Association of leukemic molecular profile with efficacy of inotuzumab ozogamicin in adults with relapsed/refractory ALL.

The phase 3 INO-VATE trial demonstrated higher rates of remission, measurable residual disease negativity, and improved overall survival for patients with relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) who received inotuzumab ozogamicin (InO) vs standard of care chemotherapy (SC). Here we examined associations between genomic alterations and the efficacy of InO. Of 326 randomized patients, 91 (InO, n=43; SC, n=48) had samples evaluable for genomic analysis. The spectrum of gene fusions and other genomic alterations observed was comparable with prior studies of adult ALL. Responses to InO were observed in all leukemic subtypes, genomic alterations, and risk groups. Significantly higher rates of complete remission (CR)/CR with incomplete count recovery rates were observed with InO vs SC in patients with BCR::ABL1-like ALL (85.7% [6/7] vs 0% [0/5] P=0.0076), with TP53 alterations (100% [5/5] vs 12.5% [1/8], P=0.0047), and in the high-risk BCR::ABL1- (BCR::ABL1-like, low hypodiploid, KMT2A-rearranged) group (83.3% [10/12] vs 10.5% [2/19]; P<0.0001). This retrospective, exploratory analysis of the INO-VATE trial demonstrated potential for benefit with InO for patients with R/R ALL across leukemic subtypes, including BCR::ABL1-like ALL, and for those bearing diverse genomic alterations. Further confirmation of the efficacy of InO in patients with R/R ALL exhibiting the BCR::ABL1-like subtype or harboring TP53 alterations is warranted. This trial was registered at www.clinicaltrials.gov as no. NCT01564784.

Haploinsufficiency of the lysosomal sialidase NEU1 results in a model of pleomorphic rhabdomyosarcoma in mice.

Rhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1+/-/ETV7TG/+/- mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1. These tumors share mostly features of embryonal and some of alveolar rhabdomyosarcoma. Mechanistically, we show that the transforming pathway is increased lysosomal exocytosis downstream of reduced neuraminidase 1, exemplified by the redistribution of the lysosomal associated membrane protein 1 at the plasma membrane of tumor and stromal cells. Here we exploit this unique feature for single cell analysis and define heterogeneous populations of exocytic, only partially differentiated cells that force tumors to pleomorphism and promote a fibrotic microenvironment. These data together with the identification of an adipogenic signature shared by human rhabdomyosarcoma, and likely fueling the tumor's metabolism, make this model of pleomorphic rhabdomyosarcoma ideal for diagnostic and therapeutic studies.

Genetic and clinical factors associated with obesity among adult survivors of childhood cancer: A report from the St. Jude Lifetime Cohort.

BACKGROUND: The objective of this study was to identify treatment and genetic factors associated with obesity among childhood cancer survivors. METHODS: Participants included 1996 survivors who previously received treatment for cancer at St. Jude Children's Research Hospital and who survived ≥10 years from diagnosis (median age at diagnosis, 7.2 years; median age at follow-up, 32.4 years). Obesity was defined as a body mass index ≥30 kg/m(2) . The factors associated with adult obesity were identified by subgroup-specific (cranial radiation [CRT] exposure status) multivariable logistic regression. Single nucleotide polymorphisms (SNPs) associated with obesity were identified by subgroup-specific, exploratory, genome-wide association analyses using a 2-stage resampling approach with a type I error rate of 5 × 10(-6) . RESULTS: Forty-seven percent of survivors who received CRT and 29.4% of those who did not receive CRT were obese at evaluation. In multivariable analyses, abdominal/pelvic radiation exposure was associated with decreased prevalence of obesity among survivors regardless of CRT status (P < .0001). The odds of obesity were increased among survivors who received CRT who had also received glucocorticoids (P = .014) or who were younger at diagnosis (P = .013). Among the survivors who had received CRT, 166 SNPs were associated with obesity. The strongest association was observed with reference SNP rs35669975 (P = 3.3 × 10(-8) ) on segment 33.3 of the long arm of chromosome 13 (13q33.3), approximately 30 kb downstream of FAM155A (family with sequence similarity 155, member A). SNPs within the glycine receptor α3 (GLRA3) gene and near the sex-determining region Y box 11 (SOX11) and cadherin 18 type 2 (CDH18) genes also were identified. These genes have been implicated in neural growth, repair, and connectivity. CONCLUSIONS: Obesity in childhood cancer survivors remains associated with previous exposure to CRT and glucocorticoids. Genetic variants related to neural connectivity may modify the risk of obesity among survivors who receive CRT. Validation of these findings in independent cohorts is required.

