Irinotecan and temozolomide in combination with dasatinib and rapamycin versus irinotecan and temozolomide for patients with relapsed or refractory neuroblastoma (RIST-rNB-2011): a multicentre, open-label, randomised, controlled, phase 2 trial.

BACKGROUND: Neuroblastoma is the most common extracranial solid tumour in children. Relapsed or refractory neuroblastoma is associated with a poor outcome. We assessed the combination of irinotecan-temozolomide and dasatinib-rapamycin (RIST) in patients with relapsed or refractory neuroblastoma. METHODS: The multicentre, open-label, randomised, controlled, phase 2, RIST-rNB-2011 trial recruited from 40 paediatric oncology centres in Germany and Austria. Patients aged 1-25 years with high-risk relapsed (defined as recurrence of all stage IV and MYCN amplification stages, after response to treatment) or refractory (progressive disease during primary treatment) neuroblastoma, with Lansky and Karnofsky performance status at least 50%, were assigned (1:1) to RIST (RIST group) or irinotecan-temozolomide (control group) by block randomisation, stratified by MYCN status. We compared RIST (oral rapamycin [loading 3 mg/m2 on day 1, maintenance 1 mg/m2 on days 2-4] and oral dasatinib [2 mg/kg per day] for 4 days with 3 days off, followed by intravenous irinotecan [50 mg/m2 per day] and oral temozolomide [150 mg/m2 per day] for 5 days with 2 days off; one course each of rapamycin-dasatinib and irinotecan-temozolomide for four cycles over 8 weeks, then two courses of rapamycin-dasatinib followed by one course of irinotecan-temozolomide for 12 weeks) with irinotecan-temozolomide alone (with identical dosing as experimental group). The primary endpoint of progression-free survival was analysed in all eligible patients who received at least one course of therapy. The safety population consisted of all patients who received at least one course of therapy and had at least one post-baseline safety assessment. This trial is registered at ClinicalTrials.gov, NCT01467986, and is closed to accrual. FINDINGS: Between Aug 26, 2013, and Sept 21, 2020, 129 patients were randomly assigned to the RIST group (n=63) or control group (n=66). Median age was 5·4 years (IQR 3·7-8·1). 124 patients (78 [63%] male and 46 [37%] female) were included in the efficacy analysis. At a median follow-up of 72 months (IQR 31-88), the median progression-free survival was 11 months (95% CI 7-17) in the RIST group and 5 months (2-8) in the control group (hazard ratio 0·62, one-sided 90% CI 0·81; p=0·019). Median progression-free survival in patients with amplified MYCN (n=48) was 6 months (95% CI 4-24) in the RIST group versus 2 months (2-5) in the control group (HR 0·45 [95% CI 0·24-0·84], p=0·012); median progression-free survival in patients without amplified MYCN (n=76) was 14 months (95% CI 9-7) in the RIST group versus 8 months (4-15) in the control group (HR 0·84 [95% CI 0·51-1·38], p=0·49). The most common grade 3 or worse adverse events were neutropenia (54 [81%] of 67 patients given RIST vs 49 [82%] of 60 patients given control), thrombocytopenia (45 [67%] vs 41 [68%]), and anaemia (39 [58%] vs 38 [63%]). Nine serious treatment-related adverse events were reported (five patients given control and four patients given RIST). There were no treatment-related deaths in the control group and one in the RIST group (multiorgan failure). INTERPRETATION: RIST-rNB-2011 demonstrated that targeting of MYCN-amplified relapsed or refractory neuroblastoma with a pathway-directed metronomic combination of a multkinase inhibitor and an mTOR inhibitor can improve progression-free survival and overall survival. This exclusive efficacy in MYCN-amplified, relapsed neuroblastoma warrants further investigation in the first-line setting. FUNDING: Deutsche Krebshilfe.

Physical Activity as a Treatment for Cancer-Related Fatigue in Children, Adolescents and Young Adults: A Systematic Review.

