Clinical trial designs for rare diseases: Studies developed and discussed by the International Rare Cancers Initiative

BACKGROUND: The past three decades have seen rapid improvements in the diagnosis and treatment of most cancers and the most important contributor has been research. Progress in rare cancers has been slower, not least because of the challenges of undertaking research.

SETTINGS: The International Rare Cancers Initiative (IRCI) is a partnership which aims to stimulate and facilitate the development of international clinical trials for patients with rare cancers. It is focused on interventional--usually randomized--clinical trials with the clear goal of improving outcomes for patients. The key challenges are organisational and methodological. A multi-disciplinary workshop to review the methods used in ICRI portfolio trials was held in Amsterdam in September 2013. Other as-yet unrealised methods were also discussed.

RESULTS: The IRCI trials are each presented to exemplify possible approaches to designing credible trials in rare cancers. Researchers may consider these for use in future trials and understand the choices made for each design.

INTERPRETATION: Trials can be designed using a wide array of possibilities. There is no 'one size fits all' solution. In order to make progress in the rare diseases, decisions to change practice will have to be based on less direct evidence from clinical trials than in more common diseases.

UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors

Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 μM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.

Time-dependent tegumental surface changes in juvenile Fasciola gigantica in response to triclabendazole treatment in goat

Triclabendazole (TCBZ), the anthelmintic drug active against both mature and immature liver flukes, was used to investigate the effect of in vivo treatment on the tegumental surface of juvenile Fasciola gigantica. Five goats were infected with 150 F. gigantica metacercariae each by oral gavage. Four of them were treated with single dose of TCBZ at 10mg/kg at four weeks post-infection. They were euthanized at 0 (untreated), 24, 48, 72 and 96h post treatment. Juvenile flukes were manually retrieved from the goat livers and processed for scanning electron microscopy. In control flukes, the anterior region was adorned with sharply pointed spines projecting away from the surface, while in the posterior region, spines become shorter and narrower, loosing serration and with the appearance of distinct furrows and papillae. The dorsal surface retained the same pattern of surface architecture similar to that of ventral surface. Flukes obtained from 24h post-treatment did not show any apparent change and were still very active. However, there were limited movements and some blebbing, swelling, deposition of tegumental secretions and some flattening displayed by the flukes of 48h post-treatment. All the worms were found dead 72h post-treatment and showed advanced level of tegumental disruptions, consisting of severe distortion of spines, sloughing off the tegument to expose the basal lamina, formation of pores and isolated patches of lesions. By 96h post-treatment, the disruption was extremely severe and the tegument was completely sheared off causing deeper lesions that exposed the underlying musculature. The disruption was more severe at posterior than anterior region and on ventral than dorsal surface. The present study further establishes the time-course of TCBZ action in vivo with 100% efficacy against the juvenile tropical liver fluke.

Proteomics and in silico approaches to extend understanding of the glutathione transferase superfamily of the tropical liver fluke Fasciola gigantica

Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member.

Donor type microchimerism is an infrequent event following liver transplantation and is not associated with graft acceptance

Donor-type microchimerism, the presence of a minority population of donor-derived haematopoietic cells following solid organ transplantation, has been postulated as a mechanism for induction of donor-specific graft tolerance. The stability, frequency, and relevance of microchimerism with respect to long-term outcome, however, remains uncertain. Using a polymerase chain reaction (PCR)-based method of microsatellite analysis of highly polymorphic short tandem repeat sequences (STRs) to detect donor-type cells, DNA from 11 patients was analyzed prospectively at specific time points for 12 months following liver transplantation, and from a further six patients retrospectively 2 years after liver transplantation. Using a panel of STRs, transient peripheral blood donor microchimerism was detected in 2 of 11 patients at a single time-point following transplantation, but persistent evidence of donor-derived cells was not observed during the study period. Analysis of DNA extracted from skin and duodenum in two patients likewise failed to show donor-type cells at these sites. None of the six patients in the retrospective arm showed donor microchimerism, resulting in an overall detection rate of 1.58%. These results suggest that donor microchimerism following liver transplantation is an infrequent event, and that the generation of graft tolerance is independent of microchimerism.

