bluuebunny/crossref_metadata_2025 huggingface dataset:
Hsp-90 Inhibition Decreases Survival of BCR-ABL T315I Leukemic Stem Cells in Mice.
Authors: Peng Cong , Brain Julia , Hu Yiguo , Kong Linghong , Goodrich Ami , Grayzel David , Pak Roger , Read Margaret , Li Shaoguang
Published: October 2019

Although advances have been made in the development of novel molecularly targeted drugs, a major therapeutic challenge in the treatment of patients with Philadelphia chromosome positive (Ph+) leukemia includes understanding how to target the leukemic stem cell. We used the bone marrow transplant (BMT) model of chronic myelogenous leukemia (CML) to study effects of imatinib mesylate and the novel, orally active heat shock protein 90 (Hsp90) inhibitor, IPI-504, on leukemic stem cells, based on our observation that unlike imatinib, IPI-504, prolongs survival in a murine model of drug-resistant T315I BCR-ABL-induced CML. We first identified BCR-ABL-expressing hematopoietic stem cells (HSCs) (Lin-c-Kit+Sca-1+) in mouse bone marrow as CML stem cells, as these cells sorted out by FACS from primary CML mice are sufficient to confer leukemia in recipient mice. We then investigated the effects of imatinib and IPI-504 on survival of leukemic stem cells from BCR-ABL T315I induced CML. Bone marrow cells from mice with T315I-induced CML were cultured under conditions that support survival and growth of stem cells, with or without IPI-504 or imatinib. FACS analysis of GFP+Lin-c-Kit+Sca-1+ cells showed that imatinib treatment did not lower the percentage and the number of leukemia stem cells, whereas IPI-504 treatment had a dramatic inhibitory effect on this population (p<0.001) at therapeutically achievable doses. To determine whether IPI-504 attenuates development of leukemia by specifically inhibiting stem cell survival, GFP+Lin-c-Kit+Sca-1+ cells were sorted from bone marrow of mice with BCR-ABL T315I-induced CML, and cultured with a placebo or IPI-504. When these cells were transferred into lethally-irradiated recipient mice, FACS analysis showed that myeloid leukemia cells were present in mice receiving the placebo-treated leukemic stem cells but not in mice receiving the IPI-504 treated leukemic stem cells. To examine whether IPI-504 inhibits leukemia stem cells in vivo, mice with BCR-ABL-T315I-induced CML were treated with a placebo, imatinib, or orally administered IPI-504 for six days. Bone marrow cells were analyzed by FACS for GFP+Lin-c-Kit+Sca-1+ cells. Consistent with the in vitro results, imatinib treatment did not lower the percentage and number of leukemia stem cells, as compared with the untreated group, whereas IPI-504 treatment had a dramatic inhibitory effect on the stem cells. Analysis of bone marrow from non-leukemic mice treated with IPI-504 for two weeks showed no change in levels of Lin-c-Kit+Sca-1+ cells, indicating that IPI-504 treatment did not inhibit survival of normal HSCs. These results provide a rationale for use of an Hsp90 inhibitor as a first-line treatment to inhibit leukemia stem cells and prevent emergence of imatininb-resistant clones in patients.
Immunomodulatory Effects of STI571 in Chronic Myeloid Leukaemia.
Authors: Bund Dagmar , Yang Ting , Buhmann Raymund , Kolb Hans-Jochem
Published: October 2019

Background: The tyrosine kinase inhibitor imatinib (imatinib, STI571, Glivec, and Gleevec) is highly effective in the treatment of chronic myeloid leukaemia (CML) and has already been shown to be effective in the setting of allogeneic stem cell transplantation. But until now, less is known with respect to its immunomodulating effects.

Objective: In the present survey we investigated, whether imatinib could modify the antigen-presenting capacity of myeloid cells and in turn affects the cellular immune response.

Method: For this purpose, patient derived chronic myeloid cells were incubated with different concentrations of imatinib (0, 1, 2, or 5microM), characterized for their antigen-presenting profile and their stimulatory capacity in the context of HLA-matched and mismatched T-cells. After 5 days, the proliferative immune response was evaluated in presence or absence of different concentrations of imatinib and altered effector-to-target ratios. Thereby, proliferation was detected via a CFDA, SE (5,6-carboxyfluorescein diacetate succinimidyl ester) based assay.

Result: The proliferative capacity of the T- cells (allogeneic, HLA-mismatched) was inhibited by imatinib in a dose-dependent manner. Also, the expression of the activation markers was reduced in the presence of the different STI571 concentrations. Moreover, myeloid blasts were sensitized for T cell mediated effector functions by pre-treatment with increasing concentrations of imatinib.

Conclusion: Taken together, imatinib can interfere with the T cellular immune response in vitro, and its impact on graft-versus-leukemia (GvL) and graft-versus-host (GvH) reactions will be further investigated.
SPAN-XB Core Promoter Sequence Is Regulated in Myeloma Cells by Specific CpG Dinucleotides Associated with MeCP2 Protein.
Authors: Wang Zhiqing , Zhang Jian , Zhang Yana , Srivenugopal Kalkunte , Lim Seah H.
Published: October 2019

SPAN-Xb is a spermatid-specific protein encoded by the SPAN-XB gene on chromosome Xq27.1. It is a novel Cancer-Testis antigen in multiple myeloma. We recently demonstrated that SPAN-Xb expression in myeloma cells is regulated through promoter methylation and could be upregulated by IL-7 and GM-CSF. In this present study, we set out to investigate the mechanism of SPAN-XB expression and the promoter association with the methyl-CpG binding protein, MeCP2. Elucidation of these interactions is likely to shed light on potential therapeutic strategies to upregulate antigen levels that could be used to improve the outcome of SPAN-Xb-based tumor vaccines.

