Africa). Mass spectrometry profiling identified latent-transforming growth factor β binding-protein 2 (LTBP2) and Osteopontin (OPN) as being significantly elevated in HCC versus CLD and controls. These two proteins were further analyzed by ELISA in a total of 684 plasma samples, including 183 HCC, 274 CLD and 227 asymptomatic controls. When combined, LTBP2 and OPN showed an area under the receiver operating curve of 0.85 in distinguishing HCC from CLD in subjects with AFP

Impulsive-compulsive disorders (ICD) in patients with Parkinson’s disease (PD) have been described as behavioral or substance addictions including hypersexuality, gambling, or compulsive medication use of the dopamine replacement therapy (DRT). A remaining challenge is to understand the neuroadaptations leading to reward bias in PD patients under DRT. To this end, the appetitive effect of the D2/D3 agonist pramipexole was assessed after chronic exposure to l-dopa in an alpha-synuclein PD rat model. Association of progressive nigral loss and chronic l-dopa was required to observe a pramipexole-induced place preference. This behavioral outcome was inhibited by metabotropic glutamate receptor 5 (mGluR5) antagonism while transcriptional profiling highlighted regulations potentially related to the context of psychostimulant addiction. This study provides evidences strongly suggesting that PD-like lesion and l-dopa therapy were concomitant factors involved in striatal remodeling underlying the pramipexole-induced place preference. Molecular and pharmacological data suggest a key involvement of the glutamatergic pathway in this behavioral outcome.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting 5% of the elderly population. PD diagnosis is still based on the identification of neuromotor symptoms although nonmotor manifestations emerge years prior to diagnosis. The discovery of biomarkers at the earliest stages of PD is of extreme interest. miRNAs have been considered potential biomarkers for neurodegenerative diseases, but only a limited number have been found to be PD related. This review focuses on the current findings in the field of circulating miRNAs in PD and the challenges surrounding clinical utility and validation. We briefly describe the more established circulating biomarkers in PD and provide a more thorough review of miRNAs differentially expressed in PD. We highlight their potential for being considered as biomarkers for diagnosis while emphasizing the challenges for adequate validation of the findings and how miRNAs can be envisioned in a clinical setting satisfying regulatory bodies.

Infection by hepatitis B virus (HBV) and dietary exposure to aflatoxin B1 (AFB1) are the main risk factors for the development of chronic liver disease and hepatocellular carcinoma (HCC). How these factors cooperate is still largely unknown. AFB1 activation leads to DNA adduction and mutagenesis, with a specific mutation at codon 249 in TP53 (p.R249S). So far, only limited studies have addressed the effects of AFB1 on HBV replication. We have analysed the effects of both risk factors on p53 induction during HBV infection in HepaRG, a cell line with hepatocyte-like morphology that metabolizes AFB1 and supports HBV infection. Exposure to AFB1 up to 5 µM induced a downregulation of HBV replication after 48 h, as measured by a decrease in viral antigens in the culture medium (HBsAg, HBeAg and large envelope protein) and in intracellular levels of HBV transcripts, DNA and HBsAg. Conversely, HBV infection did not significantly modify AFB1-DNA adduct formation or repair as assessed by immunodot-blot assay, and the induction of p53 in response to AFB1 was similar in infected and non-infected HepaRG cells. Overall, our results suggest that AFB1 exposure decreases HBV replication, whereas DNA damage by AFB1 and subsequent p53 induction is not affected by the presence of the virus. Thus, in HepaRG cell line, AFB1 and HBV do not cooperate to increase DNA damage by AFB1. Further studies on the effects of both factors in a context of chronicity are needed to better understand synergistic effects.

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event – A Full Immersion Bioanalytical Week – specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.

