The Role of Ten-Eleven Translocation Proteins in Inflammation.

Ten-eleven translocation proteins (TET1-3) are dioxygenases that oxidize 5-methyldeoxycytosine, thus taking part in passive and active demethylation. TETs have shown to be involved in immune cell development, affecting from self-renewal of stem cells and lineage commitment to terminal differentiation. In fact, dysfunction of TET proteins have been vastly associated with both myeloid and lymphoid leukemias. Recently, there has been accumulating evidence suggesting that TETs regulate immune cell function during innate and adaptive immune responses, thereby modulating inflammation. In this work, we pursue to review the current and recent evidence on the mechanistic aspects by which TETs regulate immune cell maturation and function. We will also discuss the complex interplay of TET expression and activity by several factors to modulate a multitude of inflammatory processes. Thus, modulating TET enzymes could be a novel pharmacological approach to target inflammation-related diseases and myeloid and lymphoid leukemias, when their activity is dysregulated.

The glucose transporter GLUT3 controls T helper 17 cell responses through glycolytic-epigenetic reprogramming.

Metabolic reprogramming is a hallmark of activated T cells. The switch from oxidative phosphorylation to aerobic glycolysis provides energy and intermediary metabolites for the biosynthesis of macromolecules to support clonal expansion and effector function. Here, we show that glycolytic reprogramming additionally controls inflammatory gene expression via epigenetic remodeling. We found that the glucose transporter GLUT3 is essential for the effector functions of Th17 cells in models of autoimmune colitis and encephalomyelitis. At the molecular level, we show that GLUT3-dependent glucose uptake controls a metabolic-transcriptional circuit that regulates the pathogenicity of Th17 cells. Metabolomic, epigenetic, and transcriptomic analyses linked GLUT3 to mitochondrial glucose oxidation and ACLY-dependent acetyl-CoA generation as a rate-limiting step in the epigenetic regulation of inflammatory gene expression. Our findings are also important from a translational perspective because inhibiting GLUT3-dependent acetyl-CoA generation is a promising metabolic checkpoint to mitigate Th17-cell-mediated inflammatory diseases.

Epigenetic histone modulation contributes to improvements in inflammatory bowel disease via EBI3.

Ulcerative colitis (UC) is characterized by relapsing-remitting inflammatory episodes paralleled by varying cytokine levels, suggesting that switching epigenetic processes might be involved. However, the epigenetic impact on cytokine levels in colitis is mostly unexplored. The heterodimeric interleukin (IL)-12 cytokine family have various functions in both pro- and anti-inflammatory processes. The family member IL-35 (EBI3/IL-12p35) was recently reported to play an anti-inflammatory role in UC. Therefore, we aimed to investigate a possible epigenetic regulation of the IL-35 subunits in vitro and in vivo, and to examine the epigenetic targeting of EBI3 expression as a therapeutic option for UC. Exposure to either the pro-inflammatory TNFα or to histone deacetylase inhibitors (HDACi) significantly increased EBI3 expression in Human Colon Epithelial Cells (HCEC) generated from healthy tissue. When applied in combination, a drastic upregulation of EBI3 expression occurred, suggesting a synergistic mechanism. Consequently, IL-35 was increased as well. In vivo, the intestines of HDACi-treated wild-type mice exhibited reduced pathological signs of colitis compared to non-treated colitic mice. However, the improvement by HDACi treatment was completely lost in Ebi3-deficient mice (Ebi3-/-). In fact, HDACi appeared to exacerbate the disease phenotype in Ebi3-/-. In conclusion, our results reveal that under inflammatory conditions, EBI3 is upregulated by the epigenetic mechanism of histone acetylation. The in vivo data show that the deficiency of EBI3 plays a key role in colitis manifestation. Concordantly, our data suggest that conditions promoting histone acetylation, such as upon HDACi application, improve colitis by a mechanism involving the local formation of the anti-inflammatory cytokine IL-35.

