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Checkpoint kinase 1 (CHK1) is a key component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent DNA damage response pathway that protect cells from replication stress, a cell intrinsic phenomenon enhanced by oncogenic transformation. Here, we show that CHK1 is overexpressed and hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). CHEK1 mRNA is highly abundant in patients of the proliferative T-ALL subgroup and leukemia cells exhibit constitutively elevated levels of the replication stress marker phospho-RPA32 and the DNA damage marker γH2AX. Importantly, pharmacologic inhibition of CHK1 using PF-004777736 or CHK1 short hairpin RNA-mediated silencing impairs T-ALL cell proliferation and viability. CHK1 inactivation results in the accumulation of cells with incompletely replicated DNA, ensuing DNA damage, ATM/CHK2 activation and subsequent ATM- and caspase-3-dependent apoptosis. In contrast to normal thymocytes, primary T-ALL cells are sensitive to therapeutic doses of PF-004777736, even in the presence of stromal or interleukin-7 survival signals. Moreover, CHK1 inhibition significantly delays in vivo growth of xenotransplanted T-ALL tumors. We conclude that CHK1 is critical for T-ALL proliferation and viability by downmodulating replication stress and preventing ATM/caspase-3-dependent cell death. Pharmacologic inhibition of CHK1 may be a promising therapeutic alternative for T-ALL treatment. [ABSTRACT FROM AUTHOR]

Specific defects in DNA repair pathways are reflected by DNA microsatellite instability (MSI) and play an important role in carcinogenesis. Reported frequencies in gastric non-Hodgkin's lymphomas (NHL) vary from 14% to as high as 90%. Another form of genetic instability in tumours is allelic imbalance (AI) due to loss or gain of genetic material at a specific chromosomal region. This might point to the presence of a tumour suppressor gene or oncogene. We examined both MSI and AI in 26 gastric lymphomas (10 low-grade and 13 high-grade MALT lymphomas and three cases lacking MALT features and categorised as diffuse large B cell lymphoma (DLCL)). Tumour components and normal cells (epithelium, muscle) were microdissected from paraffin-embedded resection samples. Contrary to other studies we did not observe frequent MSI when investigating 18 different loci distributed over 12 chromosomes. Microsatellite instability of a single locus was found in 1/10 (10%) low-grade MALT lymphomas and 2/13 (15%) high-grade MALT lymphomas. These data indicate that DNA mismatch repair genes do not play a role in the pathogenesis of these lymphomas. Allelic imbalance was detected in 60% (6/10) of low-grade MALT lymphomas, in 62% (8/13) of high-grade MALT lymphoma and in 67% (2/3) of DLCL. In high-grade lymphomas more loci showed AI (one to seven loci, with a mean of 2.5 loci per case) than in the low-grade lymphomas (one to two loci, with a mean of 1.3 loci per case), possibly reflecting an increased genomic instability. [ABSTRACT FROM AUTHOR]

The t(14;18) is the most frequent chromosomal aberration observed in follicular non-Hodgkin's lymphoma (NHL), and less frequently in diffuse large cell lymphoma (DLCL). The t(14;18) does not affect overall survival in follicular NHL or DLCL. It provides a unique lymphoma-specific marker that can be used to quantify residual disease during treatment, and may forward evaluation of new treatment protocols. The t(14;18) results in the deregulation of bcl-2, a proto-oncogene that protects against induction of programmed cell death (PCD). Nowadays, a whole family of bcl-2-related genes has been identified and consist of members which can either protect or promote induction of PCD. The family members form protein dimers, and the relative abundance of specific dimers regulates the cellular sensitivity to death signals (the rheostat model). Aberrant expression of different family members in several types of malignancies alter the cellular rheostat and thereby promote cellular resistance to chemotherapy. [ABSTRACT FROM AUTHOR]

