Deletions of IKZF1 and SPRED1 are associated with poor prognosis in a population-based series of pediatric B-cell precursor acute lymphoblastic leukemia diagnosed between 1992 and 2011.

Despite the favorable prognosis of childhood acute lymphoblastic leukemia (ALL), a substantial subset of patients relapses. As this occurs not only in the high risk but also in the standard/intermediate groups, the presently used risk stratification is suboptimal. The underlying mechanisms for treatment failure include the presence of genetic changes causing insensitivity to the therapy administered. To identify relapse-associated aberrations, we performed single-nucleotide polymorphism array analyses of 307 uniformly treated, consecutive pediatric ALL cases accrued during 1992-2011. Recurrent aberrations of 14 genes in patients who subsequently relapsed or had induction failure were detected. Of these, deletions/uniparental isodisomies of ADD3, ATP10A, EBF1, IKZF1, PAN3, RAG1, SPRED1 and TBL1XR1 were significantly more common in B-cell precursor ALL patients who relapsed compared with those remaining in complete remission. In univariate analyses, age (≥10 years), white blood cell counts (>100 × 10(9)/l), t(9;22)(q34;q11), MLL rearrangements, near-haploidy and deletions of ATP10A, IKZF1, SPRED1 and the pseudoautosomal 1 regions on Xp/Yp were significantly associated with decreased 10-year event-free survival, with IKZF1 abnormalities being an independent risk factor in multivariate analysis irrespective of the risk group. Older age and deletions of IKZF1 and SPRED1 were also associated with poor overall survival. Thus, analyses of these genes provide clinically important information.

Genomic heterogeneity in acute leukemia.

Acquired genetic aberrations are the underlying cause of leukemogenesis in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). The karyotypes of AML and ALL cases are generally quite simple as seen by chromosome banding analysis, with few genetic changes and a limited number of subclones. However, investigations using fluorescence in situ hybridization, loss of heterozygosity analysis, single-nucleotide polymorphism arrays, and, most recently, massively parallel sequencing have challenged this view. In particular, comparison of diagnostic and relapse samples, modeling in transgenic mice, and whole-exome and whole-genome sequencing have indicated that widespread genomic heterogeneity, which is masked by a dominant clone, may be present in AML and ALL. In the present review, our current knowledge of genomic heterogeneity in acute leukemia is summarized.

Relapsed childhood high hyperdiploid acute lymphoblastic leukemia: presence of preleukemic ancestral clones and the secondary nature of microdeletions and RTK-RAS mutations.

Although childhood high hyperdiploid acute lymphoblastic leukemia is associated with a favorable outcome, 20% of patients still relapse. It is important to identify these patients already at diagnosis to ensure proper risk stratification. We have investigated 11 paired diagnostic and relapse samples with single nucleotide polymorphism array and mutation analyses of FLT3, KRAS, NRAS and PTPN11 in order to identify changes associated with relapse and to ascertain the genetic evolution patterns. Structural changes, mainly cryptic hemizygous deletions, were significantly more common at relapse (P<0.05). No single aberration was linked to relapse, but four deletions, involving IKZF1, PAX5, CDKN2A/B or AK3, were recurrent. On the basis of the genetic relationship between the paired samples, three groups were delineated: (1) identical genetic changes at diagnosis and relapse (2 of 11 cases), (2) clonal evolution with all changes at diagnosis being present at relapse (2 of 11) and (3) clonal evolution with some changes conserved, lost or gained (7 of 11), suggesting the presence of a preleukemic clone. This ancestral clone was characterized by numerical changes only, with structural changes and RTK-RAS mutations being secondary to the high hyperdiploid pattern.

Sleep problems and the need for parental night-time attention in children with physical disabilities.

