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Chromosome studies in a phenotypically normal 40-year-old man with Philadelphia (Ph) chromosome-positive chronic myelocytic leukemia (CML) and a constitutional Robertsonian translocation are reported. The possibility of carriers of Robertsonian translocations having an increased risk to develop Ph-positive CML is mentioned.
The study of chromosome banding pattern of leukaemic cells in 15 patients with CML revealed t(9;22) in all cases. Similar additional chromosome abnormalities were observed in the terminal stage of the disease in 5 of 9 patients with aneuploid cell lines. The most frequent abnormalities were i(17q) and trisomy 8. The regularities of karyotype evolution in the terminal stage of CML are discussed.
The CML patients with so called masked Ph1-chromosome have been reviewed. Although the importance of c-sis and c-abl oncogenes is gaining popularity yet their role in the genesis of CML remain obscure. Patients with masked Ph1-chromosomes where chromosome 9 is not involved in the translocation(s) will provide a clue to the role of c-abl and/or c-sis in oncogenesis.
We have studied the clinical courses of 69 patients with blastic crises of Philadelphia chromosome positive CML to identify parameters that were associated with an increased response rate or survival. Cytogenetic analysis at the time of blastic transformation revealed additional chromosome changes in 70% of the patients tested. Bone marrow fibrosis was detected in 58% of evaluable patients. Lymphoblastic transformation was seen in 28% of the patients tested with cell surface marker analysis. The value of 5'-nucleotidase as a marker for distinguishing lymphoid from non-lymphoid blast crisis was confirmed. Of 57 evaluable patients, 23 (40%) responded to therapy (CR/PR longer than 14 days). Median survival was 75 days. Longer survival was related to the following factors: Ph1-chromosome as the only detectable cytogenetic abnormality; lymphoblastic transformation; no bone marrow fibrosis; high percentage of blasts and promyelocytes in the bone marrow, and response to therapy. No prognostic significance was associated with age, sex, Tdt, LDH, spleen size, duration of the chronic phase of the disease, white blood cell count, Hb, platelet count and percentages of basophils, eosinophils, erythroblasts and blasts and promyelocytes in the peripheral blood. These data confirm the poor prognosis of patients with blastic crisis of CML treated by conventional chemotherapy.
To evaluate treatment-related changes of the reticulin stain-measured fibrosis in Ph(1+)-CML, a clinicopathological study was performed on sequential trephine biopsies of the bone marrow following either interferon (IFN) or busulfan (BU) monotherapy. Using the monoclonal antibody CD61 for the identification of megakaryopoiesis and Gomori's silver impregnation method, number of megakaryocytes and density of argyrophilic (reticulin and collagen) fibers were determined by morphometry. We studied specimens from 26 patients with IFN-alpha 2b (including nine patients with additional IFN gamma) therapy and from 23 patients who had received BU. In both groups, repeated bone marrow biopsies (total 125) revealed a significant increase in the fiber content, as well as in the number of megakaryocytes during treatment. To assess the dynamics of myelofibrosis more precisely, computation of differences in the degree of fiber density between the first and last examination was carried out. Regarding the considerable variations in the biopsy intervals, a so-called myelofibrosis progression index (MPI) was calculated. Following this rationale, we were able to demonstrate that, in comparison to the BU-group, speed of progression of bone marrow fibrosis was significantly increased in CML patients treated with IFN. Preliminary statistical analysis indicated a relationship between myelofibrosis on admission, which was always associated with increased growth of megakaryocytes, and the MPI with survival. Even when these parameters were regarded, prognosis was significantly more favorable in the IFN-treated patients. The failure of IFN and BU to inhibit the evolution of myelofibrosis may be related to several conversely acting pathomechanisms. Among others, the inability of both therapeutic agents to reduce the number of megakaryocytes more effectively should be taken into consideration.
