Semimechanistic Physiologically-Based Pharmacokinetic/Pharmacodynamic Model Informing Epcoritamab Dose Selection for Patients With B-Cell Lymphomas.

Epcoritamab is a CD3xCD20 bispecific antibody (bsAb) that induces T-cell-mediated cytotoxicity against CD20-positive B cells. Target engagement and crosslinking of CD3 and CD20 (trimer formation) leads to activation and expansion of T cells and killing of malignant B cells. The primary objective of the dose-escalation part of the phase I/II trial of epcoritamab was to determine the maximum tolerated dose, recommended phase II dose (RP2D), or both. For bsAbs, high target saturation can negatively affect trimer formation. The unique properties and mechanisms of action of bsAbs require novel pharmacokinetic (PK) modeling methods to predict clinical activity and inform RP2D selection. Traditional PK/pharmacodynamic (PD) modeling approaches are inappropriate because they may not adequately predict exposure-response relationships. We developed a semimechanistic, physiologically-based PK/PD model to quantitatively describe biodistribution, trimer formation, and tumor response using preclinical, clinical PK, biomarker, tumor, and response data from the dose-escalation part of the phase I/II trial. Clinical trial simulations were performed to predict trimer formation and tumor response in patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL). Model-predicted trimer formation plateaued at doses of 48 to 96 mg. Simulation results suggest that the 48-mg dose may achieve optimal response rates in DLBCL and FL. Exposure-safety analyses showed a flat relationship between epcoritamab exposure and risk of cytokine release syndrome in the dose range evaluated. This novel PK/PD modeling approach guided selection of 48 mg as the RP2D and provides a framework that may be applied to other CD3 bsAbs.

Safety, tolerability, and activity of mesenchymal stem cells versus placebo in multiple sclerosis (MESEMS): a phase 2, randomised, double-blind crossover trial.

BACKGROUND: Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, have been proposed as a promising therapeutic option for people with multiple sclerosis on the basis of their immunomodulatory and neuroprotective properties. The MEsenchymal StEm cells for Multiple Sclerosis (MESEMS) study was devised to evaluate the safety, tolerability, and activity of autologous MSCs derived from bone marrow and infused intravenously in patients with active multiple sclerosis. METHODS: MESEMS is a randomised phase 2 trial done at 15 sites in nine countries. Patients (aged 18-50 years) with active relapsing-remitting or progressive multiple sclerosis were included if they had a disease duration of 2-15 years since onset of multiple sclerosis and an Expanded Disability Status Scale score of 2·5-6·5. Patients were randomly assigned (1:1), according to a crossover design, to receive a single intravenous dose of autologous bone marrow-derived MSCs followed by placebo at week 24, or to receive placebo followed by autologous MSCs at week 24, with a follow-up visit at week 48. Primary objectives were to test safety and activity of MSC treatment. The primary safety endpoint was to assess the number and severity of adverse events within each treatment arm. The primary efficacy endpoint was the number of gadolinium-enhancing lesions (GELs) counted over week 4, 12, and 24 compared between treatment groups. The primary efficacy endpoint was assessed in the full analyis set, after all participants completed the week 24 visit. Efficacy endpoints were evaluated using a predefined statistical testing procedure. Safety was monitored throughout the study by recording vital signs and adverse events at each visit. FINDINGS: From July 16, 2012, until July 31, 2019, 144 patients were randomly assigned to first receive early intravenous infusion of autologous bone marrow-derived MSCs (n=69) or placebo (n=75). MSC treatment did not meet the primary endpoint of efficacy on the total number of GELs accumulated from baseline to week 24 (rate ratio [RR] 0·94, 95% CI 0·58-1·50; p=0·78). 213 adverse events were recorded, similarly distributed between groups (93 cases recorded in 35 [51%] of 69 patients treated first with MSCs vs 120 cases in 42 [56%] of 75 patients infused first with placebo). The most frequent adverse events reported were infection and infestations, with a total of 54 (25%) of 213 adverse events (18 [19%] of 93 in the early-MSC group and 36 [30%] of 120 in the delayed-MSC group). Nine serious adverse events were reported in seven patients treated with placebo versus none in the MSC group. All serious adverse events were considered to be unrelated to the treatment infusion. No deaths were reported during the study. INTERPRETATION: Bone marrow-derived MSC treatment was safe and well tolerated but did not show an effect on GELs, an MRI surrogate marker of acute inflammation, in patients with active forms of multiple sclerosis, at week 24. Thus, this study does not support the use of bone marrow-derived MSCs to treat active multiple sclerosis. Further studies should address the effect of MSCs on parameters related to tissue repair. FUNDING: Fondazione Italiana Sclerosi Multipla (FISM), the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS), and the Multiple Sclerosis International Federation (MSIF) for centralised activities. Individual trials participating in the MESEMS network are funded by the following agencies: FISM and Compagnia di San Paolo (Italy); The Danish Multiple Sclerosis Society, The Toyota Foundation, and Danish Blood Donors' Research Foundation (Denmark); the Spanish Health Research Institute Carlos 3 and the Andalusian Public Foundation Progreso y Salud (Spain); the Royan Institute for Stem Cell Biology and Technology (Iran); the Spinal Cord Injury and Tissue Regeneration Centre Salzburg, Paracelsus Medical University, and Salzburg (Austria); the Fondation pour l'aide à la recherche sur la sclérose en plaques (ARSEP), French Muscular Dystrophy Association (AFM)-Telethon (France); the UK Multiple Sclerosis Society and the UK Stem Cell Foundation (UK); and the Multiple Sclerosis Society of Canada and The Multiple Sclerosis Scientific Research Foundation and Research Manitoba (Canada).