The kidney is a complex excretory organ playing a crucial role in various physiological processes such as fluid and electrolyte balance, control of blood pressure, removal of waste products, and drug disposition. Drug-induced kidney injury (DIKI) remains a significant cause of candidate drug attrition during drug development. However, the incidence of renal toxicities in preclinical studies is low, and the mechanisms by which drugs induce kidney injury are still poorly understood. Although some in vitro investigational tools have been developed, the in vivo assessment of renal function remains the most widely used methodology to identify DIKI. Stand-alone safety pharmacology studies usually include assessment of glomerular and hemodynamic function, coupled with urine and plasma analyses. However, as renal function is not part of the ICH S7A core battery, such studies are not routinely conducted by pharmaceutical companies. The most common approach consists in integrating renal/urinary measurements in repeat-dose toxicity studies. In addition to the standard analyses and histopathological examination of kidneys, novel promising urinary biomarkers have emerged over the last decade, offering greater sensitivity and specificity than traditional renal parameters. Seven of these biomarkers have been qualified by regulatory agencies for use in rat toxicity studies.

The kidney is a complex excretory organ playing a crucial role in various physiological processes such as fluid and electrolyte balance, control of blood pressure, removal of waste products, and drug disposition. Drug-induced kidney injury (DIKI) remains a significant cause of candidate drug attrition during drug development. However, the incidence of renal toxicities in preclinical studies is low, and the mechanisms by which drugs induce kidney injury are still poorly understood. Although some in vitro investigational tools have been developed, the in vivo assessment of renal function remains the most widely used methodology to identify DIKI. Stand-alone safety pharmacology studies usually include assessment of glomerular and hemodynamic function, coupled with urine and plasma analyses. However, as renal function is not part of the ICH S7A core battery, such studies are not routinely conducted by pharmaceutical companies. The most common approach consists in integrating renal/urinary measurements in repeat-dose toxicity studies. In addition to the standard analyses and histopathological examination of kidneys, novel promising urinary biomarkers have emerged over the last decade, offering greater sensitivity and specificity than traditional renal parameters. Seven of these biomarkers have been qualified by regulatory agencies for use in rat toxicity studies.

Balter, Vincent Nogueira da Costa, Andre Bondanese, Victor Paky Jaouen, Klervia Lamboux, Aline Sangrajrang, Suleeporn Vincent, Nicolas Fourel, Francois Telouk, Philippe Gigou, Michelle Lecuyer, Christophe Srivatanakul, Petcharin Brechot, Christian Albarede, Francis Hainaut, Pierre eng Research Support, Non-U.S. Gov't 2015/01/15 06:00 Proc Natl Acad Sci U S A. 2015 Jan 27;112(4):982-5. doi: 10.1073/pnas.1415151112. Epub 2015 Jan 12.; International audience; The widespread hypoxic conditions of the tumor microenvironment can impair the metabolism of bioessential elements such as copper and sulfur, notably by changing their redox state and, as a consequence, their ability to bind specific molecules. Because competing redox state is known to drive isotopic fractionation, we have used here the stable isotope compositions of copper ((65)Cu/(63)Cu) and sulfur ((34)S/(32)S) in the blood of patients with hepatocellular carcinoma (HCC) as a tool to explore the cancer-driven copper and sulfur imbalances. We report that copper is (63)Cu-enriched by approximately 0.4 per thousand and sulfur is (32)S-enriched by approximately 1.5 per thousand in the blood of patients compared with that of control subjects. As expected, HCC patients have more copper in red blood cells and serum compared with control subjects. However, the isotopic signature of this blood extra copper burden is not in favor of a dietary origin but rather suggests a reallocation in the body of copper bound to cysteine-rich proteins such as metallothioneins. The magnitude of the sulfur isotope effect is similar in red blood cells and serum of HCC patients, implying that sulfur fractionation is systemic. The (32)S-enrichment of sulfur in the blood of HCC patients is compatible with the notion that sulfur partly originates from tumor-derived sulfides. The measurement of natural variations of stable isotope compositions, using techniques developed in the field of Earth sciences, can provide new means to detect and quantify cancer metabolic changes and provide insights into underlying mechanisms.

