Fluorescence in situ hybridization: a sensitive method for trisomy 8 detection in bone marrow specimens.

Trisomy 8 is a common anomaly in bone marrow (BM) cells of patients with myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS), or acute nonlymphocytic leukemia (ANLL). We studied the efficacy of fluorescence in situ hybridization (FISH) detection of trisomy 8 in patients with MPD, MDS, or ANLL using directly labeled fluorescent alpha-satellite and whole chromosome paint (WCP) DNA probes specific for chromosome 8. Using FISH, we analyzed interphase nuclei and metaphase spreads from randomized series of BM specimens from normal individuals and patients with varying proportions of trisomy 8 as determined by conventional cytogenetic analysis. The BM of all normal donors contained less than or equal to 2.0% nuclei with 3 interphase FISH signals and less than or equal to 1 metaphase with 3 WCP FISH signals. Ninety-five percent and 98% of BM specimens with at least two metaphase cells with trisomy 8 by cytogenetic analysis contained greater than 2.0% nuclei with 3 interphase FISH and greater than 2 metaphases with 3 WCP FISH signals, respectively. Thirteen patients had 1 in 20 or 1 in 30 metaphase cells with trisomy 8 by conventional cytogenetic studies. Of these patients, four had greater than 2.0% nuclei with 3 interphase FISH signals. The BM of all four patients contained positive metaphase FISH results. We then studied the usefulness of FISH analysis to detect occult trisomy 8 by analyzing BM nuclei from 144 patients who had MPD, MDS, or ANLL and either 20 normal metaphase cells or an abnormal karyotype without trisomy 8. Seven patients had greater than 2.0% nuclei with 3 interphase FISH signals (range, 2.10% to 3.40%) and six patients had 2 or more cells with trisomy 8 upon metaphase FISH or extensive conventional cytogenetic analysis. Our results show that interphase and metaphase FISH analyses are useful methods to detect trisomy 8 cells in BM specimens, especially for specimens with normal or uncertain conventional cytogenetic results.     

Origin of stroma cells in long-term bone marrow cultures from patients with acute myeloid leukemia

 Bone marrow stroma cells from patients with acute myeloid leukemia (AML) display a variety of functional abnormalities. In order to determine whether this is related to an imbalance in the proportion of different stroma cell types or to integration of leukemic progeny into the regulatory cell network, stroma layers were established in mycophenolic acid-treated long-term marrow cultures from 16 patients with AML and 42 controls and analyzed by means of simultaneous membrane immunofluorescence and interphase cytogenetics. Macrophages were identified by CD14 expression, fibroblasts by staining with the AS02 antibody, and malignant cells by leukemia-specific numerical chromosome aberrations, including monosomy 7 and trisomy 8. Compared with normal controls, there was a slight decrease in the proportion of stroma fibroblasts (52±27% versus 77±5%) in 10-week-old cultures from patients with AML. Two of five AML patients with trisomy 8 and both patients with monosomy 7 had evidence of leukemic stroma cells. Most malignant cells were CD14+ macrophages (3.8–98.1% of all CD14+ cells), but some were AS02+ (2.8–5.2%). AML stroma layers showed a reduced capacity to support the growth of normal hematopoietic cells in standard two-stage long-term cultures, but this was unrelated to the presence or absence of leukemic stroma elements. In conclusion, AML populations vary with respect to their ability to produce a malignant microenvironment. Functional defects in the hematopoietic microenvironment, however, are not limited to AML patients with cytogenetically abnormal stroma cells, but extend to cases without evidence of malignant stroma cells. Received: December 9, 1998 / Accepted: February 2, 1999     

Clonal CD5-positive B lymphocytes in myelodysplastic syndrome with systemic vasculitis and trisomy 8