The macroeconomic impact of non-communicable diseases in China and India: Estimates, projections, and comparisons

This study provides estimates of the macroeconomic impact of non-communicable diseases (NCDs) inChina and India for the period 2012–2030. Our estimates are derived using the World Health Organization’sEPIC model of economic growth, which focuses on the negative effects of NCDs on labor supply andcapital accumulation. We present results for the five main NCDs (cardiovascular disease, cancer, chronicrespiratory disease, diabetes, and mental health). Our undiscounted estimates indicate that the cost ofthe five main NCDs will total USD 23.03 trillion for China and USD 4.58 trillion for India (in 2010 USD).For both countries, the most costly domain is cardiovascular disease. Our analyses also reveal that thecosts are much larger in China than in India mainly because of China’s higher and steeper income trajectory,and to a lesser extent its older population. Rough calculations also indicate that WHO’s best buys foraddressing the challenge of NCDs are highly cost-beneficial

Abundant genetic overlap between blood lipids and immune-mediated diseases indicates shared molecular genetic mechanisms

Epidemiological studies suggest a relationship between blood lipids and immune-mediated diseases, but the nature of these associations is not well understood. We used genome-wide association studies (GWAS) to investigate shared single nucleotide polymorphisms (SNPs) between blood lipids and immune-mediated diseases. We analyzed data from GWAS (n~200,000 individuals), applying new False Discovery Rate (FDR) methods, to investigate genetic overlap between blood lipid levels [triglycerides (TG), low density lipoproteins (LDL), high density lipoproteins (HDL)] and a selection of archetypal immune-mediated diseases (Crohn's disease, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, celiac disease, psoriasis and sarcoidosis). We found significant polygenic pleiotropy between the blood lipids and all the investigated immune-mediated diseases. We discovered several shared risk loci between the immune-mediated diseases and TG (n = 88), LDL (n = 87) and HDL (n = 52). Three-way analyses differentiated the pattern of pleiotropy among the immune-mediated diseases. The new pleiotropic loci increased the number of functional gene network nodes representing blood lipid loci by 40%. Pathway analyses implicated several novel shared mechanisms for immune pathogenesis and lipid biology, including glycosphingolipid synthesis (e.g. FUT2) and intestinal host-microbe interactions (e.g. ATG16L1). We demonstrate a shared genetic basis for blood lipids and immune-mediated diseases independent of environmental factors. Our findings provide novel mechanistic insights into dyslipidemia and immune-mediated diseases and may have implications for therapeutic trials involving lipid-lowering and anti-inflammatory agents.

Selecting the best targeted agent in first-line treatment of unresectable liver metastases from colorectal cancer:does the bench have the answers?

For physicians facing patients with organ-limited metastases from colorectal cancer, tumor shrinkage and sterilization of micrometastatic disease is the main goal, giving the opportunity for secondary surgical resection. At the same time, for the majority of patients who will not achieve a sufficient tumor response, disease control remains the predominant objective. Since FOLFOX or FOLFIRI have similar efficacies, the challenge is to define which could be the most effective targeted agent (anti-EGFR or anti-VEGF) to reach these goals. Therefore, a priori molecular identification of patients that could benefit from anti-EGFR or anti-VEGF monoclonal antibodies (i.e. the currently approved targeted therapies for metastatic colorectal cancer) is of critical importance. In this setting, the KRAS mutation status was the first identified predictive marker of response to anti-EGFR therapy. Since it has been demonstrated that tumors with KRAS mutation do not respond to anti-EGFR therapy, KRAS status must be determined prior to treatment. Thus, for KRAS wild-type patients, the choices that remain are either anti-VEGF or anti-EGFR. In this review, we present the most updated data from translational research programs dealing with the identification of biomarkers for response to targeted therapies.

