De novo appearance of the ph-1 chromosome in a previously monosomic bone marrow (45,XX,-6): conversion of a myeloproliferative disorder to acute myelogenous leukemia

Bone marrow examination of a patient with a myeloproliferative disorder revealed monosomy for chromosome No. 6 (45,XX,-6). Two months later, during blastic crisis, reinvestigation of the bone marrow showed the presence of the Ph-1 chromosome in the previously aneuploid cell line (45,XX,-6,-22,+Ph-1). This case differs from those previously published in that the Ph-1 chromosome appeared de novo during the development of frank acute myelogenous leukemia.     

Autografting with cultured marrow in chronic myeloid leukemia: results of a pilot study [see comments]

Incubation of chronic myeloid leukemia (CML) marrow for 10 days in vitro causes a marked and selective loss of very primitive Philadelphia chromosome (Ph)+ as compared with Ph- progenitors. We have autografted 22 patients with CML (16 in first chronic phase [group 1] and 6 with more advanced disease [group 2]) with marrow treated in this way to facilitate restoration of Ph- hematopoiesis after intensive therapy. Hematologic recovery to greater than 0.5 x 10(9)/L neutrophils occurred in 16 patients, and to greater than 20 x 10(9)/L platelets in 15 of 21 evaluable patients at a median of 29 and 48 days postautograft, respectively. Regenerating marrow cells were 100% Ph- in 13 patients and 75% to 94% Ph- in 3. Between 4 and 36 months (median 12) postautograft, Ph+ cells became detectable in all but 1 (who died in remission) of the 13 patients who achieved complete cytogenetic remission. Four of 7 evaluable patients treated with low-dose interferon alpha were returned to complete cytogenetic remission. Thirteen group 1 patients (81%) are alive 1.0 to 5.7 years (median 2.6) after autografting: 4 in complete cytogenetic remission, 2 in hematologic remission, 6 in chronic phase, and 1 in myeloid blast phase. Three group 2 patients (50%) are alive at 2.6, 3.8, and 4.3 years after autografting: 1 in partial cytogenetic remission, 1 in chronic phase, and 1 in accelerated phase. Thus, autografts of cultured marrow can result in prolonged restoration of Ph- hematopoiesis for some patients with CML.     

Isochromosome 17 in a case of eosinophilic leukaemia. An abnormality common to eosinophilic and neutrophilic cells.

In a patient with eosinophilic leukaemia, serial chromosome studies using the Giemsa banding technique revealed a similar marker chromosome, identified as an isochromosome 17, in all bone marrow metaphases analysed. There was no Ph-1-chromosome. The cytogenetic data support the view that eosinophilic leukaemia is a specific disease entity, and suggest that both eosinophilic and neutrophilic cells are involved in the leukaemic process.     

Immunophenotypes and Outcome of Philadelphia Chromosome-Positive and -Negative Thai Adult Acute Lymphoblastic Leukemia

Little evidence exists in Asian countries regarding the incidence, immunologic characteristics, and clinical outcomes of adult patients with Philadelphia chromosome-positive (Ph+) and -negative (Ph-) acute lymphoblastic leukemia (ALL). In this study, we prospectively studied 324 Thai adult acute leukemia patients, 79 (24%) of whom were identified as having ALL. Immunophenotyping was performed by 5-parameter flow cytometry, and karyotyping was conducted by standard banding methods. The Ph chromosome was detected in 18.5% of cases. The mean age of Ph+ ALL patients was 29 years (50% male), and that of Ph- ALL patients was 33 years (62% female). The Ph+ ALL patients had significantly higher white blood cell (WBC) counts (mean, 93 x 10(9)/L), with 67% having WBC counts higher than 50 x 10(9)/L. In contrast, most Ph- ALL patients had WBC counts lower than 50 x 10(9)/L (mean, 36 x 10(9)/L; P < .05). CD10 and CD34 were more highly expressed in the Ph+ ALL patients (mean expression, 83% and 87%, respectively) than in the Ph- ALL patients (45% and 57%; P < .005). The aberrant expression of myeloid antigens, including CD33 and CD13, was also significantly observed in the Ph+ ALL patients. The median survival time of Ph+ ALL patients was 8 months, compared with 22 months for the Ph- ALL patients. In conclusion, immunophenotyping results showed that Ph+ ALL in Thai adults arises from B-cells at an earlier stage of development. Extreme leukocytosis, a younger age, male sex, high expression levels of CD10 and CD34, aberrant myeloid antigens, and poorer rates of survival appeared to be associated with the Ph chromosome in Thai adult ALL cases. The incidence of the Ph chromosome among Thai adult ALL patients was not different from that found in Western countries.     

