Acute lymphoblastic leukemia (ALL) in infants (< 1 year) is characterized by a poor prognosis and a high incidence of MLL translocations. Several studies demonstrated the unique gene expression profile associated with MLL-rearranged ALL, but generally small cohorts were analyzed as uniform patient groups regardless of the type of MLL translocation, whereas the analysis of translocationnegative infant ALL remained unacknowledged. Here we generated and analyzed primary infant ALL expression profiles (n ‫؍‬ 73) typified by translocations t(4;11), t(11;19), and t(9;11), or the absence of MLL translocations. Our data show that MLL germline infant ALL specifies a gene expression pattern that is different from both MLL-rearranged infant ALL and pediatric precursor B-ALL. Moreover, we demonstrate that, apart from a fundamental signature shared by all MLL-rearranged infant ALL samples, each type of MLL translocation is associated with a translocation-specific gene expression signature. Finally, we show the existence of

Acute lymphoblastic leukemia (ALL) diagnosed in the first month of life (congenital ALL) is very rare. Although congenital ALL is often assumed to be fatal, no studies have been published on outcome except for case reports. The present study reports the outcome of 30 patients with congenital ALL treated with the uniform Interfant-99 protocol, a hybrid regimen combining ALL treatment with elements designed for treatment of acute myeloid leukemia. Congenital ALL was characterized by a higher white blood cell count and a strong trend for higher incidence of MLL rearrangements and CD10-negative B-lineage ALL compared with older infants. Induction failure rate was 13% and not significantly different from that in older infants (7%, P ‫؍‬ .14), but relapse rate was significantly higher in congenital ALL patients (2-year cumulative incidence [ IntroductionAcute lymphoblastic leukemia (ALL) in infants (up to 1 year of age) is known to be biologically different from ALL in older children diagnosed with ALL. ALL in infants is more often associated with a higher tumor load at diagnosis, 1,2 a rearrangement in the mixed lineage leukemia (MLL) gene, and very immature B-cell phenotype (pro-B ALL) without CD10 expression. 1-3 Infant ALL cells are more resistant to several standard chemotherapeutic agents, 3,4 and the disease is also characterized by a poorer prognosis compared with older children. [5][6][7][8][9][10][11][12][13][14] Congenital ALL is diagnosed at birth or within the first month of life and is very rare. Although it is assumed to be inevitably fatal and the toxicity of the chemotherapeutic agents in these very young infants is unclear, to the best of our knowledge, no series have been published on congenital ALL except for case reports. Bresters et al 15 reviewed 24 patients with congenital ALL diagnosed over 25 years who were described in case reports: all patients died.We recently reported the results of a large international collaborative trial, Interfant-99, in infants younger than 1 year with ALL. 14 Here, we detail the outcome and characteristics of 30 patients with congenital ALL who received uniform therapy with curative intent. Methods PatientsThe Interfant-99 trial design, the inclusion criteria, and recruitment methods have been published earlier. 14 Individual study groups obtained ethics approval from all participating institutions, and patient informed consent was obtained in accordance with the Declaration of Helsinki. Of the 518 infants diagnosed with ALL, which account for approximately 3% of the ALL population, 35 patients were younger than 1 month, confirming the rarity of congenital ALL (ϳ 2 children per every 1000 with ALL). The present study reports on 30 cases that were treated with Interfant-99 (Figure 1). ProceduresEnrolled patients were stratified into standard-risk and high-risk groups on the basis of their response to 1 week of daily systemic prednisone (at a dose of 60 mg/m 2 ) and 1 intrathecal dose of methotrexate. Patients were classified as standard risk if their peripheral bloo...

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Acute lymphoblastic leukemia in infants (< 1 year-of-age) is characterized by a high incidence of MLL rearrangements. Recently, direct targets of the MLL fusion protein have been identified. However, functional validation of the identified targets remained unacknowledged. In this study, we identify CDK6 as a direct target of the MLL fusion protein and an important player in the proliferation advantage of MLL-rearranged leukemia. CDK6 mRNA was significantly higher expressed in MLL-rearranged infant ALL patients compared with MLL wild-type ALL patients (P < 0.001). Decrease of MLL-AF4 and MLL-ENL fusion mRNA expression by siRNAs resulted in downregulation of CDK6, affirming a direct relationship between the presence of the MLL fusion and CDK6 expression. Knockdown of CDK6 itself significantly inhibited proliferation in the MLL-AF4-positive cell line SEM, whereas knockdown of the highly homologous gene CDK4 had virtually no effect on the cell cycle. Furthermore, we show in vitro sensitivity of MLL-rearranged leukemia cell lines to the CDK4/6-inhibitor PD0332991, inducing a remarkable G 1 arrest, and downregulation of its downstream targets pRB1 and EZH2. We therefore conclude that CDK6 is indeed a direct target of MLL fusion proteins, playing an important role in the proliferation advantage of MLL-rearranged ALL cells.