BACKGROUND: Cancer-related fatigue (CRF) is one of the most common and distressing symptoms in paediatric oncology. Based on previous studies, physical activity interventions are considered to be effective in reducing CRF in adult cancer patients. AIM: The aim of this systematic review is to investigate whether physical activity interventions can reduce CRF in paediatric patients undergoing cancer treatment. METHODOLOGY: A systematic literature search was conducted in PubMed and Sport-Discus in October 2021 to identify intervention studies examining the effects of physical activity on CRF in cancer patients ≤ 21 years of age. Their methodological quality was assessed using the JBI Critical Appraisal Tool. RESULTS: A total of 20 studies (seven randomized-controlled, six quasi-experimental and seven single-arm intervention trials) were included in the review. Nine studies reported significant positive effects of physical activity interventions on CRF in group comparison or within groups. Eleven trials reported no significant changes in CRF. CONCLUSION: Physical activity as a therapeutic intervention in paediatric oncology may have the potential to reduce CRF in childhood cancer patients undergoing cancer treatment. Further high-quality studies with large samples are needed to verify these results and to assess the interdependence of dose and response of physical activity interventions.

Identification of an Immunogenic Medulloblastoma-Specific Fusion Involving EPC2 and GULP1.

Medulloblastoma is the most common malignant brain tumor in children. Immunotherapy is yet to demonstrate dramatic results in medulloblastoma, one reason being the low rate of mutations creating new antigens in this entity. In tumors with low mutational burden, gene fusions may represent a source of tumor-specific neoantigens. Here, we reviewed the landscape of fusions in medulloblastoma and analyzed their predicted immunogenicity. Furthermore, we described a new in-frame fusion protein identified by RNA-Seq. The fusion involved two genes on chromosome 2 coding for the enhancer of polycomb homolog 2 (EPC2) and GULP PTB domain containing engulfment adaptor 1 (GULP1) respectively. By qRT-PCR analysis, the fusion was detected in 3 out of 11 medulloblastoma samples, whereby 2 samples were from the same patients obtained at 2 different time points (initial diagnosis and relapse), but not in other pediatric brain tumor entities. Cloning of the full-length sequence indicated that the fusion protein contains the N-terminal enhancer of polycomb-like domain A (EPcA) of EPC2 and the coiled-coil domain of GULP1. In silico analyses predicted binding of the neoantigen-derived peptide to HLA-A*0201. A total of 50% of the fusions described in the literature were also predicted to produce an immunogenic peptide. The EPC2-GULP1 fusion peptide was able to induce a de novo T cell response characterized by interferon gamma release of CD8+ cytotoxic T cells in vitro. While the functional relevance of this fusion in medulloblastoma biology remains to be clarified, our data support an immunotherapeutic approach for pediatric medulloblastoma patients carrying the EPC2-GULP1 fusion and other immunogenic fusions.

Tumor Lipids of Pediatric Papillary Renal Cell Carcinoma Stimulate Unconventional T Cells.

Papillary renal cell carcinoma (PRCC) is a rare entity in children with no established therapy protocols for advanced diseases. Immunotherapy is emerging as an important therapeutic tool for childhood cancer. Tumor cells can be recognized and killed by conventional and unconventional T cells. Unconventional T cells are able to recognize lipid antigens presented via CD1 molecules independently from major histocompatibility complex, which offers new alternatives for cancer immunotherapies. The nature of those lipids is largely unknown and α-galactosylceramide is currently used as a synthetic model antigen. In this work, we analyzed infiltrating lymphocytes of two pediatric PRCCs using flow cytometry, immunohistochemistry and qRT-PCR. Moreover, we analyzed the CD1d expression within both tumors. Tumor lipids of PRCC samples and three normal kidney samples were fractionated and the recognition of tumor own lipid fractions by unconventional T cells was analyzed in an in vitro assay. We identified infiltrating lymphocytes including γδ T cells and iNKT cells, as well as CD1d expression in both samples. One lipid fraction, containing ceramides and monoacylglycerides amongst others, was able to induce the proliferation of iNKT cells isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and of one matched PRCC patient. Furthermore, CD1d tetramer stainings revealed that a subset of iNKT cells is able to bind lipids being present in fraction 2 via CD1d. We conclude that PRCCs are infiltrated by conventional and unconventional T cells and express CD1d. Moreover, certain lipids, present in pediatric PRCC, are able to stimulate unconventional T cells. Manipulating these lipids and T cells may open new strategies for therapy of pediatric PRCCs.

CpG-Islands as Markers for Liquid Biopsies of Cancer Patients.