MEF2C is a selectively expressed transcription factor involved in different transcriptional complexes. Originally identified as an essential regulator of muscle development, ectopic expression of MEF2C as a result of chromosomal rearrangements is now linked to leukemia. Specifically, high MEF2C expression has been linked to mixed lineage leukemia-rearranged acute myeloid leukemia as well as to the immature subgroup of T-cell acute lymphoblastic leukemia. This review focuses on the role of MEF2C in the hematopoietic system and on aberrant MEF2C expression in human leukemia. [ABSTRACT FROM AUTHOR]

Children with Down's syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL) and have a low frequency of established genetic aberrations. We aimed to determine which genetic abnormalities are involved in DS ALL. We studied the frequency and prognostic value of deletions in B-cell development genes and aberrations of janus kinase 2 (JAK2) and cytokine receptor-like factor 2 (CRLF2) using array-comparative genomic hybridization, and multiplex ligation-dependent probe amplification in a population-based cohort of 34 Dutch Childhood Oncology Group DS ALL samples. A population-based cohort of 88 DS samples from the UK trials was used to validate survival estimates for IKZF1 and CRLF2 abnormalities. In total, 50% of DS ALL patients had 1 deletion in the B-cell development genes: PAX5 (12%), VPREB1 (18%) and IKZF1 (35%). JAK2 was mutated in 15% of patients, genomic CRLF2 rearrangements in 62%. Outcome was significantly worse in patients with IKZF1 deletions (6-year event-free survival (EFS) 45±16% vs 95±4%; P=0.002), which was confirmed in the validation cohort (6-year EFS 21±12% vs 58±11%; P=0.002). This IKZF1 deletion was a strong independent predictor for outcome (hazard ratio EFS 3.05; P=0.001). Neither CRLF2 nor JAK2 were predictors for worse prognosis. If confirmed in prospective series, IKZF1 deletions may be used for risk-group stratification in DS ALL. [ABSTRACT FROM AUTHOR]

NK-like (NKL) homeobox genes code for transcription factors, which can act as key regulators in fundamental cellular processes. NKL genes have been implicated in divergent types of cancer. In this review, we summarize the involvement of NKL genes in cancer and leukemia in particular. NKL genes can act as tumor-suppressor genes and as oncogenes, depending on tissue type. Aberrant expression of NKL genes is especially common in T-cell acute lymphoblastic leukemia (T-ALL). In T-ALL, 8 NKL genes have been reported to be highly expressed in specific T-ALL subgroups, and in ∼30% of cases, high expression is caused by chromosomal rearrangement of 1 of 5 NKL genes. Most of these NKL genes are normally not expressed in T-cell development. We hypothesize that the NKL genes might share a similar downstream effect that promotes leukemogenesis, possibly due to mimicking a NKL gene that has a physiological role in early hematopoietic development, such as HHEX. All eight NKL genes posses a conserved Eh1 repressor motif, which has an important role in regulating downstream targets in hematopoiesis and possibly in leukemogenesis as well. Identification of a potential common leukemogenic NKL downstream pathway will provide a promising subject for future studies. [ABSTRACT FROM AUTHOR]

Aberrant activation of the NOTCH1 pathway by inactivating and activating mutations in NOTCH1 or FBXW7 is a frequent phenomenon in T-cell acute lymphoblastic leukemia (T-ALL). We retrospectively investigated the relevance of NOTCH1/FBXW7 mutations for pediatric T-ALL patients enrolled on Dutch Childhood Oncology Group (DCOG) ALL7/8 or ALL9 or the German Co-Operative Study Group for Childhood Acute Lymphoblastic Leukemia study (COALL-97) protocols. NOTCH1-activating mutations were identified in 63% of patients. NOTCH1 mutations affected the heterodimerization, the juxtamembrane and/or the PEST domains, but not the RBP-J-κ-associated module, the ankyrin repeats or the transactivation domain. Reverse-phase protein microarray data confirmed that NOTCH1 and FBXW7 mutations resulted in increased intracellular NOTCH1 levels in primary T-ALL biopsies. Based on microarray expression analysis, NOTCH1/FBXW7 mutations were associated with activation of NOTCH1 direct target genes including HES1, DTX1, NOTCH3, PTCRA but not cMYC. NOTCH1/FBXW7 mutations were associated with TLX3 rearrangements, but were less frequently identified in TAL1- or LMO2-rearranged cases. NOTCH1-activating mutations were less frequently associated with mature T-cell developmental stage. Mutations were associated with a good initial in vivo prednisone response, but were not associated with a superior outcome in the DCOG and COALL cohorts. Comparing our data with other studies, we conclude that the prognostic significance for NOTCH1/FBXW7 mutations is not consistent and may depend on the treatment protocol given. [ABSTRACT FROM AUTHOR]

