Adaptive Designs with Discrete Test Statistics and Consideration of Overrunning.

BACKGROUND: Interim analyses are used in clinical trials in order to enable early decisions for medical, ethical, and economic reasons. However, it appears unfeasible to stop a trial during such an interim analysis. New patients will thus enter the trial while the interim analysis is ongoing. Moreover, depending on the event kinetics of the specific disease, the trial design, and the corresponding endpoints, some patients might still be unevaluable at the interim analysis due to not yet completed follow-up. Occurrence of these types of patients is characteristic for sequentially analyzed trials. Such patients are referred to as interim patients. In trials with multiple primary endpoints, another type of interim patients occurs. If some but not all null hypotheses can be rejected at the interim analysis, the trial might be continued to a second stage in order to answer the remaining questions. These second stage patients, however, provide new data to all trial questions including the already rejected ones and thus formally act as interim patients regarding the already rejected null hypotheses. Although all kinds of interim patients are not part of the interim analysis, the data collected on those patients have to be sent to the office of regulatory affairs and will be analyzed. If a smaller or contrasting treatment effect is observed in interim patients, this might lead to a withdrawal of an earlier superiority proof. OBJECTIVES: Presently, interim patients and their data are usually not considered in the confirmatory test. We offer a strategy to deal with interim patients in sequentially analyzed trials with discrete test statistics. The method covers sequentially analyzed single- and multi-arm trials with one or multiple primary endpoints. METHODS: When planning adaptive designs, it is common practice to assume that the stage-wise p-values are independent and standard uniformly distributed under the null hypothesis. In the context of discrete test statistics, this implies conservative tests. We provide an algorithm which iteratively optimizes an initially given design while adjusting for both discreteness of test statistics and interim patients. The algorithm is described verbally, graphically and formally to facilitate immediate implementation in computer software. RESULTS: The optimized design exploits the aspired significance level better and is more powerful than the initial one. The algorithm applies to fixed sample and planned flexible adaptive designs for single- and multi-arm trials with one or multiple primary endpoints. The benefit increases with the number of interim patients. CONCLUSIONS: When planning a trial with interim analyses, the rules for decisions must be adjusted to interim patients. Otherwise, the test procedure is conservative resulting in loss of power. This is essential in situations where the number of interim patients is important compared to the first stage, particularly in trials with multiple primary endpoints.

Pediatric Nodular Lymphocyte-predominant Hodgkin Lymphoma: Treatment Recommendations of the GPOH-HD Study Group.

Nodular lymphocyte-predominant Hodgkin lymphoma (nLPHL) is a very rare disease in childhood and adolescence. In Germany, about 15 newly diagnosed patients present with this disease annually; this number comprises less than 10% of all pediatric Hodgkin lymphoma cases. Since the EuroNet-PHL-LP1 trial for early stage nLPHL patients stopped recruiting in Germany in October 2014, the GPOH-HD writing committee reviewed the literature and decided to deliver treatment recommendations for childhood and adolescent nLPHL patients. These guidelines shall be applicable to young nLPHL patients in European countries that will no longer be able to participate in nLPHL trials for young patients. Therefore, the EuroNet-PHL-nLPHL-registry will be installed to provide quality assured central review of staging and response assessment for registered patients by the Central Review Board of EuroNet-PHL in Halle/Leipzig, Germany.

Cardiomyopathy in Duchenne Muscular Dystrophy: Current Value of Clinical, Electrophysiological and Imaging Findings in Children and Teenagers.

