Identification of HDAC4 as a potential therapeutic target and prognostic biomarker for ZFTA-fused ependymomas.

Members of the HDAC family are predictive biomarkers and regulate the tumorigenesis in several cancers. However, the role of these genes in the biology of intracranial ependymomas (EPNs) remains unexplored. Here, an analysis of eighteen HDACs genes in an EPN transcriptomic dataset, revealed significantly higher levels of HDAC4 in supratentorial ZFTA fusion (ST-ZFTA) compared with ST-YAP1 fusion and posterior fossa EPNs, while HDAC7 and SIRT2 were downregulated in ST-ZFTA. HDAC4 was also overexpressed in ST-ZFTA as measured by single-cell RNA-Seq, quantitative real time-polymerase chain reaction, and immunohistochemistry. Survival analyses showed a significantly worse outcome for EPNs with higher HDAC4 and SIRT1 mRNA levels. Ontology enrichment analysis showed an HDAC4-high signature consistent with viral processes while collagen-containing extracellular matrix and cell-cell junction were enriched in those with an HDAC4-low signature. Immune gene analysis demonstrated a correlation between HDAC4 expression and low levels of NK resting cells. Several small molecules compounds targeting HDAC4 and ABCG2, were predicted by in silico analysis to be effective against HDAC4-high ZFTA. Our results provide novel insights into the biology of the HDAC family in intracranial ependymomas and reveal HDAC4 as a prognostic marker and potential therapeutic target in ST-ZFTA.

Early Mortality in Children and Adolescents with Acute Promyelocytic Leukemia: Experience of the Boldrini Children's Center.

INTRODUCTION: Acute promyelocytic leukemia (APL) is currently considered a highly curable disease. However, an early death (ED) remains one of the main causes of APL treatment failure. PATIENTS AND METHODS: In this retrospective study, we aimed to analyze the clinical characteristics of 91 children and adolescents with APL, who were consecutively registered at the (name of institution removed) Children's Center from January 1, 1998 to December 31, 2017. Data were assessed for age, sex, ethnicity, body mass index percentile, initial white blood cell count, peripheral blood blast count, and platelet count, hemoglobin value, partial thromboplastin time, prothrombin time, fibrinogen level, serum creatinine level, APL morphology subtype (classic vs. hypogranular variant M3v), and FLT3 gene mutations. RESULTS: ED occurred in 12 of 91 (13.1%) patients and was mainly related to cerebral thromboembolism. Overall 66% of deaths occurred in the second week after diagnosis. ED was associated with white blood cell ≥10×10 cells/L (odds ratio of 8.44; 95% confidence interval [CI]=1.48-48.26; P=0.0016), initial promyelocytes ≥20×10/L (odds ratio of 9.29; 95% CI=2.45-35.8; P=0.001), morphologic subtype M3v (odds ratio of 3.63; 95% CI=1.04-12.64; P=0.043), and creatinine serum levels >0.7 mg/dL (odds ratio of 6.78; 95% CI=1.83-25.13; P=0.004). In multivariate analyses, ED was associated with initial peripheral promyelocytes ≥20×10 blasts/L and creatinine serum levels >0.7 mg/dL. CONCLUSIONS: EDs were mainly caused by thrombohemorrhagic events and occurred within the second week after diagnosis. High peripheral promyelocytes and creatinine levels were predictors of ED in APL.

Adrenocortical tumors associated with the TP53 p. R337H germline mutation can be identified during child-care consultations / Tumores adrenocorticais associados à mutação germinativa TP53 p. R337H podem ser identificados durante as consultas de puericultura

Abstract Objective: To evaluate the clinical features associated with adrenocortical hormone overexpression and familial cancer profiling as potential markers for early detection of adrenocortical tumors in children from South and Southeast Brazil. Methods: The clinical manifestations and anthropometric measurements of 103 children diagnosed with adrenocortical tumors were analyzed. Results: Between 1982 and 2011, 69 girls and 34 boys diagnosed with adrenocortical tumors were followed-up for a median time of 9.0 years (0-34 years). Signs of androgen overproduction alone (n = 75) or associated with cortisol (n = 18) were present in 90.3%. TP53 p.R337H mutation was found in 90.5% of patients. Stages I, II, III, and IV were observed in 45.6%, 27.2%, 19.4%, and 7.8% of patients, respectively. At diagnosis, there were no significant differences in height (p = 0.92) and weight (p = 0.22) among children with adrenocortical tumors, but children with virilization alone had significantly higher height-for-age Z-scores (0.92 ± 1.4) than children with hypercortisolism alone or combined (−0.32 ± 1,8; p = 0.03). The five-year overall survival was 76.7% (SD ± 4.2). Patients with advanced-stage disease had a significantly worse prognosis than those with limited disease (p < 0.001). During follow-up, ten of 55 p.R337H carrier parents developed cancer, whereas none of the 55 non-carriers did. Conclusions: Signs of adrenocortical hormone overproduction appear early, even in cases with early-stage. These signs can be identified at the physical examination and anthropometric measurements. In southern Brazil, pediatric adrenocortical tumor is a sentinel cancer for detecting families with germline p.R337H mutation in TP53 gene.