We previously showed that the putative SPAN-XB promoter resides within exon 1 and contains 433 bp, starting from position −546 to position −104 upstream of the translational start site. Using a panel of truncated promoter constructs generated from the 3′ and 5′ ends of the promoter gene, we localized the core sequence of SPAN-XB promoter to the 73 base pairs at the 3-end of the promoter, a region that lacks CpG dinucleotides within the full length promoter. There are 11 CpG dinucleotides within the putative SPAN-XB promoter. We previously found that DNA methylation provides the primary regulatory mechanism for SPAN-XB gene expression.

We also identified that hypomethylation at positions −310, −307, −299 and −221 strongly predicted for SPAN-XB expression, suggesting the involvement of these four CpG dinucleotides in the regulation of SPAN-XB gene expression through DNA methylation. In the present study, using reporter gene expression assays, we found that the core promoter function is significantly modulated by these adjacent CpG sequences so that mutation of the CpG dinucleotides outside the core sequence resulted in changes in the promoter function. We also previously demonstrated by bisulfite conversion and sequence analysis the association between methylation at specific CpG dinucleotides with repression of SPAN-XB gene. Here, we extended our study to determine whether or not the methylated cytosine binding protein, MeCP2, interacts with the SPAN-XB promoter gene in myeloma cells. Chromatin immunoprecipitation assays revealed a specific association of the MeCP2 with SPAN-XB promoter, and MeCP2 binding strongly correlated with repression of the SPAN-XB gene in myeloma cell lines and CD138-enriched fresh myeloma cells. Reactivation of the SPAN-XB gene by 5-azacytidine treatment resulted in the loss of MeCP2 from this site. We, therefore, conclude that SPAN-XB promoter consists of a core sequence and a regulatory element; the core element resides within the 73 base pairs at the 3′ end of the full length promoter. SPAN-XB gene expression by the core sequence is regulated in myeloma cells by specific CpG nucleotides and associated with MeCP2 binding to the promoter sequence.
PU.1 Is a Downstream Target of SOX4 in Myeloid Cells.
Authors: Aue Georg , Du Yang , Dunbar Cynthia E. , Jenkins Nancy A. , Copeland Neal G.
Published: October 2019

Mice that express 20% the normal levels of the Ets transcription factor PU.1 develop AML, unlike mice that express 50% or 80% the normal levels, indicating that PU.1 is a dosage-sensitive tumor suppressor gene. In addition, 3 of 13 AMLs induced by transplanting mice with cells transduced with a Sox4 oncogene-containing retrovirus were found to carry a Sox4 retroviral integration in one PU.1 allele, suggesting that downregulation of PU.1 may cooperate with Sox4 in AML induction. Since the other PU.1 allele remains intact in these AMLs and a 50% decrease in PU.1 expression is not sufficient to induce AML, we hypothesized that Sox4 might further downregulate PU.1 expression in these AMLs. To test this hypothesis, we transfected HL60 promyelocytes with an expression vector carrying both GFP and Sox4 cDNAs or a GFP vector control. Transfected GFP+ cells were purified by flow cytometry and PU.1 mRNA levels were analyzed by real-time RT-PCR. PU.1 mRNA levels were consistently downregulated 4 to 10 fold in cells transfected with Sox4 cDNA compared to cells transfected with the vector control, while β-actin mRNA levels were maintained constant, confirming that overexpression of Sox4 downregulates PU.1 expression in myeloid cells. The decrease of PU.1 mRNA was observed as early as 8 hours after Sox4 transfection, further suggesting that Sox4 may directly repress the PU.1 promoter in myeloid cells. Consistent with this, analysis of 2 published microarray databases comprising 401 de novo AML patient samples showed that SOX4 expression is significantly negatively correlated with PU.1 expression (coefficient: −0.337, P-value: 1E-07). Interestingly, AML FAB M1 and M2 subtypes were associated with statistically significant higher SOX4 expression levels compared to AML FAB M3, M4, M5. In order to confirm that downregulation of PU.1 cooperates with Sox4 in AML induction, we infected wild type or PU.1 heterozygous knockout bone marrow cells with the Sox4 retrovirus and then monitored the time of AML development in transplanted mice. Early results show accelerated leukemogenesis in mice transplanted with Sox4-infected PU +/− bone marrow (115 days) compared to mice receiving Sox4-infected wild type marrow (160 days). We are currently trying to identify Sox4 binding sites in the PU.1 promoter, or in an upper regulatory element that may be responsible for mediating the repression of PU.1.
Target Proteins of C/EBPa-p30 in AML: A Critical Role of Ubiquitin-Conjugating Enzyme (Ubc9) in Modulating C/EBPa-p42 Functions.
Authors: Geletu M. , Balkhi M. , Zada A. Peer , Trivedi A. , Pulikkan J. , Christopeit M. , Tenen Daniel G. , Behre G.
Published: October 2019

The mutations in the transcription factor CCAAT/enhancer binding protein-a (C/EBPa) occur in 10% of patients with acute myeloid leukemia which often leads to the expression of an N-terminal truncated 30KDa isoform of C/EBPa. The mutated 30KDa isoform has been shown to be dominant negative over wild type isoform that affects most of its biological functions. In the present study, we applied a global proteomics approach to identify the target proteins of C/EBPap30. This analysis revealed that C/EBPap30 modulates the expression of 60 different proteins notably, ubiquitin-conjugating enzyme (Ubc9), Peptidylprolyl isomerase (pin1), hnRNP A2/B1, Cacyclin binding protein, calgranulin B, and Tubulin beta5. Further, we show that in AML patients with C/EBPap30 mutations Ubc9 was predominantly upregulated. We discovered that this ligase was responsible for the enhanced sumolyation of C/EBPap42 on a lysine residue (K161) which results in its reduced transactivation in a promoter assay. When lysine (K) residue at 161 of C/EBPa-p42 was mutated to argenine residue(R), transcriptional activity of C/EBPap42 was restored. This finding was further validated by silencing the expression of Ubc9 expression which completely abrogates the sumoylation at K161 residue and further restored the transactivation and differentiation capacity of C/EBPap42. Our results demonstrate that increasing expression of Ubc9 by C/EBPap30 enhances sumolyation of C/EBPap42 and thereby inhibits its transactivation and differentiation capacity.
Identification and Characterization of Novel SET Domain-Containing Histone Lysine Methyltransferase and Implications of Histone Methylation in Acute Leukemia.
Authors: Kook Hoon , Choe Nakwon , Kee Hae-Jin , Kim Sung-Mi , Kim Ji-Young , Han Dong-Kyun , Baek Hee-Jo , Lee Kun-Soo , Hwang Tai-Ju , Seo Sang-Beom , Kook Hyun
Published: October 2019