This article discusses the genetic aspects of the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). The importance of the oncogene LMO2 in the translocation of genes leading to the development of T-ALL is described. The advantages of multiplex ligation probe amplification testing over fluorescence in situ hybridization in the treatment of T-ALL are explored.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder, in which multiple genetic abnormalities cooperate in the malignant transformation of thymocytes. About 20% of pediatric T-ALL cases are characterized by TLX3 expression due to a cryptic translocation t(5;14)(q35;q32). Although a number of collaborating genetic events have been identified in TLX3 rearranged T-ALL patients (NOTCH1 mutations, p15/p16 deletions, NUP214-ABL1 amplifications), further elucidation of additional genetic lesions could provide a better understanding of the pathogenesis of this specific T-ALL subtype. In this study, we used array-CGH to screen TLX3 rearranged T-ALL patients for new chromosomal imbalances. Array-CGH analysis revealed five recurrent genomic deletions in TLX3 rearranged T-ALL, including del(1)(p36.31), del(5)(q35), del(13)(q14.3), del(16)(q22.1) and del(19)(p13.2). From these, the cryptic deletion, del(5)(q35), was exclusively identified in about 25% of TLX3 rearranged T-ALL cases. In addition, 19 other genetic lesions were detected once in TLX3 rearranged T-ALL cases, including a cryptic WT1 deletion and a deletion covering the FBXW7 gene, an U3-ubiquitin ligase that mediates the degradation of NOTCH1, MYC, JUN and CyclinE. This study provides a genome-wide overview of copy number changes in TLX3 rearranged T-ALL and offers great new challenges for the identification of new target genes that may play a role in the pathogenesis of T-ALL. [ABSTRACT FROM AUTHOR]

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is characterized by chromosomal rearrangements possibly enforcing arrest at specific development stages. We studied the relationship between molecular-cytogenetic abnormalities and T-cell development stage to investigate whether arrest at specific stages can explain the prognostic significance of specific abnormalities. We extensively studied 72 pediatric T-ALL cases for genetic abnormalities and expression of transcription factors, NOTCH1 mutations and expression of specific CD markers. HOX11 cases were CD1 positive consistent with a cortical stage, but as 4/5 cases lacked cytoplasmatic-β expression, developmental arrest may precede β-selection. HOX11L2 was especially confined to immature and pre-AB developmental stages, but 3/17 HOX11L2 mature cases were restricted to the γδ-lineage. TAL1 rearrangements were restricted to the αβ-lineage with most cases being TCR-αβ positive. NOTCH1 mutations were present in all molecular-cytogenetic subgroups without restriction to a specific developmental stage. CALM-AF10 was associated with early relapse. TAL1 or HOX11L2 rearrangements were associated with trends to good and poor outcomes, respectively. Also cases with high vs low TAL1 expression levels demonstrated a trend toward good outcome. Most cases with lower TAL1 levels were HOX11L2 or CALM-AF10 positive. NOTCH1 mutations did not predict for outcome. Classification into T-cell developmental subgroups was not predictive for outcome.Leukemia (2008) 22, 124–131; doi:10.1038/sj.leu.2404957; published online 11 October 2007 [ABSTRACT FROM AUTHOR]

