Triosephosphate-Isomerase Deficiency: Epiphenomenon or Cause of Loin Pain Haematuria Syndrome?

A 32-year-old male patient presented the clinical picture of loin pain haematuria syndrome with pain attacks accompanied by macrohaematuria. In renal biopsy, the preglomerular vessels showed segmental wall hyalinosis in the sense of low-grade nephrosclerosis, and glomerular capillaries with slightly but diffusely thickened, non-split basal membranes on electron microscopy. Notable were irregularly deformed, different dense erythrocytes in the glomerular capillaries, and several tubular lumina. The suspicion of erythrocytic enzyme deficiency could be confirmed. The enzyme activities of the erythrocytes were predominantly normal or slightly increased; only the activity of triosephosphate isomerase, a critical key enzyme of glycolysis, was reduced to 71% (resp. 57%) of the normal level, compatible with a heterozygous carrier status that could not be found. Patients with genomic triosephosphate-isomerase deficiency have degraded enzyme activities in virtually all tissues, such as leucocytes, platelets, and muscle cells. An association with neuromuscular symptoms is also known. Thus, it is possible that smooth muscle and intrarenal vascular spasms trigger clinical symptoms consisting of flank pain and phases of macrohaematuria. An aspirin-like defect (thrombocytopathy) had previously been found in connection with epistaxis (also due to TPI deficiency?). Enalapril treatment drastically reduced the frequency of macrohaematuria and pain attacks decreased to a lesser extent.

Amsacrine combined with etoposide and methylprednisolone is a feasible and safe component in first-line intensified treatment of pediatric patients with high-risk acute lymphoblastic leukemia in CoALL08-09 trial.

BACKGROUND: The prognosis of children with acute lymphoblastic leukemia (ALL) has improved considerably over the past five decades. However, to achieve cure in patients with refractory or relapsed disease, novel treatment options are necessary. METHODS: In the multicenter trial Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (CoALL)08-09, one additional treatment element consisting of the rarely used chemotherapeutic agent amsacrine combined with etoposide and methylprednisolone (AEP) (amsacrine 2 × 100 mg/m2 , etoposide 2 × 500 mg/m2 , and methylprednisolone 4 × 1000 mg/m2 ) was incorporated into the first-line treatment of pediatric patients with poor treatment responses at the end of induction (EOI), measured by minimal residual disease (MRD). These patients were stratified into a high-risk intensified arm (HR-I), including an AEP element at the end of consolidation. Patients with induction failure (IF), that is, with lack of cytomorphological remission EOI, were eligible for hematopoietic stem cell transplantation (HSCT) after remission had been reached. These patients received AEP as a part of their MRD-guided bridging-to-transplant treatments. RESULTS: A significant improvement in probability of overall survival (pOS) was noted for the CoALL08-09 HR-I patients compared to MRD-matched patients from the preceding CoALL07-03 trial in the absence of severe or persistent treatment-related toxicities. Relapse rate and probability of event-free survival (pEFS) did not differ significantly between trials. In patients with IF, stable or improved MRD responses after AEP were observed without severe or persistent treatment-related toxicities. CONCLUSION: In conclusion, AEP is well tolerated as a component of the HR treatment and is useful in bridging-to-transplant settings.

Integrative medicine during the intensive phase of chemotherapy in pediatric oncology in Germany: a randomized controlled trial with 5-year follow up.

BACKGROUND: Integrative medicine is used frequently alongside chemotherapy treatment in pediatric oncology, but little is known about the influence on toxicity. This German, multi-center, open-label, randomized controlled trial assessed the effects of complementary treatments on toxicity related to intensive-phase chemotherapy treatment in children aged 1-18 with the primary outcome of the toxicity sum score. Secondary outcomes were chemotherapy-related toxicity, overall and event-free survival after 5 years in study patients. METHODS: Intervention and control were given standard chemotherapy according to malignancy & tumor type. The intervention arm was provided with anthroposophic supportive treatment (AST); given as anthroposophic base medication (AMP), as a base medication for all patients and additional on-demand treatment tailored to the intervention malignancy groups. The control was given no AMP. The toxicity sum score (TSS) was assessed using NCI-CTC scales. RESULTS: Data of 288 patients could be analyzed. Analysis did not reveal any statistically significant differences between the AST and the control group for the primary endpoint or the toxicity measures (secondary endpoints). Furthermore, groups did not differ significantly in the five-year overall and event-free survival follow up. DISCUSSION: In this trial findings showed that AST was able to be safely administered in a clinical setting, although no beneficial effects of AST between group toxicity scores, overall or event-free survival were shown.

