Cytogenetic differences between bone marrow and spleen in a case of agnogenic myeloid metaplasia developing blast crisis.

Chromosome studies of cells from bone marrow and spleen were performed in a patient with agnogenic myeloid metaplasia (MM) developing blast crisis after a chronic phase lasting for 6.5 years. The proportions of blast cells were roughly the same in bone marrow and spleen. In the bone marrow 43 % of the metaphases were abnormal with a marker chromosome whereas all spleen metaphases were normal. The results indicate that chromosomal changes associated with blast transformation in MM may occur in the bone marrow prior to such changes in the spleen and support the concept that bone marrow and spleen may constitute relatively separate pools of haemopoietic tissue in chronic myeloproliferative diseases.     

Dual rearrangement of immunoglobulin and T-cell receptor genes in blast crisis of CML

Dual rearrangement of immunoglobulin and T-cell antigen receptor (beta, delta) genes was demonstrated in a case of Philadelphia chromosome-positive chronic myeloid leukemia (CML) in blast crisis. The blast cells, showing L2 morphology and high activity of TdT, expressed pre-B cell (CD19+, Ia+) and myeloid (CD13+, CD34+) surface antigens but lacket T-cell antigens (CD2-, CD7-). Cytogenetic studies on bone marrow and peripheral blood revealed the Phl chromosome in all metaphases analyzed, majority of which also had the additional chromosome changes, +8, +10, +21. Furthermore, molecular analysis of the breakpoint cluster region (bcr) on chromosome 22 showed a rearrangement, confirming the CML origin of the blast cells.     

Transformation in chronic granulocytic leukaemia. Different blast cell clones in different anatomical sites.

A 45-year-old female developed blastic metamorphosis in chronic granulocytic leukaemia after 52 months of chronic phase. During the subsequent 6--7 months, lymphosarcomatous enlargements of various lymph nodes developed. The blast cells in lymph nodes differed morphologically from those in bone marrow and blood, being 'lymphoid' non-B, non-T, non-ALL cells. The karyotype of all metaphases from one lymph node was 47,XX, +21(Ph1+) being identical to the karyotype of medullary cells. However, the karyotype of all blasts from another lymph node was 47,XX,+mar(Ph1+). It is likely that the local micro-environment controlled the clonal differentiation of these subpopulations which had originated from the same Ph1-positive multipotent stem cell. In lymph nodes and other extramedullary sites blasts were primitive without differentiation, but a myeloid differentiation in the bone marrow was demonstrated morphologically and cytochemically.     

IL-4 improves the detection of cytogenetic abnormalities in multiple myeloma and increases the proportion of clonally abnormal metaphases

In the present study the incidence of abnormal karyotypes and the number and proportion of abnormal metaphases obtained in multiple myeloma (MM) using three culture conditions were compared: unstimulated culture (72 h), IL-6/GM-CSF-stimulated culture (120 h) and IL-4-stimulated culture (120 h). The three types of culture conditions were assessed simultaneously on bone marrow samples from 30 consecutive myeloma patients. In addition DNA content (DNA ploidy and cell cycle) was analysed by flow cytometry. The number of MM samples with clonal karyotypic abnormalities was higher after IL-4-stimulated cultures (53%) than it was after IL-6 + GM-CSF (37%) and unstimulated (30%) cultures. The benefit of IL-4 was also observed in cases with low numbers of plasma cells in the bone marrow, in early clinical stages and in untreated patients. In those cases in whom clonal chromosomal abnormalities were detected by the three culture methods, the cytogenetic findings were always identical. According to our results the addition of IL-4 to the cultures of bone marrow cells in MM increases the number of abnormal metaphases.     

Cytogenetic Studies in Myelomatosis

Cytogenetic studies have been carried out on bone marrow aspirates from 25 patients with myelomatosis. Abnormal stem lines were present in 7 of the patients; the remainder had a diploid chromosome complement. In most patients — also in those without an abnormal clone — some metaphases had a blurred appearance similar to that seen in bone marrow aspirates from patients with acute leukaemia. In many of the chromosome preparations obtained before cytostatic therapy some metaphases with structural aberrations of the chromosomes were seen. Evidence is presented that in the patients with abnormal stem lines in the bone marrow, the chromosome abnormalities are confined to the myeloma cells and are not found in the erythrocytic or granulocytic precursors, which thus do not seem to be involved by the neoplastic process. Based on the present results and on a review of the relevant literature some general cytogenetic features are emphasized which may contribute to a better understanding of the disorder. Especially, it is demonstrated that in myelomatosis the cytogenetic changes are much more uniform than in other malignant disorders with the exception of chronic myeloid leukaemia.     