 Bone marrow and peripheral blood from a myelodysplastic syndrome (MDS) patient with trisomy 8 and associated systemic vasculitis was investigated for clonal lymphoid lineage involvement using simultaneous metaphase and interphase fluorescence in situ hybridization (FISH) and immunocytochemistry with antibodies against CD13 (granulocytic), glycophorin A (GPA, erythroid), and the lymphocytic antigens CD3, CD5, CD20, and CD22. Trisomy 8 was detected in 55% of CD13+, 40% of GPA+, 6% of CD5+, and 5% of CD20/22+, but not in CD3+ cells. In a complementary experiment using interphase FISH on bone marrow cells sorted by flow cytometry, 13% of CD5/CD19 double-positive cells (76% purity) were found to be trisomic. The results indicate the existence of a small CD5-positive B-lymphoid clone as part of the MDS process in this patient. Since CD5/19-positive cells have been proposed to be autoantibody producing, this finding might be a clue to the pathogenesis underlying the propensity for MDS patients to develop immune-mediated complications. Received: 2 September 1996 / Accepted: 17 October 1996     

Cytogenetically aberrant cells in the stem cell compartment (CD34+lin-) in acute myeloid leukemia

Leukemia may be viewed as a clonal expansion of blast cells; however, the role of primitive cells and/or stem cells in disease etiology and progression is unclear. We investigated stem cell involvement in leukemia using fluorescence in situ hybridization (FISH), immunofluorescence labeling of hematopoietic subpopulations, and flow cytometric analysis/sorting to discriminate and quantify cytogenetically aberrant stem cells in 12 acute myeloid leukemia (AML) and three myelodysplastic (MDS) specimens. Flow cytometric analysis and sorting were used to discriminate and collect a primitive subpopulation enriched in stem cells expressing CD34+ and lacking CD33 and CD38 (CD34+lin-). A subpopulation containing progenitors and differentiating myeloid cells expressed CD34, CD33, and CD38 (CD34+lin+). Nine specimens contained less than 10% CD34+ cells and, thus, were considered to be CD34- leukemias. Mature lymphoid, myeloid, and erythroid subpopulations were sorted on the basis of antigen-linked immunofluorescence. Cytogenetically aberrant cells in sorted subpopulations were identified using FISH with enumerator probes selected on the basis of diagnosis karyotype. Cytogenetically aberrant CD34+lin- cells were present at frequencies between 9% and 99% in all specimens. CD34+lin- cytogenetically aberrant cells comprised between 0.05% and 11.9% of the marrow/blood specimens. Cytogenetically aberrant CD34+lin+ cells constituted 0.01% tp 56% of the marrow/blood population. These data demonstrate that aberrant cells are present in primitive CD34+ stem cell compartments, even in CD34- leukemias. Stem cell involvement was confirmed further by sorting lymphoid and erythroid subpopulations from eight specimens in which the predominant leukemic population lacked lymphoid/erythroid differentiation markers. In these specimens, as well as in multiple lineages, suggests involvement of a cell(s) with multilineage capabilities. The ability of aberrant CD34+lin- stem cells to contribute to clonal and compartment expansion within immunofluorescently defined subpopulations was evaluated to explore the functional phenotype of aberrant CD34+lin- cells. Analysis of compartment size and aberrant cell frequency suggests that frequency of cytogenetically aberrant stem cells is uncoupled from compartment size. These data suggest that cytogenetically aberrant cells in the primitive compartment show varying abilities to expand primitive compartments. Cytogenetically aberrant CD34+lin- cells precede the blast subpopulation in hierarchical maturation and may in some cases by considered preleukemic, requiring maturation or additional mutations before transformation (eg, compartmental expansion) occurs.     

Exacerbation of acute leukemia bearing isolated i(17q) along with proliferation of blasts with high BMI-1 expression

We report a case of acute leukemia with an isolated isochromosome 17q karyotypic abnormality, which may be transformed from myeloproliferative disease (MPD)/myelodysplastic syndrome (MDS). A 69-year-old male patient with 27% of blasts in the peripheral blood underwent hematological examinations including cytochemical staining of cells such as myeloperoxydase (MPO), surface marker study on blasts, chromosomal test and bcr-abl mRNA analysis. The cytological and molecular findings (MPO-positive, myeloid marker CD13 expression (67.3%) and megakaryocytic marker CD41 expression (24.8%)) indicated that the blasts were consistent with myeloid leukemic cells partially committed to megakaryocytes. He was diagnosed as having leukemic transformation from MPD/MDS based on history of leukocytosis and thrombocytosis, isolated i(17q), bcr-abl negative, hepatosplenomegaly, increased eosinophil/basophil count and cytologic dysplasia. Positivity of BMI-1 in CD34+ blasts was 25.8% at the diagnosis and anti-leukemic drugs including anthracyclines were effective for his disease control during 6 months. However, the CD34+ cells turned out to highly express BMI-1 (83.1%), and leukemic cells started to increase progressively following which the leukemic cells failed to respond efficiently to any anti-leukemic drugs. Thus, expression of BMI-1 was well correlated with the disease progression, growth ability of blasts and resistance to anti-cancer drugs, indicating that BMI-1 positivity in CD34+blasts is an excellent molecular marker for disease progression and prognosis in such patients.     

