Bone Marrow Of Leukemia Patients Research Articles

Abstract 5557: Stem-like T cells maintain latent anticancer activity and underly therapy resistance

Abstract Pre-cancerous cells are normally recognized and eliminated by immune cells. Cancer progresses only when this immunosurveillance system fails. Although immunotherapy has driven the most significant and exciting advances in cancer treatment in modern times, current approaches are running into barriers. An emerging area of interest includes evidence for rare but highly potent stem cell-like T cells. Stem-like T cells combine long-term persistence and high potency with immunological memory. Our recent findings reveal insights into the nature and regulation of stem-like T cells and their potential anticancer activity. Minimal residual disease (MRD) underlies therapeutic resistance, dictates treatment escalation, and predicts patient outcomes. We have shown that the Wnt and PI3K pathways promote the transformation of hematopoietic stem cells (HSCs) into chemoresistant leukemia stem cells (LSCs), which form the root of leukemia initiation and post-treatment recurrence. High-throughput screening revealed that LSCs could be targeted by low dose anthracycline treatment. Mechanistically, low dose, but not high dose anthracyclines indirectly target LSCs by inhibiting their unique properties of immune escape, allowing for their elimination by CD8+ T cells. We employed single-cell genomic and proteomic analysis to investigate immunological changes during development of HSCs, LSCs, and their progeny in response to low dose anthracycline treatment. While leukemia progression results in exhaustion of differentiated CD8+ T cells, stem-like T cells accumulate. However, low dose anthracycline treatment reverses this imbalance. Comparing MRD+ and MRD- patient samples, we found that differential proportions of stem-like T cells persist in the bone marrow of leukemia patients. While T cells of MRD- patients recover following chemotherapy induction, T cell recovery is severely attenuated in MRD+ patients. Furthermore, failure of immunological recovery is driven at least partially by a lack of stem-like T cells in MRD+ leukemia patients. Overall, our studies have revealed that low dose anthracyclines, in contrast to high dose, induce opposing, dichotomous effects on LSCs vs. HSCs and stem-like vs. differentiated T cells. Citation Format: Fang Tao, Sara McElroy, Jacqelyn Nemechek, Irina Pushel, John Szarejko, Santosh Khanal, Todd Bradley, Doug Myers, John M. Perry. Stem-like T cells maintain latent anticancer activity and underly therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5557.

FLT3, a prognostic biomarker for acute myeloid leukemia (AML): Quantitative monitoring with a simple anti‐FLT3 interaction and flow cytometric method

BackgroundOverexpression of fms‐like tyrosine kinase 3 (FLT3) protein in leukemia is highly related to poor prognosis and reduced survival rate in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Simple but efficient quantification of FLT3 protein levels on the leukemic cell surface using flow cytometry had been developed for rapid determination of FLT3 on intact cell surface.MethodsQuantitation protocol for FLT3 biomarker in clinical samples was developed and validated. Cell model selection for calibration curve construction was identified and evaluated. Selected antibody concentrations, cell density, and incubation time were evaluated for most appropriate conditions. Comparison of the developed FLT3 determination protocol with the conventional Western blot analysis was performed.ResultsEoL‐1 cell line was selected for using as positive control cells. Calibration curve (20%‐120% of FLT3 positive cells) and quality control (QC) levels were constructed and evaluated. The results demonstrated good linearity (r 2 > 0.99). The intra‐ and inter‐day precision and accuracy, expressed as the coefficient of variation (%CV) and % recovery, were <20% and fell in 80%‐120% in all cases. When compared with Western blotting results, FLT3 protein expression levels in leukemia patient's bone marrow samples were demonstrated in the same trend.ConclusionsThe effective, reliable, rapid, and economical analytical technique using the developed flow cytometric method was demonstrated for FLT3 protein determination on leukemic cell surface. This method provided a practical analysis of FLT‐3 biomarker levels which is valuable for physician decision in acute leukemia treatment.