Although the basic structure of the gastrointestinal tract (GIT) is similar across species, there are significant differences in the anatomy, physiology, and biochemistry between humans and laboratory animals, which should be taken into account when conducting a gastrointestinal (GI) assessment. Historically, the percentage of cases of drug attrition associated with GI-related adverse effects is small; however, this incidence has increased over the last few years. Drug-related GI effects are very diverse, usually functional in nature, and not limited to a single pharmacological class. The most common GI signs are nausea and vomiting, diarrhea, constipation, and gastric ulceration. Despite being generally not life-threatening, they can greatly affect patient compliance and quality of life. There is therefore a real need for improved and/or more extensive GI screening of candidate drugs in preclinical development, which may help to better predict clinical effects. Models to identify drug effects on GI function cover GI motility, nausea and emesis liability, secretory function (mainly gastric secretion), and absorption aspects. Both in vitro and in vivo assessments are described in this chapter. Drug-induced effects on GI function can be assessed in stand-alone safety pharmacology studies or as endpoints integrated into toxicology studies. In silico approaches are also being developed, such as the gut-on-a-chip model, but await further optimization and validation before routine use in drug development. GI injuries are still in their infancy with regard to biomarkers, probably due to their greater diversity. Nevertheless, several potential blood, stool, and breath biomarkers have been investigated. However, additional validation studies are necessary to assess the relevance of these biomarkers and their predictive value for GI injuries.

Colorectal cancer is an important cancer worldwide, whose etiology is not fully understood. Known causes include several genetic factors, overweight/obesity, tobacco smoking, and heavy alcohol drinking. An etiologic role of diet is highly plausible, but the evidence for specific factors, with the possible exception of processed meat, is not conclusive. Workers exposed to asbestos have been found at increased risk of colorectal cancer in several studies, but the evidence is not sufficiently strong to conclude in favor of a causal association. No other occupational factors have been linked to colorectal cancer. Liver cancer is a common cancer in many regions of the world and is the second cause of cancer-specific mortality. About 75 % of livercancers are hepatocellular carcinoma (HCC), the second most frequent type being cholangiocarcinoma (CCA). HCC develops in the context of a web of interactions between viral (HBV, HCV), environmental (alcohol, aflatoxin), and metabolic (fatty liver disease, obesity) factors. Genetic predisposition accounts for only a small fraction of the global burden of HCC. The only established occupational cause of liver cancer is vinyl chloride, which causes a rare type of neoplasm, angiosarcoma, and has also been associated with HCC. Detection and diagnosis of HCC are complicated by its occurrence in a background of chronic liver disease characterized by inflammation and cycles of hepatocyte proliferation and destruction. Markers used in clinical practice include serological and molecular markers of viral hepatitis, enzymatic tests for liver function and injury, and a growing list of plasma-based tumor markers, the current gold standard being alpha-fetoprotein (AFP). Recent research has identified molecular changes in transcriptome, microRNAome, epigenome, and, significantly, plasma proteome that pave the way to the development of a new generation of biomarkers for early detection of HCC in different etiologic contexts. © Springer-Verlag London 2014.

Chronic Hepatitis B (HB) is the main risk factor for chronic liver disease (CLD) and hepatocellular carcinoma (HCC) in many low-resource countries, where diagnosis is constrained by lack of clinical, histopathological and biomarker resources. We have used proteomics to detect plasma biomarkers that outperform α-Fetoprotein (AFP), the most widely used biomarker for HCC diagnosis in low-resource contexts. Deep plasma proteome analysis was performed in HCC patients, patients with chronic liver disease (CLD) and in HB-carrier controls from Thailand (South-East Asia) and The Gambia (West-Africa). Mass spectrometry profiling identified Latent-Transforming Growth Factor β Binding-Protein 2 (LTBP2) and Osteopontin (OPN) as being significantly elevated in HCC versus CLD and controls. These two proteins were further analysed by ELISA in a total of 684 plasma samples, including 183 HCC, 274 CLD and 227 asymptomatic controls. When combined, LTBP2 and OPN showed an area under the receiver operating curve (ROC) of 0.85 in distinguishing HCC from CLD in subjects with α-Fetoprotein (AFP) < 20 ng/mL. In a prospective cohort of 115 CLD patients from Korea, increased plasma levels of LTBP2 and/or OPN were detected in plasma collected over 2 years prior to diagnosis in 21 subjects who developed HCC. Thus, the combination of LTBP2 and OPN outperformed AFP for diagnosis and prediction of HCC and may therefore improve biomarker-based detection of HBV-related HCC.