Economics of Non-Communicable Diseases in Indonesia

The Economics of Non-Communicable Diseases in Indonesia provides new data on the economic burden of NCDs in the country, and puts it in perspective by drawing a comparison with India and China. With this new addition to the series on the economics of NCDs, the World Economic Forum aims to advance the understanding of the expected economic output loss at the country level, particularly in countries in economic and epidemiological transition. The evidence presented provides a starting point in reorienting the dialogue on investing in healthy living and NCD prevention in Indonesia towards the view that a healthy population is an important factor for sustainable growth.

Calmodulin disruption impacts growth and motility in juvenile liver fluke

Background: Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.

Methods: In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.

Results: Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.

Conclusions: These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.

Targeting the complement system for the management of retinal inflammatory and degenerative diseases

The retina, an immune privileged tissue, has specialized immune defense mechanisms against noxious insults that may exist in diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), uveoretinitis and glaucoma. The defense system consists of retinal innate immune cells (including microglia, perivascular macrophages, and a small population of dendritic cells) and the complement system. Under normal aging conditions, retinal innate immune cells and the complement system undergo a low-grade activation (parainflammation) which is important for retinal homeostasis. In disease states such as AMD and DR, the parainflammatory response is dysregulated and develops into detrimental chronic inflammation. Complement activation in the retina is an important part of chronic inflammation and may contribute to retinal pathology in these disease states. Here, we review the evidence that supports the role of uncontrolled or dysregulated complement activation in various retinal degenerative and angiogenic conditions. We also discuss current strategies that are used to develop complement-based therapies for retinal diseases such as AMD. The potential benefits of complement inhibition in DR, uveoretinitis and glaucoma are also discussed, as well as the need for further research to better understand the mechanisms of complement-mediated retinal damage in these disease states.

Severe acute liver failure as the initial manifestation of haematological malignancy.

Acute liver failure is rarely secondary to lymphoma or leukaemia and it is extremely uncommon as the initial presentation of malignancy. We report a case of a young adult patient with severe acute liver failure referred for liver transplant, in which a Burkitt acute lymphoblastic leukaemia was diagnosed by bone marrow examination. A complete recovery and long remission were obtained with chemotherapy.