Characterization of Thy-1 (CDw90) expression in CD34+ acute leukemia

Thy-1 (CDw90) is a phosphatidylinositol-anchored cell surface molecule which, when coexpressed with CD34 in normal human bone marrow, identifies a population of immature cells that includes putative hematopoietic stem cells. To date, the characterization of Thy-1 expression has been confined largely to normal tissues and cell lines. In this study, we evaluated the frequency and intensity of Thy-1 expression as defined by reactivity with the anti-Thy-1 antibody 5E10 in 38 cases of CD34+ acute leukemia (21 acute myelogenous leukemia [AML], 8 chronic myelogenous leukemia [CML] in blast crisis, and 9 acute lymphoblastic leukemia [ALL]). In 34 of 38 cases (89%) the CD34+ cells lacked expression of the Thy-1 antigen. High-density Thy-1 expression was found in 1 case of CML in lymphoid blast crisis, and low- density Thy-1 expression was identified on a portion of the leukemic cells in 2 cases of AML with myelodysplastic features, and 1 case of CML in myeloid blast crisis, suggesting a possible correlation between Thy-1 expression and certain instances of stem cell disorders such as CML and AML with dysplastic features. In contrast, the dissociation of Thy-1 and CD34 expression in the majority of acute leukemias studied suggests that the development of these leukemias occurs at a later stage than the hematopoietic stem cell. Characterization of Thy-1 expression in acute leukemia may eventually provide insights into the origin of the disease. In addition, separation of leukemic blasts from normal stem cells based on Thy-1 expression may prove useful in assessing residual disease, as well as in excluding leukemic blasts from stem cell preparations destined for autologous bone marrow or peripheral stem cell transplantation.     

Acute leukemia after a primary myelodysplastic syndrome: immunophenotypic, genotypic, and clinical characteristics.

We studied the nature of blast cells in 41 patients with acute leukemia following a previous primary myelodysplastic syndrome (MDS) by a combined multiparameter analysis including morphologic, immunophenotypic, and molecular genetic (Igs, T-cell receptor (TCR)-beta, -gamma, and -delta and the major breakpoint cluster region [M-bcr]) investigations. In addition, the clinical and hematologic characteristics according to the immunophenotype of blast cells were analyzed. Our results show that, although the granulocytic and/or monocytic lineages are those most commonly involved in these acute leukemias, other cell components, including the megakaryocytic and lymphoid, may be present (12% and 15% of the cases, respectively). Moreover, both morphologic and phenotypic studies show the frequent coexistence of two or three cell populations. Interestingly, in all cases the lymphoblastic component constantly displayed an early B phenotype (CD19+, CD10-, TdT+). Upon analyzing whether the type of MDS conditioned any differences in the immunophenotype of blast cells, we observed that, although the lymphoid lineage may be involved in all MDS subgroups, some differences emerge within the myeloid leukemic transformations. Thus, the refractory anemias with excess of blasts (RAEB) and RAEB in transformation displayed a significantly higher incidence of myeloblastic and megakaryoblastic transformations, while in the RA, RA with ring sideroblasts and chronic myelomonocytic leukemia, the granulo-monocytic phenotype predominated. In addition, our results show that the clinical and hematologic characteristics of these patients may be partially related to the immunophenotype of the blast cells. Ig heavy chain gene rearrangements were found in two of 19 patients analyzed (11%), one with a hybrid leukemia (lymphoid-myeloid) and the other with a granulo-monocytic phenotype. Two other hybrid transformations analyzed were in germline configuration. Gamma and delta gene rearrangements were found in 21% and 37% of these acute transformation, respectively. The TCR-beta and M-bcr were in germline configuration in all 19 cases studied. In summary, immunophenotype and molecular studies point to a pluripotent stem cell with preferential myeloid commitment as the target cell of leukemias following a primary MDS.     