Dose escalation of subcutaneous epcoritamab in patients with relapsed or refractory B-cell non-Hodgkin lymphoma: an open-label, phase 1/2 study.

BACKGROUND: Patients with relapsed or refractory B-cell non-Hodgkin lymphoma have few treatment options. We aimed to establish the safety and recommended phase 2 dose of epcoritamab, a novel bispecific antibody that targets CD3 and CD20 and induces T-cell-mediated cytotoxic activity against CD20+ malignant B cells. METHODS: For the dose-escalation part of this phase 1/2 study, we enrolled adults (aged ≥18 years) with relapsed or refractory CD20+ B-cell non-Hodgkin lymphoma at ten sites across four countries (Denmark, the Netherlands, the UK, and Spain). Eligible patients received priming and intermediate doses followed by full doses of subcutaneous epcoritamab administered in 28-day cycles; each subsequent cohort involved escalation of the priming, intermediate, or full dose (0·0128-60 mg). The primary objectives were to determine the maximum tolerated dose and the recommended phase 2 dose. Safety, antitumour activity, pharmacokinetics, and immune biomarkers were also assessed. This study is registered with ClinicalTrials.gov, NCT03625037, with the dose-expansion part ongoing. FINDINGS: Between June 26, 2018, and July 14, 2020, we enrolled 73 patients with relapsed, progressive, or refractory CD20+ mature B-cell non-Hodgkin lymphoma. 68 patients received escalating full doses (0·0128-60 mg) of subcutaneous epcoritamab. No dose-limiting toxic effects were observed, and the maximum tolerated dose was not reached; the full dose of 48 mg was identified as the recommended phase 2 dose. All 68 patients received at least one dose of epcoritamab and were included in safety analyses: common adverse events were pyrexia (47 patients [69%]), primarily associated with cytokine release syndrome (CRS; 40 [59%], all grade 1-2), and injection site reactions (32 [47%]; 31 grade 1). There were no grade 3 or higher CRS events. No discontinuations occurred due to treatment-related adverse events or treatment-related deaths. Overall response rate in patients with relapsed or refractory diffuse large B-cell lymphoma was 68% (95% CI 45-86), with 45% achieving a complete response at full doses of 12-60 mg. At 48 mg, the overall response rate was 88% (47-100), with 38% achieving a complete response. Patients with relapsed or refractory follicular lymphoma had an overall response rate of 90% (55-100), with 50% achieving a complete response at full doses of 0·76-48 mg. Epcoritamab induced robust and sustained B-cell depletion, and CD4+ and CD8+ T-cell activation and expansion, with modest increases in cytokine levels. INTERPRETATION: Single-agent subcutaneous epcoritamab for treatment of patients with relapsed or refractory B-cell non-Hodgkin lymphoma merits investigation in ongoing phase 2 and phase 3 studies. FUNDING: Genmab and AbbVie.