Concise Review: Adaptation of the Bone Marrow Stroma in Hematopoietic Malignancies: Current Concepts and Models.

The bone marrow stroma maintains hematopoiesis and coordinately regulates regenerative responses through dynamic interactions with hematopoietic stem and progenitor cells. Recent studies indicate that stromal components in the bone marrow of leukemia patients undergo a process of successive adaptation that in turn exerts dramatic effects on the hematopoietic stem cell compartment and promotes leukemic drug resistance. Therefore, functional changes in discrete marrow stromal populations can be considered an aspect of leukemia biogenesis in that they create an aberrant, self-reinforcing microenvironment. In this review, we will describe the current understanding of the remodeling of the hematopoietic stem cell niche following invasion by leukemia cells. We place emphasis on existing evidence of how mesenchymal stem cells and their progeny facilitate neoplastic growth and describe available models and analytical techniques to understand the conversion of the niche toward disease persistence. Stem Cells 2018;36:304-312.

Dynamic association rules for gene expression data analysis.

BackgroundThe purpose of gene expression analysis is to look for the association between regulation of gene expression levels and phenotypic variations. This association based on gene expression profile has been used to determine whether the induction/repression of genes correspond to phenotypic variations including cell regulations, clinical diagnoses and drug development. Statistical analyses on microarray data have been developed to resolve gene selection issue. However, these methods do not inform us of causality between genes and phenotypes. In this paper, we propose the dynamic association rule algorithm (DAR algorithm) which helps ones to efficiently select a subset of significant genes for subsequent analysis. The DAR algorithm is based on association rules from market basket analysis in marketing. We first propose a statistical way, based on constructing a one-sided confidence interval and hypothesis testing, to determine if an association rule is meaningful. Based on the proposed statistical method, we then developed the DAR algorithm for gene expression data analysis. The method was applied to analyze four microarray datasets and one Next Generation Sequencing (NGS) dataset: the Mice Apo A1 dataset, the whole genome expression dataset of mouse embryonic stem cells, expression profiling of the bone marrow of Leukemia patients, Microarray Quality Control (MAQC) data set and the RNA-seq dataset of a mouse genomic imprinting study. A comparison of the proposed method with the t-test on the expression profiling of the bone marrow of Leukemia patients was conducted.ResultsWe developed a statistical way, based on the concept of confidence interval, to determine the minimum support and minimum confidence for mining association relationships among items. With the minimum support and minimum confidence, one can find significant rules in one single step. The DAR algorithm was then developed for gene expression data analysis. Four gene expression datasets showed that the proposed DAR algorithm not only was able to identify a set of differentially expressed genes that largely agreed with that of other methods, but also provided an efficient and accurate way to find influential genes of a disease.ConclusionsIn the paper, the well-established association rule mining technique from marketing has been successfully modified to determine the minimum support and minimum confidence based on the concept of confidence interval and hypothesis testing. It can be applied to gene expression data to mine significant association rules between gene regulation and phenotype. The proposed DAR algorithm provides an efficient way to find influential genes that underlie the phenotypic variance.

Identification of Cytogenetically Normal Human CD34+CD38+ Hematopoietic Stem/Progenitor Cells from Inv(16)+ Leukemic Bone Marrow

