Cases Of Myelogenous Leukemia Research Articles

Variable Expression Levels of the Hematopoietic Stem Cell Surface Antigen ESAM Depict Heterogeneity and Fluctuations in Leukemic Stem Cells

Traditionally, hematopoietic stem cells (HSCs) have been regarded as a homogeneous cell population. However, many studies have shown that populations of HSCs are essentially heterogenous. Moreover, several recent studies have proposed that HSCs exhibit fluctuating biological characteristics. Although the physiological significance of this heterogeneity and fluctuation is unclear, we presume that these qualities enable HSCs to flexibly demonstrate their self-renewal or differentiation abilities dependent on physiological demands. It is, therefore, unsurprising that such heterogeneity and fluctuation is also seen in leukemic stem cells (LSCs), and that they are involved in the development of resistance to treatment.We have previously demonstrated that the endothelial-cell selective adhesion molecule (ESAM) is an important marker of HSCs in humans and mice (Blood 2009, Exp Hematol 2013), and that the expression level of this molecule indicates the activation status of HSCs (J Immunol 2012). Furthermore, we found that a subset of leukemic cells exhibit elevated levels of ESAM expression on their surfaces (Exp Hematol 2013). Approximately two-thirds of acute myelogenous leukemia cases in humans were found to be positive for ESAM expression. Since ESAM was found to be useful in monitoring the fluctuating status of HSCs after 5-FU treatment, we posited that the marker could be used to characterize LSCs.In this study, we used ESAM expression levels as an index to analyze the heterogeneity and fluctuation in LSC populations. Flow cytometric analysis of the human leukemia cell lines KG1a and CMK revealed wide-ranging heterogeneity in ESAM expression levels, with up to a 1000-fold difference in variation, even among individual cells derived from the same clone. When these cell populations were roughly fractionated into ESAM-high or ESAM-low expression groups, the ESAM-high expression group showed a higher proliferative capacity in culture. Interestingly, however, the expression levels of ESAM were not maintained indefinitely at the same level in these groups. In four weeks, both ESAM-high and ESAM-low cell fractions developed heterogeneous ESAM expression distributions similar to that in the parent cell lines. Furthermore, when single cells were sorted and cultured, each cell showed a self-renewal ability and the heterogenous nature of ESAM expression levels was reconstructed in each population derived from these single cells. These results suggest that populations of LSCs develop heterogeneity and have a fluctuating nature.When the transcriptomes of ESAM-low and ESAM-high KG1a cells were compared using RT-PCR, the expression of the transforming growth factor beta receptor type 2 (TGFBR2) gene was found to be higher in the ESAM-low cells. Reportedly, the transforming growth factor beta (TGFβ) signaling pathway is involved in various biological processes such as cell proliferation, differentiation, and migration. In tumor cells, this pathway is involved in reducing proliferation and accelerating invasion. Based on this information, it was considered likely that the TGFβ signaling pathway is involved in the heterogeneity of ESAM expression. When TGFβ1 (5 ng/ml) was added to a culture of KG1a cells (grown in RPMI 1640 medium with 10% fetal bovine serum (FBS)), the heterogeneity of ESAM expression in the population became more pronounced, mainly due to a decrease in ESAM expression in some cells.A transcriptomic analysis of cells exhibiting decreased ESAM expression after TGFβ1 stimulation showed that these cells had a higher expression of TGFBR2 than cells not exhibiting a decrease in ESAM expression. Since the presence of 10% FBS in the culture medium provides natural TGFβ1 at a concentration of ~1 ng/ml, we conducted culture experiments without FBS. When KG1a cells were cultured in serum-free medium, the ESAM-low fraction disappeared and the heterogeneity of ESAM expression could not be replicated. However, the heterogeneity of ESAM expression in KG1a cultures could be replicated in serum-free medium by the addition of 1 ng/ml TGFβ1. These results suggest that the TGFβ signaling pathway is involved in inducing heterogeneity in ESAM expression in cell populations. The fluctuation of ESAM expression levels could be caused by differences in TGF sensitivity, which in turn, may confer certain benefits to LSCs, such as drug resistance and promoting invasiveness. DisclosuresYokota:Celgene: Research Funding; MSD K.K.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Pfizer Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding. Ezoe:Taiho Pharmaceutical Co., LTD.: Research Funding. Shibayama:Novartis Pharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:MOCHIDA PHARMACEUTICAL: Speakers Bureau; Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Kanakura:Kyowa Hakko Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Fujimotoseiyaku: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Nippon Shinyaku: Research Funding; Toyama Chemical: Research Funding; Shionogi: Research Funding.

Upregulation of Leukocytic Syncytin-1 in Acute Myeloid Leukemia Patients.

BackgroundSyncytin-1, a cell membrane-localizing fusogen, is abnormally expressed in several cancers, including endometrial cancer, breast cancer, and leukemia. Although abnormal syncytin-1 expression has been detected in two-thirds of leukemia blood samples, its expression profile in acute leukemia patients has not yet been analyzed.Material/MethodsBone marrow samples from 50 acute myelogenous leukemia (AML) cases and 14 B-cell acute lymphocytic leukemia (B-cell ALL) patients were subjected to flow cytometry to assess leukocyte type distributions and leukocytic syncytin-1 surface expression. RT-PCR was applied to assess leukocytic syncytin-1 mRNA expression. Statistical analysis was applied to compare syncytin-1 expression between AML and B-cell ALL patients across blasts, granulocytes, lymphocytes, and monocytes as well as to determine clinical factors statistically associated with changes in syncytin-1 expression.ResultsThe leukocyte type distributions of the AML and B-cell ALL cohorts highly overlapped, with an observable difference in blast distribution between the 2 cohorts. The AML cohort displayed significantly greater syncytin-1 surface and mRNA expression (p<0.05). Syncytin-1 surface and mRNA expression was significantly increased across all 4 leukocyte types (p<0.05). The percentage of syncytin-1-expressing blasts was significantly greater in AML patients (p<0.05), with blasts showing the largest fold-change in syncytin-1 expression (p<0.05). M5, M5a, and M5b AML patients displayed significantly higher syncytin-1 surface expression relative to all other AML French-American-British (FAB) classifications (p<0.05).ConclusionsThese findings suggest leukocytic syncytin-1 expression may play a role in the development and/or maintenance of the AML phenotype and the acute monocytic leukemia phenotype in particular.