Vitamin C in combination with inhibition of mutant IDH1 synergistically activates TET enzymes and epigenetically modulates gene silencing in colon cancer cells.

Mutations in the enzyme isocitrate dehydrogenase 1 (IDH1) lead to metabolic alterations and a sustained formation of 2-hydroxyglutarate (2-HG). 2-HG is an oncometabolite as it inhibits the activity of α-ketoglutarate-dependent dioxygenases such as ten-eleven translocation (TET) enzymes. Inhibitors of mutant IDH enzymes, like ML309, are currently tested in order to lower the levels of 2-HG. Vitamin C (VC) is an inducer of TET enzymes. To test a new therapeutic avenue of synergistic effects, the anti-neoplastic activity of inhibition of mutant IDH1 via ML309 in the presence of VC was investigated in the colon cancer cell line HCT116 IDH1R132H/+ (harbouring a mutated IDH1 allele) and the parental cells HCT116 IDH1+/+ (wild type IDH1). Measurement of the oncometabolite indicated a 56-fold higher content of 2-HG in mutated cells compared to wild type cells. A significant reduction of 2-HG was observed in mutated cells after treatment with ML 309, whereas VC produced only minimally changes of the oncometabolite. However, combinatorial treatment with both, ML309 and VC, in mutated cells induced pronounced reduction of 2-HG leading to levels comparable to those in wild type cells. The decreased level of 2-HG in mutated cells after combinatorial treatment was accompanied by an enhanced global DNA hydroxymethylation and an increased gene expression of certain tumour suppressors. Moreover, mutated cells showed an increased percentage of apoptotic cells after treatment with non-cytotoxic concentrations of ML309 and VC. These results suggest that combinatorial therapy is of interest for further investigation to rescue TET activity and treatment of IDH1/2 mutated cancers.

Vitamin C promotes decitabine or azacytidine induced DNA hydroxymethylation and subsequent reactivation of the epigenetically silenced tumour suppressor CDKN1A in colon cancer cells.

Epigenetic silencing of tumour suppressor genes is a key hallmark of colorectal carcinogenesis. Despite this, the therapeutic potential of epigenetic agents capable of reactivating these silenced genes remains relatively unexplored. Evidence has shown the dietary antioxidant vitamin C (ascorbate) acts as an inducer of the ten-eleven translocation (TET) dioxygenases, an enzyme family that catalyses a recently described mechanism of DNA demethylation linked to gene re-expression. In this study, we set out to determine whether vitamin C can enhance the known anti-neoplastic actions of the DNA-demethylating agents decitabine (DAC) and azacytidine (AZA) in colorectal cancer cells. Administration of vitamin C alone significantly enhanced global levels of 5-hydroxymethyl-2'-deoxycytidine (5-hmdC), without altering 5-methyl-2'-deoxycytidine (5-mdC), as would be expected upon the activation of TET dioxygenases. Concomitant treatment of vitamin C with either AZA or DAC resulted in an unexpectedly high increase of global 5-hmdC levels, one that administration of any these compounds alone could not achieve. Notably, this was also accompanied by increased expression of the tumour suppressor p21 (CDKN1A), and a significant increase in apoptotic cell induction. Our in vitro data leads us to hypothesize that the reactivation of genes in colorectal cancer cells by AZA or DAC can be improved when the 5-hmdC levels are simultaneously increased by the TET activator vitamin C. The dual administration of demethylating agents and vitamin C to colorectal cancer patients, a demographic in which vitamin C deficiencies are common, may improve responses to epigenetic therapies.

Formulation and ex vivo evaluation of polymeric nanoparticles for controlled delivery of corticosteroids to the skin and the corneal epithelium.