BACKGROUND: The aim of the study was to investigate the frequency and predictors of sleep problems and the need for parental night-time attention in children with physical disabilities. METHODS: A questionnaire on sleep problems and need for parental night-time attention was completed by 505 parents of children with physical disabilities aged 1-16 years (mean 9 years 3 months) with moderate to severe motor disabilities. General characteristics of the children were analysed by frequencies and cross-tabulations. Logistic regression analysis was used to identify factors associated with sleep problems and the need for parental night-time attention. RESULTS: The results showed a high prevalence of sleep problems, which in general were chronic. Currently 48% of the children had sleep problems, of which 23% estimated the problems to be serious. About one-third (37%) needed parental night-time attention every night, and 10% needed help five times or more. No significant differences were found between younger children and school-aged children regarding sleep problems and the need for parental night-time attention. Having pain [odds ratio (OR) = 3.4] was associated with sleep problems, as was having problems eating and drinking (OR = 3) and the diagnosis of cerebral palsy (OR = 2.5) (P < 0.05). Children with muscular dystrophy (OR = 68.5), cerebral palsy (OR = 26.7) and 'other diagnosis' (OR = 18.5) were more likely to need support at night than were children with spina bifida, P < 0.001. Pain (OR = 7.6) was also associated with need for support at night, P < 0.001. CONCLUSIONS: The prevalence of sleep problems and need for parental night-time attention is high among children with physical disabilities. This in turn affects the whole family, and paediatric caregivers must keep this in mind. Besides certain diagnoses, the results suggest that children who have pain should be prioritized.

Trisomy 8 as the sole chromosomal aberration in acute myeloid leukemia and myelodysplastic syndromes.

Trisomy 8 as the sole abnormality is the most common karyotypic finding in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), occurring in approximately 5% and 10% of the cytogenetically abnormal cases, respectively. However, despite the high frequency of +8, much remains to be elucidated as regards its epidemiology, etiology, clinical impact, association with other chromosomal abnormalities, cell of origin, and functional and pathogenetic consequences. Here, we summarize and review these various aspects of trisomy 8, focusing on AMLs and MDS harboring this abnormality as a single change.

Identification of cryptic aberrations and characterization of translocation breakpoints using array CGH in high hyperdiploid childhood acute lymphoblastic leukemia.

High hyperdiploidy, characterized by non-random trisomies, is the largest cytogenetic subgroup in childhood acute lymphoblastic leukemia (ALL). It is not known whether the gained chromosomes are sufficient for leukemogenesis or if additional genetic aberrations are necessary. However, the suboptimal chromosome morphology of hyperdiploid ALLs makes detection of structural abnormalities difficult if using cytogenetic techniques; alternative methods are, therefore, needed. We performed array comparative genome hybridization (CGH) analyses, with a resolution of 100 kb, of eight cases of high hyperdiploid childhood ALL to characterize structural abnormalities found with G-banding/multicolor fluorescence in situ hybridization (FISH) and to detect novel changes. The non-centromeric breakpoints of four rearrangements, including three translocations and one 1q duplication, were narrowed down to <0.2 Mb. Furthermore, four submicroscopic imbalances involving 0.6-2.7 Mb were detected, comprising two segmental duplications involving 1q22 and 12q24.31 in one case and two hemizygous deletions in 12p13.2-31 - including ETV6 - and in 13q32.3-33.1 in another case. Notably, FISH analysis of the latter revealed an associated reciprocal t(3;13)(q?;32.2-33.1). In conclusion, the array CGH analyses revealed putative leukemia-associated submicroscopic imbalances and rearrangements in 2/8 (25%) hyperdiploid ALLs. The detection and characterization of these additional genetic aberrations will most likely increase our understanding of the pathogenesis of high hyperdiploid childhood ALL.

High-resolution genome-wide array-based comparative genome hybridization reveals cryptic chromosome changes in AML and MDS cases with trisomy 8 as the sole cytogenetic aberration.