The analysis of tumours using biomarkers in blood is transforming cancer diagnosis and therapy. Cancers are characterised by evolving genetic alterations, making it difficult to develop reliable and broadly applicable DNA-based biomarkers for liquid biopsy. In contrast to the variability in gene mutations, the methylation pattern remains generally constant during carcinogenesis. Thus, methylation more than mutation analysis may be exploited to recognise tumour features in the blood of patients. In this work, we investigated the possibility of using global CpG (CpG means a CG motif in the context of methylation. The p represents the phosphate. This is used to distinguish CG sites meant for methylation from other CG motifs or from mentions of CG content) island methylation profiles as a basis for the prediction of cancer state of patients utilising liquid biopsy samples. We retrieved existing GEO methylation datasets on hepatocellular carcinoma (HCC) and cell-free DNA (cfDNA) from HCC patients and healthy donors, as well as healthy whole blood and purified peripheral blood mononuclear cell (PBMC) samples, and used a random forest classifier as a predictor. Additionally, we tested three different feature selection techniques in combination. When using cfDNA samples together with solid tumour samples and healthy blood samples of different origin, we could achieve an average accuracy of 0.98 in a 10-fold cross-validation. In this setting, all the feature selection methods we tested in this work showed promising results. We could also show that it is possible to use solid tumour samples and purified PBMCs as a training set and correctly predict a cfDNA sample as cancerous or healthy. In contrast to the complete set of samples, the feature selections led to varying results of the respective random forests. ANOVA feature selection worked well with this training set, and the selected features allowed the random forest to predict all cfDNA samples correctly. Feature selection based on mutual information could also lead to better than random results, but LASSO feature selection would not lead to a confident prediction. Our results show the relevance of CpG islands as tumour markers in blood.

Safety and Activity of the Combination of Ceritinib and Dasatinib in Osteosarcoma.

Osteosarcoma (OS) is the second most common cause of cancer-related death in pediatric patients. The insulin-like growth factor (IGF) pathway plays a relevant role in the biology of OS but no IGF targeted therapies have been successful as monotherapy so far. Here, we tested the effect of three IGF specific inhibitors and tested ceritinib as an off-target inhibitor, alone or in combination with dasatinib, on the proliferation of seven primary OS cells. Picropodophyllin, particularly in combination with dasatinib and the combination ceritinib/dasatinib were effective in abrogating the proliferation. The ceritinib/dasatinib combination was applied to the primary cells of a 16-year-old girl with a long history of lung metastases, and was more effective than cabozantinib and olaparib. Therefore, the combination was used to treat the patient. The treatment was well tolerated, with toxicity limited to skin rush and diarrhea. A histopathological evaluation of the tumor after three months of therapy indicated regions of high necrosis and extensive infiltration of macrophages. The extension of the necrosis was proportional to the concentration of dasatinib and ceritinib in the area, as analysed by an ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS). After the cessation of the therapy, radiological analysis indicated a massive growth of the patient's liver metastases. In conclusion, these data indicate that the combination of ceritinib/dasatinib is safe and may be used to develop new therapy protocols.

Multivariate analysis on complications of central venous access devices in children with cancer and severe disease influenced by catheter tip position and vessel insertion site (A STROBE-compliant study).

CONTEXT: Reliable long-term central venous access device (CVAD) is essential for the management of pediatric patients with cancer or chronic diseases. However, there is no general consensus for optimal catheter tip location and vessel insertion site in children. OBJECTIVE: This single center study analyzes the risk of complications associated with long-term upper body CVAD and evaluates them with respect to catheter tip location as well as vessel insertion site. DESIGN: Pediatric patients who received long-term upper body CVAD from January 2008 through April 2017 and underwent radiographic documentation of the tip location were retrospectively included in the study. Data on demographics, catheter tip location on chest x-ray, intraoperative vessel insertion sites and postoperative complications were analyzed. Catheter tip location was categorized as "high" (above the right mainstem bronchus), "medium" (at the level of the bronchus), and "low" (below the right mainstem bronchus). Distance to the carina was measured as well. RESULTS: A total of 396 patients, 74.7% suffering from cancer were included in our study (mean age 6.3 ± 0.3 years). Complications occurred in about one fourth of all patients. Catheter-related blood stream infections (BSI) (n = 40, 36.4%) were most prevalent, but catheter tip position or vessel insertion site had no impact on the risk of infections. Dislodgement (n = 27, 24.6%) and occlusion (n = 11, 10.0%) were more frequent in "high" positioned catheter tips. While there was one patient who developed arrhythmia, no case of cardiac perforation, and in particular, no catheter-related death was recorded in our series. The vessel insertion site seemed to have no influence on the complication frequency of CAVDs. CONCLUSION: The catheter tip position seems to have an impact on the catheter-related complication profile in children. To avoid complications, we recommend avoiding a "high" localization of the catheter tip above the right main bronchus. "Low" catheter tip placement was associated with the lowest dislocation rate. Given the overall low complication rate, insertion and use of CVADs in children can generally be considered as safe.

