Tor Olofsson

Microarray-based classification of a consecutive series of 121 childhood acute leukemias: prediction of leukemic and genetic subtype as well as of minimal residual disease status.

Gene expression analyses were performed on 121 consecutive childhood leukemias (87 B-lineage acute lymphoblastic leukemias (ALLs), 11 T-cell ALLs and 23 acute myeloid leukemias (AMLs)), investigated during an 8-year period at a single center. The supervised learning algorithm k-nearest neighbor was utilized to build gene expression predictors that could classify the ALLs/AMLs according to clinically important subtypes with high accuracy. Validation experiments in an independent data set verified the high prediction accuracies of our classifiers. B-lineage ALLs with uncharacteristic cytogenetic aberrations or with a normal karyotype displayed heterogeneous gene expression profiles, resulting in low prediction accuracies. Minimal residual disease status (MRD) in T-cell ALLs with a high (>0.1%) MRD at day 29 could be classified with 100% accuracy already at the time of diagnosis. In pediatric leukemias with uncharacteristic cytogenetic aberrations or with a normal karyotype, unsupervised analysis identified two novel subgroups: one consisting mainly of cases remaining in complete remission (CR) and one containing a few patients in CR and all but one of the patients who relapsed. This study of a consecutive series of childhood leukemias confirms and extends further previous reports demonstrating that global gene expression profiling provides a valuable tool for genetic and clinical classification of childhood leukemias.

Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein

Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome, formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML will require full eradication of Ph chromosome-positive (Ph+) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34+ cells and also in cord blood CD34+ cells as a consequence of retroviral BCR/ABL1 expression. To test whether IL1RAP expression distinguishes normal (Ph−) and leukemic (Ph+) cells within the CML CD34+CD38− cell compartment, we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method, we sorted cells directly into drops on slides to investigate their Ph-chromosome status. Interestingly, we found that the CML CD34+CD38−IL1RAP+ cells were Ph+, whereas CML CD34+CD38−IL1RAP− cells were almost exclusively Ph−. By performing long-term culture-initiating cell assays on the two cell populations, we found that Ph+ and Ph− candidate CML stem cells could be prospectively separated. In addition, by generating an anti-IL1RAP antibody, we provide proof of concept that IL1RAP can be used as a target on CML CD34+CD38− cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell surface biomarker distinguishing Ph+ from Ph− candidate CML stem cells and opens up a previously unexplored avenue for therapy of CML.

Fusion of the BCR and the fibroblast growth factor receptor-1 (FGFR1) genes as a result of t(8;22)(p11;q11) in a myeloproliferative disorder: The first fusion gene involving BCR but not ABL

Constitutive activation of tyrosine kinases as a consequence of chromosomal translocations, forming fusion genes, plays an important role in the development of hematologic malignancies, in particular, myeloproliferative syndromes (MPSs). In this respect, the t(9;22)(q34;q11) that results in the BCR/ABL fusion gene in chronic myeloid leukemia is one of the best‐studied examples. The fibroblast growth factor receptor 1 (FGFR1) gene at 8p11 encodes a transmembrane receptor tyrosine kinase and is similarly activated by chromosomal translocations, in which three alternative genes—ZNF198 at 13q12, CEP110 at 9q34, and FOP at 6q27—become fused to the tyrosine kinase domain of FGFR1. These 8p11‐translocations are associated with characteristic morphologic and clinical features, referred to as “8p11 MPS.” In this study, we report the isolation and characterization of a novel fusion gene in a hematologic malignancy with a t(8;22)(p11;q11) and features suggestive of 8p11 MPS. We show that the breakpoints in the t(8;22) occur within introns 4 and 8 of the BCR and FGFR1 genes, respectively. On the mRNA level, the t(8;22) results in the fusion of BCR exons 1–4 in‐frame with the tyrosine kinase domain of FGFR1 as well as in the expression of a reciprocal FGFR1/BCR chimeric transcript. By analogy with data obtained from previously characterized fusion genes involving FGFR1 and BCR/ABL, it is likely that the oligomerization domain contributed by BCR is critical and that its dimerizing properties lead to aberrant FGFR1 signaling and neoplastic transformation.

Characterization of bone marrow-derived mesenchymal stromal cells (MSC) based on gene expression profiling of functionally defined MSC subsets.