MEsenchymal StEm cells for Multiple Sclerosis (MESEMS): a randomized, double blind, cross-over phase I/II clinical trial with autologous mesenchymal stem cells for the therapy of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory disease of the central nervous system with a degenerative component, leading to irreversible disability. Mesenchymal stem cells (MSC) have been shown to prevent inflammation and neurodegeneration in animal models of MS, but no large phase II clinical trials have yet assessed the exploratory efficacy of MSC for MS. METHODS/DESIGN: This is an academic, investigator-initiated, randomized, double-blind, placebo-compared phase I/II clinical trial with autologous, bone-marrow derived MSC in MS. Enrolled subjects will receive autologous MSC at either baseline or at week 24, through a cross-over design. Primary co-objectives are to test safety and efficacy of MSC treatment compared to placebo at 6 months. Secondary objectives will evaluate the efficacy of MSC at clinical and MRI levels. In order to overcome funding constraints, the MEsenchymal StEm cells for Multiple Sclerosis (MESEMS) study has been designed to merge partially independent clinical trials, following harmonized protocols and sharing some key centralized procedures, including data collection and analyses. DISCUSSION: Results will provide patients and the scientific community with data on the safety and efficacy of MSC for MS. The innovative approach utilized to obtain funds to support the MESEMS trial could represent a new model to circumvent limitation of funds encountered by academic trials. TRIAL REGISTRATION: Andalusia: NCT01745783 , registered on Dec 10, 2012. Badalona: NCT02035514 EudraCT, 2010-024081-21. Registered on 2012. Canada: ClinicalTrials.gov, NCT02239393 . Registered on September 12, 2014. Copenhagen: EudraCT, 2012-000518-13 . Registered on June 21, 2012. Italy: EudraCT, 2011-001295-19, and ClinicalTrials.gov, NCT01854957 . Retrospectively registered on May 16, 2013. London: Eudra CT 2012-002357-35, and ClinicalTrials.gov, NCT01606215 . Registered on May 25, 2012. Salzburg: EudraCT, 2015-000137-78 . Registered on September 15, 2015. Stockholm: ClinicalTrials.gov, NCT01730547 . Registered on November 21, 2012. Toulouse: ClinicalTrials.gov, NCT02403947 . Registered on March 31, 2015.

Safety and Efficacy of Mesenchymal Stem Cells for Radiation-Induced Xerostomia: A Randomized, Placebo-Controlled Phase 1/2 Trial (MESRIX).

BACKGROUND: Salivary gland hypofunction and xerostomia are major complications to head and neck radiotherapy. This trial assessed the safety and efficacy of adipose tissue-derived mesenchymal stem cell (ASC) therapy for radiation-induced xerostomia. PATIENT AND METHODS: This randomized, placebo-controlled phase 1/2 trial included 30 patients, randomized in a 1:1 ratio to receive ultrasound-guided transplantation of ASCs or placebo to the submandibular glands. Patients had previously received radiotherapy for a T1-2, N0-2A, human papillomavirus-positive, oropharyngeal squamous cell carcinoma. The primary outcome was the change in unstimulated whole salivary flow rate, measured before and after the intervention. All assessments were performed one month prior (baseline) and one and four months following ASC or placebo administration. RESULTS: No adverse events were detected. Unstimulated whole salivary flow rates significantly increased in the ASC-arm at one (33%; P = .048) and four months (50%; P = .003), but not in the placebo-arm (P = .6 and P = .8), compared to baseline. The ASC-arm symptom scores significantly decreased on the xerostomia and VAS questionnaires, in the domains of thirst (-22%, P = .035) and difficulties in eating solid foods (-2%, P = .008) after four months compared to baseline. The ASC-arm showed significantly improved salivary gland functions of inorganic element secretion and absorption, at baseline and four months, compared to the placebo-arm. Core-needle biopsies showed increases in serous gland tissue and decreases in adipose and connective tissues in the ASC-arm compared to the placebo-arm (P = .04 and P = .02, respectively). MRIs showed no significant differences between groups in gland size or intensity (P < .05). CONCLUSIONS: ASC therapy for radiation-induced hypofunction and xerostomia was safe and significantly improved salivary gland functions and patient-reported outcomes. These results should encourage further exploratory and confirmatory trials.