Cytogenetic status pre-transplant as a predictor of outcome post bone marrow transplantation for chronic myelogenous leukaemia

We have analysed pre-transplant cytogenetic findings in 418 patients with CML in pre-blastic phase who underwent allogeneic BMT between February 1981 and January 1998. Five different patient groups were identified: A = Philadelphia (Ph)+; B = Ph-, BCR-ABL+; C = variant Ph (VPh); D = Ph chromosome plus at least one of: trisomy 8, +Ph, chromosome 17 abnormalities and E = other abnormalities in addition to the Ph chromosome. There were two principal conclusions. Firstly, Ph- patients showed a better outcome, and VPh patients a worse outcome, than those with a standard Ph, both in terms of leukaemia-free survival (LFS) (76.9%, 22.1% and 31.9%) and the risk of treatment failure relative to those with a standard Ph (relative risks of 0.49 and 1.92, respectively). One contributing factor may be relapse: no Ph- patients relapsed, whereas all other groups showed similar probabilities of relapse at 5 years (range 33.0-44. 0%). Secondly, those with the additional changes of +8, +Ph and i(17q) did not show a worse outcome than those with no additional changes (5 year survival of 44.7% vs 51.8%; 5 year LFS of 40.6% vs 31.9%), whereas those with other additional changes may fare worst of all (40.4% and 16.0%, respectively). Bone Marrow Transplantation (2000) 25, 143-146.     

Characterization of primitive subpopulations of normal and leukemic cells present in the blood of patients with newly diagnosed as well as established chronic myeloid leukemia

Elevated numbers of primitive Philadelphia chromosome-positive (Ph+) progenitors, including long-term culture-initiating cells (LTC-IC) as well as colony-forming cells (CFC), have been previously described in the blood of patients with chronic myeloid leukemia (CML) in chronic phase with high white blood cell counts. In the present study, which focused primarily on an analysis of circulating progenitors present in such patients at diagnosis, we discovered the frequent and occasionally exclusive presence of circulating normal (Ph-) LTC-IC, often at levels above those seen for LTC-IC in the blood of normal individuals. The presence of detectable numbers of circulating Ph- LTC-IC was independent of the fact that the same peripheral blood samples also contained elevated numbers of predominantly or exclusively Ph+ CFC. Interestingly, both the Ph+ and Ph- LTC-IC in these samples were CD34+CD71- and variably CD38- and Thy-1+, as previously documented for LTC-IC in normal marrow. Thus, neither CD38 nor Thy-1 expression was useful for discriminating between Ph+ and Ph- LTC-IC in mixed populations. Nevertheless, an association of these phenotypes with LTC- IC function did allow highly enriched (> 5% pure) suspensions of either Ph+ or Ph- LTC-IC to be obtained from selected samples of CML blood in which the initial LTC-IC population was either predominantly Ph+ or Ph- , respectively. These findings suggest that the mechanisms causing mobilization of leukemic stem cells in untreated CML patients may affect their normal counterparts. They also indicate a possible new source of autologous cells for the support of intensive therapy of CML patients. Finally, they provide a method for obtaining the most highly purified populations of Ph+ LTC-IC described to date. This method should be useful for further analyses of the molecular activities of these very primitive neoplastic cells.     

Gene disruption of a G4-DNA-dependent nuclease in yeast leads to cellular senescence and telomere shortening.

The yeast gene KEM1 (also named SEP1/DST2/XRN1/RAR5) produces a G4-DNA-dependent nuclease that binds to G4 tetraplex DNA structure and cuts in a single-stranded region 5' to the G4 structure. G4-DNA generated from yeast telomeric oligonucleotides competitively inhibits the cleavage reaction, suggesting that this enzyme may interact with yeast telomeres in vivo. Homozygous deletions of the KEM1 gene in yeast block meiosis at the pachytene stage, which is consistent with the hypothesis that G4 tetraplex DNA may be involved in homologous chromosome pairing during meiosis. We conjectured that the mitotic defects of kem1/sep1 mutant cells, such as a higher chromosome loss rate, are also due to failure in processing G4-DNA, especially at telomeres. Here we report two phenotypes associated with a kem1-null allele, cellular senescence and telomere shortening, that provide genetic evidence that G4 tetraplex DNA may play a role in telomere functioning. In addition, our results reveal that chromosome ends in the same cells behave differently in a fashion dependent on the KEM1 gene product.     