BACKGROUND AIMS Human mesenchymal stromal cells (MSC) are promising candidates for cell therapy because of their intriguing properties (high proliferation and differentiation capacity, microenvironmental function and immune modulation). However, MSC are heterogeneous and a better understanding of the heterogeneity of the cells that form the MSC cultures is critical. METHODS Human MSC were generated in standard cultures and stained with carboxyfluorescein succinimidyl ester (CFSE) for cell division tracking. Gene expression profiling of MSC that were sorted based on functional parameters (i.e. proliferation characteristics) was utilized to characterize potential MSC subpopulations (progenitor content and differentiation capacity) and identify potential MSC subpopulation markers. RESULTS The majority of MSC had undergone more than two cell divisions (79.7+/-2.0%) after 10 days of culture, whereas 3.5+/-0.9% of MSC had not divided. MSC were then sorted into rapidly dividing cells (RDC) and slowly/non-dividing cells (SDC/NDC). Colony-forming unit-fibroblast (CFU-F) frequencies were lowest in NDC and highest in RDC with low forward-/side-scatter properties (RDC(lolo)). Comparative microarray analysis of NDC versus RDC identified 102 differentially expressed genes. Two of these genes (FMOD and VCAM1) corresponded to cell-surface molecules that enabled the prospective identification of a VCAM1(+)/FMOD(+) MSC subpopulation, which increased with passage and showed very low progenitor activity and limited differentiation potential. CONCLUSIONS These data clearly demonstrate functional differences within MSC cultures. Furthermore, this study shows that cell sorting based on proliferation characteristics and gene expression profiling can be utilized to identify surface markers for the characterization of MSC subpopulations.

A novel and cytogenetically cryptic t(7;21)(p22;q22) in acute myeloid leukemia results in fusion of RUNX1 with the ubiquitin-specific protease gene USP42

Although many of the chromosomal abnormalities in hematologic malignancies are identifiable cytogenetically, some are only detectable using molecular methods. We describe a novel cryptic t(7;21)(p22;q22) in acute myeloid leukemia (AML). FISH, 3′RACE, and RT-PCR revealed a fusion involving RUNX1 and the ubiquitin-specific protease (USP) gene USP42. The genomic breakpoint was in intron 7 of RUNX1 and intron 1 of USP42. The reciprocal chimera was not detected – neither on the transcriptional nor on the genomic level – and FISH showed that the 5′ part of USP42 was deleted. USP42 maps to a 7p22 region characterized by segmental duplications. Notably, 17 kb duplicons are present 1 Mb proximal to USP42 and 3 Mb proximal to RUNX1; these may be important in the genesis of t(7;21). This is the second cryptic RUNX1 translocation in hematologic malignancies and the first in AML. The USPs have not previously been reported to be rearranged in leukemias. The cellular context in which USP42 is active is unknown, but we here show that it is expressed in normal bone marrow, in primary AMLs, and in cancer cell lines. Its involvement in the t(7;21) suggests that deregulation of ubiquitin-associated pathways may be pathogenetically important in AML.

Induction of differentiation in a human promyelocytic leukemic cell line (HL-60): Production of granule proteins☆

Abstract The human promyelocytic leukemia cell line HL-60 could be induced to differentiate to mature myeloid cells not only with chemical inducers like DMSO, but also with conditioned media produced from human placenta and pokeweed mitogen-stimulated lymphocytes. HL-60 cells were found to be rich in myeloperoxidase but showed only a low content of other enzymes associated with primary granules formed in promyelocytes. The decrease in primary granule enzymes found during induction of differentiation may depend on dilution of granules as a result of cell division. DMSO-induced cells, contrary to cells induced with conditioned media, however, produced increasing amounts of lysozyme indicating diversity of differentiating capacity of HL-60. The absence observed of the production of lactoferrin during differentiation indicates abnormal production or composition of secondary granules expected to be formed as a result of maturation. Furthermore, the expected shift to a higher density of the maturating product was not seen, suggesting that this product is abnormal. Findings of functional capacity of differentiated HL-60 cells reminiscent of normal granulocytes but abnormal granule composition may reflect the leukemic origin which could be responsible for uncoupling of various markers of differentiation.

The lipocalin alpha1-microglobulin protects erythroid K562 cells against oxidative damage induced by heme and reactive oxygen species.

α1-Microglobulin is a 26 kDa plasma and tissue glycoprotein that belongs to the lipocalin protein superfamily. Recent reports show that it is a reductase and radical scavenger and that it binds heme and has heme-degrading properties. This study has investigated the protective effects of α1-microglobulin against oxidation by heme and reactive oxygen species in the human erythroid cell line, K562. The results show that α1-microglobulin prevents intracellular oxidation and up-regulation of heme oxygenase-1 induced by heme, hydrogen peroxide and Fenton reaction-generated hydroxyl radicals in the culture medium. It also reduces the cytosol of non-oxidized cells. Endogeneous expression of α1-microglobulin was up-regulated by these oxidants and silencing of the α1-microglobulin expression increased the cytosol oxidation. α1-microglobulin also inhibited cell death caused by heme and cleared cells from bound heme. Binding of heme to α1-microglobulin increased the radical reductase activity of the protein as compared to the apo-protein. Finally, α1-microglobulin was localized mainly at the cell surface both when administered exogeneously and in non-treated cells. The results suggest that α1-microglobulin is involved in the defence against oxidative cellular injury caused by haemoglobin and heme and that the protein may employ both heme-scavenging and one-electron reduction of radicals to achieve this.