For many subtypes of AML including cases with the inv(16), mutations that give rise to the leukemic phenotype occur, at least in part, in the hematopoietic stem/progenitor (HSPC) cell subset, as suggested by studies showing that primitive CD34+ CD38- bone marrow cells can function as leukemia-initiating cells (LIC) when transferred into immunodeficient mice. A significant challenge has been that LIC share many of the same cell-surface markers as their normal HSPC counterparts, thus making it difficult to purify and functionally characterize either subset from the bulk bone marrow of leukemia patients. Here we report the FACS analysis of several previously reported human LIC markers on bone marrow samples from inv(16) AML patients and show that a combination of TIM3, CLL1, and CD33 can significantly enrich for a rare population of CD34+ CD38- cells that lack the inv(16) fusion mRNA when tested by nested RT-PCR. Heterogeneous expression of these markers among different patient samples often causes incomplete elimination of the fusion mRNA when FACS-sorting the CD34+ CD38- population as single TIM3-, CLL1-, or CD33- subsets. The combination of TIM3 with CLL1 and/or CD33 leads to a more consistent elimination of the fusion mRNA from the FACS-sorted CD34+ CD38- subsets. Results from methylcellulose assays showed that the TIM3- CLL1- CD33- subset of CD34+CD38- cells could form multiple colony types, including CFU-GEMM, that were all negative for the fusion mRNA by RT-PCR. In contrast, colonies derived from bulk bone marrow were all positive for the fusion mRNA. The TIM3- CLL1- CD33- subset of CD34+CD38- cells displayed greater than 600-fold enrichment for progenitor activity compared to bulk bone marrow but did not form additional colonies upon serial re-plating. These results have important implications for the therapeutic targeting of inv(16)+ hematopoietic stem/progenitor cells in patients with relapsed and refractory disease and for purification of normal HSPC from leukemic bone marrow samples. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1030: Are endothelial cells essential for the development of leukemia

Abstract Background The most pronounced micro-environmental changes observed in the bone marrow of leukemia patients are endothelial proliferation and angiogenesis. The molecular basis underlying how blood vessels sustain/initiate leukemia remains poorly understood. However, this may explain why anti-angiogenic agents are effective treatments for leukemia. Aim To define whether modification of the endothelium initiates and/ or perpetuates leukemia and determine the genes responsible. Methods Sleeping Beauty (SB) is a system of insertional mutagenesis that randomly integrates transposons into genomic DNA. Endothelial-specific SB activation was achieved by driving CRE-recombinase expression via the Tie-2 promoter/enhancer. Tissues were analyzed for histology, immune profiles and genes with insertional mutations. Colony forming unit (CFU) assays were performed on magnetically selected CD45-/CD31+ endothelial cells and CD45-/CD31-/Sca1+ hematopoietic stem cells isolated from bone marrow of CRE+ and CRE- mice and the resulting colonies analyzed by flow cytometry for CD3 (lymphoid) and CD14 (myeloid) populations. Results Over 50 weeks all CRE+ mice (n=106), but none of the WT mice (n=35), became moribund and were sacrificed. At necropsy, CRE+ mice displayed peripheral leukocytosis and gross anatomical/histological modifications in spleens, livers and thymus. Molecular analyses showed amplifications of either T cell (CD3; 49%) or macrophage (CD14; 28%) populations or both (23%) in the spleen and bone marrow of CRE+ mice. These leukocytes were SB positive indicating they originated from endothelial cells, and not hematopoietic stem cells. This was confirmed by CFU assays in which elevated CD3+ and CD14+ populations were found arising from the CD45-/CD31+ cells but not CD45-/CD31-/Sca1+ cells from CRE+ mice relative to WT mice. Targeted deep sequencing identified genes related to cytokine mediated signaling and transcriptional control. Many of the identified genes are already implicated in human leukemia. Conclusions Targeting SB insertional mutagenesis to the endothelium resulted in hematological malignancy in mice indicating direct control over both myeloid and lymphoid differentiation by endothelial cells. Targeted deep sequencing identified changes to known mediators in hematological lineage specification, validating our model. Also identified were novel candidates that may contribute to the development of leukemia and offer new therapeutic targets to treat this malignancy. These data, for the first time, provide evidence for a role of the endothelium in the regulation of post-natal hematopoiesis and provide a rationale for the use of anti-angiogenic therapy in the treatment of leukemia. Citation Format: Indhu Subramanian, Emily S. Fuller, Katie Powell, Rolfe Howlett, Anthony W. Ashton, Viive M. Howell. Are endothelial cells essential for the development of leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1030. doi:10.1158/1538-7445.AM2014-1030