Although trisomy 8 as the sole chromosome aberration is the most common numerical abnormality in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), little is known about its pathogenetic effects. Considering that +8 is a frequent secondary change in AML/MDS, cryptic--possibly primary--genetic aberrations may occur in cases with trisomy 8 as the apparently single anomaly. However, no such hidden anomalies have been reported. We performed a high-resolution genome-wide array-based comparative genome hybridization (array CGH) analysis of 10 AML/MDS cases with isolated +8, utilizing a 32K bacterial artificial chromosome array set, providing >98% coverage of the genome with a resolution of 100 kb. Array CGH revealed intrachromosomal imbalances, not corresponding to known genomic copy number polymorphisms, in 4/10 cases, comprising nine duplications and hemizygous deletions ranging in size from 0.5 to 2.2 Mb. A 1.8 Mb deletion at 7p14.1, which had occurred prior to the +8, was identified in MDS transforming to AML. Furthermore, a deletion including ETV6 was present in one case. The remaining seven imbalances involved more than 40 genes. The present results show that cryptic genetic abnormalities are frequent in trisomy 8-positive AML/MDS cases and that +8 as the sole cytogenetic aberration is not always the primary genetic event.

A novel and cytogenetically cryptic t(7;21)(p22;q22) in acute myeloid leukemia results in fusion of RUNX1 with the ubiquitin-specific protease gene USP42.

Although many of the chromosomal abnormalities in hematologic malignancies are identifiable cytogenetically, some are only detectable using molecular methods. We describe a novel cryptic t(7;21)(p22;q22) in acute myeloid leukemia (AML). FISH, 3'RACE, and RT-PCR revealed a fusion involving RUNX1 and the ubiquitin-specific protease (USP) gene USP42. The genomic breakpoint was in intron 7 of RUNX1 and intron 1 of USP42. The reciprocal chimera was not detected - neither on the transcriptional nor on the genomic level - and FISH showed that the 5' part of USP42 was deleted. USP42 maps to a 7p22 region characterized by segmental duplications. Notably, 17 kb duplicons are present 1 Mb proximal to USP42 and 3 Mb proximal to RUNX1; these may be important in the genesis of t(7;21). This is the second cryptic RUNX1 translocation in hematologic malignancies and the first in AML. The USPs have not previously been reported to be rearranged in leukemias. The cellular context in which USP42 is active is unknown, but we here show that it is expressed in normal bone marrow, in primary AMLs, and in cancer cell lines. Its involvement in the t(7;21) suggests that deregulation of ubiquitin-associated pathways may be pathogenetically important in AML.

Evolutionary and functional perspectives of the major histocompatibility complex class I antigen-processing machinery.

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8+ T cells, providing the basis for immune recognition of pathogen-infected cells. Peptides generated mainly by proteasomes in the cytosol are transported into the lumen of the endoplasmic reticulum by transporters associated with antigen processing (TAP). The maturation of MHC class I molecules is controlled by a number of accessory proteins and chaperones that are to a varying degree dedicated to the assembly of MHC class I. Several newly characterised proteins have been demonstrated to play important roles in this process. This review focuses on the functional relationship and evolutionary history of the antigen-processing machinery (APM) components and MHC class I itself. These are of great interest for further elucidating the origin of the immune system and understanding the mechanisms of antigen presentation and immunology in general.

The incidence of trisomy 8 as a sole chromosomal aberration in myeloid malignancies varies in relation to gender, age, prior iatrogenic genotoxic exposure, and morphology.