Cytogenetic studies in myelomatosis.

Cytogenetic studies have been carried out on bone marrow aspirates from 25 patients with myelomatosis. Abnormal stem lines were present in 7 of the patients; the remainder had a diploid chromosome complement. In most patients - also in those without an abnormal clone - some metaphases had a blurred appearance similar to that seen in bone marrow aspirates from patients with acute leukaemia. In many of the chromosome preparations obtained before cytostatic therapy some metaphases with structural aberrations on the chromosomes were seen. Evidence is presented that in the patients with abnormal stem lines in the bone marrow, the chromosome abnormalities are confined to the myeloma cells and are not found in the erythrocytic or granulocytic precursors, which thus do not seem to be involved by the neoplastic process. Based on the present resuts and on a review of the relevant literature some general cytogenetic features are emphasized which may contribute to a better understainding of the disorder. Especially, it is demonstrated that in myelomatosis the cytogenetic changes are much more uniform than in other malignant disorders with the exception of chronic myeloid leukaemia.     

Morphology and quantitative composition of hematopoietic cells in murine bone marrow and spleen of healthy subjects

Laboratory mice play an outstanding role in modeling human development and disease. In contrast to human leukemia, the spleen is involved in almost all cases, and the bone marrow is only variably involved in murine models. Although mice have been used for medical research for over 100 years, there are only few reports with a small number of cases looking at morphology and quantitative composition of murine hematopoietic cells in the bone marrow of non-transplanted animals of most strains. To our knowledge, there is not even a single report describing the splenogram in C57BL/6J mice, one of the most commonly used strains for medical research. The present study illustrates the morphology of the hematopoietic cells in the bone marrow and spleen of non-treated C57BL/6J mice and establishes the murine myelogram from the largest healthy C57BL/6J cohort reported to date. Furthermore, we present the first murine splenogram described for C57BL/6J mice. Our study supports the acceptance of the presence of >5 % blast cells as providing clear evidence of abnormality in bone marrow like in humans. In addition, we are the first to show <1 % blast cells in the normal spleen. Interestingly, classical dysplastic changes were rare in normal healthy mice. Our study of the bone marrow and spleen of healthy non-transplanted animals provides reference ranges of each cell type and for the myeloid/erythroid ratio, which can be used to interpret preclinical gene therapy data, leukemogenesis, and hematopoiesis studies, and may improve the quality of such analyses.     

Nonclonal hemopoietic progenitor cells detected in long-term marrow cultures from a Turner syndrome mosaic with chronic myeloid leukemia

We have investigated the clonality of Ph1-negative hemopoietic progenitor cells appearing in long-term marrow cultures established with cells from a mosaic Turner syndrome patient (46,XX/45,X) with Ph1- positive chronic myeloid leukemia (CML). The Ph1-positive clone had been shown previously to have arisen from a cell of the 45,X lineage. At the time of the present study, the patient was five years post- diagnosis and had been off chemotherapy for two months following a year of treatment for lymphoid blast crisis. All analyzed unstimulated marrow metaphases and each of 23 individually analyzed erythroid and granulocyte colonies produced in assays of the starting marrow were 45,X,Ph1. Pooled granulocyte colonies from the same assays yielded four metaphases that were 45,X,Ph1 and one that was 46,XX. Very few hemopoietic progenitors were detected in long-term cultures at any time; however, all of four individually analyzed large granulocyte colonies and a pooled granulocyte colony preparation obtained from assays of 4- to 6-week-old adherent layers yielded exclusively 46,XX metaphases. These results provide evidence that non-clonal progenitors can persist in patients with CML, even after the onset and treatment of blast crisis, and that the long-term marrow culture system provides a sensitive method for detecting such cells.     