Radiation-induced vascular changes in the intracranial irradiation field in medulloblastoma survivors: An MRI study.

BACKGROUND AND PURPOSE: While survival times after treatment of medulloblastoma are increasing, little is known about radiochemotherapy (RCT)-induced cerebrovascular changes. High resolution vessel wall imaging (VWI) sequences are an emerging tool for the evaluation of cerebrovascular diseases. We performed VWI in medulloblastoma long-term survivors to screen for late sequelae of RCT. MATERIAL AND METHODS: Twenty-two pediatric medulloblastoma survivors (mean age 25.8 years (10-53 years); 16.3 years (mean) post primary RCT (range 1-45 years)) underwent 2D VWI-MRI. Vessel wall thickening, contrast enhancement and luminal narrowing were analyzed. The findings were correlated with the patients' radiation protocols. RESULTS: Vessel wall changes were observed the intracranial internal carotid artery (ICA) and the vertebrobasilar circulation (VBC) in 14 of 22 patients (63.6%). In multivariate analysis, time after RCT (OR = 1.38, p < 0.05) was strongest independent predictor for development of vessel wall alterations. The dose of radiation was not a relevant predictor. CONCLUSIONS: With longer follow-up time intracranial vessel wall changes are observed more frequently in medulloblastoma survivors. Thus VWI is a useful tool to monitor vessel wall alterations of cranially irradiated patients, creating the prerequisite for further treatment of late sequelae.

AlloHSCT in paediatric ALL and AML in complete remission: improvement over time impacted by accreditation?

Allogeneic hematopoietic stem cell transplantation (alloHSCT) has become a well-established treatment option for many patients suffering from malignant and non-malignant diseases. In the past decade, high-resolution HLA-typing, remission surveillance, pre-emptive immune intervention, and standardisation in supportive care measures have substantially improved transplant outcomes. This retrospective study evaluated transplant procedures in 162 paediatric patients with acute lymphoblastic leukaemia (n = 124) or acute myeloid leukaemia (n = 38) who received their first alloHSCT in our institution over an 11-year period. We observed a significant reduction in risk of non-relapse mortality (NRM) over time (HR = 0.34, 95% CI 0.12-0.98; P = 0.05), the 4-year NRM estimate decreased from 20% in 2005-2008 to 7% in 2012-2016 (P = 0.02) and an increase in survival after relapse. There was no significant difference in patients who received a graft from a sibling, haplo, or an unrelated donor with regard to their overall survival (P = 0.45), event-free survival (P = 0.61), and non-relapse mortality (P = 0.19). Our data suggest that a specific transplant infrastructure with a highly experienced team in an accredited transplant centre likely contributes to better transplant outcomes for acute leukaemia patients in complete remission regardless of donor type.

Susceptibility-weighted magnetic resonance imaging of cerebrovascular sequelae after radiotherapy for pediatric brain tumors.

BACKGROUND AND PURPOSE: Due to sensitive neuroimaging techniques, cerebrovascular complications such as cerebral microbleeds (CMB) and cerebral cavernous malformations (CCM) are increasingly recognized as considerable late effects after treatment for pediatric brain tumor. The aim of this study was to analyze CMB in a cohort of patients after cranial irradiation therapy for medulloblastoma or other pediatric brain tumors using susceptibility-weighted magnetic resonance imaging (SWI). MATERIALS AND METHODS: Forty former pediatric brain tumor patients were enrolled in this prospective cross-sectional study and examined by cranial MRI including SWI sequences. Cerebral microbleeds, clinical symptoms and disability were evaluated. RESULTS: Thirty-six (90%) of the examined individuals (mean follow-up age 22.2 y; mean follow-up time 13.5 y) were affected by CMB. Longer follow-up time and higher craniospinal irradiation doses correlated with higher total lesion count (p < 0.01). Thirteen patients (32.5%) presented with clinical symptoms. Individuals with CMB were more severely disabled than patients without CMB (p < 0.05). CONCLUSIONS: Cerebrovascular sequelae occur frequently after treatment for pediatric brain tumor. In this study, a remarkable part of pediatric brain tumor patients presents with CMB. As a sign of vascular damage, they can cause clinical symptoms and may correspond to neurocognitive decline. Further studies are needed to standardize MRI protocols and to improve quality of long-term follow-up.

