Proteomic studies of pediatric medulloblastoma tumors with 17p deletion.

CNS tumors are the leading cause of cancer-related death in children. Medulloblastoma is the commonest pediatric CNS malignancy, wherein, despite multimodal therapy with surgery, radiation, and chemotherapy, 5 year survival rates merely approach 60%. Until present, gene expression and cytogenetic studies have produced contradicting findings regarding the molecular background of the specific disease. Through integration of genomics, bioinformatics, and proteomics, the current study aims to shed light at the proteomic-related molecular events responsible for MBL pathophysiology, as well as to provide molecular/protein/pathway answers concerning tumor-onset. Experiments were performed on tissues collected at surgery. With 17p loss being the commonest chromosomal aberrance observed in our sample set, array-CGH were employed to first distinguish for 17p-positive cases. 2-DE coupled to mass spectrometry identification exposed the MBL-specific protein profile. Protein profiles of malignant tissues were compared against profiles of normal cerebellar tissues, and quantitative protein differences were determined. Bioinformatics, functional and database analyses, characterization, and subnetwork profiling generated information on MBL protein interactions. Key molecules of the PI3K/mTOR signaling network were identified via the techniques applied herein. Among the findings IGF2, PI3K, Rictor, MAPKAP1, S6K1, 4EBP1, and ELF4A, as part of the IGF network (implicating PI3K/mTOR), were founded to be deregulated.

Protein biomarkers distinguish between high- and low-risk pediatric acute lymphoblastic leukemia in a tissue specific manner.

The current study evaluated the differential expression detected in the proteomic profiles of low risk- and high risk- ALL pediatric patients to characterize candidate biomarkers related to diagnosis, prognosis and patient targeted therapy. Bone marrow and peripheral blood plasma and cell lysates samples were obtained from pediatric patients with low- (LR) and high-risk (HR) ALL at diagnosis. As controls, non-leukemic pediatric patients were studied. Cytogenetic analysis was carried out by G- banding and interphase fluorescent in situ hybridization. Differential proteomic analysis was performed using two-dimensional gel electrophoresis and protein identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The differential expression of certain proteins was confirmed by Western blot analysis. The obtained data revealed that CLUS, CERU, APOE, APOA4, APOA1, GELS, S10A9, AMBP, ACTB, CATA and AFAM proteins play a significant role in leukemia prognosis, potentially serving as distinctive biomarkers for leukemia aggressiveness, or as suppressor proteins in HR-ALL cases. In addition, vitronectin and plasminogen probably contributed to leukemogenesis, whilst bicaudal D-related protein 1 could afford a significant biomarker for pediatric ALL therapeutics.

Clinical significance of productive immunoglobulin heavy chain gene rearrangements in childhood acute lymphoblastic leukemia.

We analyzed the CDR3 region of 80 children with B-cell acute lymphoblastic leukemia (B-ALL) using the ImMunoGeneTics Information System and JOINSOLVER. In total, 108 IGH@ rearrangements were analyzed. Most of them (75.3%) were non-productive. IGHV@ segments proximal to IGHD-IGHJ@ were preferentially rearranged (45.3%). Increased utilization of IGHV3 segments IGHV3-13 (11.3%) and IGHV3-15 (9.3%), IGHD3 (30.5%), and IGHJ4 (34%) was noted. In pro-B ALL more frequent were IGHV3-11 (33.3%) and IGHV6-1 (33.3%), IGHD2-21 (50%), IGHJ4 (50%), and IGHJ6 (50%) segments. Shorter CDR3 length was observed in IGHV@6, IGHD7, and IGHJ1 segments, whereas increased CDR3 length was related to IGHV3, IGHD2, and IGHJ4 segments. Increased risk of relapse was found in patients with productive sequences. Specifically, the relapse-free survival rate at 5 years in patients with productive sequences at diagnosis was 75% (standard error [SE] ±9%), whereas in patients with non-productive sequences it was 97% (SE ±1.92%) (p-value =0.0264). Monoclonality and oligoclonality were identified in 81.2% and 18.75% cases at diagnosis, respectively. Sequence analysis revealed IGHV@ to IGHDJ joining only in 6.6% cases with oligoclonality. The majority (75%) of relapsed patients had monoclonal IGH@ rearrangements. The preferential utilization of IGHV@ segments proximal to IGHDJ depended on their location on the IGHV@ locus. Molecular mechanisms occurring during IGH@ rearrangement might play an essential role in childhood ALL prognosis. In our study, the productivity of the rearranged sequences at diagnosis proved to be a significant prognostic factor.