Differential Signaling through p190 and p210 Forms of BCR-ABL Fusion Proteins Revealed By Proteomic Analysis

Chromosomal translocations involving chromosome 9q34 and 22q11 generate the BCR-ABL1 fusion gene. The location of the translocation within the BCR gene dictates which exons are excluded or included in the resulting fusion with the ABL1 gene. The most common translocations produce three main protein products with molecular weights of 190, 210, and 230 kD. Interestingly, the p190 and p210 BCR-ABL1 forms are associated with different clinical characteristics. Specifically, BCR-ABL1+ acute lymphoblastic leukemia (ALL) cases typically harbor the p190 form, whereas chronic myelogenous leukemia (CML) cases typically harbor the p210 form. Despite a great deal of study over many decades devoted to the BCR-ABL1 molecule, neither phosphorylation of putative target proteins at site-specific resolution nor protein-protein interaction differences between the p190 and the p210 forms is well understood, preventing thorough understanding of the signaling programs employed by these variants.Therefore, to examine signaling differences between p190 and p210 we chose a homogeneous system to investigate global phosphorylation of tyrosine residues. Ba/F3 cell lines, which are normally dependent on IL-3 for growth, were transduced with either the p190 or p210 form of BCR-ABL1. Subsequently, these cell lines were selected for successful growth in cytokine free media, indicating that the BCR-ABL1 fusions were signaling and inducing aberrant growth. These two BCR-ABL1 cell lines, as well as the parental Ba/F3 line, were grown in SILAC (stable isotope labeling by amino acids in cell culture) media and lysates were enriched for phosphotyrosine (pTyr) peptides and submitted for mass spectrometric analysis. The resulting 3-state SILAC dataset includes quantitation on 355 pTyr sites in three pairwise comparisons; p190 vs. p210 as well as each BCR-ABL1 cell line vs. its parental control. Many of the known BCR-ABL1 associated phosphorylation sites are recapitulated in our dataset. To our knowledge we report the first data on pTyr site-specific differences between p190 and p210 forms within in the same cell type. Surprisingly, 78 pTyr sites are differentially regulated between p190 and p210, with notable examples including pTyr sites on TEC, SHC1, PEAK1, PTPN6 and LYN. Additionally, sites in the kinase domain of JAK2 and the DNA binding domain of STAT6 showed a reciprocal relationship in p190 vs. p210, suggesting the differential involvement of JAK-STAT pathway. Notably, 28 pTyr sites were differentially regulated exclusively in either p190 or p210 BCR-ABL1 Ba/F3 cells, suggesting that there may be signaling pathways unique to p190 and p210, respectively.To attempt to identify direct substrates of BCR-ABL1 and detect differential binding of p190 and p210, we used the BioID system (Roux et al., JBC 2012). In this system, a specialized biotin ligase (BirA) is used to label adjacent or interacting proteins with a biotin moiety on lysine residues. Specifically, Ba/F3 cells transduced with BirA-tagged p190 or untagged p190 were grown in SILAC media and whole cell lysates were subjected streptavidin immunoprecipitation followed by mass spectrometry. Forty-four proteins were enriched over the control and known interactors such as SHC1, DOK2, and GAB1 were detected, along with several putative novel interactors. Combining both the phosphorylation and interactome datasets revealed all these proteins are hyper-phosphorylated compared to parental cell lines, suggesting these are direct substrates or in proximal complexes with p190 BCR-ABL1. A direct comparison of p190 and p210 interactomes using BioID is pending, and will further elucidate potential differences between these important fusion protein isoforms. In sum, our data suggests that p190 and p210 have at least partially independent signaling cascades that are mediated by differential protein-protein interactions, which may help explain the observed association of p190 with BCR-ABL1+ ALL and p210 with CML. DisclosuresNo relevant conflicts of interest to declare.

Exome Sequencing in a Family with Chronic Lymphocytic Leukemia, Mantle Cell Lymphoma and Autoimmune Disease Uncovers Potential Germline Risk-Alleles