Resumo Objetivo: Avaliar as manifestações clínicas da hiperexpressão de hormônios do córtex da adrenal e câncer familiar como marcadores para a detecção precoce de tumores adrenocorticais em crianças do Sul e Sudeste do Brasil. Pacientes e métodos: Foram analisadas as manifestações clínicas e antropométricas de 103 crianças diagnosticadas com tumores adrenocorticais. Resultados: Entre 1982 e 2011, 69 meninas e 34 meninos diagnosticados com tumores adrenocorticais foram acompanhados por um tempo mediano de nove anos (0-34). Ao diagnóstico, sinais de virilização isolada (n = 75) ou associada ao cortisol (n = 18) estavam presentes em 90,3% dos pacientes; a mutação do gene TP53 p.R337H foi identificada em 90,5% dos pacientes. Os pacientes foram classificados em estádio I (45,6%), II (27,2%), III (19,4%) e IV (7,8%). Ao diagnóstico, não houve diferença significativa para as medidas de altura (p = 0,92) e de peso (p = 0,22) entre as crianças com tumores adrenocorticais, mas crianças com virilização tiveram escore-Z mais elevado para a idade (0,92 ± 1,4) do que aquelas com hipercortisolismo isolado ou combinado (−0,32 ± 1,8; p = 0,03). A sobrevida global de cinco anos foi de 76,7% (DP ± 4,2). Pacientes com estádios avançados tiveram pior prognóstico (p < 0,001). Durante o seguimento, 10 dos 55 genitores portadores da p.R337H desenvolveram câncer, enquanto que nenhum caso ocorreu entre os 55 não portadores. Conclusões: Os sinais de hiperprodução de hormônios adrenocorticais aparecem precocemente no desenvolvimento do tumor e podem ser identificados pelo exame físico e pelas medidas antropométricas na consulta pediátrica de rotina. O tumor adrenocortical pediátrico é sentinela para a detecção de câncer em famílias que segregam a mutação germinativa p.R337H do gene TP53.

Adrenocortical tumors associated with the TP53 p.R337H germline mutation can be identified during child-care consultations.

OBJECTIVE: To evaluate the clinical features associated with adrenocortical hormone overexpression and familial cancer profiling as potential markers for early detection of adrenocortical tumors in children from South and Southeast Brazil. METHODS: The clinical manifestations and anthropometric measurements of 103 children diagnosed with adrenocortical tumors were analyzed. RESULTS: Between 1982 and 2011, 69 girls and 34 boys diagnosed with adrenocortical tumors were followed-up for a median time of 9.0 years (0-34 years). Signs of androgen overproduction alone (n=75) or associated with cortisol (n=18) were present in 90.3%. TP53 p.R337H mutation was found in 90.5% of patients. Stages I, II, III, and IV were observed in 45.6%, 27.2%, 19.4%, and 7.8% of patients, respectively. At diagnosis, there were no significant differences in height (p=0.92) and weight (p=0.22) among children with adrenocortical tumors, but children with virilization alone had significantly higher height-for-age Z-scores (0.92±1.4) than children with hypercortisolism alone or combined (-0.32±1,8; p=0.03). The five-year overall survival was 76.7% (SD±4.2). Patients with advanced-stage disease had a significantly worse prognosis than those with limited disease (p<0.001). During follow-up, ten of 55 p.R337H carrier parents developed cancer, whereas none of the 55 non-carriers did. CONCLUSIONS: Signs of adrenocortical hormone overproduction appear early, even in cases with early-stage. These signs can be identified at the physical examination and anthropometric measurements. In southern Brazil, pediatric adrenocortical tumor is a sentinel cancer for detecting families with germline p.R337H mutation in TP53 gene.