The SCID mouse as a model for multiple myeloma

Summary. The SCID mouse was investigated as a potential animal model for human multiple myeloma (MM). Duplicate samples of bone marrow mononuclear cells (BMMC) and/or peripheral blood mononuclear cells (PBMC) of six MM patients in different clinical phases and one patient with monoclonal gammapathy of undetermined significance (MGUS) were injected intraperitoneally into SCID mice. Human immunoglobulins (Ig) in the SCID sera were quantified with a light-chain isotype-specific ELISA, and their monoclonality biochemically characterized, using a sensitive immunoblotting technique after agar gel electrophoresis. Successful transplantation of bone marrow derived-tumour cells in SCID mice was obtained with BMCC of two MM patients with progressive disease. Human plasma cells were detected in the mesenteric fat tissue around the pancreas and the spleen. This model in SCID mice may facilitate studies on processes involved in tumour progression and provides a new tool for therapeutic approaches in MM.     

Cytogenetic events after bone marrow transplantation for chronic myeloid leukemia in chronic phase

Forty-eight patients treated by allogeneic bone marrow transplantation (BMT) for Philadelphia (Ph) chromosome-positive chronic myeloid leukemia in chronic phase had serial cytogenetic studies of marrow performed at intervals after transplant. Twenty patients received marrow cells from donors of opposite sex. Ph+ marrow metaphases were identified in 24 of 48 (50%) of patients after BMT; they were first seen early (within 1 year) in 16 cases and late (greater than 1 year after BMT) in eight cases. Ph-positivity after BMT occurred more commonly in recipients of T-depleted than nondepleted marrow (19 of 28 v 5 of 20). In 4 cases the Ph+ metaphases were found only transiently after BMT; in 11 cases the Ph+ metaphases have persisted but hematologic relapse has not ensued; in 9 cases the finding of Ph+ metaphases coincided with or preceded hematologic relapse. Chromosomes in cells of donor origin had morphological abnormalities in two cases. No relapses were identified in cells of donor origin. Our data suggest that the relationship between cells of recipient and donor origin is complex: cure of leukemia may depend on factors that operate for some months or years after BMT.     

Marrow repopulation in mice treated with busulphan or isopropyl methane sulphonate and bone marrow

By using karyotypic analysis of female mice treated with busulphan or isopropyl methane sulphonate (IMS), and injected with male bone marrow the donor contribution to both total marrow cellularity and spleen colony forming cells (CFU-S) was assessed for up to 6 months after transplant. In the mice treated with busulphan the marrow cells yielded metaphases of which between 40% and 83% were of donor type. Between 60% and 97% of metaphases in spleen colonies formed in irradiated mice were of donor type during the 24-week study period. In contrast, mice prepared for the transplant with IMS showed no cells of donor type at any time after transplant, neither did they possess CFU-S of donor type. We were therefore led to conclude that the donor cells made no contribution to longterm engraftment in mice prepared with IMS, whilst in those prepared with busulphan they were the predominantly active haemopoietic cells. These results are consistent with a model of haemopoiesis in which the most primitive cells reside in a 'niche' where they are resistant to the effects of IMS but susceptible to the action of busulphan. Busulphan may vacate some niches to allow engraftment by transplanted marrow, whilst IMS yields no unoccupied niches for grafted cells to occupy, and cannot therefore lead to a stable chimaerism.     

Cytogenetics in multiple myeloma and plasma cell leukemia: simultaneous cytogenetic and cytologic studies in 51 patients

Summary Cytogenetic studies in patients with multiple myeloma (MM) and plasma cell leukemia (PCL) have in general been largely unsuccessful. The investigation of mitoses of nonmalignant hematopoietic precursor cells, rather than mitoses of malignant plasma cells might account for the low percentage of pathological genetic findings. We investigated bone marrow (BM) cells of 51 patients both cytogenetically and cytologically. In patients with a normal karyotype (n=39) nearly all mitoses examined cytologically (107/117) derived from granulopoietic or erythropoietic cell lineages. In contrast, 20/27 metaphases in patients with a pathological karyotype (n=12) were found to be plasma cell mitoses. These findings may explain the low rate of chromosomal rearrangements in MM and may suggest that the real abnormality rate is considerably higher.     