BACKGROUND: Progressive cardiomyopathy (CMP) is one main cause of death in DMD. This cross-sectional assessment of different cardiac diagnostic procedures focusses on preterm diagnosis of cardiac dysfunction. PATIENTS: 39 male DMD patients aged 6-20 years were included. 6 patients were still ambulatory, 21 patients received corticosteroid therapy. METHODS: All patients were investigated by ECG, Holter ECG and heart rate variability (HRV), B-type natriuretic peptide (BNP), echocardiography (TTE), tissue Doppler Imaging (TD) and magnetic resonance imaging (MRI) with Late Gadolinium enhancement (LE) and segmental wall motion analysis (WMA). RESULTS: 56% of the patients showed repolarization abnormalities and 76% altered HRV. Subnormal ventricular function was found in 25% by TTE and in 34% by MRI. TD differed from normal controls only in the apical septum. In MRI 89% of the patients showed different distribution and intensity of LE and WM restriction. The extent of LE was less in patients after steroid treatment (p<0.05). DISCUSSION: MRI with segmental LE- and WM-analysis seems to be superior to TTE and TD in exploring regional distribution and severity of damage of the myocardium. ECG and HRV abnormalities are common in DMD-patients but not tightly predictive for segmental and global left ventricular dysfunction. Targeted treatment of CMP in DMD needs prospective evaluation. CONCLUSION: A timely cardiac MRI is the most sensitive investigation for the identification of early myocardial changes in DMD which is a prerequisite for early interventions and therapeutic strategies.

Consensus of German transplant centers on hematopoietic stem cell transplantation in Fanconi anemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only curative therapy for the severe hematopoietic complications associated with Fanconi anemia (FA). In Germany, it is estimated that 10-15 transplants are performed annually for FA. However, because FA is a DNA repair disorder, standard conditioning regimens confer a high risk of excessive regimen-related toxicities and mortality, and reduced intensity regimens are linked with graft failure in some FA patients. Moreover, development of graft-versus-host disease is a major contributing factor for secondary solid tumors. The relative rarity of the disorder limits HSCT experience at any single center. Consensus meetings were convened to develop a national approach for HSCT in FA. This manuscript outlines current experience and knowledge about HSCT in FA and, based on this analysis, general recommendations reached at these meetings.

Cardiac function after repair of tetralogy of fallot: how are the atria performing? pilot study by cardiac magnetic resonance imaging.

The atria of the heart function as reservoir, conduit, and active pump and are critical for ventricular filling and cardiac output. We sought to evaluate right (RA) and left atrial (LA) function in patients after tetralogy of Fallot (TOF) repair by using cardiovascular magnetic resonance. Twelve TOF patients, age 16.7 ± 6.1 years, weight 50.9 ± 14.9 kg, were compared to 10 healthy volunteers, age 18.8 ± 6.8 years, weight 52.3 ± 20.8 kg. Both atria and both ventricles were imaged in short-axis planes by Steady State Free Precession. Volume changes and all derived atrial functional parameters were calculated from the volume/time curves obtained after segmentation on the cine images. Blood flow across the AV valves was used to define ventricular diastolic dysfunction. TOF patients showed similar maximal RA volume compared to controls, but increased volumes at mid-diastole (p < 0.05), resulting in a decreased cyclic volume change and atrial filling fraction (p < 0.01), and a decreased passive emptying volume and fraction (p < 0.01). In patients with diastolic dysfunction, conduit volume was increased (p < 0.05), and active emptying volume and fraction tended to be increased, respectively. No significant changes were found in LA, except for a decreased passive emptying fraction (p < 0.05). RA function and particularly reservoir function are impaired in TOF patients. The RA conduit/reservoir ratio is increased and reflects the lost of the ability of the atrium to fill the ventricle by pulsatile flow.

Guidelines for surveillance of individuals with constitutional mismatch repair-deficiency proposed by the European Consortium "Care for CMMR-D" (C4CMMR-D).