In seven patients with chronic myeloid leukemia (CML) and ultrastructural and morphometric study was performed on neutrophilic granulopoiesis in bone marrow trephine biopsies. Bone marrow specimens from five patients without hematological abnormalities served as controls. In stable phases of CML, abnormalities of the maturing granulocytic lineage were most conspicuously expressed by an infrequently occurring nuclear disfiguration (blebs and disturbed bridging of segments). Morphometric evaluation included the numbers of azurphil (primary) and specific (secondary) granules, the cisternal length of the endoplasmic reticulum and the area of the mitochondrial profiles. These variables could be determined in early and late myeloblasts, promyelocytes, metamyelocytes, band cells and mature polymorphonuclear granulocytes. Statistical analysis with regard to control specimens demonstrated no significant differences in the total amount of neutrophil granules or of the other cell organelles.
Point mutations within the ABL kinase domain of the BCR-ABL gene are associated with clinical resistance to imatinib mesylate in chronic myeloid leukemia (CML). To obtain more information about the association between BCR-ABL mutations and type of imatinib resistance, we studied 30 early chronic phase (CP) CML patients, commencing imatinib therapy, using a conventional sequencing technique. Seven patients treated in late CP and three patients treated in the accelerated phase were included for comparison. Blood samples were collected before and every third month during imatinib therapy. Mutations were not seen in any blood sample collected before start of therapy. During imatinib treatment, 2 of the 30 early CP patients acquired point mutations and both of them had other signs of imatinib resistance. None of the five early CP patients with a complete hematologic response (HR), but no cytogenetic response at 12 months, displayed any missense mutation. Likewise, none of 12 early CP patients with detectable BCR-ABL transcripts but in complete hematologic and cytogenetic remission at 12 months displayed any mutation. We conclude that screening early CP patients for BCR-ABL mutations before start of imatinib therapy is not cost-effective. BCR-ABL kinase domain mutations do not appear to explain cytogenetic or molecular (detectable BCR-ABL transcripts by polymerase chain reaction) disease persistence in patients otherwise in stable disease. However, in patients with signs of expanding disease burden, a search for BCR-ABL mutations is warranted.
PURPOSE: A new chemotherapy regimen was designed for leukemia to improve response to therapy and elucidate the possible underlying mechanisms responsible for its efficacy. METHODS: Cells of the chronic myelogenous leukemia (CML) cell line, K562, were treated singly, in combination, and in sequence with clinically equivalent dosages of topotecan, which targets topoisomerase I (Topo I), and etoposide and mitoxantrone, which target topoisomerase II (Topo II), to determine the best treatment. Apoptosis, early cell deaths, and cell cytotoxicities in drug-treated cells were determined with annexin V and propidium iodide staining and MTT assays, respectively. Confocal microscopy and RT-PCR showed the cellular locations and relative increases in Topo IIalpha in topotecan-treated cells. The double comet assay of individual cells showed simultaneously free Topo proteins, non-Topo-associated DNA, and Topo-DNA complexes in drug-induced DNA fragments. RESULTS: Sequential treatment with topotecan on days 1-3, followed by etoposide+mitoxantrone on days 4, 5, 9 and 10 resulted in 100% cell death whereas treatments involving administration of drugs singly or simultaneously resulted in less cell kill. The cytotoxicity results in cells treated for fewer days with the same sequential chemotherapy regimen showed the same trend, and adequate surviving cells for the experiments on the cellular and molecular mechanisms of drug action were produced. An increase in Topo IIalpha mRNA from RT-PCR 1 h after topotecan treatment was observed. Observations on K562 cells treated sequentially with topotecan followed by etoposide, mitoxantrone or etoposide+mitoxantrone were as follows: (1) Topo IIalpha protein levels increased and relocated from the cytoplasm into the nucleus as detected by confocal microscopy, (2) Topo IIalpha-DNA complexes increased and were associated with fragmented DNA (positive double comets) as detected by protein-DNA double comet assay, and (3) Topo I and Topo IIbeta proteins were not associated with fragmented DNA. Topotecan-induced Topo IIalpha protein levels correlated with increased numbers of positive double comets and reduction of cell viability. CONCLUSIONS: Our results showed that Topo IIalpha protein induction after Topo I-directed drug treatment enhanced the sensitivity of cells to subsequent exposure to Topo II-directed drugs. Timed sequential chemotherapy with topotecan followed by etoposide+mitoxantrone is an effective regimen to ablate CML cancer cells.