Controlled delivery of corticosteroids using nanoparticles to the skin and corneal epithelium may reduce their side effects and maximize treatment effectiveness. Dexamethasone-loaded ethyl cellulose, Eudragit® RS and ethyl cellulose/Eudragit® RS nanoparticles were prepared by the solvent evaporation method. Dexamethasone release from the polymeric nanoparticles was investigated in vitro using Franz diffusion cells. Drug penetration was also assessed ex vivo using excised human skin. Nanoparticle toxicity was determined by MTT and H2DCFDA assays. Eudragit® RS nanoparticles were smaller and positively charged but had a lower dexamethasone loading capacity (0.3-0.7%) than ethyl cellulose nanoparticles (1.4-2.2%). By blending the two polymers (1:1), small (105nm), positively charged (+37mV) nanoparticles with sufficient dexamethasone loading (1.3%) were obtained. Dexamethasone release and penetration significantly decreased with decreasing drug to polymer ratio and increased when Eudragit® RS was blended with ethyl cellulose. Ex vivo, drug release and penetration from the nanoparticles was slower than a conventional cream. The nanoparticles bear no toxicity potentials except ethyl cellulose nanoparticles had ROS generation potential at high concentration. In conclusion, the nanoparticles showed great potential to control the release and penetration of corticosteroids on the skin and mucus membrane and maximize treatment effectiveness.

Biocompatibility and characterization of polyglycerol-based thermoresponsive nanogels designed as novel drug-delivery systems and their intracellular localization in keratinocytes.

Novel nanogels that possess the capacity to change their physico-chemical properties in response to external stimuli are promising drug-delivery candidates for the treatment of severe skin diseases. As thermoresponsive nanogels (tNGs) are capable of enhancing penetration through biological barriers such as the stratum corneum and are taken up by keratinocytes of human skin, potential adverse consequences of their exposure must be elucidated. In this study, tNGs were synthesized from dendritic polyglycerol (dPG) and two thermoresponsive polymers. tNG_dPG_tPG are the combination of dPG with poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)) and tNG_dPG_pNIPAM the one with poly(N-isopropylacrylamide) (pNIPAM). Both thermoresponsive nanogels are able to incorporate high amounts of dexamethasone and tacrolimus, drugs used in the treatment of severe skin diseases. Cellular uptake, intracellular localization and the toxicological properties of the tNGs were comprehensively characterized in primary normal human keratinocytes (NHK) and in spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (HaCaT). Laser scanning confocal microscopy revealed fluorescently labeled tNGs entered into the cells and localized predominantly within lysosomal compartments. MTT assay, comet assay and carboxy-H2DCFDA assay, demonstrated neither cytotoxic or genotoxic effects, nor any induction of reactive oxygen species of the tNGs in keratinocytes. In addition, both tNGs were devoid of eye irritation potential as shown by bovine corneal opacity and permeability (BCOP) test and red blood cell (RBC) hemolysis assay. Therefore, our study provides evidence that tNGs are locally well tolerated and underlines their potential for cutaneous drug delivery.

Poly[acrylonitrile-co-(N-vinyl pyrrolidone)] nanoparticles - Composition-dependent skin penetration enhancement of a dye probe and biocompatibility.

Nanoparticles can improve topical drug delivery: size, surface properties and flexibility of polymer nanoparticles are defining its interaction with the skin. Only few studies have explored skin penetration for one series of structurally related polymer particles with systematic alteration of material composition. Here, a series of rigid poly[acrylonitrile-co-(N-vinyl pyrrolidone)] model nanoparticles stably loaded with Nile Red or Rhodamin B, respectively, was comprehensively studied for biocompatibility and functionality. Surface properties were altered by varying the molar content of hydrophilic NVP from 0 to 24.1% and particle size ranged from 35 to 244nm. Whereas irritancy and genotoxicity were not revealed, lipophilic and hydrophilic nanoparticles taken up by keratinocytes affected cell viability. Skin absorption of the particles into viable skin ex vivo was studied using Nile Red as fluorescent probe. Whilst an intact stratum corneum efficiently prevented penetration, almost complete removal of the horny layer allowed nanoparticles of smaller size and hydrophilic particles to penetrate into viable epidermis and dermis. Hence, systematic variations of nanoparticle properties allows gaining insights into critical criteria for biocompatibility and functionality of novel nanocarriers for topical drug delivery and risks associated with environmental exposure.

