Chronic myelocytic leukemia (CML): failure to detect residual normal committed stem cells in vitro.

Granulocytic colonies grown in culture from marrow and peripheral blood from five patients with Ph1-positive CML and heterozygous at the G-6-PD locus were analyzed for G-6-PD in order to identify CFU-C that do not arise from the CML clone. The patients had both B and A enzymes in normal tissues, but their CML clones typed as B. Whereas about 50% of colonies from normal subjects heterozygous as the G-6-PD locus show type-A G-6-PD and 50% type B, only two of the 1308 colonies from the CML patients had type-A G-6-PD. These data provide little evidence for persistence of normal committed stem cells in CML, a finding in contrast to that made previously in polycythemia vera, another clonal stem cell myeloproliferative disorder.     

Human mixed cell colonies: unicellular or multicellular origin–analysis by G-6-PD

Marrow and peripheral blood cells from normal women heterozygous (GdB/GdA) at the X-chromosome-linked glucose-6-phosphate dehydrogenase (G-6-PD) locus were cultured at cell concentrations ranging from 2 X 10(4)/ml to 4 X 10(5)/ml to test formally the plating conditions necessary for reliable enumeration of multipotent stem cells (CFU-mix). The culture system was rigorously tested by plating cells obtained after velocity sedimentation and the G-6-PD enzyme type of individual colonies was determined. At cell concentrations less than or equal to 7.5 X 10(4)/ml for marrow and less than or equal to 1 X 25 X 10(5)/ml for peripheral blood, mixed-cell colonies had either type A or type B enzyme, but not both. At higher cell concentrations, significant numbers of colonies showed both enzyme types and therefore arose from more than one cell. These studies demonstrate that enumeration of CFU-mix by in vitro colony assay is accurate only at low cell concentrations. Studies of haematopoietic differentiation relying on in vitro colony assays of multipotent stem cells must be carefully analysed in light of these data.     

Cytogenetic study of myeloid and erythroid colonies in chronic myeloid leukaemia with stable Ph1 mosaicism

We have investigated two chronic myeloid leukaemic (CML) patients with a stable Ph1 mosaicism to see whether the Ph1 positive cells belonged to more than one lineage. Individual erythroid and granulocytic colonies were cytogenetically examined. Ph1-positive and Ph1-negative colonies were found both in the erythroid and granulocytic lineage supporting the contention that CML with a stable Ph1 mosaicism as well as classical CML involves a pluripotent stem cell.     

Incidence of involvement of the B and T lymphocyte lineages in chronic myelogenous leukemia

Peripheral blood specimens were obtained from 22 patients with Philadelphia chromosome (Ph1) positive chronic myelogenous leukemia (CML) (16 in chronic phase, 2 in an accelerated phase, and 4 in blast crisis). Studies were performed to determine the frequency of the presence of the Ph1 chromosome in cells of lymphoid lineages. Rosetted (E+) lymphocytes (T lymphocytes) from nine patients in chronic phase and one patient in blast crisis were stimulated with T cell growth factor interleukin 2 (IL-2) and/or phytohemagglutinin (PHA). All ten patients had sufficient T lymphocyte metaphases for analysis and of a total of 461 metaphases examined, only one contained the Ph1 chromosome. Nucleated cells of density less than 1.077 g/mL were infected with Epstein-Barr virus (EBV). Following infection, cell lines were established from individual colonies attached to egg albumin- coated Lab-Tek slide chambers (clonal cell lines) or from suspension culture in 96-well tissue culture cluster dishes (nonclonal cell lines). Cell surface and intracellular marker analysis confirmed the B lymphocyte phenotype of all the cell lines examined. B lymphoblastoid cell lines were established from 16 of the 22 patients. All lines from 12 patients were Ph1-negative. From two chronic phase patients, both Ph1-positive and Ph1-negative lines were established. From one patient in an accelerated phase, only Ph1-positive lines were established. From another patient in blast crisis (of myeloblastic phenotype), only Ph1- positive lines were established initially; however, five months later, after the patient had been treated with mitoxantrone, only Ph1-negative lines were derived from this patient. Based on these results, it appears that most B cells and mature T cells in most CML patients are Ph1-negative, but that about 25% of patients have predominantly Ph1- positive B cells or a mixture of Ph1-positive and Ph1-negative B cells that are capable of growing as established cell lines after transformation with EBV.     

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.     

Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macrophage.