The use of viscoelastic haemostatic assays in goal-directing treatment with allogeneic blood products - A systematic review and meta-analysis.

BACKGROUND: Management of the critically bleeding patient can be encountered in many medical and surgical settings. Common for these patients is a high risk of dying from exsanguination secondary to developing coagulopathy. The purpose of this meta-analysis was to systematically review and assess randomised controlled trials (RCTs) performed on patients in acute need for blood transfusions due to bleeding to evaluate the effect of viscoelastic haemostatic assay (VHA) guidance on bleeding, transfusion requirements and mortality. METHODS: PubMed and EMBASE were searched for RCTs that 1) randomised patients into receiving transfusions based on either a VHA-guided (thromboelastography [TEG] or rotational thromboelastometry [ROTEM]) algorithm (intervention group) or at the clinician's discretion and/or based on conventional coagulation tests (control group) and 2) adequately reported on the outcomes bleeding and/or transfusions and/or mortality. Data on bleeding, transfusions and mortality were extracted from each trial and included in a meta-analysis. RESULTS: Fifteen RCTs (n = 1238 patients) were included. Nine trials referred to cardiothoracic patients, one to liver transplantation, one to surgical excision of burn wounds and one to trauma. One trial was conducted with cirrhotic patients, one with patients undergoing scoliosis surgery while one trial randomised treatment in post-partum females presenting with bleeding. The amount of transfused red blood cells (RBCs), fresh frozen plasma (FFP) and bleeding volume was found to be significantly reduced in the VHA-guided groups, whereas no significant difference was found for platelet transfusion requirements or mortality.

First-in-man mesenchymal stem cells for radiation-induced xerostomia (MESRIX): study protocol for a randomized controlled trial.

BACKGROUND: Salivary gland hypofunction and xerostomia are major complications following radiotherapy for head and neck cancer and may lead to debilitating oral disorders and impaired quality of life. Currently, only symptomatic treatment is available. However, mesenchymal stem cell (MSC) therapy has shown promising results in preclinical studies. Objectives are to assess safety and efficacy in a first-in-man trial on adipose-derived MSC therapy (ASC) for radiation-induced xerostomia. METHODS: This is a single-center, phase I/II, randomized, placebo-controlled, double-blinded clinical trial. A total of 30 patients are randomized in a 1:1 ratio to receive ultrasound-guided, administered ASC or placebo to the submandibular glands. The primary outcome is change in unstimulated whole salivary flow rate. The secondary outcomes are safety, efficacy, change in quality of life, qualitative and quantitative measurements of saliva, as well as submandibular gland size, vascularization, fibrosis, and secretory tissue evaluation based on contrast-induced magnetic resonance imaging (MRI) and core-needle samples. The assessments are performed at baseline (1 month prior to treatment) and 1 and 4 months following investigational intervention. DISCUSSION: The trial is the first attempt to evaluate the safety and efficacy of adipose-derived MSCs (ASCs) in patients with radiation-induced xerostomia. The results may provide evidence for the effectiveness of ASC in patients with salivary gland hypofunction and xerostomia and deliver valuable information for the design of subsequent trials. TRIAL REGISTRATION: EudraCT, Identifier: 2014-004349-29. Registered on 1 April 2015. ClinicalTrials.gov, Identifier: NCT02513238 . First received on 2 July 2015. The trial is prospectively registered.

Mesenchymal stem cells improve locomotor recovery in traumatic spinal cord injury: systematic review with meta-analyses of rat models.