Clofarabine increases the eradication of minimal residual disease of primary B-precursor acute lymphoblastic leukemia compared to high-dose cytarabine without improvement of outcome. Results from the randomized clinical trial 08-09 of the Cooperative Acute Lymphoblastic Leukemia Study Group.

Novel treatment strategies are needed to improve cure for all children with acute lymphoblastic leukemia (ALL). To this end, we investigated the therapeutic potential of clofarabine in primary ALL in trial CoALL 08-09 (clinicaltrials gov. identifier: NCT01228331). The primary study objective was the minimal residual disease (MRD)- based comparative assessment of cytotoxic efficacies of clofarabine 5x40 mg/m2 versus high-dose cytarabine (HIDAC) 4x3g/m2, both in combination with PEG-ASP 2,500 IU/m2 as randomized intervention in early consolidation. The secondary objective was an outcome analysis focused on treatment arm dependence and MRD after randomized intervention. In B-cell precursor (BCP)-ALL, eradication of MRD was more profound after clofarabine compared to cytarabine, with 93 versus 79 of 143 randomized patients per arm reaching MRD-negativity (c2 test P=0.03, leftsided P [Fisher's exact test]=0.04). MRD status of BCP-ALL after randomized intervention maintained its prognostic relevance, with a significant impact on event-free survival (EFS) and relapse rate. However, no difference in outcome regarding EFS and overall survival (OS) between randomized courses was observed (5-year EFS: clofarabine 85.7, SE=4.1 vs. HIDAC 84.8, SE=4.7 [P=0.96]; OS: 95.7, SE=1.9 vs. 92.2, SE=3.2 [P=0.59]), independent of covariates or overall risk strata. Severe toxicities between randomized and subsequent treatment elements were also without significant difference. In conclusion, clofarabine/PEG-ASP is effective and safe, but greater cytotoxic efficacy of clofarabine compared to HIDAC did not translate into improved outcomes indicating a lack of surrogacy of post-intervention MRD at the trial level as opposed to the patient level, which hampers a broader implementation of this regimen in the frontline treatment of ALL.

Primary mismatch repair deficient IDH-mutant astrocytoma (PMMRDIA) is a distinct type with a poor prognosis.

Diffuse IDH-mutant astrocytoma mostly occurs in adults and carries a favorable prognosis compared to IDH-wildtype malignant gliomas. Acquired mismatch repair deficiency is known to occur in recurrent IDH-mutant gliomas as resistance mechanism towards alkylating chemotherapy. In this multi-institutional study, we report a novel epigenetic group of 32 IDH-mutant gliomas with proven or suspected hereditary mismatch repair deficiency. None of the tumors exhibited a combined 1p/19q deletion. These primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) were histologically high-grade and were mainly found in children, adolescents and young adults (median age 14 years). Mismatch repair deficiency syndromes (Lynch or Constitutional Mismatch Repair Deficiency Syndrom (CMMRD)) were clinically diagnosed and/or germline mutations in DNA mismatch repair genes (MLH1, MSH6, MSH2) were found in all cases, except one case with a family and personal history of colon cancer and another case with MSH6-deficiency available only as recurrent tumor. Loss of at least one of the mismatch repair proteins was detected via immunohistochemistry in all, but one case analyzed. Tumors displayed a hypermutant genotype and microsatellite instability was present in more than half of the sequenced cases. Integrated somatic mutational and chromosomal copy number analyses showed frequent inactivation of TP53, RB1 and activation of RTK/PI3K/AKT pathways. In contrast to the majority of IDH-mutant gliomas, more than 60% of the samples in our cohort presented with an unmethylated MGMT promoter. While the rate of immuno-histochemical ATRX loss was reduced, variants of unknown significance were more frequently detected possibly indicating a higher frequency of ATRX inactivation by protein malfunction. Compared to reference cohorts of other IDH-mutant gliomas, primary mismatch repair-deficient IDH-mutant astrocytomas have by far the worst clinical outcome with a median survival of only 15 months irrespective of histological or molecular features. The findings reveal a so far unknown entity of IDH-mutant astrocytoma with high prognostic relevance. Diagnosis can be established by aligning with the characteristic DNA methylation profile, by DNA-sequencing-based proof of mismatch repair deficiency or immunohistochemically demonstrating loss-of-mismatch repair proteins.