Low-dose methotrexate in sickle-cell disease: a pilot study with rationale borrowed from rheumatoid arthritis.

BACKGROUND: Inflammation is a major feature of sickle cell disease (SCD). Low-dose methotrexate (MTX) has long been used in chronic inflammatory diseases. This pilot study examined the MTX effect on acute vaso-occlusive pain crises (VOC) in SCD patients. METHODS: Fourteen adults on hydroxyurea with severe and refractory VOC received one intramuscular injection of 10 mg of MTX per week for 12 weeks. A single weekly dose of 5 mg of leucovorin was administered orally 48 h after each MTX injection. The primary outcome was reduction in number/intensity of acute pain episodes. The secondary outcomes were improvement of quality of life (QOL) and reduction of the inflammatory status. RESULTS: MTX did not significantly change the median VOC frequency (12 before vs 10.5 during treatment, P = 0.6240) or the median McGill pain index (45 at week 0 vs 39.5 at week 12, P = 0.9311). However, there was a decrease of ≥50% in chronic pain resulting from avascular osteonecrosis (AVN) in 5 out of 7 patients with radiologic evidence of AVN, with the perception of longer pain-free periods. There was a 44.4% median gain in physical function in the SF-36 QOL questionnaire (P = 0.0198). MTX treatment up-regulated two C-X-C motif chemokines (CXCL), CXCL10 (P = 0.0463) and CXCL12 (P < 0.0001), without significant effect on 14 additional plasma inflammatory markers. Adverse events: One individual had fever of unknown origin. Respiratory tract infections were recorded in five patients. Among the latter, one also had dengue fever and another had a central venous line infection and died of pneumonia and septic shock. Three patients with previous history of hydroxyurea-induced hematological toxicity developed low blood platelet counts while receiving simultaneously MTX and hydroxyurea. CONCLUSIONS: Although MTX did not reduce acute VOC frequency/intensity, it decreased chronic pain and led to QOL improvement. Trial registration http://www.who.int/ictrp/en/ and http://www.ensaiosclinicos.gov.br, RBR-2s9xvn, 19 December 2016, retrospectively registered.

Contribution of the TP53 R337H mutation to the cancer burden in southern Brazil: Insights from the study of 55 families of children with adrenocortical tumors.

BACKGROUND: The tumor protein p53 (TP53) arginine-to-histidine mutation at codon 337 (R337H) predisposes children to adrenocortical tumors (ACTs) and, rarely, to other childhood tumors, but its impact on adult cancer remains undetermined. The objective of this study was to investigate the frequency and types of cancer in relatives of children with ACT who carry the TP53 R337H mutation. METHODS: TP53 R337H testing was offered to relatives of probands with ACT. The parental lineage segregating the R337H mutation was identified in all families. The frequency and distribution of cancer types were compared according to R337H status. The authors' data also were compared with those publicly available for children with TP53 mutations other than R337H. RESULTS: The mean and median follow-up times for the probands with ACT were 11.2 years and 9.7 years (range, 3-32 years), respectively. During this time, cancer was diagnosed in 12 of 81 first-degree relatives (14.8%) carrying the R337H mutation but in only 1 of 94 noncarriers (1.1%; P = .0022). At age 45 years, the cumulative risk of cancer was 21% (95% confidence interval, 5%-33%) in carriers and 2% (95% confidence interval, 0%-4%) in noncarriers (P = .008). The frequency of cancer was higher in the R337H segregating lineages than in the nonsegregating lineages (249 of 1410 vs 66 of 984 individuals; P < .001). Breast and gastric cancer were the most common types. CONCLUSIONS: TP53 R337H carriers have a lifelong predisposition to cancer with a bimodal age distribution: 1 peak, represented by ACT, occurs in the first decade of life, and another peak of diverse cancer types occurs in the fifth decade. The current findings have implications for genetic counseling and surveillance of R337H carriers. Cancer 2017;123:3150-58. © 2017 American Cancer Society.

Shorter Maintenance Therapy in Childhood Acute Lymphoblastic Leukemia: The Experience of the Prospective, Randomized Brazilian GBTLI ALL-93 Protocol.

