Application of clinical and molecular profiling data to improve patient outcomes in psoriatic arthritis.

Achieving a good outcome for a person with Psoriatic Arthritis (PsA) is made difficult by late diagnosis, heterogenous clinical disease expression and in many cases, failure to adequately suppress inflammatory disease features. Single-centre studies have certainly contributed to our understanding of disease pathogenesis, but to adequately address the major areas of unmet need, multi-partner, collaborative research programmes are now required. HIPPOCRATES is a 5-year, Innovative Medicines Initiative (IMI) programme which includes 17 European academic centres experienced in PsA research, 5 pharmaceutical industry partners, 3 small-/medium-sized industry partners and 2 patient-representative organizations. In this review, the ambitious programme of work to be undertaken by HIPPOCRATES is outlined and common approaches and challenges are identified. It is expected that, when completed, the results will ultimately allow for changes in the approaches to diagnosing, managing and treating PsA allowing for better short-term and long-term outcomes.

Improving outcomes in Psoriatic Arthritis Psoriatic Arthritis (PsA) is a form of arthritis which is found in approximately 30% of people who have the skin condition, Psoriasis. Frequently debilitating and progressive, achieving a good outcome for a person with PsA is made difficult by late diagnosis, disease clinical features and in many cases, failure to adequately control features of inflammation. Research studies from individual centres have certainly contributed to our understanding of why people develop PsA but to adequately address the major areas of unmet need, multi-centre, collaborative research programmes are now required. HIPPOCRATES is a 5-year, Innovative Medicines Initiative (IMI) programme which includes 17 European academic centres experienced in PsA research, 5 pharmaceutical industry partners, 3 small-/medium-sized industry partners and 2 patient representative organisations (see appendix). In this review, the ambitious programme of work to be undertaken by HIPPOCRATES is outlined and common approaches and challenges are identified. The participation of patient research partners in all stages of the work of HIPPOCRATES is highlighted. It is expected that, when completed, the results will ultimately allow for changes in the approaches to diagnosing, managing and treating PsA allowing for improvements in short-term and long-term outcomes.

Comparison of pharmacokinetics, safety and tolerability of secukinumab administered subcutaneously using different delivery systems in healthy volunteers and in psoriasis patients.

AIMS: The aim of the study was to compare the pharmacokinetics (PK), safety and tolerability of secukinumab with different devices for subcutaneous (s.c.) administration of 2 mL. METHODS: A phase 1 study in healthy subjects with 6 devices to administer 2 mL injection volumes was conducted to evaluate the serum PK, safety and tolerability of secukinumab following single s.c. injection of 300 mg in the abdomen (either side) or in the thigh (either leg). Primary PK endpoints were maximum observed serum concentration and area under the serum concentration-time curve. The impact of device, site and side of injection on serum exposure was evaluated. In a phase 3 study in psoriasis patients, PK of secukinumab was evaluated following multiple s.c. injections of 300 mg by either 2 × 1-mL prefilled syringe or 1 × 2-mL prefilled syringe. RESULTS: Mean serum concentration-time profiles for administration as 2 × 1 mL injections or as 1 × 2 mL injections were similar. With an injection volume of 2 mL, perceived injection pain was not different from 2 × 1 mL injections. A nonclinically significant difference in PK endpoints was observed between thigh and abdomen. Results with a 2 mL prefilled syringe in a 1-year phase 3 study in patients confirmed PK results observed in the phase 1 study. CONCLUSION: Collective evidence from both studies demonstrated that 2-mL injections of secukinumab into the abdomen or thigh using different devices resulted in comparable PK characteristics and were all well tolerated without noticeable local reactions.

Secukinumab Treatment Does Not Alter the Pharmacokinetics of the Cytochrome P450 3A4 Substrate Midazolam in Patients With Moderate to Severe Psoriasis.

This open-label disease-drug-drug interaction study assessed whether blockade of the interleukin (IL)-17A pathway by secukinumab and subsequent downregulation of inflammatory cytokines like IL-6 or high-sensitivity C-reactive protein affects the pharmacokinetics (PKs) of a sensitive probe substrate of the cytochrome P450 3A4 isoform (CYP3A4). The PKs of midazolam, metabolized by CYP3A4, was evaluated before and after 7 and 35 days of treatment initiation of subcutaneous secukinumab at a dose of 300 mg weekly in 24 patients with moderate-to-severe psoriasis. Although demonstrating the expected decrease in downstream inflammatory cytokines, secukinumab had no clinically relevant effects on the PKs of midazolam, provided substantial clinical benefit, and was generally well tolerated. In summary, blockade of IL-17A signaling in patients with moderate-to-severe psoriasis does not significantly affect CYP3A4 enzyme activities and, therefore, the use of secukinumab is unlikely to influence the PKs of CYP3A4 substrates.