The Mi-2 homolog Mit1 actively positions nucleosomes within heterochromatin to suppress transcription.

Mit1 is the putative chromatin remodeling subunit of the fission yeast Snf2/histone deacetylase (HDAC) repressor complex (SHREC) and is known to repress transcription at regions of heterochromatin. However, how Mit1 modifies chromatin to silence transcription is largely unknown. Here we report that Mit1 mobilizes histone octamers in vitro and requires ATP hydrolysis and conserved chromatin tethering domains, including a previously unrecognized chromodomain, to remodel nucleosomes and silence transcription. Loss of Mit1 remodeling activity results in nucleosome depletion at specific DNA sequences that display low intrinsic affinity for the histone octamer, but its contribution to antagonizing RNA polymerase II (Pol II) access and transcription is not restricted to these sites. Genetic epistasis analyses demonstrate that SHREC subunits and the transcription-coupled Set2 histone methyltransferase, which is involved in suppression of cryptic transcription at actively transcribed regions, cooperate to silence heterochromatic transcripts. In addition, we have demonstrated that Mit1's remodeling activity contributes to SHREC function independently of Clr3's histone deacetylase activity on histone H3 K14. We propose that Mit1 is a chromatin remodeling factor that cooperates with the Clr3 histone deacetylase of SHREC and other chromatin modifiers to stabilize heterochromatin structure and to prevent access to the transcriptional machinery.

Prox1 ablation in hepatic progenitors causes defective hepatocyte specification and increases biliary cell commitment.

The liver has multiple functions that preserve homeostasis. Liver diseases are debilitating, costly and often result in death. Elucidating the developmental mechanisms that establish the liver's architecture or generate the cellular diversity of this organ should help advance the prevention, diagnosis and treatment of hepatic diseases. We previously reported that migration of early hepatic precursors away from the gut epithelium requires the activity of the homeobox gene Prox1. Here, we show that Prox1 is a novel regulator of cell differentiation and morphogenesis during hepatogenesis. Prox1 ablation in bipotent hepatoblasts dramatically reduced the expression of multiple hepatocyte genes and led to very defective hepatocyte morphogenesis. As a result, abnormal epithelial structures expressing hepatocyte and cholangiocyte markers or resembling ectopic bile ducts developed in the Prox1-deficient liver parenchyma. By contrast, excessive commitment of hepatoblasts into cholangiocytes, premature intrahepatic bile duct morphogenesis, and biliary hyperplasia occurred in periportal areas of Prox1-deficient livers. Together, these abnormalities indicate that Prox1 activity is necessary to correctly allocate cell fates in liver precursors. These results increase our understanding of differentiation anomalies in pathological conditions and will contribute to improving stem cell protocols in which differentiation is directed towards hepatocytes and cholangiocytes.

Emergence of polyclonal FLT3 tyrosine kinase domain mutations during sequential therapy with sorafenib and sunitinib in FLT3-ITD-positive acute myeloid leukemia.

PURPOSE: To evaluate the clinical activity of sequential therapy with sorafenib and sunitinib in FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD)-positive acute myelogenous leukemia (AML) and monitor the emergence of secondary FLT3 tyrosine kinase domain (TKD) mutations during treatment. EXPERIMENTAL DESIGN: Six children with relapsed/refractory AML were treated with sorafenib in combination with clofarabine and cytarabine, followed by single-agent sorafenib if not a candidate for transplantation. Sunitinib was initiated after sorafenib relapse. Bone marrow samples were obtained for assessment of FLT3 TKD mutations by deep amplicon sequencing. The phase of secondary mutations with ITD alleles was assessed by cloning and sequencing of FLT3 exons 14 through 20. Identified mutations were modeled in Ba/F3 cells, and the effect of kinase inhibitors on FLT3 signaling and cell viability was assessed. RESULTS: Four patients achieved complete remission, but 3 receiving maintenance therapy with sorafenib relapsed after 14 to 37 weeks. Sunitinib reduced circulating blasts in two patients and marrow blasts in one. Two patients did not respond to sorafenib combination therapy or sunitinib. FLT3 mutations at residues D835 and F691 were observed in sorafenib resistance samples on both ITD-positive and -negative alleles. Deep sequencing revealed low-level mutations and their evolution during sorafenib treatment. Sunitinib suppressed leukemic clones with D835H and F691L mutations, but not D835Y. Cells expressing sorafenib-resistant FLT3 mutations were sensitive to sunitinib in vitro. CONCLUSIONS: Sunitinib has activity in patients that are resistant to sorafenib and harbor secondary FLT3 TKD mutations. The use of sensitive methods to monitor FLT3 mutations during therapy may allow individualized treatment with the currently available kinase inhibitors.

