Incidence and prognostic impact of U2AF1 mutations and other gene alterations in myelodysplastic neoplasms with isolated 20q deletion.

BACKGROUND: In myelodysplastic neoplasms (MDS), the 20q deletion [del(20q)] is a recurrent chromosomal abnormality that it has a high co-occurrence with U2AF1 mutations. Nevertheless, the prognostic impact of U2AF1 in these MDS patients is uncertain and the possible clinical and/or prognostic differences between the mutation type and the mutational burden are also unknown. METHODS: Our study analyzes different molecular variables in 100 MDS patients with isolated del(20q). RESULTS & CONCLUSIONS: We describe the high incidence and negative prognostic impact of U2AF1 mutations and other alterations such as in ASXL1 gene to identify prognostic markers that would benefit patients to receive earlier treatment.

Comparative genetic study of intratumoral heterogenous MYCN amplified neuroblastoma versus aggressive genetic profile neuroblastic tumors.

Intratumoral heterogeneous MYCN amplification (hetMNA) is an unusual event in neuroblastoma with unascertained biological and clinical implications. Diagnosis is based on the detection of MYCN amplification surrounded by non-amplified tumor cells by fluorescence in situ hybridization (FISH). To better define the genetic features of hetMNA tumors, we studied the Spanish cohort of neuroblastic tumors by FISH and single nucleotide polymorphism arrays. We compared hetMNA tumors with homogeneous MNA (homMNA) and nonMNA tumors with 11q deletion (nonMNA w11q-). Of 1091 primary tumors, 28 were hetMNA by FISH. Intratumoral heterogeneity of 1p, 2p, 11q and 17q was closely associated with hetMNA tumors when analyzing different pieces for each case. For chromosome 2, 16 cases showed 2p intact, 4 focal gain at 2p24.3 and 8 MNA. The lengths of the smallest regions of overlap (SROs) for 2p gains and 1p deletions were between the SRO lengths observed in homMNA and nonMNA w11q- tumors. Co-occurrence of 11q- and +17q was frequently found with the largest SROs for both aberrations. The evidence for and frequency of different genetic subpopulations representing a hallmark of the hetMNA subgroup of NB indicates, on one hand, the presence of a considerable genetic instability with different SRO of either gains and losses compared with those of the other NB groups and highlights and, on the other hand, the need for multiple sampling from distant and macroscopically and microscopically distinct tumor areas. Narrowing down the different SRO for both deletions and gains in NB groups would be crucial to pinpointing the candidate gene(s) and the critical gene dosage with prognostic and therapeutic significance. This complexity of segmental chromosomal aberration patterns reinforces the necessity for a larger cohort study using FISH and pangenomic techniques to develop a suitable therapeutic strategy for these patients.

Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification.

BACKGROUND: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis. METHODS: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol. RESULTS: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P=0.04). A significant correlation (P=0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS=46% vs 75%, P=0.023; OS=66.8% vs 100%, P=0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P=0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P=0.018). CONCLUSIONS: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

Segmental chromosomal alterations lead to a higher risk of relapse in infants with MYCN-non-amplified localised unresectable/disseminated neuroblastoma (a SIOPEN collaborative study).

BACKGROUND: In neuroblastoma (NB), the presence of segmental chromosome alterations (SCAs) is associated with a higher risk of relapse. METHODS: In order to analyse the role of SCAs in infants with localised unresectable/disseminated NB without MYCN amplification, we have performed an array CGH analysis of tumours from infants enrolled in the prospective European INES trials. RESULTS: Tumour samples from 218 out of 300 enroled patients could be analysed. Segmental chromosome alterations were observed in 11%, 20% and 59% of infants enroled in trials INES99.1 (localised unresectable NB), INES99.2 (stage 4s) and INES99.3 (stage 4) (P<0.0001). Progression-free survival was poorer in patients whose tumours harboured SCA, in the whole population and in trials INES99.1 and INES99.2, in the absence of clinical symptoms (log-rank test, P=0.0001, P=0.04 and P=0.0003, respectively). In multivariate analysis, a SCA genomic profile was the strongest predictor of poorer progression-free survival. CONCLUSION: In infants with stage 4s MYCN-non-amplified NB, a SCA genomic profile identifies patients who will require upfront treatment even in the absence of other clinical indication for therapy, whereas in infants with localised unresectable NB, a genomic profile characterised by the absence of SCA identifies patients in whom treatment reduction might be possible. These findings will be implemented in a future international trial.

Comparative study of MLPA-FISH to determine DNA copy number alterations in neuroblastic tumors.

Neuroblastoma tumor cells show complex combinations of genetic aberrations, and to date many different methods have been used for their detection. To apply genome-wide techniques, such as Multiplex Ligation-dependent Probe Amplification (MLPA), in routine diagnosis their validation is appropriate and necessary. DNA copy number alterations in 129 cases of neuroblastic tumors were detected using MPLA, and the results validated by Fluorescence In Situ Hybridization (FISH) (MYCN gene, 1p36, 11q and 17q). Kappa index values showed very good concordance between the two techniques in detecting homogeneous MYCN amplification (1); 11q deletion (0.908) and 17q gain (0.922). The validation results showed that MLPA is a highly efficient technique for diagnosis based on the genetic aberrations in relevant regions in neuroblastoma, showing a high concordance with FISH.