AIM: Maintenance therapy is an important phase of the childhood ALL treatment, requiring 2-year long therapy adherence of the patients and families. Weekly methotrexate with daily 6-mercaptopurine (6MP) constitutes the backbone of maintenance therapy. Reduction in the maintenance therapy could overweight problems related with poverty of children with ALL living in limited-income countries (LIC). OBJECTIVE: To compare, prospectively, the EFS rates of children with ALL treated according to two maintenance regimens: 18 vs. 24 months duration. MATERIALS AND METHODS: From October 1993 to September 1999, 867 consecutive untreated ALL patients <18 years of age were treated according to the Brazilian Cooperative Group for Childhood ALL Treatment (GBTLI) ALL-93 protocol. Risk classification was based exclusively on patient's age and leukocyte count (NCI risk group) and clinical extra medullary involvement of the disease. Data were analyzed by the intention-to-treat approach. RESULTS: Fourteen patients (1.6%) were excluded: wrong diagnosis (n = 7) and previous corticosteroid (n = 7). Of the 853 eligible patients, 421 were randomly allocated, at study enrollment, to receive 18-month (group 1) and 432 to receive 24-month (group 2) maintenance therapy. Complete remission rate was achieved in 96% of the patients (817/853). Twenty-eight patients (3.4%) died during the induction phase. Thirty-four patients (4.0%) were lost to follow-up. The overall EFS was 66.1 ± 1.7% at 15 years. No difference was seen according to maintenance: EFS15y was 65.8 ± 2.3% (group 1) and 66.3 ± 2.3% (group 2; p = 0.79). No difference between regimens was detected after stratifying the analyses according to factors associated with adverse prognosis in this study (age group <1 year or >10 years and high WBC at diagnosis). Overall death in remission rate was 6.85% (56 patients). Deaths during maintenance were 13 in group 1 and 12 in group 2, all due to infection. Over 15 years of follow-up, two patients both from group 2 presented a second malignancy (Hodgkin's disease and thyroid carcinoma) after 8.3 and 11 years off therapy, respectively. CONCLUSION: Six-month reduction of maintenance therapy in ALL children treated according to the GBTLI ALL-93 protocol provided the same overall outcome as 2-year duration regimen.

Unraveling the expression of the oncogene YAP1, a Wnt/beta-catenin target, in adrenocortical tumors and its association with poor outcome in pediatric patients.

BACKGROUND: Overexpression of the oncogene yes-associated-protein-1 (YAP1) is associated with increased cell proliferation in human cancers. YAP1 is a potential target of the Wnt/beta-catenin pathway, which plays an important role in adrenocortical tumors (ACT). The role of YAP1 in adrenocortical tumorigenesis has not been assessed. AIMS: To evaluate YAP1 expression in normal adrenals and pediatric ACT and its association with disease outcome. To investigate the interaction between YAP1 and the Wnt/beta-catenin pathway in adrenocortical cells. RESULTS: Strong YAP1 staining was present in fetal adrenals and pediatric ACT but weak in postnatal adrenals. In pediatric ACT, YAP1 mRNA overexpression was associated with death, recurrent/metastatic disease and lower overall survival. The inhibition of the Wnt/beta-catenin pathway increased YAP1 mRNA expression. siYAP1 increased CTNNB1/beta-catenin expression and nuclear staining regardless of DLV2, moreover, it decreased cell growth and impaired cell migration. MATERIALS AND METHODS: We assessed in 42 pediatric ACT samples the YAP1 protein expression by immunohistochemistry and mRNA expression by RT-qPCR and analyzed their association with outcome. As controls, we resort 32 fetal and postnatal normal adrenals for IHC and 10 normal adrenal cortices for RT-qPCR. The interaction between YAP1 and the Wnt/beta-catenin pathway was assessed in NCI-H295 adrenocortical cells by inhibiting the TCF/beta-catenin complex and by knocking down YAP1. CONCLUSION: YAP1 overexpression is a marker of poor prognosis for pediatric patients with ACT. In adrenocortical cells, there is a close crosstalk between YAP1 and Wnt/beta-catenin. These data open the possibility of future molecular therapies targeting Hippo/YAP1 signaling to treat advanced ACT.

Lymphotoxin-ß receptor in microenvironmental cells promotes the development of T-cell acute lymphoblastic leukaemia with cortical/mature immunophenotype.