Protein and mRNA expression of CTGF, CYR61, VEGF-C and VEGFR-2 in bone marrow of leukemia patients and its correlation with clinical features

This study was aimed to investigate the mRNA and protein expression of CTGF, CYR61, VEGF-C and VEGFR-2 in bone marrow of patients with leukemia, and to analyze the role and clinical significance of these 4 factors in genesis and development of leukemia, infiltration and metastasis of leukemic cells. A total of 100 cases of newly diagnosed leukemia, 26 cases of acute leukemia in complete remission and 30 controls were enrolled in this study. The mononuclear cells of bone marrow were collected, the mRNA and protein expression levels of CTGF, CYR61, VEGF-C, VEGFR-2 in leukemia patients and controls were detected by real time PCR and Western blot, respectively. The results showed that the mRNA and protein expression levels of above mentioned 4 factors were significantly higher than those in control (P < 0.05), only CTGF mRNA expression in AL patients after complete remission showed statistical difference as compared with control (P < 0.05), but the expression of CTGF mRNA showed statistical significance in different bone marrow hyperplasia of acute leukemia (P < 0.05). The expression level of CTGF protein showed difference in different chromosome karyotypes of leukemia (P < 0.05). The expression levels of CYR61 and VEGF-C proteins showed statistical difference in different bone marrow hyperplasia of acute leukemia (P < 0.05). The expression level of CTGF, CYR61, VEGF-C mRNA and protein in CML group were higher than that in control group. The expression levels of CTGF and CYR61 protein were higher than that in control. The mRNA and protein expression levels of above-mentioned 4 factors in sex and infiltration lf leukemic cells did not show statistical significance(P < 0.05). In correlative analysis, the mRNA expressions of above mentioned 4 factors were positively correlated with bone marrow blast count(P < 0.05), the protein expression of CTGF, CYR61 and VEGF-C were positively correlated with bone marrow blast count. It is concluded that the CTGF, CYR61, VEGF-C and VEGFR-2 mRNA and protein play a role in acute leukemia. In acute leukemia (AML/ALL), the expression of above mentioned factor was high, but except VEGFR-2. Most of them were positively correlated with bone marrow blast count. Joint block of these angiogenesis-related factors is likely to play an important role in targeting treatment of leukemia.

Abstract 773: WT1 regulation of Cyclin A1 in leukemia.

Abstract We have observed high levels of Wilms Tumor 1 (WT1) expression in acute leukemias, suggesting that expression of this zinc finger transcription factor might contribute to leukemia development. WT1 has also been used as a marker for minimal residual disease, and patients who relapse often show high WT1 expression in peripheral blood lymphocytes. WT1 levels in the bone marrow of leukemia patients are found to be higher than in normal marrow or peripheral blood. Thus, we predict that WT1 plays a leukemogenic role by regulating expression of growth control genes. Cyclin A1 (CCNA1) is a cell cycle protein expressed in highly proliferative tissues such as the testis and bone marrow, where it is found in both hematopoietic stem cells and leukemia cells. We hypothesized that WT1 up-regulates the expression of CCNA1, potentially enhancing proliferation of leukemic blasts. To determine whether WT1 regulated CCNA1 expression we analyzed the CCNA1 promoter region using the MatInspector software and identified two possible WT1 binding sites. WT1 binding at the CCNA1 promoter in chromatin of K562 leukemic cells was demonstrated by Chromatin Immunoprecipitation (ChIP) analysis using both end point and quantitative real-time PCR (qRT-PCR). Transcriptional activation of the CCNA1 gene by WT1 was tested by transfection of K562 cells with the transcriptionally active WT1 isoform (-KTS) and analysis of RNA expression. Analysis of cDNA by qRT-PCR showed an upregulation of CCNA1 transcripts in WT1 transfected cells. However, upregulation was not observed following transfection with a mutant form of the WT1 that lacks the DNA binding domain (due to a frameshift mutation in exon 7). CCNA1 up-regulation may be relatively specific for leukemia cells, as prostate cancer cells transfected with WT1 showed no changes in CCNA1 levels. To address the clinical relevance of WT1 expression in pediatric acute leukemia, qRT-PCR was used to measure WT1 and CCNA1 transcript levels in leukemic bone marrow, relative to non-neoplastic bone marrow samples. Results showed higher levels of WT1 expression in Acute Myeloid Leukemia (AML) in comparison to Acute Lymphoid Leukemia (ALL). In particular, an examination of AML-M3 bone marrow samples showed high expression of both WT1 and CCNA1, consistent with WT1 upregulation of CCNA1. Additionally, in pediatric ALL samples with elevated CCNA1 levels, we also observed elevated WT1 levels. Overall these findings support the hypothesis that WT1 may up-regulate CCNA1 expression in bone marrow progenitors potentially promoting oncogenic transformation. WT1 may also contribute to leukemogenesis through transcriptional regulation of other growth control genes (such as VEGF, bcl2, and myc) shown to be regulated by WT1 in other tumor types. Ultimately this approach of identifying WT1 target genes up-regulated in leukemic blasts could reveal new biomarkers and identify biologically-defined therapeutic targets. Citation Format: Sony Pandey, Mustafa Moazam, Douglas Snyder, Steven J. Kuerbitz, Gail C. Fraizer. WT1 regulation of Cyclin A1 in leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 773. doi:10.1158/1538-7445.AM2013-773