Prognostic impact of distinct genetic entities in pediatric diffuse glioma WHO-grade II-Report from the German/Swiss SIOP-LGG 2004 cohort.

Reports on pediatric low-grade diffuse glioma WHO-grade II (DG2) suggest an impaired survival rate, but lack conclusive results for genetically defined DG2-entities. We analyzed the natural history, treatment and prognosis of DG2 and investigated which genetically defined sub-entities proved unfavorable for survival. Within the prospectively registered, population-based German/Swiss SIOP-LGG 2004 cohort 100 patients (age 0.8-17.8 years, 4% neurofibromatosis [NF1]) were diagnosed with a DG2. Following biopsy (41%) or variable extent of resection (59%), 65 patients received no adjuvant treatment. Radiologic progression or severe neurologic symptoms prompted chemotherapy (n = 18) or radiotherapy (n = 17). Multiple lines of salvage treatment were necessary for 19/35 patients. Five years event-free survival dropped to 0.44, while 5 years overall survival was 0.90 (median observation time 8.3 years). Extensive genetic profiling of 65/100 DG2 identified Histone3-K27M-mutation in 4, IDH1-mutation in 11, BRAF-V600-mutation in 12, KIAA1549-BRAF-fusions in 6 patients, while the remaining 32 tumor tissues did not show alterations of these genes. Progression to malignant glioma occurred in 12 cases of all genetically defined subgroups within a range of 0.5 to 10.8 years, except for tumors carrying KIAA1549-BRAF-fusions. Histone3-K27M-mutant tumors proved uniformly fatal within 0.6 to 2.4 years. The current LGG treatment strategy seems appropriate for all DG2-entities, with the exemption of Histone3-K27M-mutant tumors that require a HGG-related treatment strategy. Our data confirm the importance to genetically define pediatric low-grade diffuse gliomas for proper treatment decisions and risk assessment.

Synovial sarcoma disease characteristics and primary tumor sites differ between patient age groups: a report of the Cooperative Weichteilsarkom Studiengruppe (CWS).

BACKGROUND: Older age is associated with worse outcome in synovial sarcoma (SS) patients. Differences in disease presentation among distinct age groups, however, are currently unknown. METHODS: SS patients < 21 years registered in consecutive CWS trials over the period of 1981-2018 were evaluated. Characteristics were analyzed according to age groups using the Fisher's exact test. RESULTS: The study population included 432 SS patients. Disease characteristics differed according to age groups of children (0-12 years, n = 176), adolescents (13-16 years, n = 178), and young adults (17-21 years, n = 78). The proportion of invasive tumors (T2) was significantly higher in older patients: children 33%, adolescents 39% and young adults 54%, p = 0.009805. Similarly, the proportion of tumors > 10 cm was higher (13%, 21%, 31%; p = 0.005657) whereas conversely, the proportion of small tumors < 3 cm was lower in older patients (29%, 24%, 6%; p = 0.000104). The presence of metastases at first diagnosis was also highest in older patients (6%, 10%, 21%, p = 0.000963). Notably, the proportion of thigh tumors was higher in older patients (p = 0.04173), whereas the proportion of head-neck tumors was lower in older patients (p = 0.08896). CONCLUSIONS: The rates of large, invasive tumors and the presence of metastases are significantly associated with older patient age. Localization to the thigh is more frequent in older patients. DISCUSSION: The causes for these variations require further exploration.

Genotyping circulating tumor DNA of pediatric Hodgkin lymphoma.