A case of T cell prolymphocytic leukemia involving blast transformation

We report a case of T cell prolymphocytic leukemia (T-PLL) involving blast transformation. At the initial diagnosis, most peripheral blood cells demonstrated proliferation of indolent T cell small cell variants, i.e., small to medium prolymphocytes with inconspicuous nucleoli and a normal karyotype. These cells were positive for surface CD4, CD5, and CD7, and cytoplasmic CD3, but negative for surface CD3 and CD8 and cytoplasmic terminal deoxynucleotidyl transferase (TdT). The T cell receptor (TCR) Cβ1 gene was rearranged in the cells. Large prolymphocytes with prominent nucleoli, irregular nuclei, and cytoplasmic vacuoles that exhibited chromosome 8 trisomy were observed about 1.5 years later. The CD4+CD8− single positive effector memory T cells transformed into surface CD4+CD8+ double positive precursor T cells. The clonal TCR gene rearrangement patterns of these cells were identical throughout the clinical course, suggesting clonal blast transformation. The CD4+CD8+ cells demonstrated increased chromosome 8 trisomy combined with complex chromosome abnormalities with t(14;14)(q11.2;q32) containing a 14q32 chromosome after transformation. T cell leukemia 1a (TCL1a) (14q32.1) may be implicated in this case. The TCL1a oncoprotein is expressed in approximately 70% of T-PLL cases. The disease gradually developed resistance to chemotherapy, and the patient died of the disease. It is known that indolent T-PLL can become aggressive. Therefore, similar transformations may occur in other aggressive T-PLL cases, particularly those involving trisomy 8 and TCL1a.     

Myelodysplastic syndrome/acute myeloid leukemia supervening previously untreated chronic B-lymphocytic leukemia: demonstration of the concomitant presence of two different malignant clones by immunologic and molecular analysis

 The development of myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML) has rarely been observed in patients with chronic B-lymphocytic leukemia (B-CLL). So far, the discussion concerning the pathogenesis of the simultaneous occurrence of these two malignancies has been speculative, opposing the theory of two separate malignant clones to the theory of a common stem cell malignancy. We describe the case of a 77-year-old woman who developed MDS after 8 years of an indolent course of B-CLL. The diagnosis of MDS was based on bone marrow (BM) morphology, showing the typical picture of a refractory anemia with excess of blasts (RAEB). The clinical course of MDS was aggressive, terminating in AML within only 6 months. Immunophenotyping of BM and peripheral blood (PB) cells revealed a CD34⁺/CD13⁺/CD33^–/CD19^– blast cell population and a CD19⁺/CD5⁺ B-cell population with kappa light chain restriction. Molecular analysis of PB and BM demonstrated the presence of an immunoglobulin heavy chain (IgH) gene rearrangement by polymerase chain reaction (PCR) amplification of genomic DNA with three different pairs of consensus primers. Cell-sorting experiments showed that the IgH gene rearrangement was present only in the CD19⁺/CD34^– B-cell population, but not in the CD34⁺/CD19^– blast cells. Furthermore, X-chromosome inactivation pattern analysis revealed two differently methylated cell populations. These experiments demonstrate the concomitant existence of two different clones in a patient with CLL-MDS/AML. Received: 30 October 1996 / Accepted: 8 January 1997     

Acute lymphoid leukemias following either a previous chronic myelogenous leukemia or myelodysplastic syndrome: Phenotypic and genomic differences

We have analyzed the immunological and genomic characteristics of the lymphoid blast cells present in secondary lymphoid leukemias following either a previous chronic myelogenous leukemia (CML) or myelodysplastic syndrome (MDS). Twenty-one of 107 secondary leukemias analyzed displayed a lymphoid phenotype (15 after a CML and 6 after a MDS). Most of the lymphoid blast crises of CML (73%) correspond to pure lymphoid transformation, all of them having a common acute lymphoblastic leukemia (ALL) phenotype (CD10+). By contrast, in all MDS cases, lymphoid blast cells coexisted with another myeloid component (hybrid leukemias) and showed an early B phenotype. IgH and TCR-γ gene rearrangements were detected in the CML-lymphoid blast crisis (86% of cases) more frequently than in the MDS transformations (33%). The TCR-β gene was in germ line configuration in all cases while TCR-δ gene rearrangements were detected in four cases, all of them corresponding to a previous diagnosis of CML. These results show the existence of both immunophenotypic and genomic differences between the lymphoid transformations of either CML or MDS, which could reflect differences at the stage of maturation of the target cell in these transformations.     