Glucose-6-phosphate dehydrogenase (G-6-PD) isoenzymes types of granulocytes were determined in eight women with chronic myelocytic leukemia (CML). The patients were heterozygous at the X-linked G-6-PD locus for the common gene, GdB, and a variant, such as GdA, so that both B and A enzyme types were found in skin cells. In contrast to these normal cells, only one G-6-PD type was found in CML granulocytes. The fact that such single-enzyme phenotypes are found in CML granulocytes, but not in nonleukemic granulocytes, provides strong evidence that the disease has a clonal origin. Single-enzyme phenotypes were also found in erythrocytes, platelets and cultured blood macrophages indicating that these cells have a common stem cell which is the site of the abnormality in CML.In the one studied patient, no evidence was found for involvement of cultured marrow fibroblasts. Clonal origin of CML virtually excludes cell recruitment as a sole pathogenetic mechanism. Either the leukemia arises as a consequence of a rare initial event in a single cell, or a series of events occurs in a clone such that it evolves into CML, or both.     

Ph1-negative T lymphocytic colonies in agar cultures of peripheral blood in chronic myeloid leukemia

T lymphocytic colony formation by peripheral lymphocytes separated by discontinuous albumin gradient centrifugation was evaluated in 8 patients with Philadelphia (Ph1)-positive chronic myeloid leukemia (CML). Colonies were obtained using a liquid-on-agar culture system recently introduced (PHA overlayer-leukocyte feeder layer assay) which has been shown to be simple and reliable. The pattern of colony growth in CML and in normal controls was similar, the peak ranging from the 4th to the 6th day. Also the morphological aspects of colonies did not differ in the two groups. Cells recovered from CML lymphocytic colonies were shown to belong to T cell lineage, as they are able to form spontaneous E-rosettes and to respond to mitogenic stimulation in vitro. In contrast, cells recovered from all other cultured fractions failed to display these properties. Cytogenetic analysis showed that T colony cells were Ph1-negative whereas the chromosome anomaly was found in nonlymphoid colonies of the same patients, thus suggesting a nonclonal origin of T lymphocytes in CML.     

In vitro restoration of polyclonal hematopoiesis in a chronic myelogenous leukemia after in vitro treatment with 4- hydroperoxycyclophosphamide

Bone marrow cells of a 45-year-old female with Philadelphia chromosome (Ph1)-positive, early-phase chronic myelogenous leukemia (CML), who was heterozygous for the glucose-6-phosphate dehydrogenase (G6PD) locus, were pretreated in vitro with 4-hydroperoxycyclophosphamide (4-HC) and tested for G6PD activity in several colony formation assays and for karyotypic abnormalities. All cells within the mixed (CFU-GEMM), the erythroid burst (BFU-E), and the granulocyte-macrophage (CFU-GM) colonies expressed type A and type B G6PD activity and a normal karyotype, whereas untreated cells expressed type A G6PD and the Ph1 chromosome. This reversal of G6PD activity type and the disappearance of the Ph1 chromosome in colonies grown from 4-HC-treated cells indicate that this cytotoxic agent spares a residual normal stem cell population in bone marrow cells of early-phase CML patients. This finding, in turn, suggests a therapeutic approach in CML based on in vitro chemotherapy of autologous bone marrow grafts.     

Clonal Karyotype Abnormalities in Erythroid and Granulocyte-Monocyte Precursors in Polycythaemia Vera and Myelofibrosis

To determine whether clonal karyotype abnormalities seen in myeloproliferative disorders originate in cells of only one or of more blood cell lines, committed erythroid and granulocyte-monocyte progenitor cells were cultured in vitro by the methyl cellulose assay, and chromosome studies were carried out on the colonies. 3 patients were studied, 1 with polycythaemia vera and 2 with myelofibrosis, which was preceded by polycythaemia vera in 1 of the cases. All analysable karyotypes from both erythroid and granulocyte-monocyte colonies in every patient showed the clonal karyotype aberration which had been demonstrated in the bone marrow or blood of the patient. This finding is evidence of the involvement of a multipotent stem cell in the clonal proliferation of these diseases.     

The chromosomes and causation of human cancer and leukemia: XXXVIII. Cytogenetic experience in Ph1-negative chronic myelocytic leukemia (CML).

Among 300 patients with chronic myelocytic leukemia (CML) followed at our institute during the last ten years, 36 (12%) were thought to have Ph1-negative CML. In eight of these patients, chromosomal abnormalities were found in the leukemic cells; in four, the karyotypic abnormalities were established with banding techniques. The data of the present study and a review of the literature regarding chromosomal changes in Ph1-negative CML indicate that: 1) no characteristic or consistent karyotypic change is present in Ph1-negative CML and that diploidy is more common in this than any other leukemia; 2) the most common changes involve group C chromosomes (particularly +8); and 3) a missing Y is less common in Ph1-negative CML than in its Ph1-positive counterpart. The karyotypic changes in Ph1-negative CML resemble more those encountered in Ph1-positive CML than in acute myeloblastic leukemia (AML). The much shorter survival of the Ph1-negative CML patients vs that of the Ph1-positive group was again substantiated, and some of the previously reported clinical and laboratory findings unique to Ph1-negative CML were confirmed. On the basis of the cytogenetic findings it is concluded that Ph1-negative CML appears to be an entity unto itself.     