Lynch syndrome (LS) is an autosomal dominant disorder caused by a defect in one of the DNA mismatch repair genes: MLH1, MSH2, MSH6 and PMS2. In the last 15 years, an increasing number of patients have been described with biallelic mismatch repair gene mutations causing a syndrome referred to as 'constitutional mismatch repair-deficiency' (CMMR-D). The spectrum of cancers observed in this syndrome differs from that found in LS, as about half develop brain tumours, around half develop digestive tract cancers and a third develop haematological malignancies. Brain tumours and haematological malignancies are mainly diagnosed in the first decade of life, and colorectal cancer (CRC) and small bowel cancer in the second and third decades of life. Surveillance for CRC in patients with LS is very effective. Therefore, an important question is whether surveillance for the most common CMMR-D-associated cancers will also be effective. Recently, a new European consortium was established with the aim of improving care for patients with CMMR-D. At a workshop of this group held in Paris in June 2013, one of the issues addressed was the development of surveillance guidelines. In 1968, criteria were proposed by WHO that should be met prior to the implementation of screening programmes. These criteria were used to assess surveillance in CMMR-D. The evaluation showed that surveillance for CRC is the only part of the programme that largely complies with the WHO criteria. The values of all other suggested screening protocols are unknown. In particular, it is questionable whether surveillance for haematological malignancies improves the already favourable outcome for patients with these tumours. Based on the available knowledge and the discussions at the workshop, the European consortium proposed a surveillance protocol. Prospective collection of all results of the surveillance is needed to evaluate the effectiveness of the programme.

Effective childhood cancer treatment: the impact of large scale clinical trials in Germany and Austria.

In Germany and Austria, more than 90% of pediatric cancer patients are enrolled into nationwide disease-specific first-line clinical trials or interim registries. Essential components are a pediatric cancer registry and centralized reference laboratories, imaging review, and tumor board assistance. The five-year overall survival rate in countries where such infrastructures are established has improved from <20% before 1950 to >80% since 1995. Today, treatment intensity is tailored to the individual patient's risk to provide the highest chances of survival while minimizing deleterious late effects. Multicenter clinical trials are internationalized and serve as platforms for further improvements by novel drugs and biologicals.

Hepatic 3D cultures but not 2D cultures preserve specific transporter activity for acetaminophen-induced hepatotoxicity.

Primary human hepatocytes (PHH) are the "gold standard" for in vitro toxicity tests. However, 2D PHH cultures have limitations that are due to a time-dependent dedifferentiation process visible by morphological changes closely connected to a decline of albumin production and CYP450 activity. The 3D in vitro culture corresponds to in vivo-like tissue architecture, which preserves functional characteristics of hepatocytes, and therefore can at least partially overcome the restrictions of 2D cultures. Consequently, several drug toxicities observed in vivo cannot be reproduced in 2D in vitro models, for example, the toxic effects of acetaminophen. The objective of this study was to identify molecular differences between 2D and 3D cultivation which explain the observed toxicity response. Our data demonstrated an increase in cell death after treatment with acetaminophen in 3D, but not in 2D cultures. Additionally, an acetaminophen concentration-dependent increase in the CYP2E1 expression level in 3D cultures was detected. However, during the treatment with 10 mM acetaminophen, the expression level of SOD gradually decreased in 3D cultures and was undetectable after 24 h. In line with these findings, we observed higher import/export rates in the membrane transport protein, multidrug resistance-associated protein-1, which is known to be specific for acetaminophen transport. The presented data demonstrate that PHH cultured in 3D preserve certain metabolic functions. Therefore, they have closer resemblance to the in vivo situation than PHH in 2D cultures. In consequence, 3D cultures will allow for a more accurate hepatotoxicity prediction in in vitro models in the future.

Mouse hepatoma cell lines differing in aryl hydrocarbon receptor-mediated signaling have different activities for glucuronidation.

For studies on the aryl hydrocarbon receptor (AhR)-dependent toxicity of the mycotoxins alternariol (AOH) and alternariol methyl ether (AME), three mouse hepatoma (Hepa-1) cell lines with intact and with compromised AhR signaling were compared with respect to their activities for hydroxylation, methylation, and glucuronidation. Whereas the activities of cytochrome P450-mediated monooxygenase and catechol-O-methyl transferase were very low and did not differ between the three cell lines, a pronounced difference was observed for UDP-glucuronosyl transferase activity, which was much higher in Hepa-1c1c4 than in c1c7 and c1c12 cells. In all three cell types, the rate of glucuronidation of AOH was about four times higher than that of AME. Whereas AME caused a concentration-dependent G2/M arrest in each cell line, AOH arrested Hepa-1c1c7 and c1c12 cells but not c1c4 cells. However, Hepa-1c1c4 cells were arrested by AOH when ß-glucuronidase was added to the incubation medium in order to reverse the formation of AOH glucuronides. We conclude that the failure of AOH to cause cell cycle inhibition in Hepa-1c1c4 cells is due to its efficient glucuronidation. The considerable UDP-glucuronosyl transferase activity of Hepa-1c1c4 cells should be taken into account when other compounds are studied in this cell line. Moreover, we demonstrate that differences in glucuronide formation between cell types can be overcome by the addition of ß-glucuronidase to the cell culture medium.