Evolutionally conserved SET domains, which methylate histone lysine residues, and thereby methylation of histones have been implicated in diverse malignancies including leukemias or multiple myelomas. Here we describe novel SET domain-containing proteins with histone methyltransferase (HMTase) activity and their characteristics. Using bioinformatics for homology screening, SET-domain containing proteins named WHISTLE, WHSC1-like 1 isoform 9 with methyltransferase activity to lysine, and RE-IIBP, interleukin-5 response element II binding protein, were identified. By mass spectrometric and immunoblot analysis, we demonstrated that WHISTLE dimethylates H9K4 and di-, and tri-methylates H3K27, while RE-IIBP methylates only H9K27. WHISTLE and RE-IIBP repressed transcription of the SV40 and IL-5 promoter activity and they recruited histone deacetylase. Chromatin immunoprecipitation analysis revealed that shRNA-mediated knockdown of RE-IIBP reduces histone methylation on the IL-5 promoter. Both proteins induce apoptosis in leukemic cells via caspase-3 activation. In acute lymphoblastic leukemia patients, the expression of RE-IIBP and WHISTLE was increased, which was accompanied with increase in the methylation histone 3. These data illustrate the important regulatory role of novel SET domain proteins with HMTase activity in transcriptional regulation and apoptosis, thereby pointing to the critical role in leukemogenesis.
Association of cis-Acting rs530 of the ETS2 Transcriptional Factor Gene with High-Risk Acute Myelogenous Leukemia (AML) and Allelic Expression Imbalance Assessment.
Authors: Lee Il-Kwon , Choi Jeong-Hwa , Kim Hee Nam , Kim Yeo-Kyeoung , Park Kyeong-Soo , Lee Je-Jung , Shin Myung-Geun , Choi Chan , Kook Hoon , Ju Hwang Tai , Park Moo-Rim , Jo Deog-Yeon , Won Jong-Ho , Kim Hyeoung Joon
Published: October 2019

We have previously implicated ETS2 in the etiopathogenesis of acute myeloid leukemia through case-control study by revealing that polymorphisms of ETS2, a hematopoietic transcription factor gene is associated with increased risk to acute myelogenous leukemia (AML). Two SNPs out of 7 genotyped sites, rs711 and rs530 were shown to be associated with increased risk to AML with the odds ratio (OR) of 1.76 (95% C.I. 1.2–2.5, p=0.0019) for rs711 and 1.67 (95% C.I. 1.3–2.2, p=0.0003) for rs530 relative to wild type genotypes respectively. Haplotype and linkage disequilibrium (LD) map was estimated, but haplotype association was not found with statistical significance. Korean LD structure was similar to Japanese LD, but least similarity was shown with LDs from African (Yoruba in Ibadan, Nigeria). Since these two SNPs are located in the 3′ UTR region we simulated the change in secondary structure in silico of the 3′ UTR region with two variants introduced in the sequence. Most dramatic change in the secondary structure was observed in the rs530 containing domain suggesting this variant of being cis-acting genetic variant. Real time Q-PCR and western blot analysis showed that expression of ETS2 decreased in individuals with heterozygous or mutant homozygous genotypes, showing most abundant expression with two wild type copies of rs530, less expression with the rare homozygous or heterozygous genotype. Sequencing cDNA of 55 heterozygous AML patients revealed mRNA expression imbalance in 13 cases (24%) effectively reverting heterozygous genotype to homozygous wild type mRNA species. The detection of a discrepancy between the mRNA alleles of rs530 clearly proves cis-acting effect of rs530. However there was not a case in 51 healthy control samples suggesting differing transcript levels derived from the two alleles of an autosomal gene is disease-specific phenomena. Taken together, our results suggest that two polymorphic variants in the 3′ UTR region predispose individual to high-risk AML by inducing change in mRNA expression as a cis-acting variant.
Integrated CGH- and Epression Array Profiling of Mantle Cell Lymphoma.
Authors: Schraders Margit , Jares Pedro , Bea Silvia , Schoenmakers Eric F.P.M. , Van Krieken Joannes H.J.M. , Campo Elias , Groenen Patricia J.T.A.
Published: October 2019