Role of cytokines in leukemic type growth of myelodysplastic CD34+ cells

The clonal growth of progenitor cells from myelodysplastic syndromes (MDS) can be subdivided into four growth patterns: (1) normal, (2) no growth or low plating efficiency, (3) low colony and high cluster number, and (4) normal or high colony number with a large number of clusters. The former two (1 and 2) can be referred to as nonleukemic patterns and latter two (3 and 4) as leukemic. In a search for a role for cytokines in leukemic-type growth of MDS progenitor cells, marrow CD34+ cells were purified up to 94% for 8 normal individuals and 88% for 12 MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). The purified CD34+ cells were cultured for 14 days with various combinations of cytokines, including recombinant human macrophage colony-stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte-macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3), and stem cell factor (SCF; a ligand for c-kit) in serum-free medium. The clonal growth of MDS CD34+ cells supported by a combination of all of the above cytokines was subdivided into the two patterns of leukemic or nonleukemic, and then the role of individual or combined cytokines in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. Evidence we obtained showed that SCF plays a central role in the leukemic-type growth of MDS CD34+ cells and that G-CSF, GM-CSF; and/or IL-3 synergize with SCF to increase undifferentiated blast cell colonies and clusters over that seen in normal CD34+ cells. SCF is present in either normal or MDS plasma at a level of nanograms per milliliter, and this physiologic concentration of SCF can stimulate progenitor cells. This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blast cells. This depends, at least in part, on cytokines such as G-CSF, GM-CSF, IL-3, and SCF.     

Surface phenotype and Ig heavy-chain gene usage in chronic B-cell leukemias: expression of myelomonocytic surface markers in CD5- chronic B-cell leukemia

We investigated the surface expression of leukocyte differentiation antigens and the Ig heavy-chain variable region (VH) gene family use in leukemic cells from 26 Japanese patients with chronic B-cell leukemias with special reference to CD5 antigen expression. CD5 was expressed on leukemic cells in 21 of 26 cases (CD5+) but not in 5 cases (CD5-). Myelomonocytic marker, CD13 antigen was expressed on the leukemic cells in all 5 CD5- cases but in none of CD5+ cases. Leukemic cells in CD5- cases also expressed CD11b antigen more frequently than those in CD5+ cases (80% v 11%; P < .01). Another myeloid marker, CD33, was expressed neither on CD5+ nor CD5- leukemic cells. CD22, a restricted B-cell marker, was expressed more frequently on CD5- leukemic cells than on CD5+ leukemic cells (80% v 33%; P < .05). Another restricted B-cell activation marker, CD23, was expressed at similar frequency in both the CD5+ and CD5- groups (67% v 60%). Although CD45RA was expressed on the majority of leukemic B cells, the CD45RA expression level was significantly higher among CD5- cases than CD5+ cases (P < .01). In the analysis of VH gene expressed in chronic B-cell leukemias by polymerase chain reaction amplification, CD5+ cases preferentially used VH4 family members (48%; 10 of 21). CD5- cases, on the other hand, mainly used VH3 family (80%; 4 of 5). Thus, from our present observation of an albeit limited patient population, we have found an association between VH gene family use and CD5 antigen expression in chronic B-cell leukemias. We have also shown the differential expression of myelomonocytic markers in the CD5+ and CD5- chronic B-cell leukemias. These result are in agreement with previous suggestions that CD5 positivity is the hallmark for distinct clinical entity commonly referred to in the literature as chronic lymphocytic leukemia.     