We used hybrid capture-targeted next-generation sequencing of circulating cell-free DNA (ccfDNA) of pediatric Hodgkin lymphoma (PHL) patients to determine pathogenic mechanisms and assess the clinical utility of this method. Hodgkin-Reed/Sternberg (HRS) cell-derived single nucleotide variants, insertions/deletions, translocations and VH-DH-JH rearrangements were detected in pretherapy ccfDNA of 72 of 96 patients. Number of variants per patient ranged from 1 to 21 with allele frequencies from 0.6 to 42%. Nine translocation breakpoints were detected. Genes involved in JAK/STAT, NFkB and PI3K signaling and antigen presentation were most frequently affected. SOCS1 variants, mainly deletions, were found in most circulating tumor (ct) DNAs, and seven of the nine translocation breakpoints involved SOCS1. Analysis of VH-DH-JH rearrangements revealed an origin of PHL HRS cells from partially selected germinal center B cells. Amounts of pretherapy ctDNA were correlated with metabolic tumor volumes. Furthermore, in all ccfDNA samples of 43 patients with early response assessment quantitative qPET < 3, indicative of a favorable clinical course, ctDNA was not detectable. In contrast, in five of six patients with qPET > 3, indicative of an unfavorable clinical course, ctDNA remained detectable. ccfDNA analysis of PHL is thus a suitable approach to determine pathogenic mechanisms and monitor therapy response.

Phase I/II intra-patient dose escalation study of vorinostat in children with relapsed solid tumor, lymphoma, or leukemia.

BACKGROUND: Until today, adult and pediatric clinical trials investigating single-agent or combinatorial HDAC inhibitors including vorinostat in solid tumors have largely failed to demonstrate efficacy. These results may in part be explained by data from preclinical models showing significant activity only at higher concentrations compared to those achieved with current dosing regimens. In the current pediatric trial, we applied an intra-patient dose escalation design. The purpose of this trial was to determine a safe dose recommendation (SDR) of single-agent vorinostat for intra-patient dose escalation, pharmacokinetic analyses (PK), and activity evaluation in children (3-18 years) with relapsed or therapy-refractory malignancies. RESULTS: A phase I intra-patient dose (de)escalation was performed until individual maximum tolerated dose (MTD). The starting dose was 180 mg/m2/day with weekly dose escalations of 50 mg/m2 until DLT/maximum dose. After MTD determination, patients seamlessly continued in phase II with disease assessments every 3 months. PK and plasma cytokine profiles were determined. Fifty of 52 patients received treatment. n = 27/50 (54%) completed the intra-patient (de)escalation and entered phase II. An SDR of 130 mg/m2/day was determined (maximum, 580 mg/m2/day). n = 46/50 (92%) patients experienced treatment-related AEs which were mostly reversible and included thrombocytopenia, fatigue, nausea, diarrhea, anemia, and vomiting. n = 6/50 (12%) had treatment-related SAEs. No treatment-related deaths occurred. Higher dose levels resulted in higher Cmax. Five patients achieved prolonged disease control (> 12 months) and showed a higher Cmax (> 270 ng/mL) and MTDs. Best overall response (combining PR and SD, no CR observed) rate in phase II was 6/27 (22%) with a median PFS and OS of 5.3 and 22.4 months. Low levels of baseline cytokine expression were significantly correlated with favorable outcome. CONCLUSION: An SDR of 130 mg/m2/day for individual dose escalation was determined. Higher drug exposure was associated with responses and long-term disease stabilization with manageable toxicity. Patients with low expression of plasma cytokine levels at baseline were able to tolerate higher doses of vorinostat and benefited from treatment. Baseline cytokine profile is a promising potential predictive biomarker. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01422499. Registered 24 August 2011.

Results of CoALL 07-03 study childhood ALL based on combined risk assessment by in vivo and in vitro pharmacosensitivity.