We identified a Caucasian family with dense distribution of B-cell non-Hodgkin lymphoma (NHL) and autoimmune disease (Figure 1). Within the family, three siblings and their mother have B-cell NHL (three with chronic lymphocytic leukemia [CLL]; one with mantle cell lymphoma [MCL]), and four family members have autoimmune diseases (rheumatoid arthritis or multiple sclerosis). In addition, two family members have melanoma. To investigate the genetic factors involved in B-cell NHL in this family, we exome sequenced nine individuals, including two unaffected spouses as technical-artifact controls. Following best-practice guidelines, we performed joint variant calling, including an additional 135 European 1000Genome exomes as a set of background controls. [Display omitted] After variant quality control, a total of 676,137 sequence variants were identified. We also performed individual genotype quality control based on a minimum read depth of 10 and genotype quality of 40, resulting in 554,690 variants. For our initial prioritization, we required that variants be: absent in the spouse controls, have minor allele frequency (MAF) ≤ 0.05 in the 1000Genome exomes, and be shared by all four B-cell NHL cases. This resulted in 73 variants of potential interest.Of the 73 variants, seven were predicted to be damaging by either PROVEAN or SIFT, two by both algorithms and by PolyPhen2. These two potentially damaging variants reside in the genes CCDC144A and RRS1. The gene CCDC144A is located at chromosome 17p11.2, a known breakpoint region of the 17p deletion often seen in CLL tumor cells. The variant identified in this gene was rare (MAF = 0.001 in the NHLBI Exome Sequencing Project [ESP]) and was also seen in two additional family members (one with rheumatoid arthritis; a second with rheumatoid arthritis and melanoma). The gene RRS1 is located at chromosome 8q13.1, and has been shown to be important for proper chromosomal organization during mitosis. The variant identified in this gene was relatively rare (MAF = 0.041 in ESP) and was seen in the same, two additional family members. Of the remaining five variants predicted damaging by only one algorithm, of interest were two variants in the SGK223 gene on chromosome 8p23.1. SGK223 is a kinase and a component in the BCR-ABL1 signaling network that is present in most chronic myelogenous leukemia cases and a quarter of adult acute lymphoblastic leukemia cases. Both of the variants in SGK223 were also seen in three additional family members (one with rheumatoid arthritis; a second with rheumatoid arthritis and melanoma; a third with melanoma).In addition to prioritization by predicted function of coding variants, we explored the 73 variants for overlap with findings from published germ-line investigations of B-cell NHL. This identified one variant in the ACOXL gene at chromosome 2q13. The variant is relatively rare (MAF = 0.017 in dbSNP) and lies in the same gene and intron (17,016 base pairs upstream) as rs13401811, the associated SNP (p = 2.08 x 10-18) reported in a genomewide association study (GWAS) for CLL.Our ongoing analyses and prioritization of the variants in this extraordinary family reveal overlap with signals coming from GWAS studies and suggest some potentially damaging variants in genes not previously implicated in NHL. The potential risk-alleles identified in our preliminary findings could shed new light about the genes and genetic factors involved in NHL. DisclosuresNo relevant conflicts of interest to declare.

NRAS:BCL-2 Complex Localization Determines Anti-Apoptotic Features Associated with Progressive Disease in Myelodysplastic Syndromes (MDS)

AbstractAbstract 3835 Background:Activation of the RAS pathway plays an important role in the pathogenesis of myeloid malignancies. RAS mutations occur in some 10% of MDS and acute myelogenous leukemia (AML) cases. We previously showed in mouse models of NRASD12/BCL-2 MDS/AML that RAS and BCL-2 co-operate in vivo. These genes co-localize at the plasma membrane in the MDS model (MRP8NRASD12/MMTVLTRTetoBCL-2) with increased apoptosis and in mitochondria in the AML post-MDS model (MRP8[NRASD12/BCL-2] with reduced apoptosis (Omidvar Cancer Res 2007). Here, we screened MDS patients for the cellular co-localizations of RAS:BCL-2 and the consequent apoptosis status. Methods:39 MDS and AML post-MDS patients were studied, including (WHO classification): 3 RCMD-RS, 3 RCMD, 1 RARS, 8 RAEB I, 9 RAEB II, 4 CMML I, 1 CMML II, 10 AML post-MDS and 2 normal individuals. 17 patients had cytogenetic abnormalities. Bone marrow (BM) mononuclear cells (MNC) were assayed for RAS:BCL-2 localization by confocal and immunofluorescence microscopy. IL-3 dependent mouse hematopoietic FDCP-1 cells were infected with NRASD12 or BCL-2 alone or in combination. Co-localization was measured using anti-NRAS, anti-BCL-2, anti-mitochondrial (Tom 20) and anti-ezrin or anti-wheat germ agglutinin (WGA) plasma membrane antibodies. Apoptosis in patient samples was detected using the Mebstain apoptosis kit and visualized using FITC-dUTP on cytospun cells; apoptosis in the mice was detected by single photon emission computed tomography (SPECT) using technicium labeled annexin-V (99mTc-AnnexinV). NRAS mutations were analyzed by exon direct sequencing or by WAVE, a denaturing high-performance liquid chromatography (DHPLC) based assay. Lineage negative Lin-/Sca-1+/c-Kit+ (LSK) spleen cells were purified by automacs followed by flow sorting. Western blots were probed with anti-caspase 3 and 9 specific antibodies. Results:RAS and BCL-2 were co-expressed in the BM (MNC) of all 39 cases. The intensity (low or high) of RAS:BCL-2 co-localization measured by confocal microscopy significantly correlated with % BM blasts (p=0.0283) and WHO classification (p<0.0001); and was particularly high in 11 of the 12 cases (2 RAEBII, 9 AML post-MDS) with >15% marrow blasts. The intensity of the complex was independent of karyotype and NRAS mutation (found in 3 of 30 (10%) patient samples). The 2 normal samples had low intensities of RAS:BCL-2 co-localizations and wild type NRAS. Further analysis of RAS and BCL-2 co-localizations was carried out in 13 of these patients. In 6 of the 7 patients with BM blasts <15%, more than 10% of cells with apoptotic features (APO+) were seen and the RAS:BCL-2 complex was found at the plasma membrane. On the other hand, 4 of the 6 patients with BM blasts ≥15% showed no apoptotic features (APO-) and a strong co-localization signal of the RAS:BCL-2 complex at the mitochondria. In the remaining 2 patients with BM blasts ≥15% both mitochondria and plasma membrane localization patterns were observed, suggesting a mixed cell population. LSK enriched spleen cells from the AML post-MDS mouse model, and the FDCP-1 cell lines expressing mutant NRAS or BCL-2 alone or in combination were subjected to ligand activated caspase-3 and 9 assays revealed that when BCL-2 is co-localized with NRASD12 at the plasma membrane the anti-apoptotic protective effect of BCL-2 appears to be lost (p<0.0001). This is consistent with the pro-caspase-9-mediated apoptotic features observed in the MDS model determined by Western blotting and further supported by in vivo SPECT apoptosis imaging observed in this model and in low risk MDS patients by FITC-dUTP assays. Conclusion:These findings illustrate the prognostic impact of the intensity of the RAS:BCL-2 complex in MDS/AML post-MDS patients, and further suggest that localization of RAS:BCL-2 at the plasma membrane correlates with less advanced disease while its presence at mitochondria correlates with more advanced MDS. Therapeutic targeting of the RAS:BCL-2 complex could potentially prevent transformation in MDS patients. Disclosures:No relevant conflicts of interest to declare.