Novel loss of function mutation in KRIT1/CCM1 is associated with distinctly progressive cerebral and spinal cavernous malformations after radiochemotherapy for intracranial malignant germ cell tumor.

PURPOSE: Cerebrospinal cavernous malformations (CCMs) are vascular lesions characterized by dilated and leaky capillary caverns. CCMs can cause seizures, focal neurological deficits or acute intracranial hemorrhage; however, most patients are asymptomatic. CCMs occur either sporadically or as a familial autosomal-dominant disorder. We present a clinical and molecular study of a patient with distinctive cerebral and spinal cavernous malformations following radiochemotherapy for a malignant brain tumor. METHODS: The patient had multiple magnet resonance imaging (MRI) examinations of his brain and spine following radiochemotherapy for a primary intracranial germ cell tumor (GCT), as part of his oncologic follow-up. The MRI sequences included susceptibility-weighted imaging (SWI). The coding exons and their flanking intronic regions of KRIT1/CCM1 gene were analyzed for mutations by polymerase chain reaction (PCR) and direct sequencing. RESULTS: MRI revealed numerous cerebral and spinal microhemorrhages and pronounced cavernous malformations that progressed with subsequent follow-up imaging. Genetic analysis demonstrated a novel heterozygous KRIT1/CCM1 two base pair deletion (c.1535_1536delTG) in exon 14. This deletion leads to a frameshift with a premature stop codon at nucleotide position 1553 and a highly likely loss of function of the KRIT1 protein. CONCLUSION: We describe a patient with a novel heterozygous germ line loss of function mutation in KRIT1, which is associated with rapid-onset and highly progressive CCMs after radiochemotherapy for a malignant brain tumor.

Prospective analysis of long-term renal function in survivors of childhood Wilms tumor.

BACKGROUND: Considering the improved outcome, a better understanding of the late effects in Wilms tumor survivors (WT-S) is needed. This study was aimed at evaluating renal function and determining the prevalence of clinical and subclinical renal dysfunction in a cohort of WT-S using a multimodal diagnostic approach. METHODS: Thirty-seven WT-S were included in this prospective cross-sectional single center study. To evaluate renal function, glomerular filtration rate (GFR) and urinary protein excretion were assessed. Additionally, kidney sonomorphology and blood pressure were analyzed. RESULTS: All examined WT-S (mean age 28.7 years, mean follow-up 24.8 years) had been treated with a combination of surgery and chemotherapy; 59.5% had received adjuvant radiotherapy. Impaired glomerular renal function was detected in a considerable proportion of WT-S, with age-adjusted cystatin-based GFR estimation below age norm in 55.9%. A lower cystatin-based estimated GFR (eGFR) correlated with longer follow-up time and higher irradiation dose. In 5 patients (13.5%) albuminuria was identified. Analysis of sonomorphology detected compensatory contralateral renal hypertrophy in 83.3% of WT-S. Chronic kidney disease (CKD) ≥ stage II was present in 55.9% of WT-S. Blood pressure measurements revealed arterial hypertension in 15 (40.5%) WT-S (newly diagnosed n=10). In 24.3% both CKD ≥ stage II and arterial hypertension were determined. CONCLUSION: Even though WT-S are believed to carry a low risk for end-stage renal disease, in this study, a remarkable number of WT-S presented with previously unidentified subclinical signs of renal function impairment and secondary morbidity. Therefore, it is important to continue regular follow-up, especially after transition into adulthood.

MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L.

The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3, and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSCs). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSCs. Inactivation of Dot1l led to downregulation of direct MLL-AF9 targets and an MLL translocation-associated gene expression signature, whereas global gene expression remained largely unaffected. Suppression of MLL translocation-associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.

MLL-rearranged B lymphoblastic leukemias selectively express the immunoregulatory carbohydrate-binding protein galectin-1.

