Dragana Janic

Clinical and molecular profile of a new series of patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: Inconsistent correlation between forkhead box protein 3 expression and disease severity

BACKGROUND Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is an autoimmune genetic disorder caused by mutation of the forkhead box protein 3 gene (FOXP3), a key regulator of immune tolerance. OBJECTIVE We sought to provide clinical and molecular indicators that facilitate the understanding and diagnosis of IPEX syndrome. METHODS In 14 unrelated affected male subjects who were given diagnoses of IPEX syndrome based on FOXP3 gene sequencing, we determined whether particular FOXP3 mutations affected FOXP3 protein expression and correlated the molecular and clinical data. RESULTS Molecular analysis of FOXP3 in the 14 subjects revealed 13 missense and splice-site mutations, including 7 novel mutations. Enteropathy, generally associated with endocrinopathy and eczema, was reported in all patients, particularly in those carrying mutations within FOXP3 functional domains or mutations that altered protein expression. However, similar genotypes did not always result in similar phenotypes in terms of disease presentation and severity. In addition, FOXP3 protein expression did not correlate with disease severity. CONCLUSION Severe autoimmune enteropathy, which is often associated with increased IgE levels and eosinophilia, is the most prominent early manifestation of IPEX syndrome. Nevertheless, the disease course is variable and somewhat unpredictable. Therefore genetic analysis of FOXP3 should always be performed to ensure an accurate diagnosis, and FOXP3 protein expression analysis should not be the only diagnostic tool for IPEX syndrome.

Intensive Chemotherapy for Childhood Acute Lymphoblastic Leukemia: Results of the Randomized Intercontinental Trial ALL IC-BFM 2002

PURPOSE From 2002 to 2007, the International Berlin-Frankfurt-Münster Study Group conducted a prospective randomized clinical trial (ALL IC-BFM 2002) for the management of childhood acute lymphoblastic leukemia (ALL) in 15 countries on three continents. The aim of this trial was to explore the impact of differential delayed intensification (DI) on outcome in all risk groups. PATIENTS AND METHODS For this trial, 5,060 eligible patients were divided into three risk groups according to age, WBC, early treatment response, and unfavorable genetic aberrations. DI was randomized as follows: standard risk (SR), two 4-week intensive elements (protocol III) versus one 7-week protocol II; intermediate risk (IR), protocol III × 3 versus protocol II × 1; high risk (HR), protocol III × 3 versus either protocol II × 2 (Associazione Italiana Ematologia Oncologia Pediatrica [AIEOP] option), or 3 HR blocks plus single protocol II (Berlin-Frankfurt-Münster [BFM] option). RESULTS At 5 years, the probabilities of event-free survival and survival were 74% (± 1%) and 82% (± 1%) for all 5,060 eligible patients, 81% and 90% for the SR (n = 1,564), 75% and 83% for the IR (n = 2,650), and 55% and 62% for the HR (n = 846) groups, respectively. No improvement was accomplished by more intense and/or prolonged DI. CONCLUSION The ALL IC-BFM 2002 trial is a good example of international collaboration in pediatric oncology. A wide platform of countries able to run randomized studies in ALL has been established. Although the alternative DI did not improve outcome compared with standard treatment and the overall results are worse than those achieved by longer established leukemia groups, the national results have generally improved.

Recommendations for the management of the haematological and onco-haematological aspects of Gaucher disease

Current knowledge of the haematological and onco‐haematological complications of type 1 Gaucher disease has been reviewed with the aim of identifying best clinical practice for treatment and disease management. It was concluded that: (i) Awareness of typical patterns of cytopenia can help clinicians distinguish haematological co‐morbidities. (ii) Red blood cell studies and complete iron metabolism evaluation at baseline are recommended. (iii) Haemoglobin levels defining anaemia should be raised and used in Gaucher disease treatment and monitoring. (iv) Surgeons should be aware of potential bleeding complications during surgery in Gaucher patients.

Retroperitoneal kaposiform hemangioendothelioma with tufted angioma‐like features in an infant with Kasabach–Merritt syndrome

Kasabach–Merritt syndrome denotes profound thrombocytopenia and coagulopathy in an infant with a vascular tumor. A retroperitoneal vascular tumor with an unusual combination of histopathological features is reported, and compared with vascular lesions described in the reported cases of Kasabach–Merritt syndrome in the literature. A large retroperitoneal tumor that had expanded through the sigmoid mesocolon into the sigmoid colon wall was resected from an 8‐month‐old infant with fully developed Kasabach–Merritt syndrome. Histological examination revealed a combination of venous (cavernous) malformation, kaposiform hemangioendothelioma and tufted angioma‐like areas. Cellular tumor components (especially tufted angioma‐like parts) infiltrated the wall of the sigmoid colon to the submucosal level. Immunohistochemical staining with antibodies to the Ki‐67 antigen and proliferating cell nuclear antigen showed a low proliferative activity, whereas the antiapoptotic bcl‐2 protein was expressed diffusely in tumor cells. This is the first reported case of a vascular tumor with tufted angioma‐like elements found in the retroperitoneum, and the first reported in combin‐ation with kaposiform hemangioendothelioma and venous malformation in the same lesion. Considering the immu‐nohistochemical results and overlapping histological features, it may be considered that tufted angioma and kaposiform hemangioendothelioma represent different growth patterns or stages in the development of a single type of hemangioma.