Effects of the glycine reuptake inhibitors bitopertin and RG7118 on glycine in cerebrospinal fluid: results of two proofs of mechanism studies in healthy volunteers.

RATIONALE: Hypofunction of NMDA receptors has been implicated in neuropsychiatric disorders including schizophrenia. NMDA receptor neurotransmission can be enhanced through inhibition of glycine reuptake by the glycine transporter type 1 (GlyT1). OBJECTIVES: The primary objective of these studies was to explore the relationship between plasma exposure and glycine cerebrospinal fluid (CSF) concentrations following administration of bitopertin and RG7118 in healthy volunteers. METHODS: The bitopertin study comprised four dose levels (3, 10, 30 and 60 mg) administered once daily for 10 days. In the RG7118 study, placebo, 15 or 30 mg RG7118 was administered once daily for 28 days. CSF samples were taken on day -2 and day 10, and day -1 and day 26 for bitopertin and RG7118, respectively. RESULTS: Twenty-two and 24 subjects participated in the bitopertin and RG7118 study, respectively. In the bitopertin study, CSF glycine concentrations showed a dose-dependent increase from baseline to day 10. The geometric mean ratios (coefficient of variation) of AUC0-12 h on day 10 over baseline were 1.3 (17 %), 1.3 (49 %), 1.7 (18 %) and 2.3 (14 %) after 3, 10, 30 and 60 mg, respectively. In the RG7118 study, the geometric mean ratio of glycine concentration (CV) on day 26 at 6 h post-dose over time-matched baseline was approx. 1.9 (24 and 15 %) for 15 and 30 mg. CONCLUSIONS: The mechanism of action of bitopertin and RG7118, i.e. inhibition of glycine reuptake in the brain, was confirmed. The maximal increase observed in healthy volunteers was similar to the one observed in animals showing the good translatability of this biomarker.

The expanding spectrum of COL2A1 gene variants IN 136 patients with a skeletal dysplasia phenotype.

Heterozygous COL2A1 variants cause a wide spectrum of skeletal dysplasia termed type II collagenopathies. We assessed the impact of this gene in our French series. A decision tree was applied to select 136 probands (71 Stickler cases, 21 Spondyloepiphyseal dysplasia congenita cases, 11 Kniest dysplasia cases, and 34 other dysplasia cases) before molecular diagnosis by Sanger sequencing. We identified 66 different variants among the 71 positive patients. Among those patients, 18 belonged to multiplex families and 53 were sporadic. Most variants (38/44, 86%) were located in the triple helical domain of the collagen chain and glycine substitutions were mainly observed in severe phenotypes, whereas arginine to cysteine changes were more often encountered in moderate phenotypes. This series of skeletal dysplasia is one of the largest reported so far, adding 44 novel variants (15%) to published data. We have confirmed that about half of our Stickler patients (46%) carried a COL2A1 variant, and that the molecular spectrum was different across the phenotypes. To further address the question of genotype-phenotype correlation, we plan to screen our patients for other candidate genes using a targeted next-generation sequencing approach.

Mutation Update for COL2A1 Gene Variants Associated with Type II Collagenopathies.

Mutations in the COL2A1 gene cause a spectrum of rare autosomal-dominant conditions characterized by skeletal dysplasia, short stature, and sensorial defects. An early diagnosis is critical to providing relevant patient care and follow-up, and genetic counseling to affected families. There are no recent exhaustive descriptions of the causal mutations in the literature. Here, we provide a review of COL2A1 mutations extracted from the Leiden Open Variation Database (LOVD) that we updated with data from PubMed and our own patients. Over 700 patients were recorded, harboring 415 different mutations. One-third of the mutations are dominant-negative mutations that affect the glycine residue in the G-X-Y repeats of the alpha 1 chain. These mutations disrupt the collagen triple helix and are common in achondrogenesis type II and hypochondrogenesis. The mutations resulting in a premature stop codon are found in less severe phenotypes such as Stickler syndrome. The p.(Arg275Cys) substitution is found in all patients with COL2A1-associated Czech dysplasia. LOVD-COL2A1 provides support and potential collaborative material for scientific and clinical projects aimed at elucidating phenotype-genotype correlation and differential diagnosis in patients with type II collagenopathies.