Formulation and comparative in vitro evaluation of various dexamethasone-loaded pH-sensitive polymeric nanoparticles intended for dermal applications.

pH-sensitive nanoparticles have a great potential for dermal and transfollicular drug delivery. In this study, pH-sensitive, dexamethasone-loaded Eudragit® L 100, Eudragit® L 100-55, Eudragit® S 100, HPMCP-50, HPMCP-55 and cellulose acetate phthalate nanoparticles were prepared by nanoprecipitation and characterized. The pH-dependent swelling, erosion, dissolution and drug release kinetics were investigated in vitro using dynamic light scattering and Franz diffusion cells, respectively. Their toxicity potential was assessed by the ROS and MTT assays. 100-700nm nanoparticles with high drug loading and entrapment efficiency were obtained. The nanoparticles bear no toxicity potential. Cellulose phthalates nanoparticles were more sensitive to pH than acrylates nanoparticles. They dissolved in 10mM pH 7.5 buffer and released>80% of the drug within 7h. The acrylate nanoparticles dissolved in 40mM pH 7.5 buffer and released 65-70% of the drug within 7h. The nanoparticles remained intact in 10 and 40mM pH 6.0 buffers (HPMCP nanoparticles dissolved in 40mM pH 6.0 buffer) and released slowly. The nanoparticles properties could be modulated by blending the different polymers. In conclusion, various pH-sensitive nanoparticles that could release differently on the skin surface and dissolve and release in the hair follicles were obtained.

Stratum corneum targeting by dendritic core-multishell-nanocarriers in a mouse model of psoriasis.

Inflammatory disorders of the skin pose particular therapeutic challenges due to complex structural and functional alterations of the skin barrier. Penetration of several anti-inflammatory drugs is particularly problematic in psoriasis, a common dermatitis condition with epidermal hyperplasia and hyperkeratosis. Here, we tested in vivo dermal penetration and biological effects of dendritic core-multishell-nanocarriers (CMS) in a murine skin model of psoriasis and compared it to healthy skin. In both groups, CMS exclusively localized to the stratum corneum of the epidermis with only very sporadic uptake by Langerhans cells. Furthermore, penetration into the viable epidermis of nile red as a model for lipophilic compounds was enhanced by CMS. CMS proved fully biocompatible in several in vitro assays and on normal and psoriatic mouse skin. The observations support the concept of CMS as promising candidates for drug delivery in inflammatory hyperkeratotic skin disorders in vivo.

Specific uptake mechanisms of well-tolerated thermoresponsive polyglycerol-based nanogels in antigen-presenting cells of the skin.

Engineered nanogels are of high value for a targeted and controlled transport of compounds due to the ability to change their chemical properties by external stimuli. As it has been indicated that nanogels possess a high ability to penetrate the stratum corneum, it cannot be excluded that nanogels interact with dermal dendritic cells, especially in diseased skin. In this study the potential crosstalk of the thermoresponsive nanogels (tNGs) with the dendritic cells of the skin was investigated with the aim to determine the immunotoxicological properties of the nanogels. The investigated tNGs were made of dendritic polyglycerol (dPG) and poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)), as polymer conferring thermoresponsive properties. Although the tNGs were taken up, they displayed neither cytotoxic and genotoxic effects nor any induction of reactive oxygen species in the tested cells. Interestingly, specific uptake mechanisms of the tNGs by the dendritic cells were depending on the nanogels cloud point temperature (Tcp), which determines the phase transition of the nanoparticle. The study points to caveolae-mediated endocytosis as being the major tNGs uptake mechanism at 37°C, which is above the Tcp of the tNGs. Remarkably, an additional uptake mechanism, beside caveolae-mediated endocytosis, was observed at 29°C, which is the Tcp of the tNGs. At this temperature, which is characterized by two different states of the tNGs, macropinocytosis was involved as well. In summary, our study highlights the impact of thermoresponsivity on the cellular uptake mechanisms which has to be taken into account if the tNGs are used as a drug delivery system.