Phosphatidylethanolamines glycoxidation: products and significance

As fosfatidiletanolaminas constituem a segunda classe de fosfolípidos mais abundantes nos organismos. Elas estão presentes nas membranas biológicas e nas lipoproteínas. As alterações estruturais dos fosfolípidos, ocorrem devido ao stress oxidativo e podem manisfestar-se em alterações das suas propriedades e funções. Já são conhecidas algumas condições fisiopatológicas, nas quais os fosfolípidos oxidados estão envolvidos, por exemplo sinalização celular, resposta imunitária, apoptose e doenças relacionadas com o envelhecimento. Por esse motivo, o interesse no estudo dos fosfolípidos oxidados e suas funções tem crescido nos últimos anos. Contudo, a maioria dos estudos realizados, focam a oxidação das fosfatidilcolinas, tendo sido dedicada pouca atenção a outras classes de fosfolipídos, como as fosfatidiletanolaminas. As fosfatidiletanolaminas, podem ainda sofrer outras modificações, devido ao grupo amina livre presente na cabeça polar, como por exemplo a glicação. As fosfatidiletanolaminas glicadas já foram detectadas em condições de hiperglicemia, em pacientes diabéticos, e tem correlação com a hemoglobina glicada. Sabe-se que a glicação de biomoléculas, pode aumentar as modificações oxidativas, que por sua vez, podem ser responsáveis pelo estado inflamatório, existente na diabetes mellitus. Tanto o stress oxidativo, como a inflamação estão relacionados com a diabetes e as suas complicações. A espectrometria de massa tem sido utilizada como uma importante tecnologia na detecção e caracterização de modificações oxidativas de fosfolípidos. Assim, neste trabalho pretendeu-se estudar as modificações oxidativas induzidas em fosfatidiletanolaminas glicadas, e os seus efeitos biológicos nos monócitos e células dendríticas do sangue periférico. Pretendeu-se ainda, estudar as alterações que ocorreram nas espécies de fosfatidiletanolaminas do fígado de ratos diabéticos. Os resultados obtidos permitiram identificar vários produtos de oxidação de fosfatidiletanolaminas glicadas, nomeadamente novos produtos formados pela oxidação da cabeça polar glicada. A oxidação na cabeça polar glicada foi, ainda, confirmada pela realização de experiências com spin traps combinadas com espetrometria de massa. Posteriormente, as fosfatidiletanolaminas oxidadas, glicadas e glicoxidadas demonstraram ter efeitos pró-inflamatórios, confirmados pelo aumento da estimulação monócitos e de células dendríticas, expresso no aumento do número de células produtoras de citocinas em comparação com o estado basal. As diferentes modificações de fosfatidiletanolaminas induziram estímulos distintos nos dois tipos de células. Sendo as fosfatidiletanolaminas glicadas e as glicoxidadas, os compostos que induziram um maior estímulo. Estes resultados sugeriram que as fosfatidiletanolaminas glicadas e as glicoxidadas podem estar associadas com o estado inflamatório que decorre da hiperglicemia crónica. Ainda, a avaliação do perfil lipídico de extratos de fígado de ratos diabéticos demonstrou que a hiperglicemia induz inúmeras alterações das espécies de fosfatidiletanolaminas e das espécies de outras classes de fosfolípidos, em simultâneo com sinais de lesão hepática. Em conclusão, este trabalho demonstra a relação existente entre, a hiperglicémia, o stress oxidativo, a glicação e oxidação de fosfatidiletanolaminas e ainda a inflamação e compliações diabéticas. Portanto a contribuição da lipidómica é importante para compreender os efeitos prejudiciais da hiperglicemia não controlada, e por isso, merece ser mais explorado.

Volatile terpenoids and C13 norisoprenoids in wines: development of rapid methods of analysis and evaluation of the sesquiterpenoids biological potential

