Growth characteristics in vitro of myeloid leukaemic cells with t(6;9).

We report an analysis of in vitro growth characteristics of leukaemic cells from five patients with t(6;9)(p23;q34). Consistent with other reports of this abnormality, our patients were comparatively young (median age at diagnosis, 29 years), and responded poorly to conventional treatment (median survival from diagnosis, 10 months). During active disease the CFU-GM growth patterns were characterized by an abundance of granulocytic aggregates (mostly 20-100 cells in size) whose leukaemic origin was confirmed by cytogenetic analysis. During remission induction, colonies derived from regenerating normal progenitor cell colonies could be distinguished from those derived from persisting leukaemic cells on the basis of differences in size, morphology, in situ staining characteristics, and karyotype. Remission growth patterns were those of a normal bone marrow. Our findings add to a growing recognition that the t(6;9) identifies a subset of leukaemic patients with distinctive clinical, haematologic, molecular, and in vitro growth characteristics for whom conventional treatment offers little hope of cure or long survival.

Deletion 20q in association with Philadelphia chromosome positive acute lymphoblastic leukemia. A report of two cases.

Cytogenetic deletions involving the long arm of chromosome 20 are thought to be characteristic of myeloid disorders. We report clinical and cytogenetic observations of two adult patients with Philadelphia chromosome positive acute lymphoblastic leukemia: one with a smaller 20q deletion that was the sole cytogenetic abnormality in a persisting remission clone, the other with a larger 20q deletion that was a late addition to the leukemic clone at disease relapse following allogeneic bone marrow transplantation.

Molecular heterogeneity at the breakpoints of smaller 20q deletions.

Deletions of the long arm of chromosome 20 [del(20q)] are recurring abnormalities in patients with myeloid disorders. Although variable in size, these deletions are usually interstitial. With the object of defining a commonly deleted region for smaller 20q deletions, we used quantitative Southern blot analysis complemented by restriction fragment length polymorphism (RFLP) analysis to determine the copy number at 15 loci spanning 20q. The proximal breakpoints of three such deletions were found to separate HCK and the growth hormone releasing factor (GHRF) locus near the centromeric boundary of band 20q11.2. The distal breakpoints were localized to the vicinity of the D20S22 locus in band q13.1. A candidate tumor suppressor gene, RBL2, and the SRC oncogene were both located within the commonly deleted region. Six loci in terminal region q13.2-q13.3 were conserved on these del(20q) chromosomes, thereby confirming that the deletions were interstitial. Molecular heterogeneity at one and possibly both deletion breakpoints rules out the pathological involvement of loci at these sites. Instead, loss of a tumor suppressor locus from within the commonly deleted region may contribute to deregulated hemopoiesis.

Identification of three RFLPs at the HCK locus on chromosome 20.

New HindIII, RsaI and TaqI restriction fragment length polymorphisms (RFLPs) within the haemopoietic cell kinase gene in chromosome band 20q11.2 are described. These RFLPs provide a useful marker for linkage analysis in proximal 20q.

Oligoclonal B-cell leukemia characterized by spontaneous cell division and telomere association.

Cytogenetic analysis of unstimulated cultures from a female patient with chronic B-cell leukemia (CLL) revealed three cytogenetically distinct clones, suggesting that the patient's leukemia was oligoclonal. Immunoglobulin heavy chain gene rearrangement studies revealed 1 germline and 4 rearranged bands, indicative of an oligoclonal leukemic population. Further evidence of oligoclonality was provided by X-linked RFLP studies. This is the first report of oligoclonality in CLL demonstrated by cytogenetic, immunoglobulin gene rearrangement, and X-chromosome inactivation studies. In addition to oligoclonality, the patient's leukemic cells exhibited telomere association, a Robertsonian translocation, and clonal evolution, suggesting an underlying genomic instability.

Localization of the SRC oncogene to chromosome band 20q11.2 and loss of this gene with deletion (20q) in two leukemic patients.

In situ hybridization of the pHul-c-src probe to metaphase cells from three normal donors and two leukemic patients showed significant labeling in the proximal region of the long arm of chromosome 20q, with modal peaks of grains consistently at band 20q11.2. A secondary peak of grains was detected in the region 20q13.2-qter, the localization of SRC suggested by previous in situ studies. The exact localization of SRC is important for understanding the del(20q) chromosomal abnormality in myeloid neoplasias. Chromosome in situ hybridization and genomic studies showed loss of one allele of SRC in two patients with the deletion (20q). These results differ from previously published findings and suggest heterogeneity of the breakpoint at 20q11.2 in interstitial deletions of 20q, which characterize myeloid disorders.

