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either PFS (P ¼ 0.63) or OS (P ¼ 0.52), nor did IgV H mutational status. However, patients with mutated IgV H had a longer treatment-free survival compared with those with unmutated IgV H (Figure 3).The overall response rate of 64% in this cohort of 126 patients with previously untreated B-CLL is comparable to results reported with intravenous fludarabine. Using the same response criteria, Rossi et al. 2 reported an overall response rate of 80% with the same schedule of oral fludarabine in a smaller group of previously untreated B-CLL patients but the proportion of patients in Rai stage III or IV in our study was higher (31% versus 22%) than in the Rossi trial.This study again confirms that the efficacy and toxicity of oral fludarabine are comparable to that of IV fludarabine given on a similar schedule. Although several randomized trials have confirmed a better overall and complete response rate for the combination of fludarabine and cyclophosphamide compared with fludarabine alone, no differences in the OS were observed. 7,8 In the British study, the oral formulation of fludarabine was introduced in both fludarabine arms when the drug became available, and differences in response rates between the two routes of administration did not appear significant. Oral fludarabine is an effective treatment for patients with previously untreated B-CLL, with less health resource utilization compared with IV administration. In an older population, the hematologic toxicity of the combination of fludarabine with cyclophosphamide and the absence of a demonstrable survival advantage, may favor initial therapy with single agent fludarabine and deferring combination therapy. In fludarabine-based combination therapies, the oral formulation should be considered an acceptable alternative. Conflict of interestThe authors declare no conflict of interest.

AS and PYJ contributed equally to this work. Acknowledgments: The authors would like to thank the contribution of patients and the clinical teams involved in providing primary leukemia samples, Dr. Miguel Abecasis for providing thymic specimens, and Dr. Adolfo Ferrando for providing some of the NOTCH sequencing primers. Funding: this work was supported by grants from Fundação para a Ciência e a Tecnologia (FCT; POCI/SAU-OBS/58913 and PTDC/SAU-OBD/69974), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 08/10034-1), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 401122/2005-0). AS and PYJ have FCT SFRH and FAPESP PhD scholarships, respectively. Manuscript received on May 27, 2009. Revised version arrived on September 18, 2009. Manuscript accepted on October 8, 2009. Correspondence: João T. Barata, Cancer Biology Unit, Instituto de Medicina Molecular, Lisbon University Medical School, Av. Prof. Egas Moniz, 1649 .pt The Online version of this article has a Supplementary Appendix.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. Haematologica. 2010;95:674-678. doi:10.3324/haematol.2009 This is an open-access paper. ABSTRACT 674haematologica | 2010; 95(4) © F e r r a t a S t o r t i F o u n d a t i o nIn some cases, exon 26 and exon 27 were amplified and sequenced from genomic DNA with the intronic primers Notch26F and Notch26R, and Notch27F and Ex27R743, respectively. The region spanning TAD and PEST domains of exon 34 was amplified with primers Notch34TF and Notch34PR, and the resulting 855 bp fragment was sequenced with primers Notch34TF and Ex34PestF. The PEST coding region was also amplified with primers Ex34PestF and Ex34PestR, followed by semi-nested PCR with primers Notch34PF and Ex34PestR, and sequenced with primer Ex34PestR. This strategy covered all mutational hot-spot regions previously reported for NOTCH1. 1,6,7 Primer sequences and the PCR protocol are shown in the Online Supplementary Appendix. All mutations were c...