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1426 MLL-rearranged acute lymphoblastic leukemia represents a highly aggressive and clinically unfavorable type of childhood leukemia, displaying unique gene expression signatures. Nevertheless, the overwhelming number of differentially expressed genes made it difficult to elucidate the actual “drivers” of the leukemia. However, recent advances demonstrated that MLL fusion proteins recruit the histone methyltransferase DOT1L, leading to H3K79 methylation. Hence, genomic regions displaying aberrant enrichment of H3K79 methylation are prone to mark genes transcriptionally activated by the MLL fusion protein itself. Based on this concept, two independent studies recently identified gene signatures consisting of genes likely to represent direct MLL fusion targets. Yet, functional validation of such genes so far remains unacknowledged. In the present study we confirmed that CDK6 (cyclin-dependent protein kinase 6) represents a direct target of MLL-AF4 in t(4;11)-positive ALL cells. In contrast to its functional homologue CDK4, ChIP-sequencing analysis showed the presence of both MLL and AF4, as well as H3K79 methylation at the CDK6. Moreover, CDK6 mRNA appeared significantly (p<0.001) higher expressed in primary MLL- rearranged infant ALL patient samples when compared with other childhood ALL subtypes without translocations of the MLL gene. Next, using RNA interference, we performed MLL-AF4 and MLL-ENL knockdown experiments in ALL cell lines bearing these corresponding fusion transcripts, resulting in CDK6 down-regulation, whereas CDK4 expression was unaffected. These results emphasize that CDK6 is indeed a genuine transcriptional target of the MLL fusion protein itself. Moreover, direct knockdown of CDK6 itself significantly inhibited proliferation in MLL-rearranged ALL cells, whereas knockdown of CDK4 virtually had no effect on the cell cycle in these cells. Taken together we conclude that CDK6 up-regulation in MLL-rearranged ALL is directly mediated by the MLL fusion itself and provides these cells with a proliferation advantage. Disclosures: No relevant conflicts of interest to declare.

Introduction MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is characterized by high relapse rates and a dismal prognosis. To facilitate the discovery of novel therapeutic targets, we here searched for genes directly influenced by the repression of various MLL fusions.MethodsFor this, we performed gene expression profiling after siRNA-mediated repression of MLL-AF4, MLL-ENL, and AF4-MLL in MLL-rearranged ALL cell line models. The obtained results were compared with various already established gene signatures including those consisting of known MLL-AF4 target genes, or those associated with primary MLL-rearranged infant ALL samples.ResultsGenes that were down-regulated in response to the repression of MLL-AF4 and MLL-ENL appeared characteristically expressed in primary MLL-rearranged infant ALL samples, and often represented known MLL-AF4 targets genes. Genes that were up-regulated in response to the repression of MLL-AF4 and MLL-ENL often represented genes typically silenced by promoter hypermethylation in MLL-rearranged infant ALL. Genes that were affected in response to the repression of AF4-MLL showed significant enrichment in gene expression profiles associated with AF4-MLL expressing t(4;11)+ infant ALL patient samples.ConclusionWe conclude that the here identified genes readily responsive to the loss of MLL fusion expression potentially represent attractive therapeutic targets and may provide additional insights in MLL-rearranged acute leukemias.

PURPOSE Adjuvant use of Neutral Argon Plasma (PlasmaJet Surgical Device) during cytoreductive surgery (CRS) for advanced-stage epithelial ovarian cancer improves surgical outcomes. The aim of this study is to examine the costs of adjuvant use of the PlasmaJet during surgery compared with conventional CRS in advanced-stage epithelial ovarian cancer. MATERIALS AND METHODS The patients were randomly assigned to surgery with or without the PlasmaJet. Analysis of the intra- and extramural health care costs was performed. Costs were divided into three categories: costs of the diagnostic phase (T1), inpatient care up to discharge including costs of surgery (T2), and outpatient care including chemotherapy until 6 weeks after the last cycle of chemotherapy (T3). RESULTS Overall, 327 patients underwent CRS (surgery with PlasmaJet: n = 157; conventional surgery: n = 170). The mean total health costs were significantly higher for CRS with adjuvant use of PlasmaJet compared with conventional CRS (€19,414 v €18,165, P = .017). Costs are divided into costs of the diagnostic phase (€2,034 v €1,974, P = .890), costs of inpatient care (€10,956 v €9,556, P = .003), and costs of outpatient care (€6,417 v €6,628, P = .147). CONCLUSION Mean total health care costs of the use of PlasmaJet in CRS were significantly higher than those for conventional CRS. This difference is fully explained by the additional surgery costs of the use of PlasmaJet. However, surgery with the use of the PlasmaJet leads to a significantly higher percentage of complete CRS and a halving of stomas. A cost-effectiveness analysis will be performed once survival data are available (funded by ZonMw, Trial Register NL62035.078.17).