Mantle cell lymphoma (MCL) is characterized by the translocation t(11;14)(q13;q32), which leads to the overexpression of cyclin D1. The cyclin D1-overexpression by itself is however not sufficient for lymphoma development. To identify other genes that are deregulated in MCL, we have performed integrated profiling of high-resolution array-based comparative genomic hybridization (array-CGH) with RNA-expression array analysis on the same set of MCL cases. DNA-alterations of MCL cases were assessed using a 3.6k BAC array with a resolution of 800 kb. On the same cases, RNA-expression array analysis was performed using the GeneChip® Human Genome U133 Plus 2.0 Array from Affymetrix. Integration of genomic-and transcription profiling data was performed using statistical tools. With array-CGH, we identified regions of chromosomal gain or loss and determined minimal common regions (mcr’s) by identifying the smallest region of overlap present in at least 36% of the cases. Integration of array-CGH and expression profiling was performed for genes located within the mcr’s and unsupervised clustering of gene-expression values within each mcr was performed. For several mcr’s, e.g. 1p22.1–31.1, 6q23.2–27 and 11q22.3–23.3, the clustering tree showed two groups, one with the chromosomal aberration and one without. Also, we observed that only a subset of genes located within a cytogenetic anomaly has a concomitant change in mRNA expression level. These genes are regulated by DNA-copy number (gene dosage). Amongst these, we identified several “hypothetical genes” and genes, which encode proteins involved in mitochondrial protein synthesis, the DNA damage repair pathway and the cAMP regulated pathway. This study shows the potential of the integrated profiling approach for identifying genes that are regulated by gene dosage (DNA-copy number). We anticipate that the genes we identified are important for MCL and it’s characteristic features like the low apoptosis rate and the chemotherapy-resistance.
Mouse B Cell Lineage Neoplasms: Comparative Analyses of Gene Expression Using High Throughput Real Time RT-PCR and Oligonucleotide Microarrays.
Authors: Shin Dong-Mi , Shaffer Daniel J. , Roopenian Derry C. , Morse Herbert
Published: October 2019

Gene expression profiling of human and mouse B cell lineage neoplasms using microarrays has provided a powerful means for:identification of previously unrecognized subsets of a single histologically-defined disease category;relating a specific disease to a previously unrecognized cell of origin; andestablishing disease subsets defined by characteristic host-tumor interactions.

Other anticipated returns including the rapid identification of new therapeutic targets have fallen short of expectations. In addition, discrepancies among results obtained using different platforms pose a major obstacle to bringing this approach into common clinical practice. Quantitation of gene expression by qPCR provides an attractive alternative approach but has been limited by the lack of high-throughput methodologies. We have approached this issue by developing qPCR methods that assay 384 “cancer genes” in a single plate, covering a wide range of targets from pathways implicated in the development of hematopoietic as well as solid tumors. We used this system to compare RNA prepared from normal spleens, mouse PCT and two lymphoma types with strikingly similar cytologies but differing origins - high-grade splenic marginal zone lymphomas (MZL) and centroblastic DLBCL (CBL). Terminally differentiated PCT were readily distinguished from MZL and CBL and MZL from CBL. Statistical analyses identified subsets of genes that distinguished each normal or tumor set from all the others and these are being validated against new groups of each tumor type. The same initial cases were also evaluated using oligonucleotide microarrays, providing instructive similarities and differences in measures of shared genes. From these comparisons, we have identified potentially important signaling pathways, such as Notch, Wnt and IL6, as well as drugable targets. These are being evaluated in studies of cultured cell lines and can be extended to in vivo tests of efficacy in mice bearing transplanted or primary tumors.
Tonic B-Cell Receptor Signaling Promotes the Survival of Diffuse Large B-Cell Lymphomas: Identification of SYK as a Rational Treatment Target.
Authors: Chen Linfeng , Juszczynski Przemyslaw , Hitoshi Yasumichi , Chen Wen , Kutok Jeffery L. , Shipp Margaret A.
Published: October 2019

The role of BCR-mediated survival signals in diffuse large B-cell lymphoma (DLBCL) remains undefined. B-cell antigen receptor (BCR) signaling induces receptor oligomerization and Ig alpha/beta ITAM phosphorylation. Thereafter, the protein tyrosine kinase (PTK) SYK is recruited and activated, initiating downstream events and amplifying the original BCR signal. Recent studies indicate that BCRs also transmit low-level survival signals in the absence of receptor engagement and highlight the critical role of SYK in this “tonic” BCR signaling. We recently found that SYK is a major substrate of the protein tyrosine phosphatase, PTPROt. PTPROt-mediated dephosphorylation and inactivation of SYK inhibited DLBCL proliferation and induced apoptosis in the absence of BCR cross-linking. These observations suggested that tonic BCR signaling may be an important survival mechanism in certain DLBCLs and identified SYK as a possible rational therapeutic target. For these reasons, we assessed the efficacy of a recently developed ATP-competitive inhibitor of SYK, R406, in a panel of 8 DLBCL cell lines. R406 inhibited the proliferation of 6 DLBCL cell lines at IC50s of 0.9–1.8 micromolar. The SYK inhibitor was cytotoxic (30–85% apoptotic cells) at low micromolar doses in 6 of these lines. To delineate the relationship between tonic BCR signaling, associated SYK activity and R406 responsiveness, we assessed SYK phosphorylation (pTYR 352 and 525/526) in the DLBCL cell line panel. Upon BCR signaling, SYK is phosphorylated at TYR 352; thereafter, the kinase undergoes autophosphorylation at TYR 525/526, which is required for SYK activity. In sensitive DLBCL cell lines, R406 specifically inhibited TYR 525/526 phosphorylation and additional downstream signals (p-ERK). In addition, all 6 R406-sensitive DLBCL cell lines exhibited basal SYK 352 phosphorylation whereas the 2 R406-resistant lines lacked pTyr 352 phosphorylation. Basal SYK pTYR 352 could be detected by immunoblotting total SYK immunoprecipitates with a phospho-specific SYK 352 antibody or using the same pTYR 352 antibody for intracellular phospho-specific flow cytometry. We conclude that :tonic BCR signaling is an important survival mechanism in many DLBCLs;SYK is a rational therapeutic target in these tumors;R406 is a promising SYK inhibitor; andSYK pTYR 352 is an excellent indicator of tonic BCR signaling and predictor of R406 sensitivity in DLBCL.
Bone Marrow Fibrosis Evaluation in Childhood Acute Lymphoblastic Leukaemia: Correlation to Biological Factors and Treatment Response.
Authors: Norén-Nyström Ulrika , Roos Goran , Bergh Anders , Thörn Ingrid , Lonnerholm Gudmar , Forestier Erik
Published: October 2019