BackgroundMolecular alterations occur frequently in T-ALL and the potential impact of those abnormalities on outcome is still controversial. The current study aimed to test whether NOTCH1 mutations and additional molecular abnormalities would impact T-ALL outcome in a series of 138 T-ALL paediatric cases.MethodsT-ALL subtypes, status of SIL-TAL1 fusion, ectopic expression of TLX3, and mutations in FBXW7, KRAS, PTEN and NOTCH1 were assessed as overall survival (OS) and event-free survival (EFS) prognostic factors. OS and EFS were determined using the Kaplan-Meier method and compared using the log-rank test.ResultsThe frequencies of mutations were 43.5% for NOTCH1, while FBXW7, KRAS and PTEN exhibited frequencies of 19.1%, 9.5% and 9.4%, respectively. In 78.3% of cases, the coexistence of NOTCH1 mutations and other molecular alterations was observed. In multivariate analysis no statistical association was revealed between NOTCH1 mutations and any other variable analyzed. The mean length of the follow-up was 68.4 months and the OS was 50.7%. SIL-TAL1 was identified as an adverse prognostic factor. NOTCH1 mutation status was not associated with outcome, while the presence of NOTCH1 complex mutations (indels) were associated with a longer overall survival (p = 0.031) than point mutations.ConclusionNOTCH1 mutations alone or in combination with FBXW7 did not impact T-ALL prognosis. Nevertheless, complex NOTCH1 mutations appear to have a positive impact on OS and the SIL-TAL1 fusion was validated as a negative prognostic marker in our series of T-ALL.

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 ≥10×109 cells/L (odds ratio of 8.44; 95% confidence interval [CI]=1.48-48.26; P=0.0016), initial promyelocytes ≥20×109/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 ≥20×109 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.

IntroductionMinimal residual disease is an important independent prognostic factor that can identify poor responders among patients with acute lymphoblastic leukemia.ObjectiveThe aim of this study was to analyze minimal residual disease using immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements by conventional polymerase chain reaction followed by homo-heteroduplex analysis and to compare this with real-time polymerase chain reaction at the end of the induction period in children with acute lymphoblastic leukemia.MethodsSeventy-four patients diagnosed with acute lymphoblastic leukemia were enrolled. Minimal residual disease was evaluated by qualitative polymerase chain reaction in 57 and by both tests in 44. The Kaplan–Meier and multivariate Cox methods and the log-rank test were used for statistical analysis.ResultsNine patients (15.8%) were positive for minimal residual disease by qualitative polymerase chain reaction and 11 (25%) by real-time polymerase chain reaction considering a cut-off point of 1 × 10−3 for precursor B-cell acute lymphoblastic leukemia and 1 × 10−2 for T-cell acute lymphoblastic leukemia. Using the qualitative method, the 3.5-year leukemia-free survival was significantly higher in children negative for minimal residual disease compared to those with positive results (84.1% ± 5.6% versus 41.7% ± 17.3%, respectively; p-value = 0.004). There was no significant association between leukemia-free survival and minimal residual disease by real-time polymerase chain reaction. Minimal residual disease by qualitative polymerase chain reaction was the only variable significantly correlated to leukemia-free survival.ConclusionGiven the difficulties in the implementation of minimal residual disease monitoring by real-time polymerase chain reaction in most treatment centers in Brazil, the qualitative polymerase chain reaction strategy may be a cost-effective alternative.

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Pediatric tumors share few recurrent mutations and are instead characterized by copy number alterations (CNAs). The cell-free DNA (cfDNA) is a prominent source for the detection of cancer-specific biomarkers in plasma. We profiled CNAs in the tumor tissues for further evaluation of alterations in 1q, MYCN and 17p in the circulating tumor DNA (ctDNA) in the peripheral blood at diagnosis and follow-up using digital PCR. We report that among the different kinds of tumors (neuroblastoma, Wilms tumor, Ewing sarcoma, rhabdomyosarcoma, leiomyosarcoma, osteosarcoma and benign teratoma), neuroblastoma presented the greatest amount of cfDNA, in correlation with tumor volume. Considering all tumors, cfDNA levels correlated with tumor stage, metastasis at diagnosis and metastasis developed during therapy. In the tumor tissue, at least one CNA (at CRABP2, TP53, surrogate markers for 1q and 17p, respectively, and MYCN) was observed in 89% of patients. At diagnosis, CNAs levels were concordant between tumor and ctDNA in 56% of the cases, and for the remaining 44%, 91.4% of the CNAs were present only in cfDNA and 8.6% only in the tumor. Within the cfDNA, we observed that 46% and 23% of the patients had MYCN and 1q gain, respectively. The use of specific CNAs as targets for liquid biopsy in pediatric patients with cancer can improve diagnosis and should be considered for monitoring of the disease response.