PURPOSE: Patients with mixed lineage leukemia (MLL)-rearranged B-lymphoblastic leukemias (B-ALL) have an unfavorable prognosis and require intensified treatment. Multiple MLL fusion partners have been identified, complicating the diagnostic evaluation of MLL rearrangements. We analyzed molecular markers of MLL rearrangement for use in rapid diagnostic assays and found the immunomodulatory protein, Galectin-1 (Gal-1), to be selectively expressed in MLL-rearranged B-ALL. EXPERIMENTAL DESIGN: Transcriptional profiling of ALL subtypes revealed selective overexpression of Gal-1 in MLL-rearranged ALLs. For this reason, we analyzed Gal-1 protein expression in MLL-germline and MLL-rearranged adult and infant pediatric B-ALLs and cell lines by immunoblotting, immunohistochemistry, and intracellular flow cytometry of viable tumor cell suspensions. Because deregulated gene expression in MLL-rearranged leukemias may be related to the altered histone methyltransferase activity of the MLL fusion protein complex, we also analyzed histone H3 lysine 79 (H3K79) dimethylation in the LGALS1 promoter region using chromatin immunoprecipitation. RESULTS: Gal-1 transcripts were significantly more abundant in MLL-rearranged B-ALLs. All 32 primary MLL-rearranged B-ALLs exhibited abundant Gal-1 immunostaining, regardless of the translocation partner, whereas only 2 of 81 germline-MLL B-ALLs expressed Gal-1. In addition, Gal-1 was selectively detected in newly diagnosed MLL-rearranged B-ALLs by intracellular flow cytometry. The LGALS1 promoter H3K79 was significantly hypermethylated in MLL-rearranged B-ALLs compared with MLL-germline B-ALLs and normal pre-B cells. CONCLUSION: In B-ALL, Gal-1 is a highly sensitive and specific biomarker of MLL rearrangement that is likely induced by a MLL-dependent epigenetic modification.

Rapid detection of common pathogenic Aspergillus species by a novel real-time PCR approach.

Invasive aspergillosis is a major cause of morbidity and mortality in immunocompromised and critically ill patients. Standard culture based methods for the diagnosis of Aspergillus infections have limited sensitivity and specificity and are time consuming. The recent availability of novel molecular based diagnostic techniques offers the potential of rapid, highly sensitive and specific pathogen detection. In this study, we aimed to develop a diagnostic assay to detect simultaneously common pathogenic Aspergillus species including Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus in whole blood specimen. A real-time PCR/probe set assay was designed to amplify the 18sRNA genomic region of Aspergillus. In addition to a previously probe set validated for the detection of A. fumigatus ('Asp.fum'), two other probe sets ('Asp.ter'; 'Asp.nig') were synthesised with 100% sequence homology to A. terreus or A. niger. Specificity testing demonstrated amplification of Aspergillus DNA, but not DNA from a panel of other common pathogenic fungi and bacteria and/or human DNA. Sensitivity testing in serial dilution assays revealed a lower detection limit of 1-5 CFU ml(-1) whole blood. However, no single probe set was most sensitive for all Aspergillus species tested and a combination of all three probe sets was necessary to achieve maximal overall assay sensitivity. Furthermore, inclusion of a subsequent probe melting temperature (Tm) analysis demonstrated species-specific Tm-patterns, useful to differentiate simultaneously between the different Aspergillus species detected. We describe a highly specific and sensitive real-time PCR approach to detect and differentiate the most common pathogenic Aspergillus species in a rapid 'same day' assay.

HOXA9 is required for survival in human MLL-rearranged acute leukemias.

Leukemias that harbor translocations involving the mixed lineage leukemia gene (MLL) possess unique biologic characteristics and often have an unfavorable prognosis. Gene expression analyses demonstrate a distinct profile for MLL-rearranged leukemias with consistent high-level expression of select Homeobox genes, including HOXA9. Here, we investigated the effects of HOXA9 suppression in MLL-rearranged and MLL-germline leukemias using RNA interference. Gene expression profiling after HOXA9 suppression demonstrated co-down-regulation of a program highly expressed in human MLL-AML and murine MLL-leukemia stem cells, including HOXA10, MEIS1, PBX3, and MEF2C. We demonstrate that HOXA9 depletion in 17 human AML/ALL cell lines (7 MLL-rearranged, 10 MLL-germline) induces proliferation arrest and apoptosis specifically in MLL-rearranged cells (P = .007). Similarly, assessment of primary AMLs demonstrated that HOXA9 suppression induces apoptosis to a greater extent in MLL-rearranged samples (P = .01). Moreover, mice transplanted with HOXA9-depleted t(4;11) SEMK2 cells revealed a significantly lower leukemia burden, thus identifying a role for HOXA9 in leukemia survival in vivo. Our data indicate an important role for HOXA9 in human MLL-rearranged leukemias and suggest that targeting HOXA9 or downstream programs may be a novel therapeutic option.