Intracellular expression of CD61 precedes surface expression

 We report a case of acute myeloid leukemia which we have classified as acute megakaryoblastic leukemia because of intracytoplasmic expression of CD61. Light microscopy demonstrated agranular blasts which, by cytochemical staining, were negative for myeloperoxidase. Using flow cytometry, the blast cells were seen to be positive for HLA-DR, CD7, CD13, CD33, and CD34, thus revealing their myeloid origin. Immunophenotyping on fixed blood smears additionally showed positive reaction with the CD61 antibody, demonstrating the megakaryoblastic differentiation of the blasts. After permeabilization of the cell membrane, the intracytoplasmic CD61 expression was confirmed by flow cytometry. Cytogenetic analysis disclosed a del(7)(q21–22). Our findings suggest that cytoplasmic expression of CD61 may precede the cell-surface expression of this antigen and should therefore be investigated in all cases of acute leukemias with undifferentiated morphology and negative cytochemistry to set apart early FAB-M7 from FAB-M0. Received: February 8, 1999 / Accepted: May 5, 1999     

Interphase cytogenetic analysis of in vivo differentiation in the myelodysplasia of Down syndrome

In Down syndrome, acute megakaryoblastic leukemia (AMKL) occurs frequently during the first 4 years of life and is usually preceded by a period of myelodysplasia (MDS), often associated with chromosomal abnormalities. Archival peripheral blood and/or bone marrow films of six patients with Down syndrome and MDS whose leukemic cells contained monosomy 7 or trisomy 8 were studied to determine whether the abnormal precursors produce mature cells in vivo. Using fluorescence in situ hybridization (FISH) of interphase nuclei with chromosome-specific centromere probes for either chromosome 7 or 8, we were able to determine which cells had one, two, or three signals indicative of one, two, or three no. 7 or 8 chromosomes. In five patients with trisomy 8, 80% to 100% (94.5% +/- 6.2%) of the megakaryoblasts had three signals using a chromosome 8 probe; in one patient with monosomy 7, 96.5% of the megakaryoblasts had one signal using a chromosome 7 probe. In all six patients, the myeloid and lymphoid series did not have evidence of the chromosomal abnormality present in the blasts. In three of five patients with trisomy 8, three signals were observed in 27%, 33%, and 41% of normoblasts, respectively. These data are evidence that the abnormal cell in MDS is a progenitor cell with the potential of forming cells of megakaryocyte and erythroid lineages.     

Expression of the hematopoietic growth factor receptor FLT3 (STK- 1/Flk2) in human leukemias

Normal expression of the hematopoietic growth factor receptor FLT3 (STK- 1@Flk2) is limited to CD34+ stem/progenitor cells. We have evaluated the expression of FLT3 by RNase protection assay and Western blotting in 161 primary bone marrow (BM) samples from patients with leukemia. FLT3 RNA was found to be expressed at a higher level than in normal BM controls in 33 of 33 B-lineage acute leukemias, 11 of 12 acute myeloid leukemias (AMLs), and 3 of 11 T-cell acute leukemias (T-ALLs). Expression of FLT3 RNA was also observed in some cases of blast crisis CML. The FLT3 signal resulted from expression on the leukemic blasts, and was not caused by increased FLT3 expression on normal CD34+ stem/progenitor cells in the leukemic samples. To determine if FLT3 protein was also overexpressed, proteins were extracted from leukemic BM samples and screened by Western blotting with anti-FLT3 antisera. FLT3 protein was not detected in normal BM controls, but was found in 14 of 14 B-lineage ALLs, 36 of 41 AMLs, and 1 of 4 T-ALLs. Stimulation of patient samples with FLT3 ligand resulted in autophosphorylation of the FLT3 receptor, suggesting the receptor is functional in these cells. These data show that FLT3 RNA and protein are aberrantly expressed by AML and ALL cells in that CD34 expression and FLT3 expression are no longer synchronous, and suggest the possibility that overexpression of FLT3 could play a role in the survival and/or proliferation of malignant clones in acute myeloid and lymphoid leukemias.     