Difference of cell lineage expression of haematopoietic progenitor cells in Philadelphia-positive acute lymphoblastic leukaemia and chronic myelogenous leukaemia

It is still difficult to clinically distinguish Philadelphia (Ph1)-positive acute lymphoblastic leukaemia (ALL) from Ph1-positive chronic myelogenous leukaemia (CML) in lymphoid crisis. In this study we tried to discriminate between these two disorders by simultaneous analyses of cell morphology and karyotype in single in vitro colonies. We studied three patients with Ph1-positive ALL and four with Ph1-positive CML in various phases of the disease. Bone marrow and peripheral blood cells obtained directly from all seven patients showed abnormal karyotypes including Ph1-chromosomes. Normal karyotypes were found in a small proportion of cells from two ALL patients, but none were found in any from the CML patients. The patients' mononuclear cells (MNCs) were plated at 1-5 x 10(4)/ml in semi-solid medium containing methylcellulose plus phytohaemagglutinin-stimulated leucocyte conditioned medium and erythropoietin. After 9-14 d cultivation, granulocyte-macrophage, erythroid and mixed colonies obtained were used for simultaneous analysis of cell morphology and karyotype. Morphological examination showed that these colonies contained neutrophils, eosinophils, basophils, macrophages and/or erythroblasts in various combinations. No lymphoblast colonies were obtained under the culture conditions used. Cytogenetic examination revealed that all metaphase cells observed in colonies obtained from Ph1-positive ALL patient MNCs had a normal karyotype, whereas those in colonies from Ph1-positive CML patient MNCs had abnormal karyotypes, including Ph1 chromosomes, suggesting that the difference between the two disorders involved a difference in cell lineage. Our results showed that this method was a practicable method for distinguishing Ph1-positive ALL from Ph1-positive CML in lymphoid crisis.     

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.     

Chromosomes and causation of human cancer and leukemia. XXI. Cytogenetically unusual cases of leukemia.

Three male patients with leukemia were found with banding techniques to have unusual cytogenetic pictures in the cells of their marrow, spleen or blood. Case No. 1 (78 yr old) was that of a Ph1-negative CML with a missing Y in the blood (cultured without PHA) and marrow cells. The patient is still alive and responding to therapy. Case No 2 (54 yr old) was considered prior to admission to have either CML or AML, but was shown, in fact, to be in the blastic phase of CML; all the cells in his marrow and spleen were Ph1-positive, but with no evidence of a translocation. Other karyotypic findings (+8, +11, +13, +21) frequently encountered in the blastic phase of CML were present in the cells of this patient. Case No. 3 (50 yr old) with AML was shown to have a Ph1 resulting from a standard translocation, i.e., [t(9;22) (q34;q11)], in a substantial number of the cells in the marrrow and blood (cultured without PHA). The implications of these unusual findings are discussed in relation to the chromosomal pictures usually encountered in these states.     

Karyotype evolution and multilineage involvement of Philadelphia chromosome-positive clones in blastic transformation of two patients with chronic myelocytic leukemia

Simultaneous analysis of the cell morphology and karyotypes on single colonies was carried out in two patients with Philadelphia chromosome (Ph1)-positive chronic myelocytic leukemia in blastic transformation in order to clarify the origin of leukemic cells involved. Patient no. 1 was in a typical myeloblastic transformation and patient no. 2 in "basophilic transformation." Both patients exhibited karyotype evolution in blastic phase (BP), so that we could differentiate BP clones with additional chromosomal abnormalities from chronic phase (CP) clones with only Ph1 among single colonies. The number of single colonies yielding two or more analyzable metaphases was 18 in patient no. 1, and 19 in patient no. 2. Among these colonies, only three in patient no. 1 and none in patient no. 2 were from CP clones and 15 in patient no. 1 and 19 in patient no. 2 were from BP clones. Morphological examination revealed that not only blasts but also mature neutrophils, eosinophils, basophils, macrophages, and erythroblasts were derived from BP clones. These results suggested that (1) BP clones developed at the pluripotent stem cell level, (2) additional chromosomal abnormalities were not restricted to occur in a specific cell line representative in BP; and (3) BP clones, if not all, may retain capacity for maturation and differentiation.     