Traumatic spinal cord injury (SCI) is a devastating event with huge personal and societal costs. A limited number of treatments exist to ameliorate the progressive secondary damage that rapidly follows the primary mechanical impact. Mesenchymal stem or stromal cells (MSCs) have anti-inflammatory and neuroprotective effects and may thus reduce secondary damage after administration. We performed a systematic review with quantitative syntheses to assess the evidence of MSCs versus controls for locomotor recovery in rat models of traumatic SCI, and identified 83 eligible controlled studies comprising a total of 1,568 rats. Between-study heterogeneity was large. Fifty-three studies (64%) were reported as randomised, but only four reported adequate methodologies for randomisation. Forty-eight studies (58%) reported the use of a blinded outcome assessment. A random-effects meta-analysis yielded a difference in behavioural Basso-Beattie-Bresnahan (BBB) locomotor score means of 3.9 (95% confidence interval [CI] 3.2 to 4.7; P<0.001) in favour of MSCs. Trial sequential analysis confirmed the findings of the meta-analyses with the upper monitoring boundary for benefit being crossed by the cumulative Z-curve before reaching the diversity-adjusted required information size. Only time from intervention to last follow-up remained statistically significant after adjustment using multivariate random-effects meta-regression modelling. Lack of other demonstrable explanatory variables could be due to insufficient meta-analytic study power. MSCs would seem to demonstrate a substantial beneficial effect on locomotor recovery in a widely-used animal model of traumatic SCI. However, the animal results should be interpreted with caution concerning the internal and external validity of the studies in relation to the design of future clinical trials.

Enrichment of autologous fat grafts with ex-vivo expanded adipose tissue-derived stem cells for graft survival: a randomised placebo-controlled trial.

BACKGROUND: Autologous fat grafting is increasingly used in reconstructive surgery. However, resorption rates ranging from 25% to 80% have been reported. Therefore, methods to increase graft viability are needed. Here, we report the results of a triple-blind, placebo-controlled trial to compare the survival of fat grafts enriched with autologous adipose-derived stem cells (ASCs) versus non-enriched fat grafts. METHODS: Healthy participants underwent two liposuctions taken 14 days apart: one for ASC isolation and ex-vivo expansion, and another for the preparation of fat grafts. Two purified fat grafts (30 mL each) taken from the second liposuction were prepared for each participant. One graft was enriched with ASCs (20 × 10(6) cells per mL fat), and another graft without ASC enrichment served as a control. The fat grafts were injected subcutaneously as a bolus to the posterior part of the right and left upper arm according to the randomisation sequence. The volumes of injected fat grafts were measured by MRI immediately after injection and after 121 days before surgical removal. The primary goal was to compare the residual graft volumes of ASC-enriched grafts with those of control grafts. This study is registered at www.clinicaltrialsregister.eu, number 2010-023006-12. FINDINGS: 13 participants were enrolled, three of whom were excluded. Compared with the control grafts, the ASC-enriched fat grafts had significantly higher residual volumes: 23·00 (95% CI 20·57-25·43) cm(3) versus 4·66 (3·16-6·16) cm(3) for the controls, corresponding to 80·9% (76·6-85·2) versus 16·3% (11·1-21·4) of the initial volumes, respectively (p<0·0001). The difference between the groups was 18·34 (95% CI 15·70-20·98) cm(3), equivalent to 64·6% (57·1-72·1; p<0·0001). No serious adverse events were noted. INTERPRETATION: The procedure of ASC-enriched fat grafting had excellent feasibility and safety. These promising results add significantly to the prospect of stem cell use in clinical settings, and indicate that ASC graft enrichment could render lipofilling a reliable alternative to major tissue augmentation, such as breast surgery, with allogeneic material or major flap surgery. FUNDING: Danish Cancer Society, Centre of Head and Orthopaedics Rigshospitalet, and Moalem Weitemeyer Bendtsen.

Pooled human platelet lysate versus fetal bovine serum-investigating the proliferation rate, chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use.