We conducted a clinical trial and report the long-term outcome of 773 children with acute lymphoblastic leukemia upon risk-adapted therapy accrued in trial CoALL 07-03 (from the Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia). In a 2-step stratification, patients were allocated to receive either low- or high-risk treatment, based on initial white blood cell count, age, and immunophenotype. A second stratification was performed according to the results of in vitro pharmacosensitivity toward prednisolone, vincristine, and asparaginase (PVA score) and in vivo response after induction therapy (minimal residual disease [MRD]). Therapy was reduced for both risk groups in patients with a low PVA score or negative MRD result, and intensified in patients with a high PVA score. Overall outcome improved significantly compared with the predecessor CoALL 06-97 trial, with identical therapy backbone despite treatment reduction in 15.8% of patients (10-year probability of event-free survival, 83.5% vs 73.9%; overall survival, 90.7% vs 83.8%). Outcome for patients in the reduced treatment arms was superior to that of patients in the standard arms, associated with a profound reduction in frequency and severity of infectious complications. Importantly, we observed a lack of correlation between in vitro and in vivo drug response, as well as a lower predictive value of in vitro drug testing, reflecting an intrinsic limitation of this methodology that prevents its use for treatment stratification in future trials. In conclusion, it might be possible to reduce chemotherapy in children with acute lymphoblastic leukemia selected by stringent in vivo measurement of MRD without jeopardizing overall outcome.

Daunorubicin during delayed intensification decreases the incidence of infectious complications - a randomized comparison in trial CoALL 08-09.

Anthracyclines are integral components of antileukemic treatment. Apart from cardiotoxicity, myelosuppression and infectious complications have been described for doxorubicin (DOX) and daunorubicin (DNR) as predominant side effects, but little is known about their differential toxicities. To address the question whether DNR is associated with a lower rate of infectious complications compared with DOX, 307 children with newly diagnosed acute lymphoblastic leukemia, enrolled in trial CoALL 08-09, were randomized to receive either DOX 30 mg/m2 (n = 153) or DNR 36 mg/m2 (n = 154) in delayed intensification. Hematologic toxicities and stomatitis were less frequent in the DNR group resulting in a significantly lower rate of infections in the DNR arm (27% vs. 59%, p < .0001). Survival was equal in both arms (95% SE 2%) (p = .55), with an insignificant difference in the relapse rate (RR 0.12 (SE = 0.03) in the DOX arm vs. 0.16 (SE = 0.04) in the DNR arm; p = .37; Hazard ratio 1.3; 95% confidence interval 0.7-2.6). In conclusion, DNR given in delayed intensification is associated with a lower incidence of infectious complications without loss of efficacy.

Disseminated oligodendroglial-like leptomeningeal tumors: preliminary diagnostic and therapeutic results for a novel tumor entity [corrected].

Pediatric tumors of the central nervous system composed of oligoid tumor cells showing diffuse leptomeningeal spread without a primary mass lesion seem to represent a novel tumor entity. The terms "diffuse leptomeningeal glioneural tumor" or-preferably-"disseminated oligodendroglial-like leptomeningeal tumor of childhood" (DOGLT) were proposed. Four patients were identified with clinico-neuropathologic findings compatible with DOGLT and a mean follow-up time of 54 months was determined. Seven different biopsies obtained from the four patients were histologically evaluated. Clinical course, diagnostic measures, histopathologic and radiologic features and treatment suggestions were recorded, on the basis of which diagnostic and therapeutic algorithm was proposed. Patients with DOGLT presented with hydrocephalus as first symptom, requiring neurosurgical therapy. Open arachnoid biopsy was necessary to confirm diagnosis. The oligoid cells in a desmoplastic or focally myxoid matrix showed OLIG2-, MAP2-, S-100 and rare HuC/HuD protein-immunopositivity. IDH1 (R132H)- and CD99-immunohistochemistry was negative in all patients. None of the evaluable biopsies of three patients showed chromosome 1p/19q deletion, neither as isolated nor combined allelic loss. Chemotherapy according to the SIOP-LGG 2004 standard induction and consolidation protocol resulted in complete response and partial response, respectively, in 50 % of the patients. However, after discontinuation of chemotherapy, two patients experienced tumor progression and one of them succumbed to the disease after 19 months. Radiological criteria as well as preliminary treatment results are presented after observation of four clinical cases. Prognosis and long-term clinical courses remain to be observed.