High incidence of biallelic point mutations in the Runt domain of the AML1/PEBP2 alpha B gene in Mo acute myeloid leukemia and in myeloid malignancies with acquired trisomy 21

The AML1 gene, situated in 21q22, is often rearranged in acute leukemias through t(8;21) translocation, t(12;21) translocation, or less often t(3;21) translocation. Recently, point mutations in the Runt domain of the AML1 gene have also been reported in leukemia patients. Observations for mutations of the Runt domain of the AML1 gene in bone marrow cells were made in 300 patients, including 131 with acute myeloid leukemia (AML), 94 with myelodysplastic syndrome (MDS), 28 with blast crisis chronic myeloid leukemia (CML), 3 with atypical CML, 41 with acute lymphoblastic leukemia (ALL), and 3 with essential thrombocythemia (ET). Forty-one of the patients had chromosome 21 abnormalities, including t(8;21) in 6 of the patients with AML, t(12;21) in 8 patients with ALL, acquired trisomy 21 in 17 patients, tetrasomy 21 in 7 patients, and constitutional trisomy 21 (Down syndrome) in 3 patients. A point mutation was found in 14 cases (4.7%), including 9 (22%) of the 41 patients with AML of the Mo type (MoAML) (none of them had detectable chromosome 21 rearrangement) and 5 (38%) of the 13 myeloid malignancies with acquired trisomy 21 (1 M1AML, 2 M2AML, 1 ET, and 1 atypical CML). In at least 8 of 9 mutated cases of MoAML, both AML alleles were mutated: 3 patients had different stop codon mutations of the 2 AML1 alleles, and 5 patients had the same missense or stop codon mutation in both AML1 alleles, which resulted in at least 3 of the patients having duplication of the mutated allele and deletion of the normal residual allele, as shown by FISH analysis and by comparing microsatellite analyses of several chromosome 21 markers on diagnosis and remission samples. In the remaining mutated cases, with acquired trisomy 21, a missense mutation of AML1, which involved 2 of the 3 copies of the AML1 gene, was found. Four of the 7 mutated cases could be reanalyzed in complete remission, and no AML1 mutation was found, showing that mutations were acquired in the leukemic clone. In conclusion, these findings confirm the possibility of mutations of the Runt domain of the AML1 gene in leukemias, mainly in MoAML and in myeloid malignancies with acquired trisomy 21. AML1 mutations, in MoAML, involved both alleles and probably lead to nonfunctional AML1 protein. As AML1 protein regulates the expression of the myeloperoxidase gene, the relationship between AML1 mutations and Mo phenotype in AML will have to be further explored. (Blood. 2000;96:2862-2869)     

Adhesion molecules on CD 34+ hematopoietic cells in normal human bone marrow and leukemia

Summary Expression of selected adhesion molecules of the integrin and immunoglobulin family was investigated on CD 34+ leukemic cells in 19 AML and 11 ALL cases to evaluate phenotypic differences in adhesive properties of malignant hematopoietic precursor cells in comparison to normal bone marrow CD 34+ cells. Of the 2-integrin family, CD 11a was expressed on > 50% of CD 34+ cells in normal bone marrow and almost all leukemias, whereas CD 11 b and CD 11 c were not expressed on CD 34+ cells in normal bone marrow, but were found on CD 34+ blasts in some leukemias of a heterogeneous immunophenotype. Of the 1-family, CDw 49d (VLA-4) was strongly expressed on normal CD 34+ bone marrow cells and on the blasts of all 30 CD 34+ leukemic samples, whereas CDw 49 b (VLA-2) was absent on CD 34+ cells in normal bone marrow, but detected on CD 34+ cells in a few leukemias which did not constitute a clinical or phenotypic entity according to the FAB classification or immunocytological analysis. The lymphocyte-homing-associated adhesion molecule CD 44 (HCAM) and CD 58 (LFA-3) were expressed on CD 34+ cells in all investigated cases of normal and leukemic bone marrow. ICAM-1 (CD 54), the inducible receptor ligand for CD 11 a/CD 18, although present on CD 34+ cells in normal bone marrow, was lacking on blast cells of some ALL and AML cases. So far, the variable expression of 2-integrins as well as of VLA-2 and of ICAM-1 could indicate distinct differences in cell-cell or cell-matrix adhesion of leukemic cells in ALL and AML patients.     