HRAS1 and INS genes are relocated but not structurally altered as a result of the t(7;11)(p15;p15) in a clone from a patient with acute myeloid leukaemia (M4).

A patient whose leukaemic cells carried the rare t(7;11)(p15;p15) was diagnosed as having acute myelomonocytic leukaemia (AML-M4), and supports the association of this specific translocation with forms of acute myeloid leukaemia showing differentiation. Blast phase chronic myeloid leukaemia was excluded by lack of involvement of the ABL and BCR genes. Chromosome in situ hybridization studies showed that both the HRAS1 and INS genes were present on the terminal part of chromosome 11p which was translocated to chromosome 7p. Neither HRAS1 nor INS were structurally rearranged. Field inversion gel electrophoresis showed that a 400 kb fragment encompassing HRAS1 was structurally entire in leukaemic DNA. Because the INS gene, which was also translocated, is probably located proximal to HRAS1 on chromosome 11p, it is unlikely that HRAS1 was near the chromosome 11 breakpoint or involved in this leukaemia.

Involvement of immunoglobulin heavy- and light-chain (kappa) gene clusters in a human B-cell translocation, t(2;14).

The breakpoints of a translocation, t(2;14)(p11;q32), detected in an Epstein-Barr virus-transformed lymphoid B-cell line were mapped by Southern analysis, field-inversion gel electrophoresis, and in situ hybridisation. The translocation involved the immunoglobulin light-chain (kappa) locus on chromosome 2 and the heavy-chain locus on chromosome 14. The breakpoint on chromosome 2 was between VK and CK, most likely within JK. The chromosome 14 break was located within the VH cluster, no more than 220 kb 5' of the productively rearranged JH locus. The translocation probably resulted from an aberrant rearrangement of the kappa light-chain genes.

Essential thrombocythaemia and the Philadelphia chromosome.

Six adult patients presented with clinical features of essential thrombocythaemia. Five of the patients, although Ph-positive, have maintained these features without evidence of leukaemia; in one case for 9 years. A sixth patient developed leukaemic blast crisis following a persistently high platelet count over 4 years. Her cells were Ph-negative, but hybridization of gene probes to chromosomes in situ and to leukaemic DNA showed that the abl oncogene had moved to the breakpoint cluster region (bcr) on the normal chromosome 22. This patient has the same molecular gene change as occurs in some cases of Ph-negative chronic myeloid leukaemia (CML) whose leukaemic cells likewise show no evidence of chromosomal translocation. Molecular studies are essential for the correct diagnosis of these patients. The Ph genomic lesion appears to have a range of leukaemic expression which includes thrombocythaemia as well as chronic myeloid leukaemia and acute lymphatic leukaemia.

An identical t(Y;1)(q12;q21) in two patients with myelodysplastic syndromes.

Two male patients with myelodysplastic syndromes, one with refractory anemia with excess blasts (RAEB), the other with chronic myelomonocytic leukemia both had in their bone marrow and peripheral blood cells the same abnormal karyotype 46,X,-Y, + der (Y)t(Y;1)(q12;q21). This abnormality produced trisomy for the 1q21-1qter region of chromosome 1. In addition to the t(Y;1), the patient with RAEB had a del(20)(q11) abnormality in separate CFU-GM and BFUe progenitor cell populations. The t(Y;1) clone of this patient underwent chromosomal evolution with the acquisition of trisomies for chromosomes 2, 6, 8, and 9. Cytogenetic analysis of serial peripheral blood samples showed that the t(Y;1) clone and its derivatives gradually replaced that with the 20q- abnormality. Metaphase cells trisomic for chromosomes 2, 6, 8, and 9 were found predominantly in the CFU-GM population and only rarely in BFUe colonies, suggesting that chromosomal evolution was largely confined to the granulocytic lineage.

Abnormal chromosome 16 in clonogenic eosinophils from a case of acute myeloid leukemia (FAB,M2).