Underreporting of Myeloid Malignancies by United States Cancer Registries

The recent decrease in myeloid leukemia incidence may be directly attributed to changes in the population-based cancer registries 2001 guidelines, which required the capture of only one malignancy in the myeloid lineage per person and the simultaneous adoption of myelodysplastic syndrome registration in the United States. We constructed four claims-based algorithms to assess myeloid leukemia incidence, applied the algorithms to the 1999-2008 Surveillance Epidemiology and End Results (SEER)-Medicare database, and assessed algorithm validity using SEER-registered cases. Each had moderate sensitivities (75%-94%) and high specificities (>99.0%), with the 2+BCBM algorithm showing the highest specificity. On the basis of the 2+BCBM algorithm, SEER registered only 50% of the acute myelogenous leukemia cases and a third of the chronic myelogenous leukemia (CML) cases. The annual incidence of myeloid leukemia in 2005 was 26 per 100,000 persons 66 years or older, much higher than the 15 per 100,000 reported by SEER using the same sample. Our findings suggest underreporting of myeloid leukemias in SEER by a magnitude of 50% to 70% as well as validate and support the use of the 2+BCBM claims algorithm in identifying myeloid leukemia cases. Use of this algorithm identified a high number of uncaptured myeloid leukemia cases, particularly CML cases. Our results call for the commitment of more resources for centralized cancer registries so that they may improve myeloid leukemia case ascertainment, which would empower policy makers with ability to properly allocate limited health care resources.

NF1 Inactivation Revs Up Ras in Adult Acute Myelogenous Leukemia

Mutations in the Ras pathway are common in myeloid malignancies. NF1, a tumor suppressor and negative regulator of Ras, is inactivated in a subset of adult acute myelogenous leukemia (AML) cases. Loss of NF1 function sensitizes cells to inhibition of mammalian target of rapamycin (mTOR), a downstream effector of Ras activation, highlighting a potential therapeutic opportunity for some AML patients.

Abstract 1240: Functional deregulation of NF-kB and abnormal TNFa response in acute promyelocytic leukemia

Abstract Acute promyelocytic leukemia (APL) accounts for approximately 10% of acute myelogenous leukemia (AML) cases, and is characterized by accumulation of abnormal promyelocytes in patient bone marrow and peripheral blood. APL is associated with balanced chromosomal translocations involving retinoic acid receptor alpha (RARA), giving rise to fusion oncoproteins referred to as X-RARA. As deregulation of retinoid signaling is insufficient for leukemia development, our studies aim to determine other signaling pathways involved in APL by assessing the gene expression profiles and cell biology of X-RARA. We previously determined, using gene expression microarray analysis, common downstream targets of the variant APL fusion proteins NPM- and NuMA-RARA. We observed an over-representation of NF-κB target genes within this dataset. In these cells, a number of NF-κB target genes were commonly over-expressed. A subset of commonly deregulated genes were validated in our APL cell lines and 23 primary APL patient samples by real-time quantitative RT-PCR (RQ-PCR). 13/16 genes that were tested were significantly altered in APL compared to normal BM (n=11) p&amp;lt;0.05. The majority of these deregulated genes showed a progressive trend towards normal expression levels in post-treated samples. These data indicate defects in NF-κB-mediated gene expression in APL pathogenesis. We next examined NF-κB activity by assessing the expression levels of NF-κB target genes by quantitative real-time RT-PCR, in the presence and absence of TNFα. We observed that there was sustained activation of NF-κB in cells expressing NPM-RARA, as evidenced by the increased expression of NF-κB transcriptional target genes after induction by TNFα. Western analysis of protein derived from U937-NPM-RARA and NB4 cells demonstrated over-expression of NF-κB (p65) protein, as well as its transcriptional target IκBα. The increased pool of NF-κB localized to both the cytoplasm and the nucleus in U937-NPM-RARA cells as visualized by immunofluorescent confocal microscopy. Having observed deregulated expression of downstream targets of TNFα, we sought to examine the ability of X-RARA to confer resistance to TNF-mediated apoptosis. Our results in colony formation assays indicated that NPM-RARA expressing cells formed significantly more colonies, in the presence of 0-100 ng/mL TNFα, in a dose-dependent manner, compared to U937 control cells. These data suggested a greater ability on the part of NPM-RARA+ cells to survive and proliferate in the presence of TNFα. Our data provides the first evidence of the functional deregulation of the NF-κB-mediated signaling pathway and the TNFα response in cells expressing the variant APL fusion proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1240.