Fibrosis may complicate bone marrow (BM) disease and in its advanced stage negatively affect the outcome of the patients due to BM failure, particularly in myeloproliferative disorders. Studies of the potential importance of BM fibrosis are rare in childhood acute lymphoblastic leukemia (ALL). We have previously shown a prognostic impact of BM fibrosis measured as reticulin fibre density (RFD) at diagnosis in childhood ALL. To further investigate the consequence of BM fibrosis in childhood ALL in relation to immunophenotype, cytogenetic findings and minimal residual disease (MRD) we retrospectively evaluated the RFD in 139 diagnostic BM biopsies from patients with a total mean follow up time of five years and eight months from two childhood oncology centers in Sweden. Patients with pre-B-ALL showed a higher mean RFD (19.1%) compared to patients with T-ALL (11.4%, p < 0.001). This was true also when comparing high-risk (HR) pre-B-ALL patients (18.1%) with the T-ALL patients (p = 0.002). RFD correlated inversely with the white blood cell count in the HR group (r = −0.41, p = 0.009), probably reflecting our finding of low degree of fibrosis in T-ALL. The cytogenetic analysis revealed that for patients with a hyperdiploid ALL (modal chromosome number: 51–61, n: 41) in the low-risk (LR) group, mean RFD was higher for relapsed patients (22.6%) compared to patients in continuous complete remission (17.2%, p = 0.019). The probability of disease free survival for the same group using RFD cutoff of 21.1%, representing the upper third of the material, was 85%±8% for patients with hyperdiploid ALL and low RFD compared to 51%±14% for patients with hyperdiploid ALL and high RFD (p = 0.01, Figure 1). Data for 32 of the patients demonstrated an association between high RFD at diagnosis and high minimal residual disease (MRD) on day 29 after start of treatment. Patients with FACS-MRD > 10−3 day 29 displayed higher mean RFD at diagnosis (21.2%) compared to patients with FACS-MRD < 10−3 (16.7%, p = 0.027, Figure 2). When analyzing the PCR-MRD data day 29 the findings were similar. We conclude that RFD is higher in pre-B-ALL compared to T-ALL, that RFD has prognostic impact in LR patients with hyperdiploid ALL and that high RFD at diagnosis is associated to high MRD on treatment day 29. All these findings are to our knowledge novel, suggesting that evaluation of BM fibrosis at diagnosis is a potentially new therapy stratifying factor and support the need of further research on BM fibrosis in childhood ALL and expanded use of BM biopsy at diagnosis.

Figure 1 Figure 1. Figure 2 Figure 2.
Wilm’s Tumour 1 (WT1) Mutations Are Associated with FLT3-ITD Mutation and Poor Prognosis in Normal Karyotype AML.
Authors: Fitzgibbon Jude , Summers Karin , Kakkas Ioannis , Stevens Jane , Smith Matthew , Smith Lan-Lan , Molloy Gael , McDougal Finlay , Bonnet Dominique , Young Bryan D. , Lister T. Andrew
Published: October 2019

Our group has previously shown an association between acquired uniparental disomy 11p and homozygous gene mutation of Wilm’s tumour 1 (WT1) in a patient with normal karyotype acute myeloid leukemia (AML). Based on this observation the incidence of WT1 mutation was investigated in a cohort of normal karyotype AMLs. Mutation screening was performed on 70 patients (median age 55 years, range 19–78 years) by a PCR-direct sequencing approach using intronic primers flanking exons 2–10 of WT1. Mutation status was inferred from the resultant traces and confirmed by use of TOPO TA cloning and sequence analysis of the corresponding mutated clones. Mutations were detected in 7/70 (10%) patients; these typically resulted in insertion of 1–16 bp that led to the disruption of the DNA binding domain of the protein. The mutation profile of FLT3-ITD, NPM and CEBPA was also examined in this cohort of patients to compare the additional mutational events present in WT1 mutated and non-mutated cases. A significant positive association was observed between WT1 and FLT3-ITD mutation with 6/7 WT1 mutated cases having a FLT3-ITD compared to 20/63 non mutated cases (p=0.01). There was no association between mutations in WT1 and either of the good prognostic mutational markers, CEBPA and NPM. All 6 patients with both WT1 and FLT3 mutations were refractory to intensive induction chemotherapy with WT1 mutation showing a trend towards a worse overall survival when compared with the non-mutated group (p=0.07). We can conclude therefore that WT1 is mutated in 10% of normal karyotype AML, is positively associated with FLT3-ITD status and identifies a putative subgroup of normal karyotype AML who fail to achieve remission with conventional cytotoxic therapy and have a poor overall survival. Validation of this data in larger series would support the inclusion of WT1 in the current molecular risk stratification of normal karyotype AML based on CEBPA, NPM and FLT3-ITD status.
Characterisation and Prognostic Significance of WT-1 Gene Expression in Acute Myeloid Leukemia (AML).
Authors: Lane Steven W. , Saal Russell , Negus Suzanne , Mollee Peter , Mather M. , Gill Devinder , Mills Anthony K. , Bird Robert J. , Hourigan Matthew J. , Jones Mark , Marlton Paula
Published: October 2019

Background: Wilms’ tumour gene 1 (WT-1) is frequently over-expressed in adult AML and may be of prognostic value. We examined the prognostic utility of WT-1 expression in the peripheral blood, measured by RQ-PCR on diagnostic specimens, and characterised this compared to other clinical and biological predictors of survival.

Methods: RQ-PCR was performed in triplicate on samples from newly diagnosed AML patients, using the Corbett Rotorgene 3000. Values were averaged and levels expressed relative to 104 ABL copies. Multivariate stratified Cox regression, with the stratification variable cytogenetic risk group and WT-1 levels analysed as a continuous variable, was used to model the outcomes of overall survival (OS) and event free survival (EFS).