Analysis of the DNA methylome and transcriptome in granulopoiesis reveals timed changes and dynamic enhancer methylation.

In development, epigenetic mechanisms such as DNA methylation have been suggested to provide a cellular memory to maintain multipotency but also stabilize cell fate decisions and direct lineage restriction. In this study, we set out to characterize changes in DNA methylation and gene expression during granulopoiesis using 4 distinct cell populations ranging from the oligopotent common myeloid progenitor stage to terminally differentiated neutrophils. We observed that differentially methylated sites (DMSs) generally show decreased methylation during granulopoiesis. Methylation appears to change at specific differentiation stages and overlap with changes in transcription and activity of key hematopoietic transcription factors. DMSs were preferentially located in areas distal to CpG islands and shores. Also, DMSs were overrepresented in enhancer elements and enriched in enhancers that become active during differentiation. Overall, this study depicts in detail the epigenetic and transcriptional changes that occur during granulopoiesis and supports the role of DNA methylation as a regulatory mechanism in blood cell differentiation.

Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and granulocyte colony‐stimulating factor (G‐CSF) in serum during induction treatment of acute leukaemia

Summary. Granulocyte‐macrophage colony stimulating factor (GM‐CSF) and granulocyte colony‐stimulating factor (G‐CSF) are increasingly used to stimulate granulopoiesis in neutropenic patients but in most cases without any knowledge of the endogenous CSF‐levels. With the purpose to define serum levels of GM‐CSF and G‐CSF during induction chemotherapy and haematological reconstitution in patients with acute leukaemia we have used enzyme‐linked immuno‐sorbent assay (ELISA) techniques to measure these growth factors in 18 patients with acute myeloid leukaemia (AML) and eight patients with acute lymphoblastic or undifferentiated leukaemia (ALL/AUL). G‐CSF above 0·05 ng/ml was detected in 54% of the analysed AML samples, median 0·29 (range 0·05–2·80) ng/ml; and in 40% of analysed ALL/AUL samples, median 0·09 (range 0·05–3·00) ng/ml. In patients with AML there was a clear correlation between an elevated serum concentration of G‐CSF and documented infections. On the other hand, 15/18 of the patients with acute myeloid leukaemia and 8/8 patients with ALL/AUL had non‐detectable levels of GM‐CSF (<0·10 ng/ml). Two patients had measurable levels of GM‐CSF in all samples, median 0·71 (range 0·26–1·18) ng/ml and in these patients the levels successively decreased during and after chemotherapy and did not increase in response to infections. In normals detectable levels of GM‐CSF were found in 2/35 individuals and G‐CSF in 0/10 individuals.

Granulocyte function in chronic granulocytic leukaemia. I. Bactericidal and metabolic capabilities during phagocytosis in isolated granulocytes.

Summary. The ingestion, bactericidal activity and metabolism of isolated mature neutrophil leucocytes during phagocytosis was studied in 17 patients with chronic granulocytic leukaemia (CGL) with the simultaneous use of normal controls. Seven patients had received no treatment and the others had been treated previously with Busulphan. The phagocytic indices for killed yeast cells did not differ from those of the controls. A diminished bactericidal activity against E. coli was found in nine CGL cases. The bactericidal capacity closely correlated with the degree of leuco‐cytosis since patients with a WBC count of 90 ooo/tl or higher with one exception showed decreased bactericidal activities while patients with WBC counts below 90 000/μl with two exceptions showed normal bactericidal activities. The [i‐14C]‐glucose oxidation during phagocytosis was increased in four patients and decreased in three patients. Some correlation was found between abnormally high or low [i‐14C]glucose oxidation and diminished bactericidal activity. The intracellular iodination reaction during phagocytosis was decreased in 10 cases while the extracellular iodination was increased in six cases and decreased in one case. The data for granulocyte iodination did not correlate with WBC count, bactericidal capacity or [i‐14C]glucose oxidation. The time course for the bactericidal activity and granulocyte iodination seemed to deviate from the controls indicating a slow initial ingestion and/or degranulation phase. The CGL granulocyte content of myeloperoxidase was normal or increased, the lysozyme content was decreased in half of the cases while the amount of antibacterial cationic proteins was increased, normal or low. The present findings indicate a variety of abnormalities in the mature CGL granulocyte, which are not closely interrelated.