Microfluidic purification and analysis of hematopoietic stem cells from bone marrow

Hematopoietic stem cells are larger in size than other cells present in bone marrow, with the exception of monocytes. This distinguishing characteristic can be used to separate them from a whole-marrow sample. A microfluidic device was fabricated using an integrated membrane that is porous at defined areas. This allows for simultaneous valving and filtering functionality, which is crucial for preventing irreversible clogging. This device, as well as a separation procedure, was optimized in this work to enrich hematopoietic progenitor cells from diluted bone marrow of leukemia patients without any additional sample preparation. An enrichment of up to 98% was achieved with this method and the process was scaled up to 17.2 μL min(-1) of processed sample. Additionally, stem cells were stained with specific antibodies for further analysis. Using a custom-made computer program, the filter was scanned to characterize and quantify cells based on fluorescence. The results were evaluated by comparing them against the results obtained from flow cytometry, confocal microscopy, and Coulter counting.

Effects of Manisa propolis on telomerase activity in leukemia cells obtained from the bone marrow of leukemia patients

Propolis is a resinous material collected by honeybees and obtained from beehives that has anticancer effects by inducing apoptosis. The aim of this study is to investigate the effect of propolis on human telomerase reverse transcriptase (hTERT) in the leukemia cells obtained from leukemia patients. Four different bone marrow cell cultures from each of four leukemia cases were prepared. The 60 ng/ml, 30 ng/ml and 15 ng/ml working concentrations of propolis were administered to three cultures of each patient, while one culture contained only culture medium. hTERT mRNA expression levels of cells were detected at 24 h, 48 h and 72 h using the LightCycler 2.0 instrument. A significant decrease in hTERT expression levels was observed in the 60 ng/ml concentration of propolis. In conclusion,Manisa propolis may also have a potential effect on the expression of hTERT in leukemia—particularly owing to its constituent chrysin.