Brief Report: Philadelphia Chromosome in Acute Lymphocytic Leukemia

It is recognized that the Ph¹ chromosomeanomaly is characteristic of chronic granulocytic leukemia. It has been shownhowever, to occur rarely in other myeloproliferative diseases. We have reported a 53-year-old roentgenologistwith acute lymphocytic leukemia whoshowed a high percentage of Ph¹ chromosomes in his marrow cells. The possiblerole of radiation exposure in the production of the abnormal chromosome in thispatient is discussed. The data presentedis evidence against the specificity of thePh¹ chromosome for myeloproliferativedisorders.

Submitted on August 27, 1969 Revised on April 1, 1970 Accepted on April 4, 1970     

Identification of Philadelphia-negative granulocyte-macrophage colony- forming units generated by stroma-adherent cells from chronic myelogenous leukemia patients

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+ progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. On direct cytogenetic analysis, the overall mean (+/- SEM) percentage of Ph- metaphases was 3% +/- 1%. Mononuclear marrow cells from CML patients (n = 18) were incubated with mafosfamide (100 micrograms/mL) or control medium, seeded onto marrow stromal layers and allowed to adhere (2 hours, 37 degrees C). After a short-term (3-day) liquid culture, the cells were harvested, incorporated in methyl-cellulose, and individual colonies were analyzed by single colony karyotyping. The mean (+/- SEM) percentage of Ph- colonies generated from the stroma- adherent fraction was 35% +/- 6%. As compared with marrow colony- forming unit granulocyte-macrophage plated before any manipulation, the mean (+/- SEM) percentage of Ph- clones was significantly increased by stroma adherence (35% +/- 6% v 15% +/- 4%, P < or = .005) and mafosfamide (100 micrograms/mL) incubation of marrow cells before stroma adherence (58% +/- 9% v 35% +/- 6%, P < or = .005). An additive effect was observed by combining mafosfamide treatment and stroma adherence. Single-colony transfer experiments showed that 50% +/- 4% stroma-adherent and 70% +/- 4% stroma-adherent mafosfamide-treated progenitors gave rise to secondary colonies. To further characterize the stroma-adherent fraction, experiments were performed in which CD34+ marrow cells were used. The mean (+/- SEM) output of progenitors generated by 10,000 CD34+, stroma-adherent cells was 888 +/- 188 and 570 +/- 258 for untreated and mafosfamide-treated cells, respectively. Individual colonies were analyzed by single-colony karyotyping and fluorescent in situ hybridization using a biotinylated cosmid DNA probe that hybridize to abl oncogene. The CD34+, stroma-adherent fraction contained 38% +/- 14% (untreated) and 56% +/- 18% (mafosfamide-treated) (P < or = .025) Ph- progenitors. In conclusion, the present data show the possibility to select Ph- clones that (1) have a maintained capability of stroma adherence, (2) are mafosfamide resistant, (3) are derived from the CD34+ fraction, and (4) have high-replating potential.     

Transient trisomy 8 abnormality in Philadelphia-negative cells during imatinib mesylate treatment of chronic myelogenous leukemia

We investigated chronic myelogenous leukemia (CML) patients who developed trisomy 8 abnormalities in Philadelphia-negative (Ph-) cells during imatinib mesylate treatment to evaluate the clinical outcome and laboratory features. Of the 470 CML patients, 1.5% (n = 7) developed trisomy 8 chromosomal abnormalities in Ph- cells. The median interval of the first trisomy 8 observation was 12 months. Our follow-up cytogenetic evaluations revealed that six of the patients demonstrated a complete or partial cytogenetic response and that all of the six patients revealed no dysplastic changes following a bone marrow examination. Moreover, the percentage of trisomy 8 in metaphase karyotyping has decreased in five of the seven subjects. In conclusion, these results suggest that the emergence of trisomy 8 in Ph- cells is transient and not related to therapy-related myelodysplasia or acute leukemia.     

T lymphocytes lack rearrangement of the bcr gene in Philadelphia chromosome-positive chronic myelocytic leukemia

To study the possible involvement of T lymphocytes in Philadelphia chromosome (Ph)-positive chronic myelocytic leukemia (CML) we analyzed the arrangement of the bcr gene in T cell and non-T cell samples of 12 CML patients. Although all the patients showed bcr rearrangements in non-T cell fractions, T cell populations lacked respective gene recombinations. Moreover, by Southern blot analyses using T cell receptor beta chain sequences our data indicate polyclonality of T cell samples from 11 of 12 cases; in one patient a clonal T cell population could be identified. These results suggest that T lineages of most Ph- positive CML patients are not derived from pluripotent stem cells involved in leukemogenesis and thus confirm previous investigations based on cytogenetic or glucose-6-phosphate dehydrogenase analyses. The demonstration of polyclonal T cell populations may reflect persistence of stem cells committed to differentiate only into T cells.     