Genetic mapping and exome sequencing identify 2 mutations associated with stroke protection in pediatric patients with sickle cell anemia.

Stroke is a devastating complication of sickle cell anemia (SCA), occurring in 11% of patients before age 20 years. Previous studies of sibling pairs have demonstrated a genetic component to the development of cerebrovascular disease in SCA, but few candidate genetic modifiers have been validated as having a substantial effect on stroke risk. We performed an unbiased whole-genome search for genetic modifiers of stroke risk in SCA. Genome-wide association studies were performed using genotype data from single-nucleotide polymorphism arrays, whereas a pooled DNA approach was used to perform whole-exome sequencing. In combination, 22 nonsynonymous variants were identified and represent key candidates for further in-depth study. To validate the association of these mutations with the risk for stroke, the 22 candidate variants were genotyped in an independent cohort of control patients (n = 231) and patients with stroke (n = 57) with SCA. One mutation in GOLGB1 (Y1212C) and another mutation in ENPP1 (K173Q) were confirmed as having significant associations with a decreased risk for stroke. These mutations were discovered and validated by an unbiased whole-genome approach, and future studies will focus on how these functional mutations may lead to protection from stroke in the context of SCA.

Hydroxycarbamide alters erythroid gene expression in children with sickle cell anaemia.

Sickle cell anaemia (SCA) is a severe debilitating haematological disorder associated with a high degree of morbidity and mortality. The level of fetal haemoglobin (HbF) is well-recognized as a critical laboratory parameter: lower HbF is associated with a higher risk of vaso-occlusive complications, organ damage, and early death. Hydroxycarbamide treatment can induce HbF, improve laboratory parameters, and ameliorate clinical complications of SCA but its mechanisms of action remain incompletely defined and the HbF response is highly variable. To identify pathways of hydroxycarbamide activity, we performed microarray expression analyses of early reticulocyte RNA obtained from children with SCA enrolled in the HydroxyUrea Study of Long-term Effects (NCT00305175) and examined the effects of hydroxycarbamide exposure in vivo. Hydroxycarbamide affected a large number of erythroid genes, with significant decreases in the expression of genes involved in translation, ribosome assembly and chromosome organization, presumably reflecting the daily cytotoxic pulses of hydroxycarbamide. Hydroxycarbamide also affected expression of numerous genes associated with HbF including BCL11A, a key regulator of baseline HbF levels. Together, these data indicate that hydroxycarbamide treatment for SCA leads to substantial changes in erythroid gene expression, including BCL11A and other potential signalling pathways associated with HbF induction.

The inflammasome adaptor ASC regulates the function of adaptive immune cells by controlling Dock2-mediated Rac activation and actin polymerization.

The adaptor ASC contributes to innate immunity through the assembly of inflammasome complexes that activate the cysteine protease caspase-1. Here we demonstrate that ASC has an inflammasome-independent, cell-intrinsic role in cells of the adaptive immune response. ASC-deficient mice showed defective antigen presentation by dendritic cells (DCs) and lymphocyte migration due to impaired actin polymerization mediated by the small GTPase Rac. Genome-wide analysis showed that ASC, but not the cytoplasmic receptor NLRP3 or caspase-1, controlled the mRNA stability and expression of Dock2, a guanine nucleotide-exchange factor that mediates Rac-dependent signaling in cells of the immune response. Dock2-deficient DCs showed defective antigen uptake similar to that of ASC-deficient cells. Ectopic expression of Dock2 in ASC-deficient cells restored Rac-mediated actin polymerization, antigen uptake and chemotaxis. Thus, ASC shapes adaptive immunity independently of inflammasomes by modulating Dock2-dependent Rac activation and actin polymerization in DCs and lymphocytes.