Results: Samples were collected from 58 patients with AML. The median age was 66 years (range 16–82) and 30 were male. Seven had antecedent haematological disorders and 9 had received prior chemo/radiotherapy. Induction therapy was with cytarabine based regimens in 18, high dose cytarabine regimens in 20, ATRA/chemotherapy in 6 and supportive care in 14. Complete remission was achieved in 64% with 18% induction mortality. Median OS was 4 months (17 months in responders) and 27% at 2 years. The median WT-1 transcript level in diagnostic AML samples was 5122 (range 9–36125), while in normal control samples it was 3 (n=11, mean 3.6, SD 1.6).

In multivariate Cox regression, stratified by cytogenetic risk group, age (p<0.001), LDH (p<0.001) and performance status (p=0.04) retained prognostic significance for OS and EFS. Elevated peripheral blood WT1 levels were significantly associated with impaired OS (HR 1.24 per log10 transcript levels p=0.022) and EFS (HR 1.31 per log10 transcript levels p=0.0019). WT1 levels correlated with cytogenetic risk group (p=0.002 with higher levels seen in the favourable risk group), antecedent haematological disorder (p=0.05), peripheral blood blast percentage (p=0.05) and bone marrow blast percentage (p=0.01). WT1 levels were significantly higher in acute promyelocytic leukaemia (p=0.0001) compared to other AML subtypes. WT1 levels did not correlate with age (p=0.16), sex (p=0.12), prior chemo/radiotherapy (p=0.96), performance status (p=0.33) or LDH (p=0.91).

Conclusions: WT1 RQ-PCR levels are frequently elevated in peripheral blood from patients with AML at diagnosis and are associated with subtype, cytogenetic risk group, blast percentage and antecedent haematological disorder. Within specified cytogenetic risk groups, elevated WT1 levels may predict AML patients at high risk of relapse and subsequent mortality.
Presence of Unbalanced Chromosomal Translocations Independently Predicts for Treatment Failure, Short Treatment-Free (TFS) and Overall Survival (OS) in B-Cell Chronic Lymphocytic Leukemia (B-CLL) Patients Treated with Cladribine.
Authors: Van Den Neste Eric W. , Robin Valérie , Francart Julie , Hagemeijer Anne , Stul Michel , Delannoy Andre , Sonet Anne , Costantini Sabrina , Ferrant Augustin , Deneys Véronique , Robert Annie , Michaux Lucienne
Published: October 2019

Chromosomal translocations have recently emerged as an important prognostic indicator in B-CLL (Mayr et al, Blood 2006). Until now however, their value had neither been determined in patients undergoing homogeneous treatment, nor compared to that of IgVH mutational status, another major prognostic factor in B-CLL. Sixty-five B-CLL pts treated with cladribine-containing regimens were included in the present analysis. All had been investigated by conventional cytogenetic analysis using TPA as a mitogen, interphase FISH (tested loci: 11q/ATM, 12cen, 17p/P53, 13q/RB1&/D13S319), IgVH mutational status and P53 mutational screening prior to inclusion. Translocations (n= 45) were detected in 42 % of the pts, and included both balanced (n=12) and unbalanced (n=33) types. Pts with translocations were more heavily pretreated (P=.05) and presented with significantly higher incidence of complex aberrations (P<.001), 17p aberrations (P<.001) and P53 gene mutations (P<.001). IgVH genes were mutated in 43% and unmutated in 57% of the pts. The presence of unbalanced translocations was associated with significant lower response rate (P=0.004), shorter median TFS (7 vs. 36 months, logrank 22.72, P<.004) and median OS (13 vs. 69 months, logrank 16.51, P<.001), as compared to patients without translocation. In multivariate analysis, translocations, especially the unbalanced subtypes, were significantly associated with the risk of therapeutic failure (P<.000, OR: 7.6) and short TFS (P<.000, HR: 2.12). In addition, the presence of unbalanced translocations proved to be the prognostic marker with the highest negative impact on OS (P=.013, HR: 4.4). Other prognostic factors independently associated with OS were abnormal cytogenetics, high LDH, prior treatment with alkylating agents, and age. IgVH mutational status was independently associated with risk of failure and TFS, but not OS. Interestingly, the presence of translocation(s) had strong negative impact on TFS and OS in IgVH mutated pts, whereas it did not modify outcome in unmutated ones. In conclusion, in B-CLL patients treated with cladribine, the presence of chromosomal translocations is a strong prognostic indicator. This indicates that conventional cytogenetics remains an important tool in defining individual B-CLL patient’s risk.
Simultaneous Analysis of Multiple Chromosomal Aberrations in Chronic Lymphocytic Leukemia (CLL) by Multiplex Ligation-Dependent Probe Amplification (MLPA).
Authors: Stevens-Kroef Marian J.P.L. , Simons Annet , Petersen Michael Egmont , Gorissen Hanneke , Nanciu Yvonne , Buijs Arjan , Raymakers Reinier , van Kessel Ad Geurts
Published: October 2019

B cell chronic lymphocytic leukemia (B-CLL) is characterized by a highly variable clinical course. Characteristic genomic abnormalities have shown to provide highly significant prognostic information (Dohner NEJM 2000). However, karyotyping and/or FISH are laborious and expensive techniques. Although other prognostic markers (like mutation status of VH-genes and expression of CD38 and ZAP70) have been noted, these fail to identify all patients with a cytogenetic high-risk profile. We have performed multiplex ligation-dependent probe amplification (MLPA) analysis on 37 CLL patients, using the P037 and P038 kits (mrc-holland, http://www.mlpa.com/pages/indexpag.html), which allow the simultaneous identification of 56 genomic CLL-specific targets. The MLPA data obtained were compared with interphase FISH data in a blinded fashion using the ATM, centromere 12, RB1, D13S319, and TP53 specific probes (Vysis). By doing so, a good correlation was revealed in those cases in which the percentage of abnormal cells was at least 27%. We diagnosed six cases with loss of the ATM locus, six cases with trisomy 12, twenty cases with loss of chromosome 13q14 (three of them with a biallelic deletion), and three cases with loss of the TP53 locus. In three cases the genetic abnormality was detected by interphase FISH only. In these latter cases, the percentages of abnormal cells were 13%, 20% and 20%, respectively, which may explain the false negative results of the MLPA analyses. Since multiple probes were used for the 13q14 loci, a critical region of genomic loss could be identified which excluded the RB1 gene but included the KCNRG, DLEU2 and DLEU1 genes and miRNA15A. Also for the TP53 and ATM genes multiple probes were tested and, based on these results, one patient is suspected to have an intragenic ATM deletion. Also, additional abnormalities were detected by MLPA as e.g. trisomy 19.