Myelogenous leukemia in adult inbred MHC-defined miniature swine: A model for human myeloid leukemias

This manuscript reports on five cases of spontaneous myelogenous leukemia, similar to human disease, occurring within highly inbred, histocompatible sublines of Massachusetts General Hospital (MGH) MHC-defined miniature swine. In cases where a neoplasm was suspected based on clinical observations, samples were obtained for complete blood count, peripheral blood smear, and flow cytometric analysis. Animals confirmed to have neoplasms were euthanized and underwent necropsy. Histological samples were obtained from abnormal tissues and suspect lesions. The phenotype of the malignancies was assessed by flow cytometric analysis of processed peripheral blood mononuclear cells and affected tissues. Five cases of spontaneous myeloid leukemia were identified in adult animals older than 30 months of age. All animals presented with symptoms of weight loss, lethargy, and marked leukocytosis. At autopsy, all animals had systemic disease involvement and presented with severe hepatosplenomegaly. Three of the five myelogenous leukemias have successfully been expanded in vitro. The clustered incidence of disease in this closed herd suggests that genetic factors may be contributing to disease development. Myelogenous leukemia cell lines established from inbred sublines of MGH MHC-defined miniature swine have the potential to be utilized as a model to evaluate therapies of human leukemia.

Clinical Performance of JAK2 V617F Mutation Detection Assays in a Molecular Diagnostics Laboratory

The presence of the JAK2 V617F mutation is now part of clinical diagnostic algorithms, and JAK2 status is routinely assessed when BCR/ABL- chronic myeloproliferative neoplasms (MPNs) are suspected. The aim of this study was to evaluate performance of 3 screening and 1 quantitative method for JAK2 V617F detection. For the study, 43 samples (27 bone marrow aspirates and 16 peripheral blood samples) were selected. The screening assays were the JAK2 Activating Mutation Assay (InVivoScribe, San Diego, CA), JAK2 MutaScreen kit (Ipsogen, Luminy Biotech, Marseille, France), and a home-brew melting curve analysis method. Ipsogen's JAK2 MutaQuant assay was used for quantification of mutant and wild-type alleles. The limit of detection was 1% for the kit-based screening methods and 10% for the melting curve method. The JAK2 MutaQuant assay demonstrated analytic sensitivity of 0.01%. All 4 methods detected cases of BCR/ABL- MPNs and gave negative results with BCR/ABL+ chronic myelogenous leukemia, multiple myeloma, myelodysplastic syndrome, and normal cases.

Preferred co-localization of chromosome 8 and 21 in myeloid bone marrow cells detected by three dimensional molecular cytogenetics

The impact of chromosome architecture in the formation of chromosome aberrations is a recent finding of interphase directed molecular cytogenetic studies. Also positive correlation of translocation frequencies and spatial proximity of chromosomes was described. Thus, disease specific chromosomal translocations could be due to tissue specific genomic organization. However, no three-dimensional interphase fluorescence in situ hybridization (FISH) studies for the nuclear architecture of bone marrow (BM) cells have previously been done. In this study, BM of three secondary acute myelogenous leukemia (AML) cases with trisomy 8 and otherwise normal karyotype were evaluated. Bone marrow cells of one AML and one ALL (acute lymphoblastic leukemia) case, peripheral blood lymphocytes and human sperm, all of them with normal karyotype, served as controls. Multicolor banding (MCB) probes for chromosomes 8 and 21 were applied in suspension-FISH (S-FISH). Interestingly, in myeloid bone marrow cells chromosomes 8 (di- and trisomic) and 21 tended to co-localize with their homologue chromosome(s), rather than to be separated. Thus, the co-localization of chromosomes 8 and 21 might promote a translocation providing a selective advantage of t(8;21) cells in AML-M2. In summary, the concept that tissue specific spatial proximity of chromosomes leads to enhanced translocation frequencies was further supported.

Deregulation of Transcription Factors GATA-1, GATA-2 and C/EBPa in Acute Promyelocytic Leukemia.

Acute promyelocytic leukemia (APL) accounts for ~10% of all acute myelogenous leukemia (AML) cases, and is characterized by accumulation of abnormal promyelocytes in the bone marrow. APL is uniquely associated with balanced chromosomal translocations involving the retinoic acid receptor alpha (RARA) locus. A functional chimeric protein, X-RARA, is created, whose N-terminus is derived from the partner (“X”) gene, and retains its oligomerization domain. The C-terminus is derived from RARA. X-RARA contributes to the APL phenotype by interfering with granulocyte differentiation, through acquisition of novel transactivating activity and/or interference with the normal functions of RARA and X. Several studies have indicated that defective retinoid signaling, though sufficient to block myeloid differentiation, cannot induce a leukemia. Thus, pathways external to retinoid signaling must be deregulated by X-RARA in order to give rise to APL. We previously reported whole genome gene expression analysis of the hCG-NuMA-RARA transgenic mouse model (Sukhai et al, Blood, Blood , 2006: a2247), and that the NuMA-RARA fusion protein deregulated a wide array of hematopoietic and myeloid transcription factors in leukemic cells derived from transgenic mice. Here, we report that the deregulated expression of this transcription factor set (specifically, Gata-1, Gata-2, C/ebpa and Pu.1) accurately distinguished leukemic TM mice from WT animals, and was dependent upon both the presence of functional RXRA (Sukhai et al, Oncogene, 2008) and transgene dosage. We thus formulated the hypothesis that NuMA-RARA initiated the deregulation of a range of transcription factors, which in turn were responsible for deregulating pathways within the cell. In order to determine whether this transcription factor signature was unique to APL, we focused on GATA-1, GATA-2, PU.1 and C/EBPA, and examined their expression in a range of AML cell lines. Strikingly, the over-expression of GATA-1 was unique to APL cell lines and the OCI/AML4 cell line. GATA-2 was over-expressed in most cell systems tested, suggesting that its up-regulation may play a general role in leukemogenesis. C/EBPA was under-expressed in NB4 cells specifically, while PU.1 was not significantly deregulated in any cell system tested. Having identified that APL cell lines recapitulated our observation of deregulated myeloid transcription factor expression in transgenic mice, we sought to extend these studies to human patients. We analyzed the expression of GATA- 1, GATA-2, PU.1 and C/EBPA in a series of 12 APL patients, 10 AML patients with a range of diagnoses, and 12 normal BM samples. We observed specific up-regulation of GATA-1 (2.0–20.0-fold change, 7/12 patients) and GATA-2 (2.0–25.0-fold change, 9/12 patients) and under-expression of C/EBPA (0.05–0.5-fold change, 11/12 patients), in APL, but not AML, patient samples, in comparison to normal BM. The deregulated expression of these three transcription factors could accurately distinguish APL patient samples from AML and normal BM in both principle component analysis and hierarchical clustering analysis. We therefore report herein that NuMA-RARA deregulated a wide array of myeloid transcription factors, suggesting that a state of globally deregulated myeloid transcription exists in APL cells. Furthermore, a specific subset of transcription factors, GATA-1, GATA-2 and C/EBPA, can be used to specifically classify APL patient samples. These deregulated transcription factors may therefore serve as potential therapeutic targets and a means of distinguishing APL from other forms of acute myelogenous leukemia.