BACKGROUND AIMS: Because of an increasing focus on the use of adipose-derived stem cells (ASCs) in clinical trials, the culture conditions for these cells are being optimized. We compared the proliferation rates and chromosomal stability of ASCs that had been cultured in Dulbecco's modified Eagle's Medium (DMEM) supplemented with either pooled human platelet lysate (pHPL) or clinical-grade fetal bovine serum (FBS) (DMEM(pHPL) versus DMEM(FBS)). METHODS: ASCs from four healthy donors were cultured in either DMEM(pHPL) or DMEM(FBS), and the population doubling time (PDT) was calculated. ASCs from two of the donors were expanded in DMEM(pHPL) or DMEM(FBS) and cultured for the final week before harvesting with or without the addition of vascular endothelial growth factor. We assessed the chromosomal stability (through the use of array comparative genomic hybridization), the expression of ASC and endothelial surface markers and the differentiation and angiogenic potential of these cells. RESULTS: The ASCs that were cultured in pHPL exhibited a significantly shorter PDT of 29.6 h (95% confidence interval, 22.3-41.9 h) compared with those cultured in FBS, for which the PDT was 123.9 h (95% confidence interval, 95.6-176.2 h). Comparative genomic hybridization analyses revealed no chromosomal aberrations. Cell differentiation, capillary structure formation and cell-surface marker expression were generally unaffected by the type of medium supplement that was used or by the addition of vascular endothelial growth factor. CONCLUSIONS: We observed that the use of pHPL as a growth supplement for ASCs facilitated a significantly higher proliferation rate compared with FBS without compromising genomic stability or differentiation capacity.

Hemostatic resuscitation with plasma and platelets in trauma.

BACKGROUND: Continued hemorrhage remains a major contributor of mortality in massively transfused patients and controversy regarding the optimal management exists although recently, the concept of hemostatic resuscitation, i.e., providing large amount of blood products to critically injured patients in an immediate and sustained manner as part of an early massive transfusion protocol has been introduced. The aim of the present review was to investigate the potential effect on survival of proactive administration of plasma and/or platelets (PLT) in trauma patients with massive bleeding. MATERIALS AND METHODS: English databases were searched for reports of trauma patients receiving massive transfusion (10 or more red blood cell (RBC) within 24 hours or less from admission) that tested the effects of administration of plasma and/or PLT in relation to RBC concentrates on survival from January 2005 to November 2010. Comparison between highest vs lowest blood product ratios and 30-day mortality was performed. RESULTS: Sixteen studies encompassing 3,663 patients receiving high vs low ratios were included. This meta-analysis of the pooled results revealed a substantial statistical heterogeneity (I(2) = 58%) and that the highest ratio of plasma and/or PLT or to RBC was associated with a significantly decreased mortality (OR: 0.49; 95% confidence interval: 0.43-0.57; P<0.0001) when compared with lowest ratio. CONCLUSION: Meta-analysis of 16 retrospective studies concerning massively transfused trauma patients confirms a significantly lower mortality in patients treated with the highest fresh frozen plasma (FFP) and/or PLT ratio when compared with the lowest FFP and/or PLT ratio. However, optimal ranges of FFP: RBC and PLT : RBC should be established in randomized controlled trials.

Importance of mesenchymal stem cells in autologous fat grafting: a systematic review of existing studies.

Autologous fat grafting (lipofilling) enables repair and augmentation of soft tissues and is increasingly used both in aesthetic and reconstructive surgery. Autologous fat has several advantages, including biocompatibility, versatility, natural appearance, and low donor site morbidity. The main limitation is unpredictable graft resorption, which ranges from 25%-80%, probably as a result of ischaemia and lack of neoangiogenesis. To obviate these disadvantages, several studies have searched for new ways of increasing the viability of the transplanted tissue. One promising approach has been to enrich the fat graft with adipose tissue-derived mesenchymal stem cells (ASC) before transplantation. We have reviewed original studies published on fat transplantation enriched with ASC. We found four murine and three human studies that investigated the subject after a sensitive search of publications. In the human studies, so-called cell assisted lipotransfer (CAL) increased the ASC concentration 2-5 times compared with non-manipulated fat grafts, which caused a questionable improvement in survival of fat grafts, compared with that of traditional lipofilling. In contrast, in two of the murine studies ASC-concentrations were increased 1250 and 6250 times, respectively, by ASC ex vivo expansion, which resulted in considerably improved fat transplant survival as well as quality. This effect of high-level enrichment with ASC is thought to have been caused by paracrine signalling, cellular differentiation, or both. The surgical and tissue handling techniques used in lipofilling are well proved, but the added effect of high-level enrichment with ex vivo expanded ASC still needs to be investigated properly in human lipofilling studies, combined with a thorough follow up and matched control groups. In conclusion, ASC-enriched lipofilling theoretically has the potential for transforming lipofilling from a relatively unpredictable intervention into one in which the resorption rate, quality of tissue, and safety can be predicted, and possibly superior to prosthetic implantation.