Development and validation of a gas chromatography/mass spectrometry method for the assessment of genomic DNA methylation

A method for the determination of DNA global methylation, taken as the ratio (%) of 5-methylcytosine (5mCyt) versus the sum of cytosine (Cyt) and 5mCyt, via gas chromatography/mass spectrometry (GC/MS), was developed and validated. DNA (2.5 microg) was hydrolyzed with aqueous formic acid 88%, spiked with cytosine-2,4-(13)C(2),(15)N(3) and 5-methyl-(2)H(3)-cytosine-6-(2)H(1) as internal standards, and derivatized with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide and 1% tert-butyldimethylchlorosilane, in the presence of acetonitrile and pyridine. GC/MS, operating in single ion monitoring mode, separated and specifically detected all nucleobases as tert-butyldimethylsilyl derivatives, without interferences, with the exception of guanosine. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 3.2 pmol for Cyt and 0.056 pmol for 5mCyt, the latter corresponding to a methylation level of 0.41%. Intra- and inter-day precision and accuracy were below 4.0% for both analytes and methylation. The matrix absolute effect, process efficiency and coefficient of variation ranged from 96.5 to 101.2%. The matrix relative effect was below 1%. The method was applied to the analysis of different human DNAs, including: nonmethylated DNA from PCR (methylation 0.00%), hypermethylated DNA prepared using M.SssI CpG methyltransferase (methylation 18.05%), DNA from peripheral blood leukocytes of healthy subjects (N = 6, median methylation 5.45%), DNA from bone marrow of leukemia patients (N = 5, 3.58%) and DNA from myeloma cell lines (N = 4, 2.74%).

Effects of Manisa propolis on telomerase activity in leukemia cells obtained from the bone marrow of leukemia patients

Effects of Manisa propolis on telomerase activity in leukemia cells obtained from the bone marrow of leukemia patients

Vascular Endothelial Growth Factor and Its Receptor as Drug Targets in Hematological Malignancies

Angiogenesis is defined as formation of new blood vessels from the preexisting vasculature, a process which is essential for malignant tumor growth. While this has been accepted for solid forms of cancer there is now emerging evidence that progression of hematological malignancies also requires the induction of new blood vessels. Vascular endothelial growth factor (VEGF) is known to be an essential regulator of physiological and pathological angiogenesis. Numerous preclinical and clinical studies have validated VEGF as target for antiangiogenesis and anticancer therapy. With regard to hematological malignancies a stimulating effect of VEGF for proliferation, survival and migration of leukemia cells could be demonstrated. Bone marrow of leukemia patients shows an increased microvessel density as well as VEGF expression. Complete remissions in acute myeloid leukemia (AML) have been reported by targeting the receptor tyrosine kinase system of VEGF. While the pathophysiology behind the contribution of VEGF to leukemia progression is not yet completely understood, VEGF and its receptors may provide promising targets not only in solid tumors but also hematological malignancies such as AML.

Immunotargetting of the Wilms' Tumor WT1 Antigen for Dendritic Cell and B-Cell-Based Vaccination of Leukemia.

The Wilms' tumor antigen (WT1) is overexpressed in almost all leukemias and in several solid tumors. Overexpression of WT1 blocks the normal differentiation and enhances proliferation of hematopoietic progenitor cells. WT1 is also used in the detection of minimal residual disease. Using WT1-specific MHC class I tetramers, we were able to detect ex vivo low numbers of WT1-specific CD8+ T cells in the peripheral blood or bone marrow of leukemia patients, but not of healthy donors. In one particular donor we could detect up to 24% WT1 tetramer positive cells at the time of diagnosis. WT1 tetramer positive cells were present in all types of leukemia, except for CLL, and also in patients with MDS. Because WT1 plays an important role in leukemogenesis, it could serve as an antigenic target for dendritic cell-based immunotherapy. We used the mRNA electroporation strategy that allows presentation of multiple WT1 epitopes by MHC class I molecules, irrespective of the HLA haplotype. Monocyte-derived DC (Mo-DC) were electroporated with in vitro transcribed WT1 mRNA. RT-PCR and Western blot analysis showed that WT1 RNA and protein, respectively, was present for up to 5 days in WT1-electroporated DC, but not in mock- or EGFP mRNA-electroporated Mo-DC. Importantly, using a CD8+ T cell clone that secretes IFN-gamma upon recognizing the HLA-A2 immunodominant WT1126–134 epitope, we showed that WT1 mRNA-electroporated Mo-DC processed the WT1 protein via the MHC class I pathway and presented the WT1 epitope to the T cells in an HLA- and antigen-specific manner. Since Mo-DCs are a non-expandable source of antigen-presenting cells, we also used proliferating CD40-activated B (CD40-B) cells as inducers for WT1-specific T cell immunity. CD40-B cells were expanded to high numbers from a limited amount of peripheral blood and subsequently electroporated with WT1 mRNA. In T cell clone activation experiments, WT1 mRNA-electroporated CD40-B cells were as efficient as Mo-DC in presenting the WT1 epitope in a MHC class I-restricted manner. Based on these results, we are currently focusing on the in vitro (re)activation of autologous WT1-specific cytotoxic T cells of leukemia patients using WT1-loaded autologous Mo-DC or CD40-B cells and on the immunological parameters to break immune tolerance against the WT1 tumor self antigen.