Lymphotoxin-mediated activation of the lymphotoxin-ß receptor (LTßR; LTBR) has been implicated in cancer, but its role in T-cell acute lymphoblastic leukaemia (T-ALL) has remained elusive. Here we show that the genes encoding lymphotoxin (LT)-α and LTß (LTA, LTB) are expressed in T-ALL patient samples, mostly of the TAL/LMO molecular subtype, and in the TEL-JAK2 transgenic mouse model of cortical/mature T-ALL (Lta, Ltb). In these mice, expression of Lta and Ltb is elevated in early stage T-ALL. Surface LTα1 ß2 protein is expressed in primary mouse T-ALL cells, but only in the absence of microenvironmental LTßR interaction. Indeed, surface LT expression is suppressed in leukaemic cells contacting Ltbr-expressing but not Ltbr-deficient stromal cells, both in vitro and in vivo, thus indicating that dynamic surface LT expression in leukaemic cells depends on interaction with its receptor. Supporting the notion that LT signalling plays a role in T-ALL, inactivation of Ltbr results in a significant delay in TEL-JAK2-induced leukaemia onset. Moreover, young asymptomatic TEL-JAK2;Ltbr(-/-) mice present markedly less leukaemic thymocytes than age-matched TEL-JAK2;Ltbr(+/+) mice and interference with LTßR function at this early stage delayed T-ALL development. We conclude that LT expression by T-ALL cells activates LTßR signalling in thymic stromal cells, thus promoting leukaemogenesis.

Low expression of HLA-DRA, HLA-DPA1, and HLA-DPB1 is associated with poor prognosis in pediatric adrenocortical tumors (ACT).

BACKGROUND: Low expression of HLA class II antigens has been associated with more aggressive disease in several human malignancies including adult adrenocortical tumors (ACT), but their clinical relevance in pediatric ACT needs to be investigated. PROCEDURE: This study analyzed the expression profile of three class II histocompatibility genes (HLA-DRA, HLA-DPA1, and HLA-DPB1) in 58 consecutive pediatric ACT (13 adenomas and 45 carcinomas) by quantitative real time PCR and their association with clinical and biological features. HLA-DPA1 protein level was determined by immunohistochemistry. RESULTS: A significant association (P < 0.01) was observed between lower expression levels of the three genes analyzed and poor prognostic factors such as age ≥ 4 years, tumor size ≥ 200 cm(3), tumor weight ≥ 100 g, and metastatic disease; the presence of an unfavorable event and death. Underexpression of the HLA-DRA, HLA-DPA1, and HLA-DPB1 genes were associated with lower 5-year event-free survival (EFS) (P = 0.017, P < 0.001, and P = 0.017, respectively). Cox multivariate analysis showed that HLA-DPA1 was an independent prognostic factor (P = 0.029) when analyzed in association with stage IV, age and tumor size. Significantly lower EFS was also observed in patients with negative/weak immunostaining for HLA-DPA1 (P = 0.002). Similar results were observed when only patients classified as having carcinomas were analyzed. CONCLUSION: Our results suggest that lower expression of HLA-DRA, HLA-DPA1, and HLA-DPB1 genes may contribute to more aggressive disease in pediatric ACT. HLA-DPA1 immunostaining may represent potential aggressiveness marker in this tumor.

Sonic hedgehog signaling is active in human adrenal cortex development and deregulated in adrenocortical tumors.

BACKGROUND: The sonic hedgehog (SHH) pathway plays a key role in rodent adrenal cortex development and is involved in tumorigenesis in several human tissues, but data in human adrenal glands are limited. OBJECTIVES: The objectives of the study were to analyze the involvement of the SHH pathway in human adrenal development and tumorigenesis and the effects of SHH inhibition on an adrenocortical tumor (ACT) cell line. PATIENTS AND METHODS: Expression of SHH pathway components was evaluated by immunohistochemistry in 51 normal adrenals (33 fetal) and 34 ACTs (23 pediatric) and by quantitative PCR in 81 ACTs (61 pediatric) and 19 controls (10 pediatric). The effects of SHH pathway inhibition on gene expression and cell viability in the NCI-H295A adrenocortical tumor cell line after cyclopamine treatment were analyzed. RESULTS: SHH pathway proteins were present in fetal and postnatal normal adrenals and showed distinct patterns of spatiotemporal expression throughout development. Adult adrenocortical carcinomas presented with higher expression of PTCH1, SMO, GLI3, and SUFU compared with normal adult adrenal cortices. Conversely, pediatric ACTs showed lower mRNA expression of SHH, PTCH1, SMO, GLI1, and GLI3 compared with normal pediatric adrenal cortices. In vitro treatment with cyclopamine resulted in decreased GLI3, SFRP1, and CTNNB1 mRNA expression and ß-catenin staining as well as decreased cell viability. CONCLUSIONS: The SHH pathway is active in human fetal and postnatal adrenals, up-regulated in adult adrenocortical carcinomas, and down-regulated in pediatric ACTs. SHH pathway antagonism impaired cell viability. The SHH pathway is deregulated in ACTs and might provide a new target therapy to be explored.