Background: Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC. Methods: Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis. Results: Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages. Conclusions: The validated changes of expression in these proteins have the potential for development into highperformance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose definitive cutoffs and combinations for evaluation of performance.

Understanding and reducing attrition rate remains a key challenge in drug development. Preclinical and clinical safety issues still represent about 40% of drug discontinuation, of which cardiac and liver toxicities are the leading reasons. Reducing attrition rate can be achieved by various means, starting with a comprehensive evaluation of the potential safety issues associated to the primary target followed by an evaluation of undesirable secondary targets. To address these risks, a risk mitigation plan should be built at very early development stages, using a panel of in silico, in vitro, and in vivo models. While most pharmaceutical companies have developed robust safety strategies to de-risk genotoxicity and cardiotoxicity issues, partly driven by regulatory requirements; safety issues affecting other organs or systems, such as the central nervous system, liver, kidney, or gastro-intestinal system are less commonly addressed during early drug development. This paper proposes some de-risking strategies that can be applied to these target organ systems, including the use of novel biomarkers that can be easily integrated in both preclinical and clinical studies. Experiments to understand the mechanisms’ underlying toxicity are also important. Two examples are provided to demonstrate how such mechanistic studies can impact drug development. Novel trends in investigative safety are reviewed, such as computational modeling, mitochondrial toxicity assessment, and imaging technologies. Ultimately, understanding the predictive value of non-clinical safety testing and its translatability to humans will enable to optimize assays in order to address the key objectives of the drug discovery process, i.e., hazard identification, risk assessment, and mitigation.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer death worldwide, but is still lacking sensitive and specific biomarkers for diagnosis and prognosis. HCC carcinogenesis is a multi-step process that usually arises in background chronic metabolic, inflammatory and/or infectious liver disease and it exhibits a wide range of epigenetic alterations that could potentially be used as biomarkers. In this study, we applied the targeted massively parallel semiconductor sequencing to assess in plasma circulating cell-free DNA (cfDNA) the methylation of a panel of genes (FBLN1, HINT2, LAMC1, LTBP1, LTBP2, PSMA2, PSMA7, PXDN, TGFB1, UBE2L3, VIM and YWHAZ) and to evaluate the potential of these genes as novel HCC biomarkers in two different series, one from France and one from Thailand. Methylation in cfDNA was detected for FBLN1, PSMA7, PXDN and VIM, with substantial differences in methylation patterns between cases and controls for FBLN1 and VIM. Further, the average methylation level across analyzed CpG-sites was associated with higher odds of HCC for VIM (1.48 [1.02, 2.16] for French cases, and 2.18 [1.28, 3.72] for Thai cases), and lower odds of HCC for FBLN1 (0.89 [0.76, 1.03] for French cases and 0.75 [0.63, 0.88] for Thai cases). We also analyzed a set of HCC and adjacent tissues as well as The Cancer Genome Atlas’ (TCGA) data to further investigate the origin of methylation pattern in cfDNA and noted that the analysis of VIM could be improved based on the methylation pattern detected in TCGA data. Overall, our study provides evidence that changes in methylation levels of cfDNA could be used as potential cancer biomarkers and that VIM and FBLN1 methylation in cfDNA are associated with HCC and may represent useful plasma-based biomarkers for improved detection and patient surveillance. Citation Format: Reetta Holmila, Athena Sklias, David C. Muller, Davide Degli Esposti, Paule Guilloreau, James Mckay, Suleeporn Sangrajrang, Petcharin Srivatanakul, Pierre Hainaut, Philippe Merle, Andre Nogueira da Costa, Zdenko Herceg. Targeted deep sequencing of plasma circulating cell-free DNA shows VIM and FBLN1 methylation as potential biomarkers for hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4468.