Autotransplant with and without induction chemotherapy in older multiple myeloma patients: long-term outcome of a randomized trial.

Autologous transplantation is controversial for older patients with multiple myeloma. The role of age-adjusted high-dose melphalan and the impact of induction chemotherapy cycles is still unclear. A total of 434 patients aged 60-70 years were randomly assigned to 4 cycles of standard anthracycline-based induction chemotherapy or no induction. For all patients, double autologous transplantation after melphalan 140 mg/m2 (MEL140) was planned. The primary end point was progression-free survival. Of 420 eligible patients, 85% received a first transplant and 69% completed double transplantation. Treatment duration was short with a median of 7.7 months with induction chemotherapy cycles and 4.6 months without induction. On an intention-to-treat basis, median progression-free survival with induction chemotherapy cycles (207 patients) was 21.4 months versus 20.0 months with no induction cycles (213 patients) (hazard ratio 1.04, 95% confidence interval 0.84-1.28; P=0.36). Per protocol, progression-free survival was 23.7 months versus 23.0 months (P=0.28). Patients aged 65 years or over (55%) did not have an inferior outcome. Patients with low-risk cytogenetics [absence of del17p13, t(4;14) and 1q21 gains] showed a favorable overall survival and included the patients with sustained first remission. MEL140 was associated with a low rate of severe mucositis (10%) and treatment-related deaths (1%). Based on hazard ratio, the short treatment arm consisting of mobilization chemotherapy and tandem MEL140 achieved 96% of the progression-free survival, demonstrating its value as an independent component of therapy in older patients with multiple myeloma who are considered fit for autologous transplantation. (clinicaltrials.gov identifier: 02288741).

The Diagnosis and Treatment of Multiple Myeloma.

BACKGROUND: Multiple myeloma is a malignant disease of plasma cells with a worldwide incidence of 6-7 cases per 100 000 persons per year. It is among the 20 most common types of cancer in Germany. METHODS: This review is based on pertinent publications up to December 2015 that were retrieved by a selective search of PubMed employing the terms "multiple myeloma" AND "therapy" OR "diagnostic." Systematic reviews, meta-analyses, randomized controlled trials, and treatment recommendations from Germany and abroad were considered. RESULTS: The diagnostic evaluation of multiple myeloma comprises thorough history-taking and physical examination, various laboratory tests including analysis of a 24-hour urine sample, a bone-marrow biopsy, and skeletal radiography. Systemic treatment should be administered only when organ damage has been diagnosed. The type of treatment to be given is chosen individually on the basis of the patient's age, comorbidities, and risk profile. High-dose therapy with autologous stem-cell transplantation remains the treatment of choice for patients under age 70 who are otherwise in good health. For patients who are not candidates for high-dose therapy or who have had a recurrence of multiple myeloma after prior high-dose therapy, there are a number of further conventional treatment options. Patients need not only systemic antineoplastic treatment, but also supportive treatment for the prevention of treatment-induced toxicity and myeloma-associated organ damage. CONCLUSION: Recent therapeutic advances have made the treatment of multiple myeloma both more complex and more costly. In particular, the median survival of patients with multiple myeloma has been markedly prolonged through the use of targeted drugs such as proteasome inhibitors and immune modulators.

Formulation and in vitro evaluation of polymeric enteric nanoparticles as dermal carriers with pH-dependent targeting potential.