Altered expression of noncanonical Wnt pathway genes in paediatric and adult adrenocortical tumours.

CONTEXT: The role of planar cell polarity (Wnt/PCP) and calcium-dependent (Wnt/Ca) noncanonical Wnt pathways in adrenocortical tumours (ACTs) is unknown. OBJECTIVES: To investigate the gene expression of Wnt/PCP and Wnt/Ca pathways and its association with TP53 p.R337H and CTNNB1 mutations in paediatric and adult ACTs and to correlate these findings with clinical outcome. PATIENTS: Expression of noncanonical Wnt-related genes was evaluated in 91 ACTs (66 children and 25 adults) by qPCR and the expression of beta-catenin, P53 and protein effectors of Wnt/Ca (NFAT) and Wnt/PCP (JNK) by immunohistochemistry. TP53 and CTNNB1 genes were sequenced. RESULTS: TP53 p.R337H mutation frequency was higher in children (86% vs 28%), while CTNNB1 mutation was higher in adults (32% vs 6%). Mortality was higher in adults harbouring TP53 p.R337H and in children with CTNNB1 mutations. Overexpression of WNT5A, Wnt/Ca ligand, was observed in children and adults. Overexpression of MAPK8 and underexpression of PRICKLE, Wnt/PCP mediators, were observed in paediatric but not in adult cases. Cytoplasmic/nuclear beta-catenin and P53 accumulation was observed in the majority of paediatric and adult ACTs as well as NFAT and JNK. Overexpression of MAPK8 and underexpression of PRICKLE were associated with mortality in children, while overexpression of WNT5A and underexpression of PRICKLE were associated with mortality in adults. CONCLUSIONS: In our study, TP53 p.R337H and CTNNB1 mutations correlated with poor prognosis in adults and children, respectively. We demonstrate, for the first time, the activation of Wnt/PCP and Wnt/Ca noncanonical pathway genes, and their association with poor outcome in children and adults, suggesting their putative involvement in ACTs aggressiveness.

Retinoblastoma diagnosis: a proposal based on the experience of Centro Infantil Boldrini, Brazil.

Advanced disease is a risk factor for eye loss in patients with retinoblastoma (RB). We still record critical rates of enucleation, especially for unilateral RB due to advanced stages of disease at diagnosis. This retrospective study of 223 RB patient records referred to treatment at Centro Infantil Boldrini, Brazil, between 1978 and 2008, showed that 176 patients (79%) presented intraocular tumors while 47 (21%) already had extraocular involvement. At the time of diagnosis, the age of patients was 26.2 months in the group that had enucleated eyes and 13.7 months in the group that preserved both eyes. Under a multiple logistic regression model, familial history (OR = 0.195; p = .01) and age at diagnosis in months (OR = 1.047; p = .04) were significantly correlated with enucleation. Strategies to early detect RB must be changed in order to offer better chances of ocular preservation with visual function. Authors propose a systematic referral of all children to the ophthalmologist for an indirect ophthalmoscopy once a year in the first two years of life, as a measure to be adopted by all pediatricians in daily routine to early detect the tumor.

Gene expression pattern contributing to prognostic factors in childhood acute lymphoblastic leukemia.

The present study evaluated the expression profile of 19 genes previously reported in microarray studies and associated with resistance or sensitivity to vincristine (RPLP2, CD44, TCFL5, KCNN1, TRIM24), prednisolone (F8A, CDK2AP1, BLVRB, CD69), daunorubicin (MAP3K12, SHOC2, PCDH9, EGR1, KCNN4) and l-asparaginase (GPR56, MAN1A1, CLEC11A, IGFBP7, GATA3). We studied 140 bone marrow samples at diagnosis from children with acute lymphoblastic leukemia (ALL) treated according to the Brazilian Childhood Leukemia Treatment Group (GBTLI) ALL-99 protocol. The expression profiles of the genes listed above were analyzed by real-time quantitative polymerase chain reaction (PCR) and then related to the clinical and biological prognostic factors. The results showed significant associations (p ≤ 0.05) between the expression levels of genes GPR56, BLVRB, IGFBP7 and white blood cell (WBC) count at diagnosis; GATA3, MAN1A1, CD44, MAP3K12, CLEC11A, SHOC2 and CD10 B-lineage ALL; TCFL5 and bone marrow status at day 14; MAP3K12 and TRIM24 and bone marrow status at day 28; and CD69, TCFL5 and TRIM24 genes and ETV6/RUNX1 positive ALL. The up-regulation of SHOC2 was also associated with better 5-year event-free survival (EFS) in univariate and multivariate analysis (p = 0.02 and p = 0.03, respectively). These findings highlight genes that could be associated with clinical and biological prognostic factors in childhood ALL, suggesting that these genes may characterize and play a role in the treatment outcome of some ALL subsets.