Inflammasome is a central player in the induction of obesity and insulin resistance.

Inflammation plays a key role in the pathogenesis of obesity. Chronic overfeeding leads to macrophage infiltration in the adipose tissue, resulting in proinflammatory cytokine production. Both microbial and endogenous danger signals trigger assembly of the intracellular innate immune sensor Nlrp3, resulting in caspase-1 activation and production of proinflammatory cytokines IL-1ß and IL-18. Here, we showed that mice deficient in Nlrp3, apoptosis-associated speck-like protein, and caspase-1 were resistant to the development of high-fat diet-induced obesity, which correlated with protection from obesity-induced insulin resistance. Furthermore, hepatic triglyceride content, adipocyte size, and macrophage infiltration in adipose tissue were all reduced in mice deficient in inflammasome components. Monocyte chemoattractant protein (MCP)-1 is a key molecule that mediates macrophage infiltration. Indeed, defective inflammasome activation was associated with reduced MCP-1 production in adipose tissue. Furthermore, plasma leptin and resistin that affect energy use and insulin sensitivity were also changed by inflammasome-deficiency. Detailed metabolic and molecular phenotyping demonstrated that the inflammasome controls energy expenditure and adipogenic gene expression during chronic overfeeding. These findings reveal a critical function of the inflammasome in obesity and insulin resistance, and suggest inhibition of the inflammasome as a potential therapeutic strategy.

RAF gene fusion breakpoints in pediatric brain tumors are characterized by significant enrichment of sequence microhomology.

Gene fusions involving members of the RAF family of protein kinases have recently been identified as characteristic aberrations of low-grade astrocytomas, the most common tumors of the central nervous system in children. While it has been shown that these fusions cause constitutive activation of the ERK/MAPK pathway, very little is known about their formation. Here, we present a detailed analysis of RAF gene fusion breakpoints from a well-characterized cohort of 43 low-grade astrocytomas. Our findings show that the rearrangements that generate these RAF gene fusions may be simple or complex and that both inserted nucleotides and microhomology are common at the DNA breakpoints. Furthermore, we identify novel enrichment of microhomologous sequences in the regions immediately flanking the breakpoints. We thus provide evidence that the tandem duplications responsible for these fusions are generated by microhomology-mediated break-induced replication (MMBIR). Although MMBIR has previously been implicated in the pathogenesis of other diseases and the evolution of eukaryotic genomes, we demonstrate here that the proposed details of MMBIR are consistent with a recurrent rearrangement in cancer. Our analysis of repetitive elements, Z-DNA and sequence motifs in the fusion partners identified significant enrichment of the human minisatellite conserved sequence/χ-like element at one side of the breakpoint. Therefore, in addition to furthering our understanding of low-grade astrocytomas, this study provides insights into the molecular mechanistic details of MMBIR and the sequence of events that occur in the formation of genomic rearrangements.

Clinical significance of low levels of minimal residual disease at the end of remission induction therapy in childhood acute lymphoblastic leukemia.

Minimal residual disease (MRD) at the end of remission-induction therapy predicts relapse in acute lymphoblastic leukemia (ALL). We examined the clinical significance of levels below the usual threshold value for MRD positivity (0.01%) in 455 children with B-lineage ALL, using polymerase chain reaction amplification of antigen-receptor genes capable of detecting at least 1 leukemic cell per 100 000 normal mononucleated cells (0.001%). Of the 455 clinical samples studied on day 46 of therapy, 139 (30.5%) had MRD 0.001% or more with 63 of these (45.3%) showing levels of 0.001% to less than 0.01%, whereas 316 (69.5%) had levels that were either less than 0.001% or undetectable. MRD measurements of 0.001% to less than 0.01% were not significantly related to presenting characteristics but were associated with a poorer leukemia cell clearance on day 19 of remission induction therapy. Patients with this low level of MRD had a 12.7% (+/- 5.1%; SE) cumulative risk of relapse at 5 years, compared with 5.0% (+/- 1.5%) for those with lower or undetectable MRD (P < .047). Thus, low levels of MRD (0.001%-< 0.01%) at the end of remission induction therapy have prognostic significance in childhood ALL, suggesting that patients with this finding should be monitored closely for adverse events.