Abnormally differentiated CD4+ or CD8+ T cells with phenotypic and genetic features of double negative T cells in human Fas deficiency.

Accumulation of CD3(+) T-cell receptor (TCR)αß(+)CD4(-)CD8(-) double-negative T cells (DNT) is a hallmark of autoimmune lymphoproliferative syndrome (ALPS). DNT origin and differentiation pathways remain controversial. Here we show that human ALPS DNT have features of terminally differentiated effector memory T cells reexpressing CD45RA(+) (TEMRA), but are CD27(+)CD28(+)KLRG1(-) and do not express the transcription factor T-bet. This unique phenotype was also detected among CD4(+) or CD8(+) ALPS TEMRA cells. T-cell receptor ß deep sequencing revealed a significant fraction of shared CDR3 sequences between ALPS DNT and both CD4(+) and CD8(+)TEMRA cells. Moreover, in ALPS patients with a germ line FAS mutation and somatic loss of heterozygosity, in whom biallelic mutant cells can be tracked by absent Fas expression, Fas-negative T cells accumulated not only among DNT, but also among CD4(+) and CD8(+)TEMRA cells. These data indicate that in human Fas deficiency DNT cannot only derive from CD8(+), but also from CD4(+) T cells. Furthermore, defective Fas signaling leads to aberrant transcriptional programs and differentiation of subsets of CD4(+) and CD8(+) T cells. Accumulation of these cells before their double-negative state appears to be an important early event in the pathogenesis of lymphoproliferation in ALPS patients.

HD-MB03 is a novel Group 3 medulloblastoma model demonstrating sensitivity to histone deacetylase inhibitor treatment.

Medulloblastomas are the most common malignant brain tumors in childhood. Emerging evidence suggests that medulloblastoma comprises at least four distinct diseases (WNT, SHH, Group 3 and 4) with different biology, clinical presentation, and outcome, with especially poor prognosis in Group 3. The tight connection of biology and clinical behavior in patients emphasizes the need for subgroup-specific preclinical models in order to develop treatments tailored to each subgroup. Herein we report on the novel cell line HD-MB03, isolated from tumor material of a patient with metastasized Group 3 medulloblastoma, and preclinical testing of different histone deacetylase inhibitors (HDACis) in this model. HD-MB03 cells grow long term in vitro and form metastatic tumors in vivo upon orthotopic transplantation. HD-MB03 cells reflect the original Group 3 medulloblastoma at the histological and molecular level, showing large cell morphology, similar expression patterns for markers Ki67, p53, and glial fibrillary acidic protein (GFAP), a gene expression profile most closely matching Group 3 medulloblastomas, and persistence of typical molecular alterations, i.e., isochromosome 17q [i(17q)] and MYC amplification. Protein expression analysis of HDACs 2, 5, 8, and 9 as well as the predictive marker HR23B showed intermediate to strong expression, suggesting sensitivity to HDACis. Indeed, treatment with HDACis Helminthosporium carbonum (HC)-toxin, vorinostat, and panobinostat revealed high sensitivity to this novel drug class, as well as a radiation-sensitizing effect with significantly increased cell death upon concomitant treatment. In summary, our data indicate that HD-MB03 is a suitable preclinical model for Group 3 medulloblastoma, and HDACis could represent a therapeutic option for this subgroup.

Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma.

Glioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

Distinct mutations in STXBP2 are associated with variable clinical presentations in patients with familial hemophagocytic lymphohistiocytosis type 5 (FHL5).

Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically determined hyperinflammatory syndrome caused by uncontrolled immune response mediated by T-lymphocytes, natural killer (NK) cells, and macrophages. STXBP2 mutations have recently been associated with FHL5. To better characterize the genetic and clinical spectrum of FHL5, we analyzed a cohort of 185 patients with suspected FHL for mutations in STXBP2. We detected biallelic mutations in 37 patients from 28 families of various ethnic origins. Missense mutations and mutations affecting 1 of the exon 15 splice sites were the predominant changes detectable in this cohort. Patients with exon 15 splice-site mutations (n = 13) developed clinical manifestations significantly later than patients with other mutations (median age, 4.1 year vs 2 months) and showed less severe impairment of degranulation and cytotoxic function of NK cells and CTLs. Patients with FHL5 showed several atypical features, including sensorineural hearing deficit, abnormal bleeding, and, most frequently, severe diarrhea that was only present in early-onset disease. In conclusion, we report the largest cohort of patients with FHL5 so far, describe an extended disease spectrum, and demonstrate for the first time a clear genotype-phenotype correlation.

Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.

Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

Oncogenic FAM131B-BRAF fusion resulting from 7q34 deletion comprises an alternative mechanism of MAPK pathway activation in pilocytic astrocytoma.

Activation of the MAPK signaling pathway has been shown to be a unifying molecular feature in pilocytic astrocytoma (PA). Genetically, tandem duplications at chromosome 7q34 resulting in KIAA1549-BRAF fusion genes constitute the most common mechanism identified to date. To elucidate alternative mechanisms of aberrant MAPK activation in PA, we screened 125 primary tumors for RAF fusion genes and mutations in KRAS, NRAS, HRAS, PTPN11, BRAF and RAF1. Using microarray-based comparative genomic hybridization (aCGH), we identified in three cases an interstitial deletion of ~2.5 Mb as a novel recurrent mechanism forming BRAF gene fusions with FAM131B, a currently uncharacterized gene on chromosome 7q34. This deletion removes the BRAF N-terminal inhibitory domains, giving a constitutively active BRAF kinase. Functional characterization of the novel FAM131B-BRAF fusion demonstrated constitutive MEK phosphorylation potential and transforming activity in vitro. In addition, our study confirmed previously reported BRAF and RAF1 fusion variants in 72% (90/125) of PA. Mutations in BRAF (8/125), KRAS (2/125) and NF1 (4/125) and the rare RAF1 gene fusions (2/125) were mutually exclusive with BRAF rearrangements, with the exception of two cases in our series that concomitantly harbored more than one hit in the MAPK pathway. In summary, our findings further underline the fundamental role of RAF kinase fusion products as a tumor-specific marker and an ideally suited drug target for PA.

The long-term impact of in vitro drug sensitivity on risk stratification and treatment outcome in acute lymphoblastic leukemia of childhood (CoALL 06-97).

BACKGROUND: In a study of childhood acute lymphoblastic leukemia (CoALL 06-97 study), the in vitro sensitivity of the patients' cells to prednisolone, vincristine and asparaginase was introduced as a new additional risk parameter for treatment stratification. In parallel in vivo treatment response was assessed by determining the presence and extent of minimal residual disease in a subset of patients (n=224). Here we report the long-term impact of in vitro sensitivity-based risk stratification according to survival and compare the results of in vitro sensitivity with in vivo response. DESIGN AND METHODS: Patients with a sensitive in vitro profile were treated with a reduced intensity protocol (n=167) whereas patients defined as low risk according to conventional parameters but with a resistant in vitro profile were given intensified therapy (n=47). RESULTS: At a median follow-up of 6.8 years event-free survival was 0.80±0.03 for patients with a sensitive profile, 0.73±0.03 for those with an intermediate profile and 0.67±0.08 for those with a resistant profile (P=0.015). Overall, the treatment results of the cases stratified according to in vitro sensitivity were similar to those of the historical control group stratified based on conventional risk factors. Minimal residual disease at the end of induction was a strong predictor of outcome in B-precursor and T-cell acute lymphoblastic leukemia. There was no correlation between in vitro and in vivo treatment response in B-precursor leukemia (Spearman's r=0.13; P=0.15) in contrast to T-cell acute lymphoblastic leukemia (Spearman's r=0.63; P<0.001) CONCLUSIONS: A moderate reduction in treatment intensity for patients with a sensitive in vitro profile was possible without jeopardizing treatment outcome. However, in vitro drug testing was affected by a decrease in risk predictive power over time and was not correlated with in vivo assessment of minimal residual disease in B-precursor acute lymphoblastic leukemia. It was, therefore, abandoned in favor of the assessment of in vivo response in subsequent CoALL trials.