In vivo stem cell function of interleukin-3-induced blast cells

The treatment of mice with high doses of 5-fluorouracil (5-FU) results in an enrichment of primitive hematopoietic progenitors. Using this procedure, we obtained a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro and could differentiate into not only myeloid cells but also into lymphoid lineage cells. The phenotypes of interleukin-3 (IL-3) induced blast colony cells were Thy-1-positive and lineage-marker-negative. We examined whether these blast colony cells contained primitive hematopoietic stem cells in vivo and could reconstitute hematopoietic tissues in lethally irradiated mice. Blast colony cells could generate macroscopic visible spleen colonies on days 8 and 12, and 5 x 10(3) blast cells were sufficient to protect them from lethally irradiation. It was shown that 6 or 8 weeks after transplantation of 5 x 10(3) blast cells, donor male cells were detected in the spleen and thymus of the female recipients but not in the bone marrow by Southern blot analysis using Y-encoded DNA probe. After 10 weeks, bone marrow cells were partially repopulated from donor cells. In a congenic mouse system, donor-derived cells (Ly5.2) were detected in the thymus and spleen 6 weeks after transplantation. Fluorescence-activated cell sorter analyses showed that B cells and macrophages developed from donor cells in the spleen. In the thymus, donor-derived cells were found in CD4, CD8 double-positive, single-positive, and double-negative populations. Reconstitution of bone marrow was delayed and myeloid and lymphoid cells were detected 10 weeks after transplantation. These results indicate that IL-3-induced blast cells contain the primitive hematopoietic stem cells capable of reconstituting hematopoietic organs in lethally irradiated mice.     

Murine hematopoietic blast colony-forming cells and their progeny have distinctive membrane marker profiles

Two distinct bone marrow-derived blast colony-forming cells can generate colonies of lineage-restricted progenitor cells in agar cultures of murine bone marrow. Both cell types selectively had a Kit⁺ ScaI⁺ phenotype distinguishing them from most lineage-restricted progenitor cells. Multicentric blast colony-forming cells stimulated by stem cell factor plus interleukin-6 (IL-6) (BL-CFC-S) were separable from most dispersed blast colony-forming cells stimulated by Flt3 ligand and IL-6 (BL-CFC-F) using CD34 and Flt3R probes. Multicentric BL-CFC-S cofractionated with colony-forming units, spleen (CFU-S) supporting the possibility that the 2 cells may be identical. The colony populations generated by BL-CFC-S were similar in their phenotype and proliferative capacity to progenitor cells in whole bone marrow but the progeny of BL-CFC-F were skewed with an abnormally high proportion of Kit⁻ Flt3R⁺ cells whose clonogenic cells tended to generate only macrophage progeny. Both blast colony populations had a high percentage of GR1⁺ and Mac1⁺ cells but BL-CFC-F colonies also contained a significant population of B220⁺ and IL-7R⁺ cells relevant to the superior ability of BL-CFC-F colony cells to generate B lymphocytes and the known dependency of this process on Flt3 ligand and IL-7. The commitment events and phenotypic changes during the generation of differing progenitor cells in blast colonies can now be clonally analyzed in a convenient in vitro culture system.     

Twenty-five year survival of chronic granulocytic leukaemia with spontaneous karyotype conversion

S ummary. Following busulphan-induced bone marrow hypoplasia a woman with chronic granulocytic leukaemia has survived 25 years from diagnosis. Since the last course of busulphan therapy in 1959 she has remained in clinical and haematological remission. Repeated cytogenetic analysis of bone marrow showed Philadelphia chromosome mosaicism with a minority of abnormal metaphases till 1969. Analysis of 150 metaphases in 1982 revealed no cells containing the Philadelphia chromosome. The possible significance of this spontaneous karyotype conversion is discussed.     

The Philadelphia chromosome in human macrophages.

Three patients with chronic myelocytic leukemia in different phases of the natural history of the disease were studied. Their bone marrow cells were cultured under conditions favoring macrophage proliferation, and parallel cytogenetic and cytochemical studies were performed. All cell metaphases examined contained the Ph1 chromosome at a time when more than 80% of these metaphases were in identifiable macrophages. We conclude that the mononuclear phagocyte cell line contains the abnormal chromosome in Ph1-positive chronic myelocytic leukemia.     

Different composition of the erythropoietic tissue in bone marrow, spleen and liver in myelofibrosis.

Aspirates from bone marrow, spleen and liver were analysed in 10 untreated patients with idiopathic myelofibrosis (MF). The proportion of erythroblasts was higher in the spleen and the liver than in the bone marrow. The mitotic indices of the erythropoietic precursor cells were subnormal in the extramedullary sites and significantly lower in the liver compared with the spleen. There was a "shift to the left" within the liver erythropoiesis and a significant megaloblastosis in the spleen. The same tendencies have formerly been found in patients with chronic myeloid leukemia and it is suggested that the discrepancies may be due to differences in the microenvironment of the erythropoietic cells.     