Non-Hodgkin's lymphoma in adolescents: experiences in 378 adolescent NHL patients treated according to pediatric NHL-BFM protocols.

Age-related differences in the distribution, biology and treatment response of non-Hodgkin's lymphoma (NHL) in adolescents remain to be elucidated. The current analyses present clinical parameters and outcomes of adolescents treated in pediatric NHL-BFM trials. Patients were stratified by histological subtype: lymphoblastic lymphoma (LBL); mature B-NHL, including Burkitt's lymphoma/leukemia (BL/B-AL), diffuse B-cell lymphoma (DLBCL-CB) and mediastinal B-cell lymphoma (PMLBL); and anaplastic large cell lymphoma (ALCL). Between October 1986 and December 2007, 2915 patients were registered, including 378 (13%) adolescents (15-18 years) with BL/B-AL (n=101), ALCL (n=74), DLBCL-CB (n=55), T-LBL (n=45), PMLBL (n=24), pB-LBL (n=13) and rare or not-specified NHL subtypes (n=66). The 5-year event-free survival (EFS) was 79±2% for adolescents compared with 85±1% for patients aged <15 years (P=0.014). EFS was 83±7% for adolescents with T-LBL, 82±4% with BL/B-AL, 85±5% with DLBCL-CB, 57±10% with PMLBL and 70±6% with ALCL. According to sex, the 5-year EFS in females versus males, respectively, was 70±5 versus 83±2% overall (P=0.004), 57±17 versus 92±6% (P=0.0036) for T-LBL patients and 71±9 versus 97±3% (P=0.0067) for DLBCL-CB patients. Adolescents with NHL treated according to pediatric NHL-BFM protocols had an EFS of 79±2%, which is marginally inferior to that of children. In adolescents with T-LBL and DLBCL-CB, female sex was associated with a worse prognosis.

Glucuronidation of the mycotoxins alternariol and alternariol-9-methyl ether in vitro: chemical structures of glucuronides and activities of human UDP-glucuronosyltransferase isoforms.

Alternariol (AOH) and alternariol-9-methyl ether (AME) are major toxins produced by fungi of the genus Alternaria and are frequently found in various food items. Because AOH has three hydroxyl groups and AME two, the formation of various glucuronides must be expected. When AOH was incubated with hepatic and intestinal microsomes from rats, pigs and humans in the presence of uridine diphosphate glucuronic acid, two glucuronides were detected and tentatively identified as AOH-3-O-glucuronide and AOH-9-O-glucuronide. Under the same conditions, AME yielded predominantly AME-3-O-glucuronide and only small amounts of AME-7-O-glucuronide. The activities of all microsomes for the glucuronidation of AOH and AME were in the same range. Nine out of ten recombinant human UDP-glucuronosyltransferases (UGTs) were able to glucuronidate AOH, and eight out of ten UGTs had activity for AME. These data suggest that AOH and AME are readily glucuronidated in hepatic and extrahepatic tissues, implying that glucuronidation constitutes a major metabolic pathway in the disposition of these mycotoxins.

Activities of human recombinant cytochrome P450 isoforms and human hepatic microsomes for the hydroxylation ofAlternaria toxins.