Oncogenic IL7R gain-of-function mutations in childhood T-cell acute lymphoblastic leukemia.

Interleukin 7 (IL-7) and its receptor, formed by IL-7Rα (encoded by IL7R) and γc, are essential for normal T-cell development and homeostasis. Here we show that IL7R is an oncogene mutated in T-cell acute lymphoblastic leukemia (T-ALL). We find that 9% of individuals with T-ALL have somatic gain-of-function IL7R exon 6 mutations. In most cases, these IL7R mutations introduce an unpaired cysteine in the extracellular juxtamembrane-transmembrane region and promote de novo formation of intermolecular disulfide bonds between mutant IL-7Rα subunits, thereby driving constitutive signaling via JAK1 and independently of IL-7, γc or JAK3. IL7R mutations induce a gene expression profile partially resembling that provoked by IL-7 and are enriched in the T-ALL subgroup comprising TLX3 rearranged and HOXA deregulated cases. Notably, IL7R mutations promote cell transformation and tumor formation. Overall, our findings indicate that IL7R mutational activation is involved in human T-cell leukemogenesis, paving the way for therapeutic targeting of IL-7R-mediated signaling in T-ALL.

Wnt/beta-catenin pathway deregulation in childhood adrenocortical tumors.

CONTEXT: CTNNB1/ß-catenin mutations and activation of Wnt/ß-catenin pathway are frequent in adult adrenocortical tumors (ACT), but data on childhood ACT are lacking. OBJECTIVE: The aim of the study was to investigate the presence of Wnt/ß-catenin pathway abnormalities in childhood ACT. PATIENTS AND METHODS: Clinicopathological findings and outcome of 62 childhood ACT patients were analyzed regarding CTNNB1 mutations and the expression of Wnt-related genes (CTNNB1; WNT4, a Wnt ligand; SFRP1, DKK3, and AXIN1, Wnt inhibitors; TCF7, a transcription factor; and MYC and WISP2, target genes) by quantitative PCR and immunohistochemistry. RESULTS: CTNNB1-activating mutations were found in only four of 62 ACT (6%), all of them harboring TP53 mutation. There was association between the presence of CTNNB1 mutations and death (P = 0.02). Diffuse ß-catenin accumulation was found in 71% of ACT, even in ACT without CTNNB1 mutations. Compared to normal adrenals, ACT presented increased expression of CTNNB1 (P = 0.008) and underexpression of Wnt inhibitor genes: DKK3 (P < 0.0001), SFRP1 (P = 0.05), and AXIN1 (P = 0.04). With regard to Wnt/ß-catenin target genes, ACT presented increased expression of WISP2 but lower expression of MYC. Higher overall survival was associated with underexpression of SFRP1 (P = 0.01), WNT4 (P = 0.004), and TCF7 (P < 0.01). CONCLUSIONS: CTNNB1 mutations are not common in childhood ACT but appear to associate with poor prognosis. Nevertheless, most ACT exhibit increased expression of ß-catenin and WISP2 and reduced expression of Wnt inhibitor genes (DKK3, SFRP1, and AXIN1). Thus, in addition to CTNNB1 mutations, other genetic events affecting the Wnt/ß-catenin pathway may be involved in childhood adrenocortical tumorigenesis.

Low mRNA expression of the apoptosis-related genes CASP3, CASP8, and FAS is associated with low induction treatment response in childhood acute lymphoblastic leukemia (ALL).