Expression of HERV-K, a Human Endogenous Retrovirus, in Hematopoietic Cells.

Background: In contrast to other human endogenous retrovirus families which are highly defective, HERV-K proviruses possess all viral gene types of exogenous retroviruses. In malignancy (tumor cell lines, breast cancer, melanoma as well as leukemias and lymphomas) but also in many normal tissues, including placenta HERV-K mRNA expression has been detected. In hematopoietic stem cells, an interaction of HERV-K with elements from vectors which are used for gene therapy may lead to unforeseen consequences. The purpose of this study was to localize HERV-K mRNA expression in hematopoietic stem cells of different sources and to evaluate a potential association of HERV-expression with hematologic diseases and to define a possible risk of HERV-expression for transplantation.Methods: Quantitative real time PCR (RTQPCR) was used for detecting HERV-K expression in MNC from blood and/or bone marrow samples of patients with leukemias ( 10 MDS, 16 MPD, 14 AML and 1 CML blast crisis) and MNC from blood (n=9) and lymph nodes (n=3) from lymphoma patients. In addition, 8 blood samples and 2 bone marrow samples from healthy donors as well as 9 cord blood samples and 20 apheresates from lymphoma patients in remission containing PBSC were analyzed. Analysis of the same HERV-K gene (pol) in placenta and maternal blood as well as in genomic DNA from all above mentioned sources served as control. In HERV-K overexpressing samples expression of HERV in specific cells was tested by RNA in situ hybridisation (RISH), and combined staining with a CD34 specific monoclonal antibody.Results: Relative mRNA levels of HERV-K in apheresates as well as in blood and bone marrow samples of patients with MDS, MPD and AML from subtypes with less than 1% CD34 positive cells were in the range of less than 2% in relation to housekeeping genes. However, in bone marrow samples of two AML and a CML blast crisis (all of them with more than 50% CD34+ cells) as well as in CD34+ cells from cord blood (but not from apheresates or normal bone marrow) as well as in three lymph nodes of patients with B-cell lymphomas, an overexpression of HERV-K was detected and confirmed by RISH. Enhanced expression of HERV-K was detected in cord blood derived apoptotic CD34+ cells, whereas the relative amount of HERV in genomic DNA did not change in samples from different sources.Conclusions. Our data indicate that HERV-K may be activated in lymph nodes of lymphomas and in CD34+ cells from bone marrow of leukemia patients and from cord blood but not in stem cell apheresates from peripheral blood. In cord blood and malignancy expression appears to be associated with apoptosis. Similar amounts of HERV-K in different samples of genomic DNA of the same cells and the absence of HERV-K activation in normal bone marrow and PBSC indicate that activation of HERV-K is the result of regulation of gene expression rather than amplification. A possible specific role of HERV in the pathogenesis of hematologic diseases and in hematopoiesis (cord blood) remains to be established. Differences between peripheral blood stem cells and cord blood may support a critical stance regarding the use of cord blood derived stem cells for transplantation and gene therapy.

The leukemia-associated gene MDS1/EVI1 is a new type of GATA-binding transactivator.