Progressive Chromatin Repression and Promoter Methylation of a-catenin Correlates with AML Transformation in Patients with and without 5q Deletions.

Background: Loss of the long arm of chromosome (Ch) 5 or complete loss of Ch 5 is frequent in de novo MDS and AML. Epigenetic modifications of tumor suppressor genes, including aberrant DNA methylation, may play an important role in the progression of MDS and AML. Cell signaling is regulated by a-catenin, which forms a trimolecular complex with E-cadherin (ECAD) and b-catenin that links with actin-containing filaments of the cytoskeleton. Studies have reported reduced expression of a-catenin in MDS and AML patients with 5q deletion as compared to those without 5q deletion. Whether promoter methylation of the a-catenin gene is responsible for this decreased expression is controversial. To explore the potential role of a-catenin in the pathogenesis of AML transformation, we (a) determined the tumor specificity of methylation in AML and non- myeloid malignancies and (b) frequency of methylation in patients with −5/del(5q) MDS/AML and those with normal cytogenetics; (c) performed bioinformatics and experimental analysis of the a-catenin adhesion complex and local 5q31.1 genes to investigate mechanisms of epigenetic silencing; and (d) performed a detailed analysis of primary AML samples correlating promoter methylation, a-catenin expression, and chromatin conformation.Methods and Results: Using methylation sensitive PCR, we found that methylation of the a-catenin promoter gene was specific for myeloid malignancy. No methylation was observed in 19 acute lymphocytic leukemia cases, 20 chronic myelogenous leukemia cases, or in 99 primary gastric and esophageal samples where a-catenin has been implicated as a tumor suppressor gene. In those patients with AML and an associated 5q deletion the frequency was 31% (8/26) as compared to those without a 5q deletion with 13% (16/120). Bioinformatics analyses of the Valk et al., 2004 leukemia database provide supportive data for under expression of a-catenin in non-5/del (5q) AML cases. We quantitated a-catenin mRNA expression by Q-PCR in our cohort. Expression was lowest in AML patients with a-catenin methylation (n=9), but also in a subset of patients without promoter methylation (n=17), suggesting alternative mechanisms of inactivation. In contrast to AML, methylation of a-catenin was rare in myelodysplastic syndrome (MDS). Although p15 was methylated in over 50% of these cases as a positive control, only 2/18 MDS cases with 5q deletion (11%) and 1/13 MDS cases with 5q intact (8%) were methylated at the a-catenin promoter. The three positive cases were RAEB-2 (1) or RAEB-t (2), suggesting that a-catenin methylation may be most important in promoting transformation from MDS to AML. To explain a potential lack of correlation of methylation with decreased a-catenin in MDS and AML, we investigated a-catenin chromatin in a myeloid stem cell progression model and in primary AML samples. We performed chromatin immunoprecipitation on the CTNNA1 promoter using two active chromatin histone marks, H3K9Ac and H3K4me2 and two inactive marks, H3K9me2 and H3K27me3. In cell lines and primary leukemia samples where a-catenin was highly expressed (N=4), activation marks were present and repression marks absent. In cell lines and primary samples with low a-catenin expression and methylation of the promoter (N=4), the opposite pattern was observed. So called “bivalent” chromatin with mixed marks, no a-catenin methylation, and intermediated mRNA expression levels were observed with 7 additional cases (2 cell lines, 5 primary AML).Conclusions: Our data indicate that methylation of a-catenin is common in AML patients with 5q deletion but also observed in cases with normal chromosome 5 copy number. We propose a model of progressive inactivation of the a-catenin locus with AML transformation, with methylation representing a late stage event. The tissue-specificity of our results and in vivo chromatin observations in primary AML samples have implications for the timing and combinatorial therapy of MDS/AML with HDAC inhibitors and methylation inhibitors. Additionally, it appears that inactivation of adhesion molecules (ECAD, a-catenin) are frequent events overall in AML, suggesting a new pathway of transformation from MDS to AML.