The genetic and epigenetic material originating from tumour that can be found in body fluids of individuals with cancer harbours tumour-specific alterations and represents an attractive target for biomarker discovery. Epigenetic changes (DNA methylation, histone modifications and non-coding RNAs) are present ubiquitously in virtually all types of human malignancies and may appear in early cancer development, and thus they provide particularly attractive markers with broad applications in diagnostics. In addition, because changes in the epigenome may constitute a signature of specific exposure to certain risk factors, they have the potential to serve as highly specific biomarkers for risk assessment. While reliable detection of cancer-specific epigenetic changes has proven to be technically challenging, a substantial progress has been made in developing the methodologies that allow an efficient and sensitive detection of epigenomic changes using the material originating from body fluids. In this review we discuss the application of epigenomics as a tool for biomarker research, with the focus on the analysis of DNA methylation in biofluids.

Villar, Stephanie Ortiz-Cuaran, Sandra Abedi-Ardekani, Behnoush Gouas, Doriane Nogueira da Costa, Andre Plymoth, Amelie Khuhaprema, Thiravud Kalalak, Anant Sangrajrang, Suleeporn Friesen, Marlin D Groopman, John D Hainaut, Pierre eng Research Support, Non-U.S. Gov't 2012/06/08 06:00 PLoS One. 2012;7(6):e37707. doi: 10.1371/journal.pone.0037707. Epub 2012 Jun 4.; International audience; Primary Liver Cancer (PLC) is the leading cause of death by cancer among males in Thailand and the 3(rd) among females. Most cases are hepatocellular carcinoma (HCC) but cholangiocarcinomas represent between 4 and 80% of liver cancers depending upon geographic area. Most HCC are associated with chronic infection by Hepatitis B Virus while a G ―> T mutation at codon 249 of the TP53 gene, R249S, specific for exposure to aflatoxin, is detected in tumors for up to 30% of cases. We have used Short Oligonucleotide Mass Analysis (SOMA) to quantify free circulating R249S-mutated DNA in plasma using blood specimens collected in a hospital case:control study. Plasma R249S-mutated DNA was detectable at low concentrations (>/= 67 copies/mL) in 53 to 64% of patients with primary liver cancer or chronic liver disease and in 19% of controls. 44% of patients with HCC and no evidence of cirrhosis had plasma concentrations of R249S-mutated DNA >/= 150 copies/mL, compared to 21% in patients with both HCC and cirrhosis, 22% in patients with cholangiocarcinoma, 12% in patients with non-cancer chronic liver disease and 3% of subjects in the reference group. Thus, plasma concentrations of R249S-mutated DNA >/= 150 copies/mL tended to be more common in patients with HCC developing without pre-existing cirrhosis (p = 0.027). Overall, these results support the preferential occurrence of R249S-mutated DNA in HCC developing in the absence of cirrhosis in a context of HBV chronic infection.

The mycotoxin deoxynivalenol (DON) commonly contaminates cereal grains. It is ubiquitous in the Western European diet, although chronic, low-dose effects in humans are not well described, but immunotoxicity has been reported. In this study, two-dimensional gel electrophoresis was used to identify phosphoproteomic changes in human B (RPMI1788) and T (Jurkat E6.1) lymphocyte cell lines after exposure to modest concentrations of DON (up to 500 ng/mL) for 24 h. Proteins identified as having altered phosphorylation state post-treatment (C-1-tetrahydrofolate synthase, eukaryotic elongation factor 2, nucleoside diphosphate kinase A, heat shock cognate 71 kDa protein, eukaryotic translation initiation factor 3 subunit I and growth factor receptor-bound protein 2) are involved in regulation of metabolic pathways, protein biosynthesis and signaling transduction. All exhibited a greater than 1.4-fold change, reproducible in three separate experiments consisting of 36 gels in total. Flow cytometry validated the observations for eukaryotic elongation factor 2 and growth factor receptor-bound protein 2. These findings provide further insights as to how low dose exposure to DON may affect human immune function and may have potential as mechanismbased phosphoprotein biomarkers for DON exposure.