EVI1, located at chromosome band 3q26, encodes a 1051 amino acid zinc finger protein inappropriately expressed in the leukemic cells of 2-5% of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. The activation of EVI1 often follows a chromosomal rearrangement involving band 3q26, and the two most frequent rearrangements are the t(3;3)(q21;q26) and the inv(3)(q21q26). EVI1 exists also as a longer protein that includes 188 additional amino acids at the N-terminus, named MDS1/EVI1. Both genes are expressed at very low levels in the normal bone marrow. The genomic region between the first coding exon of MDS1/EVI1 and the first coding exon of EVI1 is 150-300 kb. The majority of the chromosomal breakpoints at the 5' end of EVI1 in the t(3;3) resulting in EVI1 activation have been mapped in this region. As a consequence of the t(3;3), the cell would be unable to express MDS1/EVI1, although it would express EVI1. We have compared the transcriptional activity of MDS1/EVI1 and EVI1, and we show that MDS1/EVI1 is a strong activator of promoters containing the AGATA motif, whereas EVI1 is a repressor. In addition, whereas EVI1 represses activation by the GATA-1 erythroid factor, MDS1/EVI1 does not, and is itself repressed by EVI1. By gene fusion to the DNA-binding domain of Gal4, we further show that the activation properties of MDS1/EVI1 are restricted to an acidic segment encoded by the second and third exons in the 5' untranslated region of EVI1. We have also examined the relative expression of the two genes in normal bone marrow and in the bone marrow of leukemia patients with 3q26 rearrangements. Our results indicate that the rearrangements at 3q26 affect expression of EVI1, but not of MDS1/EVI1. We propose that rearrangements at 3q26 involving EVI1 could result in leukemia by a two-step process involving first transcriptional disruption of MDS1/EVI1, and next by inappropriately activating expression of EVI1.

Monitoring of WT-1 gene expression in peripheral blood of patients with acute leukemia by semiquantitative RT-PCR; possible marker for detection of minimal residual leukemia.

The expression of the WT-1 gene which is found exclusively in human leukemic blasts frequently disappears from bone marrow of leukemia patients in complete remission (CR). Using semiquantitative RT-PCR, we investigated the expression of the WT-1 gene in peripheral bloods (PBs) of 33 patients with acute leukemia (AML 26; ALL 7) and monitored its expression after achievement of CR. None of the 6 normal controls expressed detectable levels of WT-1 transcripts (< 10(-4), background level), whereas 31 (93.9%) of 33 patients expressed variable levels of WT-1 transcripts (range, 10(-4) to 10(1)) at diagnosis. The level of WT-1 expression was not different between AML and ALL. By monitoring WT-1 gene expression in PB of 31 patients during CR, 5 patients relapsed (two from the 18 patients with undetectable levels of WT-1 gene expression and three from the 13 with WT-1 gene expression in low levels). Three of the 5 relapsed patients showed preceding reappearance or rise of WT-1 gene expression. From these results, we reconfirmed that the WT-1 gene is a pan-acute leukemic marker, which can be used to monitor minimal residual leukemia (MRL) after chemotherapy or in patients with CR.

Induction of lymphokine-activated killer cells against human leukemia cells in vitro

The induction of lymphokine-activated killer (LAK) cells against fresh human leukemia cells was investigated. Two thirds of the 62 leukemias examined were susceptible to the lytic effect of allogeneic IL-2 induced LAK cells in vitro. No substantial differences could be detected between myeloid or lymphoid leukemias or with regard to the FAB subtype or the immunophenotype. Culturing mononuclear cells from peripheral blood or bone marrow of leukemia patients with IL-2 resulted in an expansion of residual large granular lymphocytes and development of cytotoxic activity. The combination of IL-2 with IFN-gamma or the presence of tumor cells during the activation process led to an enhancement of LAK cell cytotoxicity. These results suggest that LAK cells may be useful in the treatment of leukemia.