Results from a large multinational clinical trial (guardian™3) using prophylactic treatment with turoctocog alfa in paediatric patients with severe haemophilia A: safety, efficacy and pharmacokinetics

Recombinant factor VIII (rFVIII) products provide a safe and efficacious replacement therapy for prophylaxis and treatment of bleeding episodes in patients with severe haemophilia A. This multinational, open‐label, non‐controlled trial investigated the safety, efficacy and pharmacokinetics (PK) of turoctocog alfa, a new rFVIII product, in a paediatric population. The primary objective was to evaluate safety. A total of 31 younger children (0–5 years) and 32 older children (6–11 years), with ≥50 exposure days to any factor VIII (FVIII) product and no history of inhibitors, received prophylaxis with turoctocog alfa (25–50 IU kg−1 every second day or 25–60 IU kg−1 three times weekly). PK assessments of turoctocog alfa and the patients’ previous FVIII product were performed in 28 patients. Mean exposure to turoctocog alfa was 60 exposure days per patient. This corresponds to approximately 4.5 months in the trial. None of the patients developed inhibitors (≥0.6 BU) and no safety concerns were raised. A total of 120 bleeding episodes (95%) were controlled with 1–2 infusions of turoctocog alfa. Based on patient reports, the success rate (defined as ‘excellent’ or ‘good’ haemostatic response) for treatment of bleeding episodes was 92%. Overall, the median annualized bleeding rate was 3.0 (interquartile range: 8.5) bleeds patient−1 year−1. PK parameters were comparable between the two age groups. In conclusion, the present large global clinical trial showed that turoctocog alfa was safe, effective in treatment of bleeding episodes and had a prophylactic effect in paediatric patients.

Analysis of thiopurine S-methyltransferase polymorphism in the population of Serbia and Montenegro and mercaptopurine therapy tolerance in childhood acute lymphoblastic leukemia

Thiopurine S-methyltransferase (TPMT) is an enzyme that converts thiopurine drugs into inactive metabolites. It is now well established that interindividual variation in sensitivity to thiopurines can be the result of the presence of genetic polymorphisms in the TPMT gene. The aim of this study was to determine the frequency and type of TPMT polymorphisms in the population of Serbia and Montenegro and to assess its relevance in the management of childhood acute lymphoblastic leukemia (ALL). Blood samples from 100 healthy adults and 100 children with ALL were analyzed for common mutations in the TPMT gene using polymerase chain reaction-based assays. The results revealed that allelic frequencies were 0.2% for TPMT*2, 3.2% for TPMT*3A, and 0.5% for TPMT*3B. A rare TPMT*3B allele was detected in 2 families. No TPMT*3C allele was found. The general pattern of TPMT-variant allele distribution as well as their frequencies in the population of Serbia and Montenegro, is similar to those determined for other Slavic and Mediterranean populations. The ability to tolerate 6-mercaptopurine (6-MP) -based maintenance therapy was used as a surrogate marker of hematologic toxicity. In the study of 50 patients with childhood ALL treated according to the BFM-like protocol, it was found that even TPMT-heterozygous patients are at greater risk of thiopurine drug-related leukopenia (mean duration of period when children missed therapy as a result of leukopenia for TPMT-heterozygous patients was 11.3 weeks vs 3.4 weeks for wild-type genotype patients, P < 0.01). In another group of 50 patients, the TPMT genotype was determined prospectively. The therapy protocol was modified considering their TPMT genotype. Administering reduced 6-MP dosages in the initial phase of maintenance allowed TPMT-heterozygous patients to later receive full protocol doses of both 6-MP and nonthiopurine therapy without omitting therapy resulting from myelotoxicity. These results justify performing TPMT genotyping before initiating thiopurine therapy in all children with ALL to minimize consequent toxicity.