A toll-like receptor 4 variant is associated with fatal outcome in children with invasive meningococcal disease.

AIMS: Toll-like receptor 4 (TLR4) is the major endotoxin signalling receptor of the innate immune system and is required for efficient recognition of bacterial infections. Here, we analysed a possible association between the TLR4 variant Asp299Gly and disease outcome in children with invasive meningococcal disease. METHODS: In total, 197 children with invasive meningococcal disease were analysed for the TLR4 Asp299Gly variant. Genotyping results were correlated with mortality, the frequency of ventilation support, application of inotropic substances, skin grafting, and limb loss. RESULTS: The overall Asp299Gly allele frequency was 9.4%. Detection of a heterozygous Asp299Gly TLR4 mutation was significantly associated with fatal outcome (non-survivor group: 31.6% vs. survivor group: 12.1%; p = 0.021) and was even more pronounced in patients with disease onset less than 24 months of age (non-survivor group: 42.8% vs. survivor group: 10.2%; p = 0.006). In this age group, ventilation support was also more frequent in patients with the Asp299Gly genotype (37.5% vs. 6.2%). CONCLUSION: Our data suggest that the heterozygous TLR4 Asp299Gly genotype is associated with an increased mortality in children with invasive meningococcal disease.

H3K79 methylation profiles define murine and human MLL-AF4 leukemias.

We created a mouse model wherein conditional expression of an Mll-AF4 fusion oncogene induces B precursor acute lymphoblastic (ALL) or acute myeloid leukemias (AML). Gene expression profile analysis of the ALL cells demonstrated significant overlap with human MLL-rearranged ALL. ChIP-chip analysis demonstrated histone H3 lysine 79 (H3K79) methylation profiles that correlated with Mll-AF4-associated gene expression profiles in murine ALLs and in human MLL-rearranged leukemias. Human MLL-rearranged ALLs could be distinguished from other ALLs by their H3K79 profiles, and suppression of the H3K79 methyltransferase DOT1L inhibited expression of critical MLL-AF4 target genes. We thus demonstrate that ectopic H3K79 methylation is a distinguishing feature of murine and human MLL-AF4 ALLs and is important for maintenance of MLL-AF4-driven gene expression.

Mixed lineage leukemia translocations and a leukemia stem cell program.

Cancer stem cells (CSC) may provide the self-renewal capacity required to sustain a tumor. One possibility is that CSC arise from the stem cell counterparts in normal tissues. Alternatively, CSC may arise from more differentiated progenitor cells found in certain tissues. In support of this idea, we showed recently that mixed lineage leukemia fusion oncoproteins can convert committed hematopoietic progenitors into leukemias, which include leukemia stem cells expressing a self-renewal associated program in the context of a differentiated myeloid cell. The findings suggest a basis to understand the pathobiology of CSC and possible strategies to attack them to undermine the self-renewal capacity of a tumor.

Age-dependent association of human mannose-binding lectin mutations with susceptibility to invasive meningococcal disease in childhood.

BACKGROUND: Mannose-binding lectin (MBL) is an important factor of the innate immune system, and MBL-initiated complement activation is an important early defense mechanism against various bacterial infections, including invasive meningococcal disease. METHODS: In a pediatric cohort (ages 2-215 months) with invasive meningococcal disease, we investigated the overall and age-stratified frequency of 3 MBL exon 1 variations (C154T, G161A, G170A), previously shown to result in markedly decreased MBL plasma concentrations, by allele specific fluorescent hybridization probe real-time PCR assays and direct sequencing. Healthy age-matched volunteers with the same ethnic background and no history of meningococcal disease served as a control group. RESULTS: The overall frequency of a MBL exon 1 variant genotype was significantly higher in patients than in controls (31.8% vs. 8.2%, P < 0.001). In the patient group with disease onset less than 24 months of age, the prevalence of MBL structural variant genotype was further increased (39.3%; P < 0.001) and most pronounced in children with disease onset less than 12 months of age (57.1%; P < 0.001) when compared with healthy controls. Analysis of clinical severity and outcome revealed no significant difference between patients with wild-type and mutant alleles. CONCLUSIONS: Our data suggest that MBL exon 1 structural variants are significantly associated with susceptibility to childhood meningococcal disease in an age-dependent manner.