Identification of disrupted AUTS2 and EPHA6 genes by array painting in a patient carrying a de novo balanced translocation t(3;7) with intellectual disability and neurodevelopment disorder.

Intellectual disability (ID) is a frequent feature but is highly clinically and genetically heterogeneous. The establishment of the precise diagnosis in patients with ID is challenging due to this heterogeneity but crucial for genetic counseling and appropriate care for the patients. Among the etiologies of patients with ID, apparently balanced de novo rearrangements represent 0.6%. Several mechanisms explain the ID in patients with apparently balanced de novo rearrangement. Among them, disruption of a disease gene at the breakpoint, is frequently evoked. In this context, technologies recently developed are used to characterize precisely such chromosomal rearrangements. Here, we report the case of a boy with ID, facial features and autistic behavior who is carrying a de novo balanced reciprocal translocation t(3;7)(q11.2;q11.22)dn. Using microarray analysis, array painting (AP) technology combined with molecular study, we have identified the interruption of the autism susceptibility candidate 2 gene (AUTS2) and EPH receptor A6 gene (EPHA6). We consider that the disruption of AUTS2 explains the phenotype of the patient; the exact role of EPHA6 in human pathology is not well defined. Based on the observation of recurrent germinal and somatic translocations involving AUTS2 and the molecular environment content, we put forward the hypothesis that the likely chromosomal mechanism responsible for the translocation could be due either to replicative stress or to recombination-based mechanisms.

Dysspondyloenchondromatosis without COL2A1 mutation: possible genetic heterogeneity.

Dysspondyloenchondromatosis is a rare form of generalized enchondromatosis associated with spinal involvement. This skeletal dysplasia is characterized by multiple enchondromas present in vertebrae as well as in metaphyseal and diaphyseal parts of the long tubular bones, post-natal short stature, and early development of kyphoscoliosis. A novel heterozygous missense mutation in COL2A1 was recently identified in a patient with dysspondyloenchondromatosis. This suggests that dysspondyloenchondromatosis might expand the already broad spectrum of type II collagenopathies. Here, we report on a young girl with features of dysspondyloenchondromatosis, specifically short stature, thoracoscoliosis, and generalized enchondromas lesions. Sanger sequencing failed to detect a mutation in COL2A1. We therefore suggest that dysspondyloenchondromatosis is a genetically heterogeneous condition.

The angiogenesis suppressor gene AKAP12 is under the epigenetic control of HDAC7 in endothelial cells.

Histone deacetylases (HDACs) are a family of 18 enzymes that deacetylate lysine residues of both histone and nonhistone proteins and to a large extent govern the process of angiogenesis. Previous studies have shown that specific inhibition of HDAC7 blocks angiogenesis both in vitro and in vivo. However, the underlying molecular mechanisms are not fully understood and hence preclude any meaningful development of suitable therapeutic modalities. The goal of the present study was to further the understanding of HDAC7 epigenetic control of angiogenesis in human endothelial cells using the proteomic approach. The underlying problem was approached through siRNA-mediated gene-expression silencing of HDAC7 in human umbilical vein endothelial cells (HUVECs). To this end, HUVEC proteins were extracted and proteomically analyzed. The emphasis was placed on up-regulated proteins, as these may represent potential direct epigenetic targets of HDAC7. Among several proteins, A-kinase anchor protein 12 (AKAP12) was the most reproducibly up-regulated protein following HDAC7 depletion. This overexpression of AKAP12 was responsible for the inhibition of migration and tube formation in HDAC7-depleted HUVEC. Mechanistically, H3 histones associated with AKAP12 promoter were acetylated following the removal of HDAC7, leading to an increase in its mRNA and protein levels. AKAP12 is responsible for protein kinase C mediated phosphorylation of signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 increasingly binds to the chromatin and AKAP12 promoter and is necessary for maintaining the elevated levels of AKAP12 following HDAC7 knockdown. We demonstrated for the first time that AKAP12 tumor/angiogenesis suppressor gene is an epigenetic target of HDAC7, whose elevated levels lead to a negative regulation of HUVEC migration and inhibit formation of tube-like structures.

Differential proteomic analysis of a human breast tumor and its matched bone metastasis identifies cell membrane and extracellular proteins associated with bone metastasis.