The T-lineage phenotype of childhood acute lymphoblastic leukaemia (ALL) is associated with an increased relapse-risk and in vitro resistance to drugs as compared to a precursor B phenotype. Antiapoptotic isoforms of p73 that lack part of the transactivation (TA) domain (DeltaTA-p73, i.e. p73Deltaex2, p73Deltaex3, p73Deltaex2/3 and DeltaN-p73) may cause resistance to anticancer agents through inhibition of p53 and/or proapoptotic p73 family members (TA-p73). We demonstrate in our study that the expression of total p73 mRNA was higher in childhood T-ALL compared to controls (P=0.004). In T-ALL, the relative contribution of antiapoptotic DeltaTA-p73 (88%) was larger than of proapoptotic TA-p73 (12%). Leukaemic cells of T-ALL patients expressing higher levels of antiapoptotic p73 were more resistant to the DNA-damaging drug daunorubicin compared to cells of patients with low or negative expression or these isoforms (P(trend)=0.045). Interestingly, p73Deltaex2 was the most abundantly expressed antiapoptotic isoform in daunorubicin-resistant patient cells (44% of total p73). No association was found between high expression of proapoptotic TA-p73 or antiapoptotic DeltaTA-p73 and relapse-risk. Our results suggest that childhood T-ALL is associated with a high expression of DeltaTA-p73. These isoforms may play a role in cellular resistance to DNA-damaging drugs in children at initial diagnosis of T-ALL. [ABSTRACT FROM AUTHOR]

Over the last decade, genetic characterization of T-cell acute lymphoblastic leukemia (T-ALL) has led to the identification of a variety of chromosomal abnormalities. In this study, we used array-comparative genome hybridization (array-CGH) and identified a novel recurrent 9q34 amplification in 33% (12/36) of pediatric T-ALL samples, which is therefore one of the most frequent cytogenetic abnormalities observed in T-ALL thus far. The exact size of the amplified region differed among patients, but the critical region encloses ∼4 Mb and includes NOTCH1. The 9q34 amplification may lead to elevated expression of various genes, and MRLP41, SSNA1 and PHPT1 were found significantly expressed at higher levels. Fluorescence in situ hybridization (FISH) analysis revealed that this 9q34 amplification was in fact a 9q34 duplication on one chromosome and could be identified in 17–39 percent of leukemic cells at diagnosis. Although this leukemic subclone did not predict for poor outcome, leukemic cells carrying this duplication were still present at relapse, indicating that these cells survived chemotherapeutic treatment. Episomal NUP214-ABL1 amplification and activating mutations in NOTCH1, two other recently identified 9q34 abnormalities in T-ALL, were also detected in our patient cohort. We showed that both of these genetic abnormalities occur independently from this newly identified 9q34 duplication.Leukemia (2006) 20, 1245–1253. doi:10.1038/sj.leu.2404247; published online 4 May 2006 [ABSTRACT FROM AUTHOR]

Cytarabine (ara-C) is the most effective agent for the treatment of acute myeloid leukaemia (AML). Aberrant expression of enzymes involved in the transport/metabolism of ara-C could explain drug resistance. We determined mRNA expression of these factors using quantitative-real-time-PCR in leukemic blasts from children diagnosed with de novo AML. Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P=0.007). In vitro sensitivity to deoxynucleoside analogues was determined using the MTT-assay. Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (rp=-0.46; P=0.001), with three-fold lower hENT1 mRNA levels in resistant patients (P=0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC50 values of cladribine (rp=-0.30; P=0.04), decitabine (rp=-0.29; P=0.04) and gemcitabine (rp=-0.33; P=0.02). Deoxycytidine kinase (dCK) and cytidine deaminase (CDA) mRNA expression seemed to correlate with in vitro sensitivity to gemcitabine (rp=-0.31; P=0.03) and decitabine (rp=0.33; P=0.03), respectively. The dCK/PN-I ratio correlated inversely with LC50 values for gemcitabine (rp=-0.45, P=0.001) and the dCK/CDA ratio seemed to correlate with LC50 values for decitabine (rp=-0.29; 0.04). In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML. [ABSTRACT FROM AUTHOR]