pH-sensitive nanoparticles which release in a controlled fashion on the skin or dissolve in the hair follicle could significantly improve treatment effectiveness and make transfollicular drug delivery a success. Dexamethasone-loaded Eudragit® L 100 nanoparticles were prepared by nanoprecipitation from an organic drug-polymer solution. Their toxicity potential was assessed using isolated human fibroblasts. pH-dependent swelling and erosion kinetics of the nanoparticles were investigated by dynamic light scattering and viscosity measurements and its effect on drug release was assessed in vitro with Franz diffusion cells. Stable, 100-550nm-sized dexamethasone-loaded Eudragit® L 100 nanoparticles with drug loading capacity and entrapment efficiency as high as 8.3% and 85%, respectively, were obtained by using polyvinyl alcohol as a stabilizer and ethanol as organic solvent. The nanoparticles showed little or no toxicity on isolated normal human fibroblasts. Dexamethasone existed in the nanoparticles as solid solution or in amorphous form. The nanoparticles underwent extensive swelling and slow drug release in media with a low buffer capacity (as low as 10mM) and a higher pH or at a pH close to the dissolution pH of the polymer (pH6) and a higher buffer capacity. In 40mM buffer and above pH6.8, the nanoparticles eroded fast or dissolved completely and thus released the drug rapidly. pH-sensitive nanoparticles which potentially release in a controlled manner on the stratum corneum but dissolve in the hair follicle could be prepared.

Tailored dendritic core-multishell nanocarriers for efficient dermal drug delivery: A systematic top-down approach from synthesis to preclinical testing.

Drug loaded dendritic core-multishell (CMS) nanocarriers are of especial interest for the treatment of skin diseases, owing to their striking dermal delivery efficiencies following topical applications. CMS nanocarriers are composed of a polyglycerol core, connected by amide-bonds to an inner alkyl shell and an outer methoxy poly(ethylene glycol) shell. Since topically applied nanocarriers are subjected to biodegradation, the application of conventional amide-based CMS nanocarriers (10-A-18-350) has been limited by the potential production of toxic polyglycerol amines. To circumvent this issue, three tailored ester-based CMS nanocarriers (10-E-12-350, 10-E-15-350, 10-E-18-350) of varying inner alkyl chain length were synthesized and comprehensively characterized in terms of particle size, drug loading, biodegradation and dermal drug delivery efficiency. Dexamethasone (DXM), a potent drug widely used for the treatment of inflammatory skin diseases, was chosen as a therapeutically relevant test compound for the present study. Ester- and amide-based CMS nanocarriers delivered DXM more efficiently into human skin than a commercially available DXM cream. Subsequent in vitro and in vivo toxicity studies identified CMS (10-E-15-350) as the most biocompatible carrier system. The anti-inflammatory potency of DXM-loaded CMS (10-E-15-350) nanocarriers was assessed in TNFα supplemented skin models, where a significant reduction of the pro-inflammatory cytokine IL-8 was seen, with markedly greater efficacy than commercial DXM cream. In summary, we report the rational design and characterization of tailored, biodegradable, ester-based CMS nanocarriers, and their subsequent stepwise screening for biocompatibility, dermal delivery efficiency and therapeutic efficacy in a top-down approach yielding the best carrier system for topical applications.

Comparison of different methods to study effects of silver nanoparticles on the pro- and antioxidant status of human keratinocytes and fibroblasts.

In times of antibiotic-resistant bacteria new strategies to avoid the septic-inducing threat of dangerous microorganisms are needed. Silver ions (Ag+) in the forms of silver nitrate or silver sulfadiazine have been used as antimicrobial agents for years. A step further was the development of micro and silver particles (AgNP). In contrast to other Ag+ ion sources, AgNP allow a sustained release of Ag+ ions, due to their high surface to volume ratio. However, AgNP are also toxic to eukaryotic cells and the mechanisms of cytotoxicity have not yet been fully elucidated. In this study, the impact of different AgNP preparations on a human keratinocyte cell line was investigated. The intracellular radical formation was confirmed by the 2',7'-dichlorodihydrofluorescein di-acetate (H2DCF-DA) assay on two cell types (HaCaT cells and normal human dermal fibroblasts) as well as by electron paramagnetic resonance (EPR) spectroscopy, which showed comparable results. EPR spectroscopy was performed for the first time for 24h in experiments using keratinocytes. Drastic changes in the mitochondrial activity were induced in cells incubated with AgNP containing high concentrations of Ag+ ions. It was also possible to show that the quantitative uptake of AgNP was dependent on the AgNP concentration. In addition, the effects of AgNP on the GSH/GSSG system were elucidated. The results showed a batch- and concentration-dependent decrease of the total glutathione concentration which correlated well with the decrease of cell viability. Furthermore, the results suggest a direct reaction of GSH molecules with Ag+ ions. In conclusion, this study proves the efficacy of the H2DCF-DA assay and the EPR spectroscopy. The investigations show that AgNP formulations containing high amounts of released Ag+ ions induce radicals in human keratinocytes and deplete them of their natural anti-oxidative molecules. On the contrary, nanoparticles prepared and stored under argon did not induce significant adverse effects, suggesting that slowing down the release of Ag+ may help to reduce AgNP-related side effects without affecting the antibacterial impact.