Partially purified platelet DNA polymerase (PDP) was able to synthesize DNA transcripts of platelet polyadenylated RNA. PDP was elevated in the earliest stages of CML and PV. In PV, successful chemotherapy resulted in rapid return of PDP to normal levels while in CML this was not the case. An hypothesis is presented proposing that PDP contributes to oncogenesis by regulating the expression of oncogenes.
Addition of serum from patients with chronic myeloid leukemia (CML) in both chronic phase and blastic transformation, to agar cultures of normal human marrow cells stimulated the growth of persisting colonies (day 35) containing either eosinophils or mast cells. Chronic phase serum stimulated an 800% increase in the total number of these colonies of which only 16% were mast cells. Serial studies using serum from 2 patients demonstrated that the proportion of mast cells increased during the progression to blastic transformation. The emergence of a greater proportion of persisting mast cell colonies and a decrease in absolute number of eosinophil colonies in agar cultures of normal marrow cells containing serum from patients with CML coincides with the emergence of blastic transformation and suggests that a significant change occurs in the absolute and relative concentration of hemopoietic growth factors in these patients.
In chronic myelogenous leukemia (CML) malignant cells are characterised by the Philadelphia chromosome (Ph), resulting from a translocation t(9;22). The position of the breakpoint within the major breakpoint cluster region (M-bcr) on chromosome 22 has been shown to correlate with the clinical course of the disease or, more recently, thrombopoietic activity. We have therefore determined the breakpoint localisation in 53 Ph-positive CML patients. Following the 5'/3'-region definition of Inokuchi et al. Leukemia Research 15, 1067 (1991) [1], 22 of our patients have 5' and 31 of our patients have 3' orientated breaks. No correlation was found between platelet counts and breakpoint localisation.
We investigated the cytotoxic activity and some aspects of the mode of action of 5-aza-2'-deoxycytidine (Aza-dC) in 21 primary cultures of leukemic cells freshly obtained from patients with chronic myeloid leukemia (CML) in blast crisis. The cytotoxic potency of Aza-dC was comparable or even greater than that of 1-beta-D-arabinofuranosylcytosine (Ara-C) in most cases, suggesting that this drug has potential in the therapy of blast crisis of CML. Drug incorporation into DNA was evaluated by exposing leukemic cells simultaneously to 3H-Aza-dC at the concentration of 0.1 micrograms/ml and 14C-thymidine (TdR) used as internal standard. Incorporation of Aza-dC into DNA was detectable in all cases. In 17 samples we evaluated the DNA integrity of leukemic cells exposed to Aza-dC using alkaline elution techniques. The drug caused a detectable amount of DNA alkali labile sites (ALS). DNA-ALS increased in cells exposed to Aza-dC concentrations from 0.1 to 1 microgram/ml but did not further increase at 10 micrograms/ml. A plateau in the levels of DNA-ALS was also seen in human K562 cells exposed to increasing concentrations of Aza-dC from 5 to 10 micrograms/ml, whereas in these cells Aza-dC incorporation into DNA increased with increasing Aza-dC concentrations. Therefore, DNA-ALS caused by Aza-dC are not simply the result of the chemical decomposition of azacytosine molecules incorporated into DNA, but are presumably the result of a saturable DNA repair mechanism (e.g., glycosylases) leading to formation of the apyrimidinic sites.
In chronic myeloid leukaemia (CML), disease progression from the initial chronic phase to the acute phase or blast crisis has previously been shown to be correlated with progressive increases in hyper-methylation of the calcitonin gene, located at chromosome 11p15. However, sequential studies of individual patients were not performed in these investigations. We have analysed 44 samples from nine patients with typical Philadelphia chromosome positive CML throughout their disease progression to determine the methylation state of the calcitonin gene at these time points. Densitometry was used to quantitate the ratio of the normal 2.0 kb Hpa II fragments, indicating normal methylation status of the gene, compared to the intensity of the abnormal, hyper-methylated, 2.6-3.1 kb Hpa II fragments. We found a gradual increase in the ratio of methylated:unmethylated calcitonin gene during chronic phase with a dramatic rise at blast crisis. Further, the ratio of the abnormal hypermethylated 3.1 kb fragments to the methylated 2.6 kb fragment resulted in the identification of a clonal expansion of abnormally methylated cells. This expansion of cells with hypermethylation of the calcitonin gene during chronic phase was shown to coincide with the presence of a mutation in the p53 gene. The data presented in this study would suggest that an increased methylation status of the calcitonin gene during disease progression may indicate the expansion of abnormal blast cell populations and subsequent progression to blast crisis.