Type B Core Binding Factor β/Smooth Muscle Myosin Heavy Chain Fusion Transcript in Myeloid Blast Phase of Chronic Myeloid Leukemia: Correlation with Nuclear and Cytoplasmic Localization of the Fusion Protein

BackgroundInv(16)(p13q22) or the rare t(16;16)(p13;q22) is characteristic of the M4Eo type of acute myeloid leukemia and results in the fusion of the core binding factor β gene (CBFB) at 16q22 with the smooth muscle myosin heavy chain gene (MYH11) at 16p13. The fusion transcript CBFB/MYH11 plays an important role in leukemogenesis. Many variations of this fusion transcript have been reported, with type A being most common and occurring in > 85% of cases. Patients and MethodsWe studied the fusion transcripts in 4 cases of chronic myelogenous leukemia in myeloid blast phase with inv(16)(p13q22). ResultsSequencing analysis revealed type B fusion transcript in 3 of the 4 cases. Immunohistochemistry demonstrated both nuclear and cytoplasmic staining for the fusion protein. ConclusionAlthough the biologic significance of this finding is unknown, the high frequency of an unusual type of fusion transcript in the chronic myelogenous leukemia cases in blast phase in which inv(16)(p13q22) first occurred at the time of blast transformation suggests that the type B transcript might play a role in disease progression. The effect of the additional smooth muscle myosin heavy chain sequences in the type B fusion on subcellular localization of the protein product is also suggested.

The role of β-catenin in chronic myeloproliferative disorders

The goal of the current study is to evaluate the role of beta-catenin in chronic myeloproliferative disorders. Expression of beta-catenin was analyzed by immunohistochemistry in formalin-fixed decalcified bone marrow biopsy specimens from 52 chronic myeloproliferative disorder cases and 6 nonchronic myeloproliferative disorder bone marrows as controls. The frequency of moderate to strong beta-catenin staining of megakaryocytes was significantly higher in polycythemia vera cases and in essential thrombocythemia cases than in chronic myelogenous leukemia cases (polycythemia vera versus chronic myelogenous leukemia, P = .002345, Fisher exact; and essential thrombocythemia versus chronic myelogenous leukemia, P = .002288), chronic idiopathic myelofibrosis cases (polycythemia vera versus chronic idiopathic myelofibrosis, P = .006707 and essential thrombocythemia versus chronic idiopathic myelofibrosis, P = .006932) or control cases (polycythemia vera versus control, P = .010489 and essential thrombocythemia versus control, P = .0113120). The erythroid and myeloid lineages showed absent to weak beta-catenin staining in most cases. In conclusion, these results indicate that the Wnt/beta-catenin signaling pathway may have a role in megakaryocytopoiesis in polycythemia vera and essential thrombocythemia. In addition, beta-catenin may be a useful marker for differentiating polycythemia vera and essential thrombocythemia from other types of chronic myeloproliferative disorders.

Analysis and comparison of clinicohematological parameters and molecular and cytogenetic response of two Bcr/Abl fusion transcripts

Different forms of p210 are produced by alternative splicing, namely b2a2 and b3a2. There have been many contrasting data establishing a relationship between the two Bcr/Abl transcripts and platelet counts and also response to treatment. However, the data published to date have been on a small group of patients. The aim of the present study was to determine whether there was any difference between clinical and hematological parameters at diagnosis between the two Bcr/Abl fusion transcripts in our population, and whether the two transcripts responded differently or similarly to imatinib treatment. RT-PCR was performed in 202 cases for detection of Bcr/Abl transcripts in newly diagnosed chronic myelogenous leukemia cases in one year. The two transcripts were compared and correlated with clinical, hematological and FISH data and with response to treatment. A total of 138 cases were of b3a2 and 64 were of b2a2 transcript. There was no correlation between the hematological parameters and the type of transcript. There was a significant association of blast crisis with b2a2, especially with myeloid blast crisis. When compared to FISH results, 10% of b3a2 were found to have a significant association with 5'Abl deletion as compared to 3% of b2a2. On analyzing the therapeutic response, we did not find any difference between the two transcripts. In conclusion, our findings confirm that the b3a2 type transcript is not significantly associated with thrombocytosis, that the short transcript, b2a2, occurs with acute phase, i.e., blast crisis, and that there is no difference in treatment response between the two transcripts. However, further studies are required to understand the molecular pathways involved in the Bcr/Abl mechanism.

The Production of Vascular Endothelial Growth Factor Is Decreased in Acute Myelogenous Leukemia Cases That Are Treated with Chemotherapeutic Agents and Show the Prolonged Bone Marrow Suppression.

Objective: There have been reported that the levels of serum vascular endothelial growth factor (VEGF) were decreased in aplastic anemia cases. We investigated VEGF system after chemotherapy to acute myelogenous leukemia (AML) cases, and determined whether VEGF system influenced the prolonged bone marrow suppression in these cases.Materials and Methods: Sera and bone marrow cells were prepared from 30 AML cases including 10 cases of AML (M3) at the onset of the disease, after chemotherapy, and the recovery periods, and the concentration of VEGF in sera of the patients and in the conditioned media obtained from bone marrow-cell cultures was measured with ELISA kit (Quantikine; R&D Systems). The expression of VEGF, VEGF receptor type-1 and VEGF receptor type-2 was analyzed with RT-PCR. The biological effect of VEGF on the bone marrow cells which showed the prolonged suppression after chemotherapy was assayed with colony-formation with or without any cytokines.Result and Discussion: As was reported previously, VEGF levels were significantly increased in M3 cases. In other types of AML cases the levels of VEGF production varied. When patients were given chemotherapy and the bone marrow suppression was prolonged, the production levels of VEGF were significantly diminished less than that observed in AML cases with normal bone marrow recovery. In M3 cases that were treated with all-trans retinoic acid and the prolonged bone marrow-suppression was observed, VEGF production was also suppressed. The expression of VEGFR-1 and -2 was observed in bone marrow cells from prolonged bone marrow suppression cases. In these cases, when bone marrow cells were cultured with VEGF, synergistic effects with G-CSF and EPO were observed with colony-formation assay. These observations indicate that VEGF works on the important role for the hematopoietic recovery after chemotherapy in AML cases.