Proteomics studies of childhood pilocytic astrocytoma.

Childhood pilocytic astrocytoma is the most frequent brain tumor affecting children. Proteomics analysis is currently considered a powerful tool for global evaluation of protein expression and has been widely applied in the field of cancer research. In the present study, a series of proteomics, genomics, and bioinformatics approaches were employed to identify, classify and characterize the proteome content of low-grade brain tumors as it appears in early childhood. Through bioinformatics database construction, protein profiles generated from pathological tissue samples were compared against profiles of normal brain tissues. Additionally, experiments of comparative genomic hybridization arrays were employed to monitor for genetic aberrations and sustain the interpretation and evaluation of the proteomic data. The current study confirms the dominance of MAPK pathway for the childhood pilocytic astrocytoma occurrence and novel findings regarding the ERK-2 expression are reported.

Proteomic analysis of childhood de novo acute myeloid leukemia and myelodysplastic syndrome/AML: correlation to molecular and cytogenetic analyses.

The aim of this study was to investigate the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML) and to provide additional data regarding the proteomic analysis of AML. The protein profiles obtained were correlated to cytogenetic and molecular analyses. Bone marrow (BM) and peripheral blood (PB) samples were obtained during MDS diagnosis, at MDS transformation to AML, at de novo AML diagnosis and 3 months following treatment. As controls, non-leukemic pediatric patients were studied. Cytogenetic and molecular analyses were carried out by G banding and polymerase chain reaction followed by sequencing, respectively. Differential proteomic analysis was performed by two-dimensional gel electrophoresis and protein identification by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. No significant correlations were noted between protein patterns and cytogenetic or molecular analyses. Certain suppressor genes, metabolic enzymes, immunoglobulins and actin-binding proteins were differentially expressed by BM or PB plasma and cell lysates compared to controls. The obtained data showed that vitamin D and gelsolin played contradicting roles in contributing and restraining leukemogenesis, while MOES, EZRI and AIFM1 could be considered as biomarkers for AML.

Duplication of Philadelphia chromosome and trisomy of chromosome 21 in a pediatric patient with acute lymphoblastic leukemia.

The evaluation of molecular and cytogenetic characteristics using novel techniques has significantly contributed into the insight of leukemia. In this study, immunoglobulin heavy chain gene rearrangements (V(H)D(H)J(H) region) were analyzed by polymerase chain reaction (PCR). Point mutations of the D835/I836 in the activation loop (AL) domain of the second tyrosine kinase domain of the fms-related tyrosine kinase 3 (FLT3) gene and NRAS (neuroblastoma cell line) point mutations were also analyzed by PCR. Furthermore, sequence analysis of the V(H)D(H)J(H) region was performed, as well as, chromosomal aberrations were identified by interphase fluorescence in situ hybridization (iFISH) in a 12.5-year-old boy with acute lymphoblastic leukemia. Positive MRD was found in bone marrow samples obtained at various time points during and after treatment completion prior to relapse. Molecular analysis of the FLT3 gene mutations revealed an acquired a G → T mutation at codon 835, which resulted to substitution of aspartate 835 for tyrosine (D835Y). Cytogenetic analysis with iFISH showed t(9;22) (q34;q11.2), with minor-BCR/ABL1 fusion gene in the majority of nuclei, while a subclone with duplication of the Philadelphia chromosome was observed. Triple signals of AML1 were detected in 80% of nuclei, which were compatible with trisomy of chromosome 21. BCR/ABL1 fusion gene, duplication of Philadelphia chromosome and persistence of MRD constitute inferior prognostic factors, while hyperdiploidy, trisomy of chromosome 21 and FLT3-AL mutations are related to better prognosis. The study of cytogenetic and molecular characteristics is essential in order to decide on the optimal treatment protocol in childhood leukemia.

Alpha-fetoprotein secretion in a craniopharyngioma. Are craniopharyngiomas part of the germ cell tumor family?