There has been a lack of direct cytogenetic studies on lymphocytes in the chronic phase of chronic myelocytic leukemia (CML). Peripheral blood mononuclear cells from seven patients in the chronic phase and on in the blastic phase of Ph1-positive CML were cultured in the presence of phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Preparations adequate for analysis were obtained from eight PHA and five PWM stimulated cultures. Cytogenetic studies on the PWM stimulated cultured cells revealed, in one patient, the presence of the Philadelphia chromosome (Ph1) in 23/25 metaphases. The PHA stimulated lymphocytes from the same patient were all Ph1 negative. Lymphocytes from PHA and PWM stimulated cultures from the other six patients in the chronic phase and the one patient in blastic crisis were all Ph1 negative. The one positive result suggests that some B lymphocytes contain the Ph1 and that there is a common hematopoietic stem cell for B lymphocytes as well as the myeloid, erythroid, and megakaryocytic series.
A case of chronic myelogenous leukemia (CML) in a young woman with a new variant Ph1-translocation--i.e., t(8;22) (q24;q12)--is described. The clinical and biological aspects of the disease did not seem to differ from those of the usual cases of CML.
A patient with CML in chronic phase was admitted to our center for bone marrow transplantation. Cytogenetic analysis of bone marrow cells revealed a Ph chromosome due to a standard t(9;22) and a Robertsonian t(14;15). This Robertsonian translocation was also found in PHA-stimulated lymphocytes of the patient's sister, the donor of the bone marrow. A chromosome study of the whole family proved the constitutional character of the t(14;15) abnormality and showed that it was inherited from the father's side. All family members were phenotypically normal with normal mental status. Female carriers of the t(14;15), as well as wives of male carriers, had a high incidence of miscarriages and early death of their offspring. The occurrence of Ph-positive CML in a patient with a Robertsonian t(14;15) might indicate increased risk for the development of leukemia in patients with this constitutional chromosome abnormality. This assumption however, is limited by the rarity of the Robertsonian translocations.
Of 175 CML patients studied, 14 variants were found, seven of which are presently described. The breakpoints involved in the translocation, other than 9q34 and 22q11, are 3p21, 5q13, 6p21, 7q22, 10q22, and 11p13. Fragile sites were investigated in some of these patients. In two cases a coincidence between fragile site location and breakpoint of the third chromosome involved in Philadelphia formation was found. This observation suggests that the fragile sites can lead to Ph variants in patients developing CML.
Nineteen patients with acute promyelocytic leukemia (APL) and one with promyelocytic blast crisis were studied by a methotrexate cell synchronization technique and by 24-hour short-term culture. Nineteen cases of APL included two cases of the microgranular variant (M3V). Except in one case of M3V, t(15;17) was detected in all patients. The breakpoints were determined as 15q22 and 17q12-21. A chronic myeloid leukemia (CML) blast crisis patient with high promyelocyte count also had a t(15;17) as well as a masked Ph chromosome. Other abnormalities, such as +8,del(7q) and an i(17q), were also observed in some patients. Our studies have indicated that 1) the translocation (15;17), characteristic of APL, was present in our population in almost all patients; 2) the presence of an identical abnormality in a promyelocytic CML blast crisis supported and confirmed the phenomenon of association of specific chromosome change with target cell type; and 3) the precise localization of breakpoints on chromosome 17 is still difficult to determine. The identification of as yet unknown genes at region 17q11-q21 and their subsequent translocation on chromosome 15 will help in assigning a precise position to these breakpoints.