Altered Expression of PP2A Regulatory Subunits in Chronic Myelogenous Leukemia: Identifying Targets for Improved Therapies.

Over 90% of chronic myelogenous leukemia (CML) cases are associated with constitutive activation of the BCR/ABL oncoprotein, which induces transformation of hematopoietic stem cells. Inhibition of BCR/ABL with imatinib has shown remarkable success in treating CML patients. However, the emergence of resistance is becoming a major clinical problem. To direct the development of improved therapies, signaling pathways activated by BCR/ABL must be identified. The aim of this project is to determine whether the activity and expression of the tumor suppressor protein phosphatase 2A (PP2A) is altered in the presence of BCR/ABL. PP2A is a serine/threonine phosphatase that is composed of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B), of which there are three different families. Each family of PP2A-B subunits has a number of isoforms that determine the substrate specificity, subcellular localization and catalytic activity of the enzyme. In this study we show that transfection of BCR/ABL into FDC-P1, a mouse factor-dependent myeloid cell line, increased tyrosine phosphorylation on the PP2A-C subunit, and this correlated with a reduction in enzymatic activity. Imatinib treatment rescued PP2A activity in wild type BCR/ABL+ cells, but not the imatinib-resistant Y253F BCR/ABL+ cells. Importantly, pharmacological enhancement of PP2A by forskolin and FTY720 resulted in reduced proliferation in both cell lines. Furthermore, we have demonstrated for the first time that BCR/ABL increases the expression of three specific PP2A-B subunit isoforms - B55α, B56α, and B56δ. It is predicted that enhanced levels of these subunits cause displacement of alternate PP2A-B isoforms to alter enzyme composition and hence the specificity of PP2A. To determine the functional role of each individual B subunit, shRNA studies are being performed. These findings confirm that functional inactivation of PP2A is an essential component of BCR/ABL-induced leukemogenesis in CML, and provide novel insight into the mechanisms by which this may occur. Elucidating the importance of each regulatory subunit may identify novel targets to develop improved therapies for CML patients.

Leukemia-Derived Fibroblast Cells Produce Vascular Endothelial Growth Factor Significantly in Chronic Myelogenous Leukemia Cases.

Objective: We reported recently that myofibroblast cells in bone marrow cells of chronic myelogenous leukemia (CML)-patients were partly derived from CML clone, which was identified after long-term bone marrow cell-cultures. The generated BCR-ABL-positive fibroblast cells produced various kinds of cytokines more than that observed in the normal clone-derived fibroblast cells. Among these cytokines, we focus on vascular endothelial growth factor (VEGF)-system in this report.Materials and Methods: Bone marrow cells were collected from chronic myelogenous leukemia cases (20 of chronic phase (CP), 3 of accelerated phase (AP), 8 of blast crisis phase (BC)), which were separated with gravity sedimentation. Obtained non-adherent mononuclear cells were cultured in DMEM with 10% FCS in the humidified 5% CO2 incubator. When cells showed morphological changes into fibroblastoid cells, cells were treated with trypsin and further cultured. The obtained stromal fibroblast cells were sub-cloned into culturing in 96 well plates, and from which RNAs were extracted. cDNAs were synthesized, and RT-PCR was performed to identify BCR-ABL fusion product. The selected clones were further analyzed on DNA levels with FISH to identify BCR-ABL. The concentration of VEGF in the patients' sera and in the conditioned media of non-adherent mononuclear cells, of fibroblast cells derived from normal clone and of fibroblast cells with BCR-ABL was measured with ELISA kit (R&D Systems). The expression of VEGF, VEGF receptor type-1 and VEGF receptor type-2 was also compared at RNA levels. The effect of anti-VEGF antibody to the culturing CML cells was determined using 3H-incorporation assay system.Results and Discussion: The levels of VEGF in sera from CML patients were elevated significantly. However, in patients with CP the expression of VEGF was not observed in non-adherent mononuclear cells. In contrast, VEGF production was observed in non-adherent mononuclear cells from the patients with AP and BC. Stromal fibroblasts produced VEGF in CP, AP and BC, in which BCR-ABL-positive fibroblast cells produced at the significant levels. The expression of VEGFR-1 and -2 was detected in non-adherent mononuclear cells from CP, AP and BC. When non-adherent mononuclear cells from CML patients were cultured onto the stromal fibroblast cells, significant additive effects were observed with 3H-thymidine incorporation assays. And when anti VEGF antibody was added to the cultures, the proliferation activity was significantly decreased. These observations indicate that in CML cases, especially in CP, VEGF plays an important role for the cellular proliferation with paracrine system.

Disseminated Intravascular Coagulation in Acute Leukemia at Presentation and During Induction Therapy

Between January 2001 and December 2003, 67 patients with acute leukemia were evaluated prospectively for hemostatic abnormality at presentation, of which 43 (64.2%) had acute lymphoblastic leukemia and 24 (35.8%) had acute myelogenous leukemia. At presentation, 27 patients (40.3%) had bleeding manifestations. Thrombocytopenia was present in 57 patients (85%), and 33(49.3%) had some abnormality of global coagulation markers. Disseminated intravascular coagulation was defined by International Society of Thrombosis and Hemostasis criteria. Disseminated intravascular coagulation was more often associated with bleeding manifestations in acute myelogenous leukemia cases than in acute lymphoblastic leukemia cases. Two patients presented disseminated intravascular coagulation on day 7 of chemotherapy, without any bleeding manifestations. Four of 15 evaluated cases who had a bleeding or infection complication after day 7 of induction therapy also had disseminated intravascular coagulation. It is recommended that all patients with leukemia be investigated for disseminated intravascular coagulation at presentation.