Vitis vinifera L., the most widely cultivated fruit crop in the world, was the starting point for the development of this PhD thesis. This subject was exploited following on two actual trends: i) the development of rapid, simple, and high sensitive methodologies with minimal sample handling; and ii) the valuation of natural products as a source of compounds with potential health benefits. The target group of compounds under study were the volatile terpenoids (mono and sesquiterpenoids) and C13 norisoprenoids, since they may present biological impact, either from the sensorial point of view, as regards to the wine aroma, or by the beneficial properties for the human health. Two novel methodologies for quantification of C13 norisoprenoids in wines were developed. The first methodology, a rapid method, was based on the headspace solid-phase microextraction combined with gas chromatography-quadrupole mass spectrometry operating at selected ion monitoring mode (HS-SPME/GC-qMS-SIM), using GC conditions that allowed obtaining a C13 norisoprenoid volatile signature. It does not require any pre-treatment of the sample, and the C13 norisoprenoid composition of the wine was evaluated based on the chromatographic profile and specific m/z fragments, without complete chromatographic separation of its components. The second methodology, used as reference method, was based on the HS-SPME/GC-qMS-SIM, allowing the GC conditions for an adequate chromatographic resolution of wine components. For quantification purposes, external calibration curves were constructed with β-ionone, with regression coefficient (r2) of 0.9968 (RSD 12.51 %) and 0.9940 (RSD of 1.08 %) for the rapid method and for the reference method, respectively. Low detection limits (1.57 and 1.10 μg L-1) were observed. These methodologies were applied to seventeen white and red table wines. Two vitispirane isomers (158-1529 L-1) and 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) (6.42-39.45 μg L-1) were quantified. The data obtained for vitispirane isomers and TDN using the two methods were highly correlated (r2 of 0.9756 and 0.9630, respectively). A rapid methodology for the establishment of the varietal volatile profile of Vitis vinifera L. cv. 'Fernão-Pires' (FP) white wines by headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (HS-SPME/GCxGC-TOFMS) was developed. Monovarietal wines from different harvests, Appellations, and producers were analysed. The study was focused on the volatiles that seem to be significant to the varietal character, such as mono and sesquiterpenic compounds, and C13 norisoprenoids. Two-dimensional chromatographic spaces containing the varietal compounds using the m/z fragments 93, 121, 161, 175 and 204 were established as follows: 1tR = 255-575 s, 2tR = 0,424-1,840 s, for monoterpenoids, 1tR = 555-685 s, 2tR = 0,528-0,856 s, for C13 norisoprenoids, and 1tR = 695-950 s, 2tR = 0,520-0,960 s, for sesquiterpenic compounds. For the three chemical groups under study, from a total of 170 compounds, 45 were determined in all wines, allowing defining the "varietal volatile profile" of FP wine. Among these compounds, 15 were detected for the first time in FP wines. This study proposes a HS-SPME/GCxGC-TOFMS based methodology combined with classification-reference sample to be used for rapid assessment of varietal volatile profile of wines. This approach is very useful to eliminate the majority of the non-terpenic and non-C13 norisoprenic compounds, allowing the definition of a two-dimensional chromatographic space containing these compounds, simplifying the data compared to the original data, and reducing the time of analysis. The presence of sesquiterpenic compounds in Vitis vinifera L. related products, to which are assigned several biological properties, prompted us to investigate the antioxidant, antiproliferative and hepatoprotective activities of some sesquiterpenic compounds. Firstly, the antiradical capacity of trans,trans-farnesol, cis-nerolidol, α-humulene and guaiazulene was evaluated using chemical (DPPH• and hydroxyl radicals) and biological (Caco-2 cells) models. Guaiazulene (IC50= 0.73 mM) was the sesquiterpene with higher scavenger capacity against DPPH•, while trans,trans-farnesol (IC50= 1.81 mM) and cis-nerolidol (IC50= 1.48 mM) were more active towards hydroxyl radicals. All compounds, with the exception of α-humulene, at non-cytotoxic levels (≤ 1 mM), were able to protect Caco-2 cells from oxidative stress induced by tert-butyl hydroperoxide. The activity of the compounds under study was also evaluated as antiproliferative agents. Guaiazulene and cis-nerolidol were able to more effectively arrest the cell cycle in the S-phase than trans,trans-farnesol and α-humulene, being the last almost inactive. The relative hepatoprotection effect of fifteen sesquiterpenic compounds, presenting different chemical structures and commonly found in plants and plant-derived foods and beverages, was assessed. Endogenous lipid peroxidation and induced lipid peroxidation with tert-butyl hydroperoxide were evaluated in liver homogenates from Wistar rats. With the exception of α-humulene, all the sesquiterpenic compounds under study (1 mM) were effective in reducing the malonaldehyde levels in both endogenous and induced lipid peroxidation up to 35% and 70%, respectively. The developed 3D-QSAR models, relating the hepatoprotection activity with molecular properties, showed good fit (R2LOO > 0.819) with good prediction power (Q2 > 0.950 and SDEP < 2%) for both models. A network of effects associated with structural and chemical features of sesquiterpenic compounds such as shape, branching, symmetry, and presence of electronegative fragments, can modulate the hepatoprotective activity observed for these compounds. In conclusion, this study allowed the development of rapid and in-depth methods for the assessment of varietal volatile compounds that might have a positive impact on sensorial and health attributes related to Vitis vinifera L. These approaches can be extended to the analysis of other related food matrices, including grapes and musts, among others. In addition, the results of in vitro assays open a perspective for the promising use of the sesquiterpenic compounds, with similar chemical structures such as those studied in the present work, as antioxidants, hepatoprotective and antiproliferative agents, which meets the current challenges related to diseases of modern civilization.