Resistance to glucocorticoids (GC) is an important adverse risk factor in the treatment of acute lymphoblastic leukemia (ALL). To induce apoptosis, GC bind to the GC receptor (GR), which is regulated by various (co)chaperone proteins such as heat-shock protein 70 (HSP-70), HSP-40, HIP (HSP-70-interacting protein), BAG-1 (BCL-2-associated gene product-1), HOP (HSP-70/HSP-90-Organizing protein), HSP-90, P-23, FKBP-51, FKBP-52 and CYP-40. In this study, we tested the hypothesis that mRNA expression levels of these molecules are determinants of GC resistance in childhood ALL. In all, 20 children with ALL cells in vitro sensitive to prednisolone (LC50<0.1?µg/ml) were compared each with a resistant patient (LC50>150?µg/ml), matched for immunophenotype, age and white blood cell count. mRNA expression levels of the (co)chaperone molecules were measured by quantitative real-time RT-PCR and normalized to GAPDH and RNaseP levels. In vitro resistance to prednisolone was measured by MTT assay. HSP-90 mRNA expression levels were 2000-fold higher as compared to HSP-70. Using matched pair analysis, mRNA expression levels of the various (co)chaperone molecules were not significantly different between in vitro-sensitive and -resistant patients. GC resistance in childhood ALL cannot be attributed to different mRNA expression levels of the investigated (co)chaperone molecules involved in GC binding and transport to the nucleus.Leukemia (2005) 19, 727-733. doi:10.1038/sj.leu.2403681 Published online 10 March 2005 [ABSTRACT FROM AUTHOR]

Summary:Donor lymphocyte infusions (DLI) are used to treat relapsed haematological diseases after allogeneic stem cell transplantation (SCT). We treated seven patients with DLI for indolent non-Hodgkin's lymphoma relapsed after SCT. In available blood and bone marrow samples, lymphoma cells were analysed by real-time quantitative polymerase chain reaction of t(14;18)-positive cells in follicular lymphoma, and by immunophenotyping in small lymphocytic lymphoma. Before DLI, three patients were treated with chemo- and/or radiotherapy, and one with rituximab. Evaluable responses to pre-DLI therapy were stable disease in one and partial remission (PR) in two patients. Six patients responded to DLI (complete remission (CR) in four and PR in two). After DLI, acute graft-versus-host disease (GVHD) occurred in 3/6 patients, classified as grade 2, whereas only limited chronic GVHD was seen (n=5). The four continuous CR are lasting for median 65+ (43-89) months. In the remaining patient, not responding to DLI, progressive disease was seen later on; chemotherapy followed by another DLI resulted in CR. In three cases, clinical responses to DLI could be substantiated by molecular or immunophenotypic analysis of lymphoma cells. We conclude that DLI is effective for treatment of indolent lymphoma relapsing after SCT.Bone Marrow Transplantation (2003) 32, 1159-1163. doi:10.1038/sj.bmt.1704290 [ABSTRACT FROM AUTHOR]

In follicular lymphoma the t(14;18) might be useful as a tumor marker in predicting the quality of the response to treatment. We investigated whether analyzing numbers of t(14;18)-positive cells in peripheral blood correlated with remission status in individual patients receiving a variety of treatments. Numbers of circulating t(14;18)-positive cells were determined by real-time polymerase chain reaction (PCR) technique. Disease parameters and response to treatment were related to the pre- and post-treatment numbers of circulating t(14;18)-positive cells for 53 follicular lymphoma patients. In these 53 patients, 70 treatment episodes were investigated. A content of more than 328 t(14;18)-positive cells per 75,000 cells prior to therapy correlated with the more advanced stage IV disease (P=0.01), bone marrow involvement (P<0.01), and overt leukemic lymphoma (P=0.04). Therapy episodes that cleared circulation from t(14;18)-positive cells with more than one log resulted in a significantly longer progression-free survival than treatment episodes with less than one log decline (26 versus 12 months, respectively) (P<0.01). After first-line treatment episodes, numbers of circulating t(14;18)-positive cells declined in fairly all cases, irrespective of the clinical response. However, for second or later lines of treatment, declining numbers of lymphoma cells correlated with a clinical remission, whereas increasing numbers of lymphoma cells were associated with clinically stable or progressive disease. From this, we conclude that quantitation of circulating t(14;18)-positive cells in peripheral blood is of only limited clinical significance in predicting treatment efficacy for the individual follicular lymphoma patient. [ABSTRACT FROM AUTHOR]

