Premature chromosome condensation in human leukemia.

Premature chromosome condensation (PCC) has previously been observed in tissue culture and is believed to arise from asynchronous mitotic activity in multinucleated cells in which the affected nucleus is in interphase and at least one nucleus is in metaphase. Such cells have been noted following fusion induced by virus infection, spontaneously, and after treatment with cytochalasin B. The phenomenon has also been observed in malignant pleural effusions, but has not previously been described as a feature of hematologic disease. In this study, we report the observations of PCC in seven patients. Six of these patients had either acute myeloblastic leukemia or acute myelomonoblastic leukemia in association with the features of erythroleukemia, i.e., leukoerythroblastic reaction in the blood, and erythroid multinuclearity, "megaloblastoid" changes, and PAS-positive staining of erythroid precursor cells in the bone marrow. In all patients, erythroid multinuclearity has been noted. However, not all patients with erythroleukemia exhibit PCC. In this series, three additional patients have had similar bone marrow morphologic changes without PCC. The finding of PCC in erythroleukemia may have important implications as to etiology of this disorder.     

Premature chromosome condensation studies in human leukemia. I. Pretreatment characteristics.

The phenomenon of premature chromosome condensation (PCC) was used to compare the bone marrow proliferation characteristics of 163 patients with various forms of leukemia prior to the initiation of new therapy. The proliferative potential index (PPI, or fraction of G1 cells in late G1 phase) and the fraction of cells in S phase was determined and compared to the type of disease and the bone marrow blast infiltrate for each patient. Previously untreated patients with acute leukemia exhibited an average PPI value three times that of normal bone marrow (37.5% for acute myeloblastic leukemia [AML], acute monomyeloblastic leukemia [AMML], or acute promyelocytic leukemia [APML] and 42% for acute lymphocytic leukemia [ALL] or acute undifferentiated leukemia [AUL]). Untreated chronic myelogenous leukemia (CML) patients showed intermediate PPI values (25.2%), whereas CML patients with controlled disease exhibited nearly normal PPI values (14.6%). On the other hand, blastic-phase CML patients exhibited PPI values closer to that observed in patients with acute leukemia (35.4%). Seven patients with chronic lymphocytic leukemia (CLL) exhibited even higher PPI values. No correlations were observed between PPI values, fraction of cells in S phase, and marrow blast infiltrate. For untreated acute disease patients, PPI values were prognostic for response only at low and high PPI values. These results suggest that the PCC-determined proliferative potential is a biologic reflection of the degree of malignancy within the bone marrow.     

Premature chromosome condensation studies in human leukemia: 4. Characterization of albumin density fractionations of bone marrow at presentation, remission, and relapse

Previous studies have suggested that the technique of premature chromosome condensation (PCC) is useful in the study of human leukemia, both as a predictive tool for course of disease and as a probe to better understand the biology of the disease process. The purpose of this study was to determine how the various subcomponents of bone marrow populations contribute to the overall PCC pattern. Thirty bone marrow aspirations from persons at various stages of disease (2 normal, 11 untreated, 12 in remission, and 5 n relapse) were fractionated according to density in albumin gradients, and the various fractions were characterized by determining the proliferative potential index (PPI, or the fraction of G1 cells in late G1) using the PCC technique. In general, normal bone marrow and marrow from patients In remission showed lower PPI values throughout the gradients, with the most dense and most mature cells yielding the lowest PPI values in the gradient. In contrast, bone marrow from newly presenting patients and from patients in relapse showed higher PPI values, with the highest PPI values found In the most dense fractions containing the most mature cells. Thus, residual mature cells from patients with active disease exhibit the malignant characteristic of accumulating in late G1 phase. It is postulated that these morphologically mature cells from patients with active disease either represent matured cells of malignant origin or represent residual normal cells pushed beyond the restriction point in early G1 phase by a putative tumor growth factor synthesized by the leukemic cell population.     