Transarterial (chemo)embolisation for unresectable hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) results in more than 600,000 deaths per year. Transarterial embolisation (TAE) and transarterial chemoembolisation (TACE) have become standard loco-regional treatments for unresectable HCC. OBJECTIVES: To assess the beneficial and harmful effects of TACE or TAE. SEARCH STRATEGY: We searched The Cochrane Hepato-Biliary Group Controlled Trials Register, The Cochrane Cancer Network register, The Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, MEDLINE, EMBASE, Science Citation Index Expanded, and The Latin American Caribbean Health Sciences Literature (LILACS) from dates of inceptions up to September 2010. SELECTION CRITERIA: We considered for inclusion all randomised trials that compared TACE or TAE versus placebo, sham, or no intervention. Co-interventions were allowed if comparable between intervention groups. Trials with inadequate randomisation were excluded. DATA COLLECTION AND ANALYSIS: For all-cause mortality, we calculated the log hazard ratio (HR) with standard error as point estimate and pooled them for meta-analysis using the inverse variance method. Sub-group analyses were performed regarding intervention regimen, trial truncation, or co-interventions. We validated the results with trial sequential analyses. We used random-effects model in all meta-analyses in anticipation of statistical heterogeneity among the trials. MAIN RESULTS: We included nine trials with 645 participants. Six trials assessed TACE versus control and three trials assessed TAE versus control. Seven trials had low risk of selection bias based on adequate generation of allocation sequence and concealment - but all these trials had other risks of bias. Three trials were stopped early due to interim inspections and one due to slow accrual. For all-cause mortality, statistical heterogeneity between trials was low to moderate (I(2)= 30%). Meta-analysis of trials with low risk of selection bias showed that TACE or TAE versus control does not significantly increase survival (HR 0.88; 95% CI 0.71 to 1.10). Two trials with low risk of selection bias, no early stopping, and no co-intervention did not establish any significant effect of TACE or TAE on overall survival (hazard ratio 1.22, 95% confidence interval 0.82 to 1.83; P = 0.33). Trial sequential analysis confirmed the absence of evidence for a beneficial effect of TACE or TAE on survival indicating the need for future randomisation of up to 383 additional participants. Substantial differences in criteria for assessing tumour response did not allow quantitative analyses. One trial investigated quality of life but did not detect any significant differences between the intervention groups. A range of adverse events including post-embolisation syndrome and serious complications were reported. AUTHORS' CONCLUSIONS: There is no firm evidence to support or refute TACE or TAE for patients with unresectable HCC. More adequately powered and bias-protected trials are needed.

Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions.

This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell maintenance and differentiation. Furthermore, a sample qPCR experiment will be shown illustrating that these techniques can be important tools in answering basic questions about hESC biology.

[Human embryonic stem cells: clinical potential and current challenges]. / Humane embryonale stamceller: potentiale og aktuelle udfordringer.

Human embryonic stem cells (hESCs) are regarded as the cornerstone of future treatment of a wide range of cellular degenerative disorders. hESCs are characterized by their pluripotency, i.e. their ability to differentiate into a wide range of cell types in the adult human body, including insulin-producing beta-cells, cardiomyocytes and neurons. Implementation in clinical settings, however, requires extensive safety studies and a solution to immunogenic issues in particular. Nonetheless, the potential outcome of regenerative medicine is likely to open avenues for treating many illnesses which today can only be treated with moderate success, if at all.