In conclusion, MLPA appears to be a useful technique for the identification of clinically relevant genetic abnormalities in a diagnostic setting.
A Novel, High-Throughput Assay for Detection of ABL T315I Mutations.
Authors: Heinrich Michael C. , Morich Jason R. , Beillard Emmanuel J. , Fuller Courtney , Galderisi Chad D. , Druker Brian J.
Published: October 2019

Mutations in the kinase domain of BCR-ABL result in impaired drug binding and are thought to be the leading cause of acquired resistance to the tyrosine kinase inhibitor imatinib (Gleevec®). While imatinib is a highly effective therapy in all stages of chronic myelogenous leukemia (CML), relapse after an initial response is common in patients with advanced disease. The T315I point mutation is one of the most common imatinib-resistant mutations and patients with this mutation are also resistant to two second generation tyrosine kinase inhibitors, dasatinib and nilotinib. Recently, the U.S. FDA approved dasatinib for treatment of imatinib-resistant, Philadelphia chromosome-positive acute and chronic leukemias. Thus, appropriate detection of this mutation is essential to optimal management of patients with imatinib resistance and may be useful for clinical trials of agents that target patients with the T315I mutation. Current methods for mutation detection, such as direct DNA sequencing, are not sensitive enough for detection of point mutations at low levels of BCR-ABL transcript. Conversely, ultrasensitive detection methods such as allele specific PCR (AS-PCR) may be too sensitive and can be plagued by false positive test results. In addition the clinical relevance/significance of mutation detection at ultra sensitive levels ( < 1% mutant) is questionable and not yet known. We developed a novel T315I mutation detection assay, using Fluorescent Resonance Energy Transfer (FRET)-based hybridization probes and melting curve analysis. BCR-ABL amplicons generated from a first round of PCR are amplified using primers flanking the ABL kinase region encoding for codon 315. The resultant amplicon is hybridized with fluorescein-labelled anchor probe and a LC Red 640-labelled T315I mutation specific probe. Wild-type and T315I mutant amplicons are distinguished by melting curve analysis (Roche Light Cycler 480™). Using a series of plasmid and cell line dilutions we determined that the sensitivity of this assay for detection of T315I mutations was 5–10%. Using patient-derived samples we were able to successfully genotype samples containing as few as 20–50 BCR-ABL transcripts. To date, the assay sensitivity and specificity are 100%. The assay is performed in a plate based format (96 or 384 wells) and commercially available software allows automated genotype assignment. This approach is suitable for high-throughput detection of T315I mutations for clinical management of CML patients and/or screening of patients to determine eligibility for clinical studies.
Clinical Analysis of Adult Acute Leukemia with Rearrangements of the 11q23/MLL: Multicenter Co-Operative Study.
Authors: Tamai Hayato , Yamaguchi Hiroki , Hamaguchi Hiroyuki , Yagasaki Humiharu , Bassyo Masami , Kobayashi Takeshi , Akiyama Hideki , Sakamaki Hisashi , Takahashi Satoshi , Tojo Arinobu , Ohmine Ken , Ozawa Keiya , Okumura Hirokazu , Nakao Shinji , Arai Ayako , Miura Osamu , Toyoda Shigeo , Gomi Seiji , Murai Yoshiro , Usui Noriko , Miyazawa Keisuke , Ohyashiki Kazuma , Takahashi Naoto , Sawada Kenichi , Kato Atsushi , Oshimi Kazuo , Inokuchi Koiti , Dan Kazuo
Published: October 2019

Background AL(acute leukemias) with 11q23 abnormalities involving the MLL (mixed lineage leukemia) gene comprise one category of recurring genetic abnormalities in the WHO classification. However, the prognosis of adult AL with 11q23/MLL between fusion partners are controversial especially in Asia.

Methods We analyzed data on 61 Japanese adult patients diagnosed as AL with 11q23/MLL abnormalities, including the translocations t(4;11), t(9;11), t(6;11), t(11;19) between 1990 and 2006.

Results Median age of the 61 patients was 48 years (range, 17–79 years). 54 patients were AML (acute myeloid leukemia) and 7 patients were ALL (acute lymphoblastic leukemia).The incidence by fusion partners in AML were as follows; t(9;11) in 29.6%, t(11;19) in 24.0%, t(6;11) in 18.5%. The incidence of patients with t(11;19) was higher than that in Caucasian and t(4;11) was lower than that in childhood. As for the FAB (French-American-British) subtype, 68.5% of the 11q23/MLL patients were M4/M5. Organ involvement was observed commonly (46.2%) in AML with 11q23/MLL patients except for t(11;19) (7.6%).The CR (complete remission) rate of AML with 11q23/MLL was not low (78.7%), but the CR duration was very short (10 months).The DFS (disease free survival) at 1year and the OS (overall survival) at 2 years were 20% and 30% in AML with t(9;11), 10% and 19% in AML with t(11;19), 10% and 30% in AML with t(6;11). In total, the DFS at 1year and the OS at 2 years were 26% and 25% in AML patients, respectively, while 50% and 50% in ALL patients, respectively. The DFS and the OS at 2 years were 44% and 53% in AML patients with 11q23/MLL who were treated with HSCT(hematopoietic stem cell transplantations), while 14% and 21% in those without HSCT respectively.