BACKGROUND: Defects in apoptosis signaling have been considered to be responsible for treatment failure in many types of cancer, although with controversial results. The objective of the present study was to assess the expression profile of key apoptosis-related genes in terms of clinical and biological variables and of the survival of children with acute lymphoblastic leukemia (ALL). PROCEDURE: The levels of mRNA expression of the apoptosis-related genes CASP3, CASP8, CASP9, FAS, and BCL2 were analyzed by quantitative real-time PCR in consecutive samples from 139 consecutive children with ALL at diagnosis treated by the Brazilian protocol (GBTLI-ALL 99). Gene expression levels and clinical and biological features were compared by the Mann-Whitney test. Event-free survival (EFS) was calculated by Kaplan-Meier plots and log-rank test. RESULTS: A significant correlation was detected between CASP3, CASP8, CASP9, and FAS expression levels (P < 0.01) in ALL samples. Higher levels of BCL2 were significantly associated with white blood cell (WBC) count <50,000/mm(3) at diagnosis (P = 0.01) and low risk group classification (P = 0.008). Lower expression levels of CASP3, CASP8 and FAS gene were associated with a poor response at day 7 according the GBTLI-ALL 99 protocol (P = 0.03, P = 0.02 and P = 0.008, respectively). There was a relationship between FAS gene expression lower than the 75th percentile and lower 5-year EFS (P = 0.02). CONCLUSION: These findings suggest an association between lower expression levels of the pro-apoptotic genes and a poor response to induction therapy at day 7 and prognosis in childhood ALL.

Benefits of the intermittent use of 6-mercaptopurine and methotrexate in maintenance treatment for low-risk acute lymphoblastic leukemia in children: randomized trial from the Brazilian Childhood Cooperative Group--protocol ALL-99.

PURPOSE To describe event-free survival (EFS) and toxicities in children with low-risk acute lymphoblastic leukemia (ALL) assigned to receive either continuous 6-mercaptopurine (6-MP) and weekly methotrexate (MTX) or intermittent 6-MP with intermediate-dose MTX, as maintenance treatment. PATIENTS AND METHODS Between October 1, 2000, and December 31, 2007, 635 patients with low-risk ALL were enrolled onto Brazilian Childhood Cooperative Group for ALL Treatment (GBTLI) ALL-99 protocol. Eligible children (n = 544) were randomly allocated to receive either continuous 6-MP/MTX (group 1, n = 272) or intermittent 6-MP (100 mg/m(2)/d for 10 days, with 11 days resting) and MTX (200 mg/m(2) every 3 weeks; group 2, n = 272). RESULTS The 5-year overall survival (OS) and EFS were 92.5% +/- 1.5% SE and 83.6% +/- 2.1% SE, respectively. According to maintenance regimen, the OS was 91.4% +/- 2.2% SE (group 1) and 93.6% +/- 2.1% SE (group 2; P = .28) and EFS 80.9% +/- 3.2% SE (group 1) and 86.5% +/- 2.8% SE (group 2; P = .089). Remarkably, the intermittent regimen led to significantly higher EFS among boys (85.7% v 74.9% SE; P = .027), while no difference was seen for girls (87.0% v 88.8% SE; P = .78). Toxic episodes were recorded in 226 and 237 children, respectively. Grade 3 to 4 toxic events for groups 1 and 2 were, respectively, 273 and 166 for hepatic dysfunction (P = .002), and 772 and 636 for hematologic episodes (P = .005). Deaths on maintenance were: seven (group 1) and one (group 2). CONCLUSION The intermittent use of 6-MP and MTX in maintenance is a less toxic regimen, with a trend toward better long-term EFS. Boys treated with the intermittent schedule had significantly better EFS.

Regulation of PTEN by CK2 and Notch1 in primary T-cell acute lymphoblastic leukemia: rationale for combined use of CK2- and gamma-secretase inhibitors.

T-cell acute lymphoblastic leukemia (T-ALL) patients frequently display NOTCH1 activating mutations and Notch can transcriptionally down-regulate the tumor suppressor PTEN. However, it is not clear whether NOTCH1 mutations associate with decreased PTEN expression in primary T-ALL. Here, we compared patients with or without NOTCH1 mutations and report that the former presented higher MYC transcript levels and decreased PTEN mRNA expression. We recently showed that T-ALL cells frequently display CK2-mediated PTEN phosphorylation, resulting in PTEN protein stabilization and concomitant functional inactivation. Accordingly, the T-ALL samples analyzed, irrespectively of their NOTCH1 mutational status, expressed significantly higher PTEN protein levels than normal controls. To evaluate the integrated functional impact of Notch transcriptional and CK2 post-translational inactivation of PTEN, we treated T-ALL cells with both the gamma-secretase inhibitor DAPT and the CK2 inhibitors DRB/TBB. Our data suggest that combined use of gamma-secretase and CK2 inhibitors may have therapeutic potential in T-ALL.