Although trisomy 8 as a sole change is one of the most common chromosomal abnormalities in myeloid malignancies, it is largely unknown if the incidence of this aberration is influenced by other factors of clinical importance. In the present study, the frequencies of isolated +8 in relation to gender, age, previous treatment with chemo- or radiotherapy, and morphologic subtype were ascertained in published, as well as in our own unpublished, cases of acute myeloid leukemia (AML; n=4,246), myelodysplastic syndromes (MDS; n=1,817), and chronic myeloproliferative disorders (MPD; n=530). The frequencies of +8 were higher in MDS and MPD than in AML (7.5% vs. 5.6%; P<0.01) and varied among the morphologic subtypes of AML and MDS (P<0.001 and P<0.05, respectively). Trisomy 8 was more common in women than in men with MPD (11% vs. 5.1%; P<0.05). Furthermore, the frequencies of +8 were higher in de novo AML and MDS than in treatment-related cases (6.0% vs. 2.8%; P<0.01 and 8.6% vs. 1.5%; P<0.001, respectively). The incidence also varied significantly with age in AML (P<0.001), being more common in elderly patients. Although the causes for this frequency heterogeneity remain to be elucidated, possible explanations may include different environmental exposures affecting the origin of +8 in AML, MDS, and MPD and the presence of different underlying cryptic primary aberrations.

Distinct differences in association of MHC class I with endoplasmic reticulum proteins in wild-type, and beta 2-microglobulin- and TAP-deficient cell lines.

In this study we have compared the interaction of human MHC class I molecules with IgG heavy chain (HC) binding protein (BiP), calnexin, calreticulin, tapasin and TAP in beta(2)-microglobulin (beta(2)m)- or TAP-deficient cells, as well as in wild-type B-LCL cells. Distinct differences between the association of HC and these endoplasmic reticulum (ER) proteins were found in the three cell lines. In the absence of beta(2)m (Daudi cells), HC associated with both BiP and calnexin. A prominent portion of HC was complexed simultaneously to both chaperones, as indicated by co-precipitation with either anti-calnexin or anti-class I antisera. In the presence of beta(2)m, but absence of TAP (T2 cells), HC could be co-precipitated with calnexin, whereas no detectable interaction with BiP could be demonstrated. This suggests that calnexin interacts with HC at a later stage than BiP. In B-LCL cells, HC-beta(2)m associated with calreticulin and tapasin, whereas no interaction with calnexin and BiP was observed. In the absence of beta(2)m, HC were rapidly degraded in the ER, while the ER retained HC were stabilized in the presence of beta(2)m, even in the absence of TAP. The dissociation of class I molecules from TAP in B-LCL cells correlated with the kinetics of appearance of class I molecules on the cell surface, suggesting that TAP retains peptide-free class I molecules in the ER. Taken together, our results suggest the model that BiP and calnexin sequentially control the folding of MHC class I, before MHC class I molecules associate with the loading complex.

Inhibition of the transcription factors AP-1 and NF-kappaB in CD4 T cells by peroxisome proliferator-activated receptor gamma ligands.

The peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, is essential for adipocyte differentiation and glucose homeostasis. PPARgamma has been found recently to regulate macrophage activation in response to mitogens and inflammation. Our study shows PPARgamma to be preferentially expressed in the nuclei of resting T cells and to increase upon activation of T cells by either anti-CD3 and anti-CD28 or phorbol myristyl acetate (PMA). We also found the PPARgamma ligand ciglitizone to attenuate the activation of T cells by inhibiting cytokine gene expression and anti-CD3 and anti-CD28 or PMA-induced proliferative responses. Inhibition of both the proliferative response and inflammatory cytokine expression in CD4 T cells was correlated with suppression of the activated transcription factors AP1 and NF-kappaB. PPARgamma ligands also strongly inhibited SEA-induced Vbeta3 T cell activation in vivo. These results, together with previous findings of the inhibitory effect of PPARgamma ligands on activated macrophages, provide clear evidence for PPARgamma as a negative regulator of the inflammatory activation of both macrophage and T cells. PPARgamma may thus be a potential therapeutic target for the treatment of autoimmunity.

Assembly of tapasin-associated MHC class I in the absence of the transporter associated with antigen processing (TAP).