Neuroblastoma in adolescents: genetic and clinical characterisation

BACKGROUND: Age at diagnosis is an important risk factor in neuroblastoma (NB) with worse prognosis in children older than 18 months. A more indolent course with long-term relapses and fatal outcome has been described in small series of adolescents. Our objective was to describe biological factors that contribute to this particular behaviour and could be helpful in their treatment. PROCEDURE: NB cases older than 10 years of age at diagnosis registered in the files of the Neuroblastoma Group of SEOP from 1992 to 2007 were included. Disease extension was classified according to the International Neuroblastoma Staging System (INSS). Tumour samples were studied according to the International Neuroblastoma Pathology Classification (INPC). Biological studies included MNA, 1p, 11q and 17q status and ploidy. RESULTS: Twenty-two patients, from 10.1 to 24.6 years old, were included. Advanced stages predominated. 14/17 patients presented unfavourable histology. None had NMA or 1p del. However, 11q del was found in 8/13 cases and 17q gain in 7/11. Overall survival (OS) and event-free survival (EFS) for the entire series at 5 years were 0.45 and 0.32, respectively. Moreover, 5-year OS and EFS for stage 4 patients were 0.33 and 0.15. CONCLUSIONS: NB in adolescents is a special subgroup characterised by high-risk prognostic features which differ from those seen in younger patients, especially in relation to genetic abnormalities. The outcome in stage 4 was worse than in younger metastatic children, outlining the need for new therapeutic approaches in this subgroup of patients. The exact cut-off to separate older patients has not yet been established and will probably be based on biology (AU)

Toll-like receptor 4 defective mice carrying point or null mutations do not show increased susceptibility to Candida albicans in a model of hematogenously disseminated infection.

We have studied the role of TLR4 in murine defenses against Candida albicans in two TLR4-defective mouse strains: C3H/HeJ mice which have defective TLR4 signaling, and TLR4-/- knockout mice. Both TLR4-defective mice strains experimentally infected with virulent C. albicans cells showed no significant difference in survival as compared with their respective controls. Recruitment of neutrophils to the peritoneal cavity of i.p. infected mice was not affected in TLR4-/-animals, but significantly enhanced in C3H/HeJ mice, compared with their control mice. In vitro production of TNF-alpha by macrophages from both types of TLR4-defective mice, in response to yeasts and hyphae of C. albicans, was not diminished as compared with production by macrophages from wild-type mice. In vitro production of TNF-alpha by yeast-stimulated splenocytes from mice intravenously infected with the low-virulence C. albicans PCA2 strain was not affected in TLR4-defective mice, but the TNF-alpha production in response to hyphae was higher in TLR4-defective than in control animals; the production of IFN-gamma by these splenocytes was similar to controls, as well as the frequency of IFN-gamma-producing CD4+T lymphocytes, indicating that TLR4-defective mice are capable of mounting a Th1 adaptive immune response. Our data indicate that TLR4 is dispensable for murine immune resistance to C. albicans.

Purification of a biologically active recombinant glyceraldehyde 3-phosphate dehydrogenase from Candida albicans.

We report here the purification of a functionally active recombinant glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Candida albicans. The GAPDH protein encoded by the TDH1 gene was obtained as a glutathione S-transferase fusion protein by expression in the vector pGEX-4T-3, and purified by affinity chromatography and thrombin digestion. The purified protein displays GAPDH enzymatic activity (42 micromol NADH min(-1) mg(-1)) as well as the laminin and fibronectin binding activities previously described. In addition, the recombinant GAPDH is covalently modified by NAD linkage; this modification is stimulated by nitric oxide and probably involves a sulfhydryl group (cysteine) residue since it is inhibited by Hg(2+) and cysteine.

Clinical strains of Candida albicans express the surface antigen glyceraldehyde 3-phosphate dehydrogenase in vitro and in infected tissues.

We have previously described the presence of an enzymatically active form of glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) in the cell surface of Candida albicans ATCC 26555 which is also a fibronectin and laminin binding protein. Immunohistochemical analysis of tissue sections from patients with disseminated candidiasis with a polyclonal antiserum to GAPDH from C. albicans (PAb anti-CA-GAPDH) revealed that the enzyme is expressed at the surface of fungal cells in infected tissues. The same PAb detected the presence of GAPDH species, with a molecular mass of approximately 33 kDa, in cell wall extracts obtained from clinical isolates of the fungus. These cell surface-bound GAPDH moieties exhibited a dose-dependent dehydrogenase activity. These results indicate that this cell surface-bound GAPDH plays a role during infection probably contributing to the attachment of fungal cells to host tissues.