Glucocorticoids (GC) are probably the most important drugs in the treatment of ALL. Despite the extensive use of GC for many years, little is known about the molecular mechanisms of sensitivity and resistance. This review summarizes the knowledge on GC cytotoxicity in leukemia. The relevance of polymorphisms, splice variants and the number and regulation of the GC receptor are discussed. The role of multidrug resistance proteins, glutathione and glutathione S-transferase is evaluated, as well as the influence of the different heat-shock chaperone (hsp 90 and 70) and co-chaperone proteins (BAG-1 and others) which form a complex together with the GC receptor. Finally, the transactivation and transrepression (via NF-κB and AP-1 binding) of a wide range of genes (like c-myc) which initiates the final apoptosis pathway are discussed and suggestions for future directions of research in ALL patients are given. [ABSTRACT FROM AUTHOR]

Expression of the multidrug resistance proteins P-glycoprotein, encoded by the MDR1 gene, multidrug resistance-associated protein (MRP1) and the lung resistance-related protein or major vault protein (LRP/MVP) is associated with clinical resistance to chemotherapy in acute myeloid leukemia (AML). Recently, the breast cancer-resistant protein (BCRP), the equivalent of mitoxantrone-resistant protein (MXR) or placental ABC transporter (ABCP), was described in AML. We investigated MDR1, MRP1, LRP/MVP and BCRP mRNA expression simultaneously in 20 paired clinical AML samples from diagnosis and relapse or refractory disease, using quantitative Taqman analysis. In addition, standard assays for P-glycoprotein expression and function were performed. BCRP was the only resistance protein that was expressed at a significantly higher RNA level (median 1.7-fold, P = 0.04) at relapsed/refractory state as compared to diagnosis. In contrast, LRP/MVP mRNA expression decreased as disease evolved (P = 0.02), whereas MDR1 and MRP1 mRNA levels were not different at relapse as compared to diagnosis. Also, at the protein level no difference of MDR1 between diagnosis and relapse was found. A significant co-expression of BCRP and MDR1 was found at diagnosis (r = 0.47, P = 0.04). The present results suggest that BCRP, but not MDR1, MRP1 or LRP/MVP is associated with clinical resistant disease in AML. [ABSTRACT FROM AUTHOR]

The behavioral and electroantennogram (EAG) responses of female Anopheles gambiae mosquitoes to pooled samples of freshly collected human sweat and human sweat incubated for 42–52 hr were tested. No behavioral or EAG response was obtained to pooled fresh sweat samples, whereas incubated pooled sweat samples produced a behavioral as well as an EAG response. GC-MS analysis of the headspace composition of the fresh sweat revealed ethanol (15.1% of the total amount of volatiles trapped), acetic acid (10.9%), and 3-hydroxy-2-butanone (9.5%) as the most abundant compounds; a wide range of ethyl esters was present as well. None of the ethyl esters was detected in the headspace collections from incubated sweat, while the relative amounts of ethanol, acetic acid, and 3-hydroxy-2-butanone were strongly reduced. In the latter collections, indole (27.9%), 1-dodecanol (22.4%), and 3-methyl-1-butanol (10%) were present in high amounts, while they were absent or present in only minor amounts in the headspace collections from fresh sweat. Geranyl acetone (6%) and 6-methyl-5-hepten-2-one (1.9%) were relatively abundant in both the fresh and incubated headspace samples. EAG responses were observed in response to indole, 6-methyl-5-hepten-2-one, and geranyl acetone. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which discusses a study on the risk for avascular necrosis of the bone in pediatric acute lymphoblastic leukemia.