Premature condensation induces breaks at the interface of early and late replicating chromosome bands bearing common fragile sites

Various studies suggest a tight relationship between chromosome rearrangements driving tumor progression and breaks at loci called common fragile sites. Most of these sites are induced after perturbation of the replication dynamics, notably by aphidicolin treatment. We have mapped the majority of these sites to the interface of R and G bands, which calls into question the previous assignment of aphidicolin-sensitive sites to R bands. This observation suggests that most of them correspond to loci that ensure the transition between early and late replicating domains. We show that calyculin A, which triggers chromosome condensation at any phase of the cell cycle but does not markedly impair replication, induces damage in the chromosomes of human lymphocytes treated in G(2) but not in G(1) phase. We demonstrate that these lesions colocalize with those induced by aphidicolin treatment. Hence, common fragile site stability is compromised, whether aphidicolin delays replication or calyculin A advances condensation. We also show that, in cells that go through an unperturbed S phase, completion of their replication and/or replication-associated chromatin reorganization occur all along the G(2) phase, which may explain their inability to condense properly after calyculin A treatment during this phase of the cell cycle.     

Long-term cytogenetic studies in acute leukemia of children; the nature of relapse.

Sequential long-term cytogenetic studies in 71 children with acute leukemia were designed to investigate the nature of relapse after prolonged remission. In the overwhelming majority of the cases the findings suggested clonal identity of the leukemic cell population in relapse with that studied at the onset of the disease, notwithstanding considerable karyotypic instability in almost half of the patients. In a small minority an independent origin of the relapse clone could not be excluded on cytogenetic grounds but was considered unlikely, since mechanisms capable of accounting for the changes observed in these patients could be demonstrated in other cases. The persistence of diploid leukemic cells in the presence of an aneuploid subclone was demonstrated in the relapse bone marrow and/or spinal fluid in all active phases of the disease. On this basis the conversion from an aneuploid to a predominantly or exclusively diploid karyotype could be visualized, and a new model of clonal evolution, involving repetitive formation of abnormal karyotypes from a surviving diploid clone could be suggested.     

Progression and survival studies in early chronic lymphocytic leukemia.

To investigate the natural history of stage A chronic lymphocytic leukemia (CLL) we reviewed 84 such patients. Among 74 cases evaluable for disease progression, 22 (29.6%) progressed to more advanced clinical stages (9 to stage B, 13 to stage C); the actuarial estimation of such an event at 4 years was 30% (95% CI: 26.3% to 33.6%). Despite a linear trend toward an increasing risk (r = .92), the hazard function analysis showed a constant pattern of progression, suggesting a lack of correlation of such an event with time (r = .04). Furthermore, disease progression when analyzed as a time-dependent variable had a clear-cut impact on survival (P less than .001). With the aim of identifying a subgroup of patients with low probability of disease progression and death, we applied to our set of patients four different proposals for subclassifying stage A. All methods were similar in terms of sample size, 5-year survival rate, and disease progression risk, suggesting that the choice between different proposals is somewhat arbitrary. Whatever the criteria are for defining "smoldering" CLL, such patients (accounting in the present study for 20.5% of overall series and 46.7% of stage A patients) should not be treated until progression occurs.     

Chromosomes and causation of human cancer and leukemia: XXVIII. Value of detailed chromosome studies on large numbers of cells in CML

Comparison of the chromosome findings obtained on routine examination (10-50 cells) of the marrows from patients with Ph¹ -positive CML with those based on a large number (110-500 cells) of metaphases in six of these patients, in whom appropriate material was available, revealed the presence of small percentages of aneuploid cells in the marrow during the chronic phase of the disease and not seen with the routine procedure. These aneuploid cells may ultimately constitute the dominant clone during the blastic phase of the chronic myelocytic leukemia (CML). Furthermore, karyotypically abnormal cells, in addition those observed on routine study, were detected in the blastic phase when a large number of cells was examined. The value and implications of these observations are discussed.     

Hand-mirror cell leukemia associated with mental retardation: immunologic, chromosome, and morphological studies.