P-selectin gene polymorphisms and risk of coronary heart disease among Tunisians

P-selectin plays a key role in inflammation and atherosclerosis, and polymorphic variants of P-selectin were implicated in the pathogenesis of atherosclerotic and inflammatory changes, including coronary heart disease (CHD) in many ethnic groups. We investigated the contribution of P-selectin promoter (−2123C/G, −1969G/A) and exon (Ser290Asn, Asn562Asp, Thr715Pro) polymorphisms to CHD genetic susceptibility among 298 Tunisian CHD patients and 339 controls. Minor allele and genotype frequencies of the five P-selectin SNPs were comparable between patients and controls, except for −2123G/G genotype which was more frequent in cases. The 715Pro allele was present at lower frequency in Tunisians than in Europeans, and was not protective of CHD. Linkage disequilibrium was seen between −1969G/A, and both Ser290Asn and Asn562Asp. Five-loci haplotype analysis did not identify any CHD-protective or CHD-susceptible haplotypes. To our knowledge, this was the first case-control study to be performed on an Arab/North-African population, and demonstrates that none of the five P-selectin polymorphisms investigated influence CHD susceptibility in Tunisian Arabs.     

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.     

Analysis of Interphase Chromosome Damage by Means of Premature Chromosome Condensation after X- and Ultraviolet-Irradiation

Sendai virus-mediated fusion between mitotic and interphase mammalian cells causes the rapid condensation of the interphase chromosomes into distinct structures, a process termed premature chromosome condensation. This phenomenon has been used to assess the immediate action of x-rays and ultraviolet light on the chromosomes of HeLa cells irradiated in the G1 phase of the life cycle. X-irradiation produces fragmented chromosomes; but even the most finely chopped fragments retain the condensed morphology characteristic of the premature chromosome condensation of unirradiated G1 cells. For doses up to about 1800 rads, the increase in the number of fragments is linearly related to the dose. One mean lethal dose (about 100 rads) yields a net increase of 10-15 fragments per G1 cell, which is considerably larger than previous estimates based on scoring of mitotic chromosomes. Incubation of irradiated cells produces a rapid (within 2 hr) reduction in the number of fragments, indicative of a rejoining process. The decrease in the number of pieces is not accompanied by unscheduled DNA synthesis detectable by radioautography. G1 chromosomes of cells irradiated with UV light in G1 phase are not appreciably fragmented but are elongated and attenuated so that they resemble the premature-chromosome-condensation chromosomes of unirradiated S-phase cells. Both the degree of "S-like" character attained by the G1 chromosomes in a cell and the percentage of the cell population displaying the G1 --> S transition increase with the dose and incubation time after irradiation. Thus, in contrast to the immediate manifestation of damage from x-rays, the maximum induction of the "S-like" state does not occur until about 2 hr after irradiation. The "S-like" chromosomes are capable of unscheduled DNA synthesis. We suggest that the difference in chromosome morphology found after UV- and x-irradiation underlies the reason why the former, but not the latter, induces unscheduled DNA synthesis in G1 cells.     

Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins.     

The G→A Mutation at Position +22 31 to the Cap Site of the β-Globin Gene as a Possible Cause for a β-Thalassemia

We describe the occurrence of a chromosome with a G→A mutation at position +22 relative to the Cap site that was found in five patients with β-thalassemia. All patients had a common type of β-thalassemia mutation on the second chromosome, namely the frameshift at codon 8 (-AA), the IVS-I-110 (G→A) and the IVS-II-1 (G→A) mutations. The β genes of two patients, including the 5′ and 3′ untranslated regions, were completely sequenced and no other mutations, except a few polymorphic sites, were observed. Dot-blot analyses failed to demonstrate this G→A mutation at +22 in nearly 400 β-thalassemia chromosomes and 180 normal chromosomes. Hetero-zygotes have the features of a high Hb A₂-β-thalassemia hetero-zygosity, although the hematological parameters might be less abnormal than observed in heterozygotes for the more common β-thalassemia mutations. The possibility has been presented suggesting that this mutation might impair the binding of mRNA to ribosomes. Another mutation in this segment of DNA, i.e. a C→G mutation at position +20, is observed exclusively on a chromosome which also carries the C→G mutation at IVS-II-745. It is postulated that the +20 C→G mutation accentuates the β-thalassemia condition caused by the IVS-II-745 mutation; the mechanism might be similar to that suggested for the G→A at +22 mutation.