Epigenetic dedifferentiation of somatic cells into pluripotency: cellular alchemy in the age of regenerative medicine?

Ever since the derivation of the first human embryonic stem cell line, hopes have persisted for the treatment of a wide range of cellular degenerative diseases. However, significant immuno-incompatibility between donor cells and recipient patients remains an unsolved challenge. Currently, three main strategies are investigated in humans to create autologous pluripotent stem cells: somatic cell nuclear transfer, cell fusion and cell extract incubation. All methods exploit the fact that a somatic genome is amenable to epigenetic dedifferentiation into a more plastic state, presumably through direct exposure to and manipulation by heterologous transcriptional factors. Epigenetic reprogramming includes profound modifications of chromatin structure, but the responsible mechanisms that work in toti- and pluripotent cells remain largely unknown. This review presents a brief introduction to stem cell terminology and epigenetics, followed by a critical examination of the predominant methodologies involved. Finally, the search for specific reprogramming factors is discussed, and obstacles for the clinical implementation of reprogrammed cells are addressed.

Evaluation in mammalian oocytes of gene transcripts linked to epigenetic reprogramming.

The mature mammalian metaphase II (MII) oocyte has a unique ability to reprogram sperm chromatin and support early embryonic development. This feature even extends to the epigenetic reprogramming of a terminally differentiated cell nucleus as observed in connection with somatic cell nuclear transfer. Epigenetic nuclear reprogramming is highly linked to chromatin structure and includes covalent modifications of DNA and core histone proteins as well as reorganization of higher-order chromatin structure. A group of conserved enzymes mediating DNA methylation, methyl-CpG-binding protein (MeCP), histone acetylation and methylation, and chromatin remodeling are extensively involved in epigenetic reprogramming in mammalian cells. Using the oligonucleotide microarray technique, the present study compared the expression levels of 86 genes associated with epigenetic reprogramming in murine in vivo matured MII oocytes with that of germinal vesicle oocytes. Correlation between biological replicates was high. A total of 57 genes with potential reprogramming effect were detected. In MII oocytes, four genes were significant up-regulated, whereas 18 were down-regulated and 35 unchanged. The significantly regulated genes were validated by real-time quantitative RT-PCR. For example, MII oocytes showed a significant down-regulation of oocyte-specific maintenance DNA methyltransferase, Dnmt1o, and up-regulation of MeCP transcript, methyl-CpG binding domain protein 2. Furthermore, histone acetyltransferases were proportionally overrepresented when compared with histone deacetylases. These data elucidate for the first time some of the mechanisms that the oocyte may employ to reprogram a foreign genome either in form of a spermatozoa or a somatic nucleus and may thus be of importance for advancing the fields of stem cell research and regenerative medicine.

Regional differences in expression of specific markers for human embryonic stem cells.

Characterization of human embryonic stem cell (hESC) lines derived from the inner cell masses of blastocysts generally includes expression analysis of markers such as OCT4, NANOG, SSEA3, SSEA4, TRA-1-60 and TRA-1-81. Expression is usually detected by immunocytochemical staining of entire colonies of hESC, using one colony for each individual marker. Four newly established hESC lines showed the expected expression pattern and were capable of differentiating into the three germ layers in vitro. Neighbouring sections of entire colonies grown for 4, 11, 21 and 28 days respectively were stained with different markers to study the regional distribution and cellular co-expression. TRA-1-60 staining defined the hESC territory at all time points analysed. This territory comprised a characteristic OCT4 and NANOG staining often in overlapping subregions. Staining intensity of nuclei varied from strong OCT4 staining to weak or absent NANOG staining, and vice versa. SSEA4 staining was only observed in small clusters or single cells and not confined to the TRA territory. Co-expression of all markers was only detected in small areas. SSEA1 expression was found exclusively outside the TRA territory. In conclusion, pronounced regional differences in the expression of markers considered specific for undifferentiated hESC may suggest the existence of different cell populations.