A novel approach for the analysis of T-cell reconstitution by using a T-cell receptor beta-based oligonucleotide microarray in hematopoietic stem cell transplantation.

OBJECTIVE: Analysis of T-cell population diversity is important to hematopoietic stem cell transplantation (HSCT). The millions of specificities in T-cell receptor (TCR) hypervariable complementarity- determining region 3 (CDR3) precludes detection of all T-cell populations by antibody-based flow cytometry. An alternative method, the TCR CDR3 spectratyping assay, involves multiple polymerase chain reaction (PCR) analyses and is interpreted only qualitatively. In this study, we designed the first TCRbeeta-based oligonucleotide microarray and investigated its specificity, clonality discrimination, sensitivity of detection, and feasibility for monitoring T-cell population diversity in HSCT. MATERIALS AND METHODS: The array contains 27 TCR Vbeta probes and 13 Jbeta probes. TCRbeta repertoire diversity was detected with single PCR, microarray hybridization system, and Spotfire analysis software. RESULTS: TCRO-based microarray provides specific sequence-based information and can distinguish T-cell monoclonal expansion within a polyclonal population. We successfully used this microarray to quantitatively and qualitatively analyze T-cell population diversity in recipients of hematopoietic stem cell transplants. CONCLUSION: This success suggests broad potential applications of the microarray for use in many other areas, including anti-tumor immunity, vaccination, autoimmunity, infectious diseases, and leukemia. By providing a single PCR-based assay to quantify multiple T-cell populations in parallel, this device will allow clinicians and researchers to rapidly perform high-throughput surveys.

Gene expression profiling of childhood adrenocortical tumors.

Pediatric adrenocortical tumors (ACT) are rare and often fatal malignancies; little is known regarding their etiology and biology. To provide additional insight into the nature of ACT, we determined the gene expression profiles of 24 pediatric tumors (five adenomas, 18 carcinomas, and one undetermined) and seven normal adrenal glands. Distinct patterns of gene expression, validated by quantitative real-time PCR and Western blot analysis, were identified that distinguish normal adrenal cortex from tumor. Differences in gene expression were also identified between adrenocortical adenomas and carcinomas. In addition, pediatric adrenocortical carcinomas were found to share similar patterns of gene expression when compared with those published for adult ACT. This study represents the first microarray analysis of childhood ACT. Our findings lay the groundwork for establishing gene expression profiles that may aid in the diagnosis and prognosis of pediatric ACT, and in the identification of signaling pathways that contribute to this disease.

Prediction of T-cell reconstitution by assessment of T-cell receptor excision circle before allogeneic hematopoietic stem cell transplantation in pediatric patients.

The extent and rapidity with which T cells are regenerated from graft-derived precursor cells directly influences the incidence of infection and the T-cell-based graft-versus-tumor effect. Measurement of T-cell receptor excision circles (TRECs) in peripheral blood is a means of quantifying recent thymic T-cell production and has been used after transplantation in many studies to estimate thymus-dependent T-cell reconstitution. We hypothesized that the quality of thymic function before transplantation affects thymus-dependent T-cell reconstitution after transplantation. We used real-time polymerase chain reaction (PCR) to quantify signal-joint TRECs (sjTRECs) before and after transplantation. T-cell reconstitution was evaluated by T-cell receptor beta (TCRbeta) CDR3 size spectratyping. We tested 77 healthy sibling donors and 244 samples from 26 pediatric recipients of allogeneic hematopoietic stem cell transplantation (AHSCT). Blood from the healthy donors contained 1200 to 155,000 sjTREC copies/mL blood. Patients who had greater than 1200 copies/mL blood before transplantation showed early recovery of sjTREC numbers and TCRbeta repertoire diversity. In contrast, patients who had fewer than 1200 copies/mL blood before transplantation demonstrated significantly slower restoration of thymus-dependent T cells. We conclude that the rate of reconstitution of thymus-dependent T cells is dependent on the competence of thymic function in the recipients before transplantation. Therefore, pretransplantation measurement of sjTREC may provide an important tool for predicting thymus-dependent T-cell reconstitution after transplantation.