The assembly of MHC class I molecules is regulated by a multi-protein complex in the endoplasmic reticules (ER) termed the loading complex. Tapasin is suggested to be one of the molecules forming this complex on the basis of its interaction with both the transporter associated with antigen processing (TAP) and MHC class I molecules. To address whether TAP is indispensable for the processing of the assembly of tapasin-associated MHC class I molecules, we studied the association of MHC class I molecules with tapasin, the assembly of tapasin-associated MHC class I with peptides and the peptide-mediated dissociation of MHC class I from tapasin in TAP-mutant T2 cells. In the absence of TAP, MHC class I heavy chain and beta(2)-microglobulin dimers were found to be properly associated with tapasin. The stable MHC class I dimer was required for its association with tapasin in the ER. In the absence of TAP, tapasin retained MHC class I molecules much longer in the ER than in the presence of TAP. This low off-rate of MHC class I from tapasin was due to the absence of high-affinity peptides in the ER of TAP-mutant cells but not to the absence of TAP per se. The introduction of peptides into permeabilized microsomes of TAP-mutant cells led to effective loading of the peptides onto tapasin-associated MHC class I and to the subsequent dissociation of MHC class I from tapasin. These results demonstrate that regulation of the assembly of tapasin-associated MHC class I is independent of the interaction of tapasin with TAP, but is dependent upon the peptides transported by TAP.

Cytokine-induced Src homology 2 protein (CIS) promotes T cell receptor-mediated proliferation and prolongs survival of activated T cells.

Members of the suppressor of cytokine signaling (SOCS) family were discovered as negative regulators of cytokine signaling by inhibition of the Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathway. Among them, cytokine-induced Src homology 2 (SH2) protein (CIS) was found to inhibit the interleukin 3- and erythropietin-mediated STAT5 signaling pathway. However, involvement of SOCS proteins in other signaling pathways is still unknown. This study shows that the expression of CIS is selectively induced in T cells after T cell receptor (TCR) stimulation. In transgenic mice, with selective expression of CIS in CD4 T cells, elevated CIS strongly promotes TCR-mediated proliferation and cytokine production in vitro, and superantigen-induced T cell activation in vivo. Forced expression of CIS also prolongs survival of CD4 T cells after TCR activation. Molecular events immediately downstream from the TCR are not changed in CIS-expressing CD4 T cells, but activation of mitogen-activated protein (MAP) kinase pathways by TCR stimulation is significantly enhanced. Together with the increased MAP kinase activation, a direct interaction of CIS and protein kinase Ctheta was also demonstrated. These results suggest that CIS is one of the important regulators of TCR-mediated T cell activation. The functions of CIS, enhancing TCR signaling and inhibiting cytokine signaling, may be important in the regulation of immune response and homeostasis.

Tapasin is required for efficient peptide binding to transporter associated with antigen processing.

The transporter associated with antigen processing (TAP) binds peptides in its cytosolic part and subsequently translocates the peptides into the lumen of the endoplasmic reticulum (ER), where assembly of major histocompatibility complex (MHC) class I and peptide takes place. Tapasin is a subunit of the TAP complex and binds both to TAP1 and MHC class I. In the absence of tapasin, the assembly of MHC class I in the ER is impaired, and the surface expression is reduced. To clarify the function of tapasin in the processing of antigenic peptides, we studied the interaction of peptide and TAP, peptide transport across the membrane of the ER, and association of peptides with MHC class I molecules in the microsomes derived from tapasin mutant cell line 721.220, its sister cell line 721.221 expressing tapasin, and their HLA-A2 transfectants. The binding of peptides to TAP in tapasin mutant 721.220 cells was significantly diminished in comparison with 721.221 cells. Impaired peptide-TAP interaction resulted in a defective peptide transport in tapasin mutant 721.220 cells. Interestingly, despite the diminished peptide binding to TAP, the transport rate of TAP-associated peptides was not significantly altered in 721.220 cells. After transfection of tapasin cDNA into 721.220 cells, efficient peptide-TAP interaction was restored. Thus, we conclude that tapasin is required for efficient peptide-TAP interaction.