Functional analysis of the role of the TPMT gene promoter VNTR polymorphism in TPMT gene transcription

AIMS Thiopurine S-methyltransferase (TPMT) activity is polymorphic, and a trimodal distribution has been demonstrated in Caucasians (low, intermediate and high methylator groups). The TPMT gene promoter contains a variable number of three GC-rich tandem repeats, namely A, B and C, ranging from three to nine in length in a A(n)B(m)C architecture. MATERIALS & METHODS Here, we investigated the influence of number and type of TPMT gene promoter tandem repeats on human TPMT gene transcription in K562 cells transiently transfected with reporter constructs bearing various variable number of tandem repeats (VNTR) and addressed the interaction of transcription factor binding to the VNTRs by electrophoretic mobility shift assays. RESULTS We found that the distribution patterns of VNTR alleles do not significantly differ among acute lymphoblastic leukemia patients, acute myeloid leukemia patients and normal individuals. We also demonstrated that the A repeat has a negative effect in TPMT gene transcription and that a positive regulatory element, identified immediately upstream to the VNTR region of the TPMT gene promoter, is indispensable for TPMT gene transcription. Our electrophoretic mobility shift assay analysis indicated that the Sp1 and Sp3 transcription factors bind to the VNTR repeats. CONCLUSION Overall, our data underline that both the number and type of VNTRs, as well as the upstream regulatory region of the TPMT gene promoter, determine the overall level of TPMT gene transcription. It remains to be seen whether these VNTRs can be employed as pharmacogenetic markers to individualize thiopurine therapy.

Challenges for children and adolescents with cancer in Europe: The SIOP-Europe agenda

In Europe, 6,000 young people die of cancer yearly, the commonest disease causing death beyond the age of 1 year. In addition, 300,000–500,000 European citizens are survivors of a childhood cancer and up to 30% of them have severe long‐term sequelae of their treatment. Increasing both cure and quality of cure are the two goals of the European paediatric haematology/oncology community. SIOPE coordinates and facilitates research, care and training which are implemented by the 18 European study groups and 23 national paediatric haematology/oncology societies. SIOPE is the European branch of the International Society of Paediatric Oncology and one of the six founding members of the European Cancer Organisation. SIOPE is preparing its strategic agenda to assure long‐term sustainability of clinical and translational research in paediatric malignancies over the next 15 years. SIOPE tackles the issues of equal access to standard care and research across Europe and improvement of long term follow up. SIOPE defined a comprehensive syllabus for training European specialists. A strong partnership with parent, patient and survivor organisations is being developed to successfully achieve the goals of this patient‐centred agenda. SIOPE is advocating in the field of EU policies, such as the Clinical Trials Regulation and the Paediatric Medicine Regulation, to warrant that the voice of young people is heard and their needs adequately addressed. SIOPE and the European community are entirely committed to the global agenda against childhood cancers to overcome the challenges to increasing both cure and quality of cure of young people with cancer. Pediatr Blood Cancer 2014;61:1551–1557.

Incidence of FLT3 and nucleophosmin gene mutations in childhood acute myeloid leukemia: Serbian experience and the review of the literature.

AbstractMutations in the fms-like tyrosine kinase 3 (FLT3) gene (internal tandem duplication (ITD) and point mutation in the tyrosine kinase domain, FLT3/D835) as well as the nucleophosmin (NPM1) gene are the most common abnormalities in adult acute myeloid leukemia (AML). Their significance in pediatric AML is still unclear. In this study we evaluated the frequency of FLT3 and NPM1 mutations in childhood AML. We also examined clinical features and outcome of these patients. FLT3 and NPM1 mutations were analysed in 42 and 37 childhood AML patients, respectively, using polymerase chain reaction (PCR) and direct sequencing. FLT3 mutations were detected in 4/42 patients (9.5%). The frequencies of FLT3/ITD and FLT3/D835 were the same, 2/42 (4.7%). NMP1 mutations were found in 1/37 patients (2.7%). FLT3 gene mutations were correlated with induction failure. Here we report the results of the study of FLT3 and NPM1 gene mutations in childhood AML patients in Serbia. Low frequencies of these molecular markers point out that these abnormalities are rare in this cohort of patients. Comparative study of data on NPM1 mutations in childhood AML revealed that various NPM1 gene mutation types are associated with childhood AML. Our findings as well as previously reported data, contributes to a hypothesis of different biology and etiology of adult and childhood AML. More extensive studies of NPM1 and FLT3 mutations in childhood AML are needed to determine their biological and clinical importance.

6-mercaptopurine influences TPMT gene transcription in a TPMT gene promoter variable number of tandem repeats-dependent manner

AIM TPMT activity is characterized by a trimodal distribution, namely low, intermediate and high methylator. TPMT gene promoter contains a variable number of GC-rich tandem repeats (VNTRs), namely A, B and C, ranging from three to nine repeats in length in an A(n)B(m)C architecture. We have previously shown that the VNTR architecture in the TPMT gene promoter affects TPMT gene transcription. MATERIALS, METHODS & RESULTS: Here we demonstrate, using reporter assays, that 6-mercaptopurine (6-MP) treatment results in a VNTR architecture-dependent decrease of TPMT gene transcription, mediated by the binding of newly recruited protein complexes to the TPMT gene promoter, upon 6-MP treatment. We also show that acute lymphoblastic leukemia patients undergoing 6-MP treatment display a VNTR architecture-dependent response to 6-MP. CONCLUSION These data suggest that the TPMT gene promoter VNTR architecture can be potentially used as a pharmacogenomic marker to predict toxicity due to 6-MP treatment in acute lymphoblastic leukemia patients.