The classical fate of metastasizing breast cancer cells is to seed and form secondary colonies in bones. The molecules closely associated with these processes are predominantly present at the cell surface and in the extracellular space, establishing the first contacts with the target tissue. In this study, we had the rare opportunity to analyze a bone metastatic lesion and its corresponding breast primary tumor obtained simultaneously from the same patient. Using mass spectrometry, we undertook a proteomic study on cell surface and extracellular protein-enriched material. We provide a repertoire of significantly modulated proteins, some with yet unknown roles in the bone metastatic process as well as proteins notably involved in cancer cell invasiveness and in bone metabolism. The comparison of these clinical data with those previously obtained using a human osteotropic breast cancer cell line highlighted an overlapping group of proteins. Certain differentially expressed proteins are validated in the present study using immunohistochemistry on a retrospective collection of breast tumors and matched bone metastases. Our exclusive set of selected proteins supports the setup of further investigations on both clinical samples and experimental bone metastasis models that will help to reveal the finely coordinated expression of proteins that favor the development of metastases in the bone microenvironment.

Metallothionein-dependent up-regulation of TGF-ß2 participates in the remodelling of the myxomatous mitral valve.

AIMS: Although an excessive extracellular matrix remodelling has been well described in myxomatous mitral valve (MMV), the underlying pathogenic mechanisms remain largely unknown. Our goal was to identify dysregulated genes in human MMV and then to evaluate their functional role in the progression of the disease. METHODS AND RESULTS: Dysregulated genes were investigated by transcriptomic, immunohistochemistry, and western blot analyses of the P2 segment collected from human idiopathic MMV during valvuloplasty (n = 23) and from healthy control valves (n = 17). The most striking results showed a decreased expression of two families of genes: the metallothioneins-1 and -2 (MT1/2) and members of the ADAMTS. The mechanistic consequences of the reduced level of MT1/2 were evaluated by silencing their expression in normal valvular interstitial cells (VICs) cultures. The knock-down of MT1/2 resulted in the up-regulation of transforming growth factor-beta 2 (TGF-ß2). Most importantly, TGF-ß2 was also found significantly increased in MMV tissues. The activation of VICs in vitro by TGF-ß2 induced a down-regulation of ADAMTS-1 and an accumulation of versican as observed in human MMV. CONCLUSION: Our studies demonstrate for the first time that MMV are characterized by reduced levels of MT1/2 accompanied by an up-regulation of TGF-ß2. In turn, increased TGF-ß2 signalling induces down-regulation of aggrecanases and up-regulation of versican, two co-operating processes that potentially participate in the development of the pathology.

Identification of novel accessible proteins bearing diagnostic and therapeutic potential in human pancreatic ductal adenocarcinoma.

Pancreas ductal adenocarcinoma (PDAC) remains a deadly malignancy with poor early diagnostic and no effective therapy. Although several proteomic studies have performed comparative analysis between normal and malignant tissues, there is a lack of clear characterization of proteins that could be of clinical value. Systemically reachable ("potentially accessible") proteins, suitable for imaging technologies and targeted therapies represent a major group of interest. The current study explores potentially accessible proteins overexpressed in PDAC, employing innovative proteomics technologies. In the discovery phase, potentially accessible proteins from fresh human normal and PDAC tissues were ex vivo biotinylated, isolated and identified using 2D-nano-HPLC-MS/MS method. The analysis revealed 422 up-regulated proteins in the tumor, of which 83 (including protein isoforms) were evaluated as potentially accessible. Eleven selected candidates were further confirmed as up-regulated using Western blot and multiple reaction monitoring protein quantification. Of these, transforming growth factor beta-induced (TGFBI), latent transforming growth factor beta binding 2 (LTBP2), and asporin (ASPN) were further investigated by employing large scale immunohistochemistry-based validations. They were found to be significantly expressed in a large group of clinical PDAC samples compared to corresponding normal and inflammatory tissues. In conclusion, TGFBI, LTBP2, and ASPN are novel, overexpressed, and potentially accessible proteins in human PDAC. They bear the potential to be of clinical value for diagnostic and therapeutic applications and merit further studies using in vivo models.

Novel comprehensive approach for accessible biomarker identification and absolute quantification from precious human tissues.