Cycochemical, morphological, immunologic, and cytogenetic studies were carried out on hand-mirror cells (HMC) from a mentally retarded patient with a constitutional chromosome abnormality, 46,XX,r(21), and acute lymphoblastic leukemia. Scanning electron and differential interference contrast microscopy showed microspikes on the uropodia, but little evidence of cellular motility, despite formation and disappearance of individual uropodia in cell suspensions. The cells rosetted with sheep erythrocytes, suggesting T-cell origin. Cells derived from the bone marrow (80% HMC) showed a high degree of polyploidy (60%) and a bimodal chromosome number of 49 (49,XX,+10,-21, +3 rings) and 94 (6 no. 10, 3 no. 18, 2 no. 21 chromosomes, 3 ring chromosomes, plus 4 copies of each other chromosome).     

Successful repeated treatment of acute myeloid leukemia in early relapse with gemtuzumab ozogamicin alone

A 68-year-old female was diagnosed with acute myeloid leukemia (AML-M2 without 8/21 translocation) in December 2006. Although a complete remission (CR) was obtained after induction chemotherapy, the first post-remission therapy was discontinued because of severe cardiovascular complications. She had a relapse of AML with CD33-positive myeloblasts which comprised 38.4 % of the bone marrow cells in November 2007. She received two courses of low-dose chemotherapy because of the previous complications. The amount of Wilm’s tumor 1 (WT1) mRNA in the peripheral blood was 13,000 copies/μg RNA after the first course of the chemotherapy, and 4.8 % myeloblasts remained in the bone marrow after the second course. She was treated with a single course of gemtuzumab ozogamicin (GO), with a subsequent CR with 0.9 % marrow myeloblasts and fewer than 50 copies of WT-1 mRNA (normal level). Thereafter, she received five courses of GO monotherapy at each occasion of early AML relapse. Hematological remission has been sustained over a period of about 24 months with the GO monotherapy alone. This case suggests that GO monotherapy is a useful salvage therapy for early relapse of CD33-positive AML in situations in which standard chemotherapy is not indicated.     

Successful immunotherapy in early relapse of acute myeloid leukemia after nonmyeloablative allogeneic stem cell transplantation

We report on a 35-year-old woman who underwent allogeneic stem cell transplantation (SCT) in second complete remission (CR) of acute myeloid leukemia (AML) after reduced-intensity conditioning with fludarabine and 2 Gy of total body irradiation. For graft-versus-host disease (GVHD) prophylaxis, cyclosporin A (CsA) and mycophenolate mofetil (MMF) were given. On day 27 after SCT complete hematological remission and donor chimerism was documented. However, in CD34(+) bone marrow cells 28% of recipient hematopoiesis persisted. On day +59 leukemic relapse occurred. After discontinuation of CsA and onset of GVHD, complete donor chimerism and hematological CR were achieved which has been maintained for 14 months.     

Action of granulocyte-macrophage colony-stimulating factors: studies using a human leukemia cell line.

Granulocyte-macrophage colony-stimulating factors (CSFs) have previously been shown to stimulate colony formation in soft agar culture by a human myelogenous leukemia cell line known as KG-1. We have used KG-1 cells as a model system to investigate the interaction of CSF with myeloid cells. We now report that exposure of KG-1 cells to human CSFs in liquid culture results in a rapid (within 3 hr) burst of RNA synthesis and, after a lag of about 10 hr, a stimulation of DNA and protein synthesis. RNA and protein synthesis were maximally stimulated about 2-fold and DNA synthesis was stimulated about 2.5-fold. The stimulation was specific; various growth factors, hormones, and mouse CSFs had no effect on KG-1 macromolecular synthesis. Treatment with CSF did not discernibly alter the morphological appearance of the KG-1 cells (primarily myeloblasts) nor did it qualitatively affect the pattern of newly synthesized proteins separable by one- and two-dimensional electrophoresis. Several myeloid leukemia cell lines that were not responsive to CSF in agar culture, including a dedifferentiated variant of KG-1, showed little or no stimulation of macromolecular synthesis upon exposure to CSF. We have used the CSF-dependent stimulation of macromolecular synthesis of KG-1 to develop a rapid, sensitive microassay for human CSFs. The assay, involving thymidine incorporation by the cells, should be useful for characterization and purification of human CSFs.