Conclusions Although there were reports that the prognosis of AML with t(9;11) was not bad in Caucasian, the present study showed poor prognosis of AML with 11q23/MLL in Japanese adult patients regardless of the fusion partners. Our study revealed the short CR duration of the AML patients with 11q23/MLL in the face of the high CR rate. AML patients with 11q23/MLL who were treated with HSCT had superior outcomes to those without HSCT (P=.04 for DFS and P=.04 for OS) .This findings suggest that treatment strategies including HSCT should be considered for AML with 11q23/MLL during the first CR.
Serum Tryptase Levels in AML at Diagnosis: A Multicenter Retrospective Study.
Authors: Cairoli Roberto , Ripamonti Carla B. , Granata Simonetta , Beghini Alessandro , Colapietro Patrizia , Grillo Giovanni , Nadali Gianpaolo , Viola Assunta , Cattaneo Chiara , Intropido Liliana , Ravelli Erika , Bertani Gianbattista , Pezzetti Laura , Nichelatti Michele , Veronese Silvio , Marocchi Alessandro , Rossi Giuseppe , Pizzolo Giovanni , Ferrara Felicetto , Nosari Annamaria , Morra Enrica
Published: October 2019

α and β-tryptase genes cluster on the short arm of human chromosome 16 and encode lineage-associated serine proteases that are abundantly expressed in mast-cells and, in trace amounts, in basophils. Under physiologic conditions no other myeloid cells express tryptases. However, in several myeloid leukemia cell lines and in AML blasts, the level of tryptase is elevated. In an attempt at correlating the levels of tryptase with cytogenetic features and the KIT and FLT3 mutational status, we analyzed serum samples collected at diagnosis from 150 AML and 57 ALL adult patients. The total serum concentration was determined by UniCAP 100 and UniCAP Tryptase Fluorenzyme Immunoassay Kit (Pharmacia-Upjohn, Uppsala, Sweden). The median value of tryptase level in the control group (50 healthy people; mean age 35 y, range 20–50; M/F= 26/24) amounted to less than 5 ng/ml, ranging from 1 to 15 ng/ml. We detected elevated tryptase levels (more than 15 ng/ml) in 66 out of 150 AML-patients (44%) and in 1 out of 57 ALL-patients (1.75%; median value 1.2 ng/ml) (p = < 0.0005, Fisher’s exact test ). In AMLs data showed that elevated tryptase values are significantly bound to patients with t(8;21) (n = 26, p = <0.0001) and inv(16) (n = 17, p = 0.035). Furthermore, we found a strong correlation between tryptase < 15 ng/ml and normal karyotype (n = 58 ; p = <0.0001). By contrast, we didn’t find any correlation between the levels of tryptase and t(15;17) (n = 12, p = 0.227), abnormal chr 5 or 7 (n = 9, p = 0.507), +8 (n = 6, p = 0.229), complex karyotype (n = 9, p = 1.000) or other abnormalities (n = 14, p = 0.778). A mutational screening for KIT (exon 2,8,10,11,and 17) and FLT3 (exon 14,15 and 20) was performed on 67/150 (45%) and 82/150 (55%) patients with AML, respectively. We recorded a significant association between high tryptase levels and the presence of KIT mutation (Fisher’s exact test: p = 0.012). No correlation was found between levels of tryptase and FLT3 mutational status (Fisher’s exact test: p = 0.803).In conclusion, data suggest that elevated serum tryptase levels at diagnosis are frequently associated with Core Binding Factor and with KIT mutated AML’s.
Role of Huntingtin Interacting Protein HIP-1 in Non-Hodgkin’s Lymphoma.
Authors: Smith Mitchell R. , Joshi Indira , Jin Fang , Bradley Sarah V. , Ross Theodora S.
Published: October 2019

We have developed a murine model for mantle cell lymphoma (muMCL) that occurs after pristane injection of Eμ-cyclin D1 transgenic mice that are > 1 year of age (Leukemia 20:891, 2006). Eμ-cyclin D1 transgenic mice are healthy, implying that cyclin D1 may be necessary, but not sufficient, for MCL decvelopment. We are using this model to identify genes that coooperate with cyclin D1 in lymphomagenensis. Comparing gene expression in B cells from spleens of these muMCL with B cells from non-pristane injected age-matched Eμ-cyclin D1 transgenic mice by differential gene expression, we identified up-regulation of RNA for the Huntingtin interacting protein HIP-1 in the muMCL. We confirmed that this translated into up-regulation at the protein level by Western blot. HIP-1 is an inositol lipid, clathrin and actin binding protein that can directly transform fibroblasts. HIP-1, located on 7q, was previously found in a fusion protein with PDGFRβ in a case of CMML with t(5;7), suggsting a role in hematopoietic malignancies. To test the generalizability of the finding of HIP-1 overexpression in lymphoma, we performed immunohistochemistry using the monoclonal anti-HIP-1 antibody 4B10 on a lymphoma tissue microarray (Cybridi). Positive antibody staining was found in 79 of 116 (68.1%) non-Hodgkin’s lymphomas (NHL). Based on:

our identification of HIP-1 as a potential cooperating gene in MCL development; overexpression of HIP-1 in many NHL; and that the presence of antibodies to HIP-1 in serum correlates with overexpression of the protein, we tested serum samples from lymphoma patients.

We found circulating HIP-1 antibodies in sera from 23 of 39 (59%) lymphoma patients. The highest antibody titer was in serum from a patient with T-ALL in remission during maintenance chemotherapy who had a cytogenetic abnormality of 7q near the site of the HIP-1 gene. Studies are ongoing to expand this data set to see if HIP-1 antibodies correlate with disease type or outcome. In summary, HIP-1 appears to be involved in lymphoma development in a murine model of MCL and is frequently overexpressed in other types of NHL. Its role in lymphomagenesis, and potentially in treatment response and prognosis needs to be further explored.