Restoration of nonclonal hematopoiesis in chronic myelogenous leukemia (CML) following a chemotherapy-induced loss of the Ph1 chromosome

After intensive chemotherapy, marrow cells of some patients with Philadelphia chromosome (Ph1) positive chronic myelogenous leukemia (CML) become partially or completely Ph1-negative. However, without a second marker for the neoplastic clone, it could not be determined if these Ph1-negative cells arose from normal progenitors or were still members of an abnormal clone. In the present study, a patient with Ph1- positive CML, also heterozygous for glucose-6-phosphate dehydrogenase (G6PD), was studied before and after intensive chemotherapy. Prior to treatment only G6PD type B was detected in the patient's red cells, platelets, and granulocytes, and all unstimulated marrow metaphases had Ph1. After four cycles of chemotherapy, 76% of marrow cells were Ph1- negative, and approximately 80% of the granulocytes were nonclonal by G6PD analysis. Thus, the frequency of nonclonal cells by G6PD analysis correlated closely with that of the Ph1-negative cells. The data indicate that intensive chemotherapy can restore nonclonal and presumably non-neoplastic hematopoiesis in CML.     

Agnogenic myeloid metaplasia: a clonal proliferation of hematopoietic stem cells with secondary myelofibrosis

The glucose-6-phosphate dehydrogenase (G-6-PD) types and chromosomes of hematopoietic and other tissues were determined in a woman with agnogenic myeloid metaplasia. The patient was heterozygous at the X- linked G-6-PD locus so that both B and A isoenzymes were found in nonhematopoietic cells. In contrast, only one G-6-PD type was found in granulocytes, red cells, and platelets. She also had a distinctive chromosome abnormality in blood cells but not in other tissues. These results indicate that agnogenic myeloid metaplasia is a disorder of a pluripotent stem cell and provide strong evidence that it is of clonal origin. In contrast to blood cells, the patient's cultured marrow "fibroblasts" had normal chromosomes and both B and A G-6-PD types, suggesting that the marrow fibrosis is a secondary abnormality. Thus, at least in this case of agnogenic myeloid metaplasia, the hematopoietic cell proliferation appears to be clonal, and, by inference, possibly neoplastic, whereas the marrow fibrosis is probably not clonal, and therefore appears to be secondary.     

Philadelphia-chromosome-negative chronic myelogenous leukemia with lymphoid stem cell blastic transformation

A patient is described who had blastic transformation of Ph1 negative chronic myelogenous leukemia (Ph1 - CML). Characterization of the leukemic cells revealed a population with a lymphoid stem cell phenotype (cALL-, TdT+, Ia+, cIgM-). This particular phenotype may be responsible for the refractoriness to vincristine and prednisone and the rapid downhill course.     

Cytogenetic studies of early myeloid progenitor compartments in Ph1-positive chronic myeloid leukaemia (CML) I. PERSISTENCE OF Ph1-NEGATIVE COMMITTED PROGENITORS THAT ARE SUPPRESSED FROM DIFFERENTIATING IN VIVO

We have cytogenetically analysed individual haemopoietic colonies to investigate the level and extent of normal stem cell suppression that occurs in patients with Philadelphia chromosome (Ph1)-positive CML. Seventeen patients were studied at diagnosis prior to the initiation of chemotherapy and five of these were studied again 1-16 months later. Another nine patients were studied for the first time 2-96 months after diagnosis and initiation of chemotherapy. No chromosomally normal metaphases were found in either direct marrow preparations or in haemopoietic colonies obtained from simultaneous assays of marrow and/or blood samples from 20 of the 26 patients studied. In the other six, chromosomally normal haemopoietic progenitors (BFU-E, CFU-C and CFU-G/E) were readily demonstrable even though in five of these patients all dividing cells in the bone marrow appeared to belong to the Ph1-positive clone at the time of study. These results indicate that the suppressive effects of clonal expansion on normal haemopoiesis are more pronounced, and apparent sooner, in the more differentiated compartments. In addition, they support the view that the original population of normal stem cells does not disappear rapidly, although their numbers may be diluted to undetectable levels depending upon the extent of clonal expansion at the stem cell level by the time of diagnosis.     

Unicellular or multicellular origin of human granulocyte-macrophage colonies in vitro

The assumption that human granulocyte-macrophage colonies have a unicellular origin and thus are true clones has been directly tested. Cells from seven females heterozygous for the common glucose-6- phosphate dehydrogenase (G-6-PD) gene (GdB) and the variant GdA were cultured in semisolid medium for granulocyte-macrophage colony growth and the enzyme type of individual colonies was determined. When the colony density was less than 20/dish, more than 95% of colonies had either type A or type B G-6-PD, but not both. At colony densities greater than 30/dish, between 15% and 75% of colonies had both enzyme types and therefore arose from more than one cell. These results are consistent with a unicellular origin for the colonies only when they are cultured at low densities. With increasing colony density, there was a greater frequency of colonies with both type A and type B activity, suggesting that accurate enumeration of committed stem cells can only be performed at low colony concentrations.