A 14-months-old girl was admitted to our hospital because of excessive irritability and abnormal eye movements over the last two months. Brain CT and MRI revealed a suprasellar cystic and partially solid mass with calcifications. The laboratory investigation revealed increased serum levels of AFP. These findings were suggestive for a brain germ cell tumor. Therefore, systemic chemotherapy was started. After two courses there was a reduction in the levels of AFP but the tumor size remained unchanged. Subtotal tumor excision was performed that revealed the presence of a craniopharyngioma. One month later there was enlargement of the cystic part of the tumor, while serum AFP was elevated. The child received again systemic chemotherapy and placement of a reservoir into the cystic part of the tumor. Analysis of the intracystic flouid revealed the presence of beta-HCG and AFP. Following that the patient received brachytherapy with intracavity yttrium placement. Three months later repeated MRI showed a decrease in the size of the cystic part, while the solid part remained unchanged. Thus, the solid part was treated by radiosurgery. One year later the patient was stable but with complete loss of vision. These observations support the theory of a germ cell tumor family, in which craniopharyngioma and germ cell tumor present the two sides of the same entity, while between them a wide variety of tumors, with variable type of secretion of AFP and/or beta-HCG, may exist.

The frequency of NPM1 mutations in childhood acute myeloid leukemia.

BACKGROUND: Mutations in the nucleophosmin (NPM1) gene have been solely associated with childhood acute myeloid leukemia (AML). We evaluated the frequency of NPM1 mutations in childhood AML, their relation to clinical and cytogenetic features and the presence of common FLT3 and RAS mutations. RESULTS: NPM1 mutations were found in 8% of cases. They involved the typical type 'A' mutation and one novel mutation characterized by two individual base pair substitutions, which resulted in 2 amino acid changes (W290) and (S293) in the NPM protein. FLT3/ITD mutations were observed in 12% of the cases and in one NPM1-mutated case bearing also t(8;21) (q22;q22). No common RAS mutations were identified. CONCLUSIONS: A relatively consistent NPM1 mutation rate was observed, but with variations in types of mutations. The role of different types of NPM1 mutations, either individually or in the presence of other common gene mutations may be essential for childhood AML prognosis.

Efficacy and safety of linezolid in immunocompromised children with cancer.

BACKGROUND: The aim of this study was to determine the safety, tolerance and efficacy of linezolid for the treatment of infections from Gram-positive bacteria in immunocompromised children with cancer. METHODS: This was a prospective non-comparative unblinded study in the Hematology/Oncology Unit over a two-year period, administering linezolid as monotherapy in children with cancer. RESULTS: Seventeen children received linezolid (30 mg/kgr: 3 i.v. per day). Mean duration of linezolid administration was 12.2 days (range, 6-38 days), while the median age of the evaluable patients was 2.2 years (range, 6 months-11.2 years). Primary diagnosis was acute lymphoblastic leukemia (nine patients), brain tumor (three patients), multi-organ Langerhans cell histiocytosis (two patients), rhabdomyosarcoma, Burkitt's lymphoma and ovarian tumor (one patient each). All patients were in the midst of chemotherapy cycles. Ten out of 17 children had positive blood cultures (methicillin-resistant Staphylococcus aureus, four patients; vancomycin-resistant Enterococcus, three patients; penicillin-resistant Streptococcus pneumoniae, three patients), while seven of the 17 had fever and vancomycin-resistant Enterococcus in stool cultures. All patients were considered clinically cured after the end of the linezolid regimen (100% efficacy). The main adverse events were thrombocytopenia grade 1-3 and anemia grade 2-3 (four and two patients, respectively). Chemotherapy-induced myelotoxicity (six patients) was not worsened during linezolid therapy. No bleeding episodes were presented. Self-limited diarrhea grade 1-2 was presented in four patients (mean duration 2 days). The total adverse event rate was 23.5%; however, there was no premature cessation of linezolid in any patient. CONCLUSIONS: Linezolid may be another effective and safe therapy to treat infections from resistant Gram-positive bacteria in immunocompromised children, even in young ages.

Desmoplastic small round cell tumor of the pleura.

Desmoplastic small round cell tumor (DSRCT) is a rare neoplasm with aggressive behavior. Usually it presents as a peritoneal mass, although other cases in various locations have been described. Since less than 10 cases of primary DSRCT in the pleura have been described, it is of interest to report a pediatric case arising from the pleura. The diagnosis was confirmed by molecular detection of the EWS/WT-1 fusion gene product. Multidisciplinary treatment with chemotherapy, radiotherapy, and surgical resection resulted in a progression-free survival time above the median survival, suggesting that this conventional approach could prove effective for this rare and very aggressive malignancy.