Genetically modified bone marrow continuously supplies anti-inflammatory cells and suppresses renal injury in mouse Goodpasture syndrome.

In chronic inflammation, macrophages and neutrophils, which are derived from bone marrow, play a pivotal role. Therefore, reconstitution of bone marrow with anti-inflammatory stem cells may modify inflammation. In this study, transplantation-based gene therapy was applied to glomerular inflammation for a long-lasting suppression of the glomerular damage seen in chronic nephritis. Bone marrow cells were harvested from male donor mice, which had received 5-fluorouracil 3 days previously, and transduced with an interleukin 1 (IL-1) receptor antagonist (IL-1Ra) or a mock gene using a retrovirus vector. After confirmation that transduced cells possessed the transgene at approximately 0.7 copies per cell and secreted recombinant IL-1Ra, these cells were infused into sublethally irradiated (6 Gy) female recipients once daily for 4 consecutive days. These female recipient mice had the male Y antigen in bone marrow, liver, and spleen, and 10% to 20% of their spleen cells possessed the transgene even 8 weeks after transplantation. Glomerulonephritis was then induced in these mice. Renal function and histology were retarded in the mice whose bone marrow was reconstituted with IL-1Ra-producing cells compared with mock transduced cells. In situ hybridization using a Y painting probe revealed that transplanted donor cells were recruited into the glomerulus upon induction of nephritis, suggesting therapeutic effects were channeled through the secretion of IL-1Ra from these cells. Furthermore, the survival rate after a second challenge with nephrotoxic antibody was significantly improved in the IL-1Ra chimera. These results suggest that reconstitution of bone marrow for continuous supply of anti-inflammatory cells may be a useful strategy for the treatment of chronic inflammation.     

Successful treatment of extramedullary blast crisis of chronic myelogenous leukemia with imatinib mesylate (STI571)

We describe a patient with chronic myelogenous leukemia (CML) who developed extramedullary blast crisis, and was successfully treated with imatinib mesylate (STI571). A 42-year-old man had been diagnosed with chronic phase Philadelphia chromosome (Ph)-positive CML and treated with interferon-alpha. He achieved partial cytogenetic response. Two years after the diagnosis, he presented with superficial lymphadenopathy in his neck and supraclavicular regions. Lymph node biopsy disclosed the infiltration of myeloblasts. Although the patient's bone marrow was without increasing blasts at that time, cytogenetic response was no longer observed. STI571 at a dose of 600 mg/day was initiated, and led to the complete disappearance of lymphadenopathy within a month and also to major cytogenetic response in the bone marrow (90% Ph-negative metaphases). Subsequently, the patient underwent allogeneic bone marrow transplantation from an HLA-matched unrelated donor and was in complete remission without evidence of extramedullary disease 12 months after transplantation.     

Phenotypic Changes in a Case of Blast Crisis of CML: Characterization by TdT,Cytochemistry, and Cytogenetics

Phenotypic changes in blast crisis of a case of Philadelphia chromosome (Ph¹)-positive chronic myelogenous leukaemia were characterized by serial terminal transferase (TdT) determinations simultaneously related to cytochemical and cytogenetic data. At the onset of the blast crisis, 90% of the blast cells were acid phosphatase-positive (focal pattern), Ph¹-positive, lymphoid cells. The TdT activity amounted to 29 units/10⁸ mononuclear cells in the peripheral blood and to 57 units/10⁸ mononuclear cells in the bone marrow. Therapy with vincristine and prednisone caused the elimination of the TdT-positive cell population. 4 months later, there was an increase in TdT-negative, myeloid blasts which was brought under control with busulfan. Cytogenetic analysis of the myeloid blasts still revealed Ph¹ positivity in 100 % of the metaphases examined and the lack of additional chromosomal abnormalities. A second relapse was again dominated by TdT-containing cells with the 46,XX,Ph¹ karyotype. This time, the patient failed to achieve remission with vincristine and prednisone. Even though the TdT activity was markedly decreased, the lymphoid blast count remained elevated and the cells showed resistance to further therapy. This failure of morphology, cytochemistry as well as cytogenetics to distinguish between the individual phenotypes emerging during the course of blast crisis of CML characterized the TdT as a cell marker of important diagnostic and therapeutically prognostic value.