GM-CSF stimulates proliferation of clonal leukemic bone marrow cells in acute myeloid leukemia (AML) in vitro

 Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to stimulate granulocytes, monocytes, and macrophages. We studied the effect of GM-CSF on (clonal) bone marrow (BM) cells obtained from AML patients after 7 days of culture in vitro: BM samples were obtained from 19 AML patients at diagnosis (DIA), from two patients with persisting disease (PERS), from eight patients in complete remission (CR), and from 12 healthy donors. Flow-cytometric comparison of differentiated, CD 15-positive cells or of CD34-positive blast cells before and after cultivation showed that the proportion of CD15-positive cells was increased in nine of 12 healthy BM samples, in 14 of 19 cases at DIA, in one of three cases during PERS, and in five of six cases in CR of AML. The proportion of CD34-positive cells was increased in one of 12 healthy BM samples, in seven of 19 cases at DIA, in one of two cases during PERS, and in three of seven cases in CR of AML. Southern blot analysis (SBA) performed in six cases during the course of AML, before and after cell culture, showed that clonal DNA increased after GM-CSF treatment in three of five cases studied at DIA, in six of nine cases studied in CR, in the one case studied at PERS, and in the one studied at relapse (REL). In one case of trisomy 8 at DIA a normal karyotype was demonstrated in CR. However, after 7 days of cultivation of the cells in GM-CSF the trisomy 8 was detected in two of 17 metaphases isolated from colony-cells from methylcellulose cultures. Our data show that a 7-day treatment of BM cells with GM-CSF induced a differentiation of healthy and leukemic BM cells in the great majority of cases. An enrichment of CD34-positive cells was not achieved in healthy BM samples. However, in 70% of the cases in CR and in 30% of the cases at DIA of AML, clonal CD34-positive cells were enriched. This means that GM-CSF stimulates ('primes') leukemic cell growth in vitro. Received: August 17, 1998 / Accepted: April 28, 1999     

Survivin expression, apoptosis and proliferation in chronic myelomonocytic leukemia

We analyzed the expression of the inhibitor of apoptosis survivin by immunocytochemistry in bone marrow cells from patients with chronic myelomonocytic leukemia (CMML) to evaluate possible abnormalities in comparison with other myelodysplastic (MDS) and myeloproliferative syndromes, and to investigate a possible correlation between survivin expression and altered apoptosis or proliferation, or relevant laboratory and clinical findings. Thirty-four patients with CMML [18 MDS-CMML and 16 myeloproliferative disorder (MPD)-CMML], 90 with MDS, 41 with acute myeloid leukemia (AML), 19 with chronic MPD and 25 control subjects were studied. In normal samples survivin was never detectable. In CMML survivin levels higher than in MDS and AML (P < 0.0001), but similar to those found in MPD were observed. In CMML and MDS apoptosis was significantly higher compared to normal controls and all other subtypes of leukemias (P < 0.0001). Proliferation did not differ significantly in normal controls, MDS and CMML; the lowest levels were observed in AML and MPD (P < 0.0001). In CMML there was no correlation between survivin expression and blast cell percentage, apoptosis or proliferation, FAB or WHO subgroup. Proliferation was higher in MDS-CMML and tended to correlate with overall survival. CMML-2 cases with higher survivin expression showed higher evolution rate and shorter survival. In conclusion, CMML is characterized by high proliferation and apoptosis. Survivin overexpression, by disrupting the balance between cell proliferation/differentiation and apoptosis, may play an important role in its pathophysiology. The detection of survivin-deregulated expression may provide a useful tool for diagnosis, prognosis and a possible target for experimental treatments.     

Progression of a myelodysplastic syndrome to pre-B acute lymphoblastic leukemia: a case report and cell lineage study

 The evolution of acute lymphoblastic leukemia from a myelodysplastic syndrome is a very uncommon event. We describe a 46-year-old man in whom refractory anemia with excess blasts (RAEB) evolved to a pre-B acute lymphocytic leukemia. Trisomy 8 was one of the cytogenetic abnormalities in the dysplastic clone and was detected in both peripheral blood and bone marrow smears of interphase cells by the fluorescence in situ hybridization (FISH) technique. Using a chromosome 8 centromeric specific DNA probe we identified the trisomy 8 to be present in lymphoblasts, erythroid precursors, myeloblasts, promyelocytes, myelocytes, metamyelocytes, granulocytes, and monocytes. Our case supports the hypothesis that in MDS the pluripotent precursor cell is affected, and we examine the potential role of FISH for the study and follow-up of some hematological diseases. Received: 9 October 1995 / Accepted: 21 December 1996