Hypermethylation of ITGA4, TFPI2 and VIMENTIN promoters is increased in inflamed colon tissue: putative risk markers for colitis-associated cancer.

PURPOSE: Epigenetic silencing of tumor suppressor genes is involved in early transforming events and has a high impact on colorectal carcinogenesis. Likewise, colon cancers that derive from chronically inflamed bowel diseases frequently exhibit epigenetic changes. But there is little data about epigenetic aberrations causing colorectal cancer in chronically inflamed tissue. The aim of the present study was to evaluate the aberrant gain of methylation in the gene promoters of VIM, TFPI2 and ITGA4 as putative early markers in the development from inflamed tissue via precancerous lesions toward colorectal cancer. METHODS: Initial screening of different cancer cell lines by using methylation-specific PCR revealed a putative colon cancer-specific methylation pattern. Additionally, a demethylation assay was performed to investigate the methylation-dependent gene silencing of ITGA4. The candidate markers were analyzed in colonic tissue specimens from patients with colorectal cancer (n = 15), adenomas (n = 76), serrated lesions (n = 13), chronic inflammation (n = 10) and normal mucosal samples (n = 9). RESULTS: A high methylation frequency of VIM (55.6 %) was observed in normal colon tissue, whereas ITGA4 and TFPI2 were completely unmethylated in controls. A significant gain of methylation frequency with progression of disease as well as an age-dependent effect was detectable for TFPI2. ITGA4 methylation frequency was high in precancerous and cancerous tissues as well as in inflammatory bowel diseases (IBD). CONCLUSION: The already established methylation marker VIM does not permit a specific and sensitive discrimination of healthy and neoplastic tissue. The methylation markers ITGA4 and TFPI2 seem to be suitable risk markers for inflammation-associated colon cancer.

Subcutaneous versus intravenous bortezomib in two different induction therapies for newly diagnosed multiple myeloma: an interim analysis from the prospective GMMG-MM5 trial.

We investigated the impact of subcutaneous versus intravenous bortezomib in the MM5 trial of the German-Speaking Myeloma Multicenter Group which compared bortezomib, doxorubicin, and dexamethasone with bortezomib, cyclophosphamide, and dexamethasone induction therapy in newly diagnosed multiple myeloma. Based on data from relapsed myeloma, the route of administration for bortezomib was changed from intravenous to subcutaneous after 314 of 604 patients had been enrolled. We analyzed 598 patients who received at least one dose of trial medication. Adverse events were reported more frequently in patients treated with intravenous bortezomib (intravenous=65%; subcutaneous=56%, P=0.02). Rates of grade 2 or more peripheral neuropathy were higher in patients treated with intravenous bortezomib during the third cycle (intravenous=8%; subcutaneous=2%, P=0.001). Overall response rates were similar in patients treated intravenously or subcutaneously. The presence of International Staging System stage III disease, renal impairment or adverse cytogenetic abnormalities did not have a negative impact on overall response rates in either group. To our knowledge this is the largest study to present data comparing subcutaneous with intravenous bortezomib in newly diagnosed myeloma. We show better tolerance and similar overall response rates for subcutaneous compared to intravenous bortezomib. The clinical trial is registered at eudract.ema.europa.eu as n. 2010-019173-16.