TheAlternaria toxins alternariol (AOH), alternariol-9-methyl ether (AME), altenuene (ALT) and isoaltenuene (iALT) undergo extensive oxidative metabolism, but the cytochrome P450 (CYP) isoforms responsible for the reported hydroxylation reactions are yet unknown. In the present study, the activities of twelve human CYP isoforms for the hydroxylation of AOH, AME, ALT and iALT at different positions have been determined. The most active monooxygenase for AOH and AME was CYP1A1, and lower activities were observed for CYP1A2, 2C19 and 3A4. Hydroxylation at C-2 of AOH and AME was the preferred reaction of most isoforms. For ALT and iALT, CYP2C19 had the highest activity, followed by 2C9 and 2D6. The dominating metabolite of all active isoforms was the 8-hydroxylated ALT and iALT. The activities of the CYP isoforms are consistent with the pattern of metabolites of theAlternaria toxins obtained with pooled human hepatic microsomes. Based on the activities of the CYP isoforms, a significant extrahepatic hydroxylation must be expectede.g. in the lung and esophagus for AOH and AME, and in the intestine and ovaries for ALT and iALT. As all major hydroxylation products are catechols, the extrahepatic metabolism ofAlternaria toxins may be of toxicological relevance.

Loss of heterozygosity on chromosome 6q14-q24 is associated with poor outcome in children and adolescents with T-cell lymphoblastic lymphoma.

Deletions of chromosome 6q have been reported in several hematological malignancies, but data are not conclusive regarding their biological and prognostic impact. Therefore, we focused on pediatric patients diagnosed with T-cell lymphoblastic lymphoma (T-LBL) treated uniformly according to the NHL-BFM95 protocol. We used loss-of-heterozygosity (LOH) analysis of 25 microsatellite markers located on chromosome 6q14-q24. Fragment-length analysis was performed on ABI-PRISM3100 Genetic-Analyzer. Eligibility criterion was > or =3 informative markers. Between April 1995 and March 2003, 185 T-LBL patients were treated according to the NHL-BFM95 protocol. Five-year event-free (EFS) and disease-free survival (DFS) were 79+/-3 and 87+/-3% (median follow-up 4.7 [1.2-10.1] years). Sixty-one patients were evaluable for LOH analysis, including 18 out of 23 patients with relapse. EFS and DFS were 67+/-6 and 69+/-6% for these 61 patients. Testing of 853 markers in the 61 patients identified the presence of LOH in 19 patients (31%): 13 of the 18 relapse patients and five of the 41 in complete remission (odds ratio 18.7, 95% confidence interval 4.7-75.3). One LOH-positive patient died from treatment-related toxicity. We conclude that LOH on chromosome 6q14-q24 may have conferred a high risk of relapse on our group of children with T-LBL treated according to the NHL-BFM95 protocol.

Mammalian cells express two VPS4 proteins both of which are involved in intracellular protein trafficking.

The yeast Vps4 protein (Vps4p) is a member of the AAA protein family (ATPases associated with diverse cellular activities) and a key player in the transport of proteins out of a prevacuolar endosomal compartment. In human cells, we identified two non-allelic orthologous proteins (VPS4-A and VPS4-B) of yeast Vps4p. The human VPS4-A and VPS4-B proteins display a high degree of sequence identity to each other (80 %) and to the yeast Vps4 protein (59 and 60 %, respectively). Yeast cells lacking a functional VPS4 gene exhibit a temperature-sensitive growth defect and mislocalise a carboxypeptidase Y-invertase fusion protein to the cell surface. Heterologous expression of human VPS4 genes in vps4 mutant yeast strains led, in the case of human VPS4-A, to a partial and, in the case of human VPS4-B, to a complete suppression of the temperature-sensitive growth defect. The vacuolar protein sorting defect of vps4 mutant yeast cells was complemented completely by heterologous expressed human VPS4-B protein, and partially by the human VPS4-A protein. Expression of mutant human VPS4-A (E228Q) and VPS4-B (E235Q) proteins, harbouring single amino acid exchanges in their AAA domains, induced dominant-negative vacuolar protein sorting defects in wild-type yeast cells in both cases. Two-hybrid experiments suggest that the human VPS4-A and VPS4-B proteins can form heteromeric complexes, and subcellular localisation experiments indicate that both human VPS4 proteins associate with endosomal compartments in yeast. Based on these results, we conclude that both human VPS4 proteins are involved in intracellular protein trafficking, presumably at a late endosomal protein transport step, similar to the Vps4p in yeast.