The identification of specific biomarkers obtained directly from human pathological lesions remains a major challenge, because the amount of tissue available is often very limited. We have developed a novel, comprehensive, and efficient method permitting the identification and absolute quantification of potentially accessible proteins in such precious samples. This protein subclass comprises cell membrane associated and extracellular proteins, which are reachable by systemically deliverable substances and hence especially suitable for diagnosis and targeted therapy applications. To isolate such proteins, we exploited the ability of chemically modified biotin to label ex vivo accessible proteins and the fact that most of these proteins are glycosylated. This approach consists of three successive steps involving first the linkage of potentially accessible proteins to biotin molecules followed by their purification. The remaining proteins are then subjected to glycopeptide isolation. Finally, the analysis of the nonglycosylated peptides and their involvement in an in silico method increased the confident identification of glycoproteins. The value of the technique was demonstrated on human breast cancer tissue samples originating from 5 individuals. Altogether, the method delivered quantitative data on more than 400 potentially accessible proteins (per sample and replicate). In comparison to biotinylation or glycoprotein analysis alone, the sequential method significantly increased the number (≥30% and ≥50% respectively) of potentially therapeutically and diagnostically valuable proteins. The sequential method led to the identification of 93 differentially modulated proteins, among which several were not reported to be associated with the breast cancer. One of these novel potential biomarkers was CD276, a cell membrane-associated glycoprotein. The immunohistochemistry analysis showed that CD276 is significantly differentially expressed in a series of breast cancer lesions. Due to the fact that our technology is applicable to any type of tissue biopsy, it bears the ability to accelerate the discovery of new relevant biomarkers in a broad spectrum of pathologies.

Combined mutation and rearrangement screening by quantitative PCR high-resolution melting: is it relevant for hereditary recurrent Fever genes?

The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase), NLRP3 (Nod like receptor family, pyrin domain containing 3) and TNFRSF1A (TNF receptor superfamily 1A), we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations) and quantitative (rearrangement) screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group) and 88 selected (retrospective group) patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.

Versican overexpression in human breast cancer lesions: known and new isoforms for stromal tumor targeting.

Proteoglycans play a key role in cancer development and progression by participating in the constitution of a specific fertile tumor microenvironment. As they are largely overexpressed in the malignant stroma, proteoglycans provide a reservoir of potential new targets for anticancer therapies, because they can serve to convey toxic payloads in the close proximity of cancer cells and subsequently destroy them. In this context, versican, a proteoglycan largely overexpressed in several solid cancers, bears the potential to be such an ideal target. As 4 main versican isoforms have been characterized, we sought to determine which isoform could represent the best target in human breast cancer. We used a series of 10 primary breast cancer lesions that were characterized as overexpressing the versican protein, when compared with matched normal breast tissues, using shotgun mass spectrometry and immunohistochemistry experiments. Quantitative polymerase chain reaction and western-blotting experiments were used to evaluate versican isoform expression in breast cancer/normal tissue pairs for which ARN quality was excellent. All known isoforms were significantly overexpressed in the malignant lesions, both at the mRNA and at the protein levels. In the course of this study, we also identified and cloned a new alternatively spliced versican isoform, referred to as V4, which was also found to be upregulated in human breast cancer. This study provides for the first time a comprehensive mRNA and protein analysis of versican isoforms expression in human breast tissues, and offers insights into which therapeutic strategy would be best suited to target versican in human breast cancer lesions.

Cell membrane proteomic analysis identifies proteins differentially expressed in osteotropic human breast cancer cells.

Metastatic breast cancer cells are characterized by their high propensity to colonize the skeleton and form bone metastases, causing major morbidity and mortality. Identifying key proteins involved in the osteotropic phenotype would represent a major step toward the development of both new prognostic markers and new effective therapies. Cell surface proteins differentially expressed in cancer cells are preferred potential targets for antibody-based targeted therapies. In this study, using cell surface biotinylation and a mass spectrometric approach, we have compared the profile of accessible cell surface proteins between the human breast cancer cell line MDA-MB-231 and its highly osteotropic B02 subclone. This strategy allowed the identification of several proteins either up- or downregulated in the osteotropic cell line, and differential protein expressions were validated using antibody-based techniques. Class I HLAs were down-regulated in the bone metastatic variant, whereas alpha(v)beta(3) integrins, among others, were consistently up-regulated in this latter cell line. These results show that comprehensive profiling of the cell surface proteome of mother cancerous cell lines and derived organ-specific metastatic cell lines provides an effective approach for the identification of potential accessible marker proteins for both prognosis and antibody-based targeted therapies.