Human immunodeficiency virus type 1 protease genotype predicts immune and viral responses to combination therapy with protease inhibitors (PIs) in PI-naive patients.

Protease genotype, as a variable in outcome to combination therapy for human immunodeficiency virus (HIV) type 1 infection, was evaluated among protease inhibitor-naive children and adolescents who had received extensive treatment with reverse-transcriptase inhibitors. After 24 weeks of combination therapy, 35% had viral and immune success (VSIS patients), 19% had viral and immune failure (VFIF patients), and 46% had viral failure but marked improvement in CD4 T cells (VFIS patients). Disease stage was the only pretherapy clinical variable associated with outcome (P=.02). Although reverse-transcriptase genotype was unrelated to outcome, pretherapy protease genotype was related significantly to therapy response (P=.005). Odds for immune or viral failure were 17.7 to 1 and 2.5 to 1, respectively, for protease genotype as a single variable. Protease genotype combined with disease stage and CD4 cell percentage predicted correct therapy response for 81% of patients (100% of VFIF, 78% of VSIS, and 75% of VFIS patiens). Naturally occurring amino acid polymorphisms in protease provide sensitive biomarkers for treatment response among inhibitor-naive patients with advanced HIV disease.

Oviductal plasminogen activator inhibitor-1 (PAI-1): mRNA, protein, and hormonal regulation during the estrous cycle and early pregnancy in the pig.

Recent identification of plasminogen activator inhibitor-1 (PAI-1) in the pig oviduct has prompted an evaluation of its mRNA, protein synthesis, and hormonal regulation during the estrous cycle and early pregnancy, defined as time prior to and after maternal recognition of pregnancy. To examine PAI-1 protein synthesis, oviductal tissue was collected from European Large White and Chinese Meishan gilts on days 0, 2, and 5 of early pregnancy, divided into three functional segments, and cultured. Culture media was collected and de novo synthesized PAI-1 analyzed by 2D-SDS-PAGE, fluorography, and densitometry. To determine hormonal regulation of PAI-1 synthesis and secretion, four groups of ovariectomized (OVX) cross-bred gilts were each treated with one of four steroid regimens (corn oil, estrogen, progesterone, or estrogen + progesterone) and tissue collected for RNA or cultured. Steady-state mRNA levels of PAI-1 were evaluated throughout the estrous cycle in cross-bred gilts. To compare steady-state PAI-1 mRNA levels between cyclic and pregnant cross-bred gilts, tissue was collected on days 0, 2, and 12. Quantitative analysis of steady-state levels of PAI-1 mRNA were analyzed by dot-blot hybridization and densitometry. A greater (P < 0.01) synthesis and secretion of PAI-1 protein was found in the isthmus portion of the oviduct relative to either the ampulla or infundibulum regardless of day of pregnancy or breed. No difference could be detected for PAI-1 protein between breeds. The Large White had a greater (P < 0.05) secretion of PAI-1 on day 2 of early pregnancy relative to other days examined. Whole oviductal tissue from cross-bred gilts was found to have a significantly greater amount of PAI-1 mRNA on days 1 and 2 compared to other days examined, while the isthmus had significantly greater levels of mRNA on days 2 and 12. A significant effect of day and segment was detected for levels of PAI-1 mRNA from cyclic and early pregnant cross-bred gilts. PAI-1 mRNA was found to be significantly greater in the isthmus than other segments, regardless of day of the estrous cycle or pregnancy. An interaction was detected for estrogen and progesterone on PAI-1 mRNA (P < 0.05) and protein (P = 0.09). Estrogen was found to inhibit PAI-1 protein synthesis and also inhibited progesterone-mediated stimulation of PAI-1 mRNA. Our results demonstrate expression of PAI-1 mRNA and protein are highest on day 2 of early pregnancy, which is consistent with its proposed function of protecting the oocyte/embryo from enzymatic degradation and/or extracellular matrix remodeling of both oviduct and early cleavage-stage embryo.