A case of acute myeloid leukemia (AML, FAB M2) is described in which the leukemic karyotype showed several numerical and structural cytogenetic abnormalities including an abnormal chromosome 16 with breakpoint at band q22, monosomy for chromosomes 5 and 7, and a single pair of double minute chromosomes. There was no patient history of treatment for a previous malignancy or occupational exposure to mutagens. Bone marrow eosinophilia was seen at presentation for refractory anemia with excess blasts in transformation and when AML was diagnosed. When bone marrow buffy coat cells were cultured in soft agar in the presence of colony stimulating factor, 19% of the colonies and 20% of the clusters were of eosinophils. Cytogenetic examination of pooled eosinophil colonies showed the marker chromosomes that identified the leukemic population.

Basophils exhibit rearrangement of the bcr gene in Philadelphia chromosome-positive chronic myeloid leukemia.

Basophils were isolated from the peripheral blood of two patients with Philadelphia positive-chronic myeloid leukemia using monoclonal antibody Bsp-1 and fluorescence activated cell sorting. DNA blot analyses demonstrated rearrangement of the breakpoint cluster region gene in the isolated basophils, which suggests their leukemic origin. Isolated T cells from these patients that were cultured for 14 days in the presence of interleukin-2 lacked rearrangement of the breakpoint cluster region gene and are therefore unlikely to be derived from the chronic myeloid leukemia clone.

Karyotypic evolution in a B-cell lymphoma.

A B-cell lymphoma in the lung of a 59-year-old woman showed a near-pentaploid karyotype and chromosomal changes indicating a t(8;14) in cells from a pleural aspirate. Cells from metastases to the skin and a second pleural aspirate were pseudodiploid, but showed separate further complex cytogenetic changes. Among these was a 14q+ chromosome, but the #8 chromosomes apparently were normal. It is suggested that there was a loss of the derivative 8q- chromosome from the t(8;14) followed by homozygozity of the normal #8 chromosome.

A characteristic CFU-GM growth pattern in acute promyelocytic leukemia.

Bone marrow and/or peripheral blood cells from 12 patients with acute promyelocytic leukemia (APL) were cultured in soft agar or methylcellulose in the presence of 15% human placental conditioned medium as a source of colony stimulating factor. Buffy coat cells, taken from ten patients when APL was diagnosed, produced a growth pattern in soft agar that was characterized by a high incidence of small, relatively uniform clusters of promyelocytes, which, when stained in situ, reacted strongly with myeloperoxidase, Sudan black B and chloroacetate esterase. In six cases, the leukemic origin of the cluster forming cells was demonstrated by the presence of the t(15;17) in these cells. During periods of complete remission the small clusters were replaced by larger and more diverse aggregates which had a normal karyotype. At relapse the small cluster growth pattern returned. The growth pattern of small clusters is more commonly associated with APL than with other types of acute myeloid leukemia.

Genomic diversity correlates with clinical variation in Ph'-negative chronic myeloid leukaemia.

The Philadelphia chromosome (Ph') is found in the blood cells of about 90% of patients with chronic myeloid leukaemia (CML) and usually results from the reciprocal chromosome translocation t(9;22). This translocation relocates the proto-oncogene c-abl, normally found on chromosome 9q34, to within the breakpoint cluster region (bcr) on chromosome 22q11 (refs 3-8). The juxtaposition of c-abl and the 5' portion of bcr appears to be the critical genomic event in CML and results in a novel 8-kilobase (kb) fused abl/bcr transcript and a c-abl-related protein of relative molecular mass 210,000 (ref.11). About 10% of adult patients diagnosed as CML lack the Ph' chromosome; they represent a heterogeneous group of disorders which are difficult to diagnose precisely. We have examined five patients with CML whose leukaemic cells have a normal karyotype. We report here that two of the patients showed the same genomic change as occurs in Ph'-positive CML, but the change resulted from a mechanism other than chromosomal translocation. The remaining three patients showed no genomic rearrangement. This genomic diversity correlated with the clinical differences between the patients.

Host origin of in vitro bone marrow fibroblasts after marrow transplantation in man.

Two female patients who received bone marrow transplants from male donors showed the donor karyotype in all blood cells cultured with phytohemagglutinin and in all cells of bone marrow aspirate after direct or 24-hour culture preparation for cytogenetic study. The donor origin of myeloid cells was demonstrated specifically by identifying the male karyotype in the dividing cells of granulocyte/macrophage colonies in soft agar. Only the host female karyotype was found in fibroblast cell populations from cultured bone marrow aspirate. Bone marrow aspirate from transplant patients contained cells of host origin and cells of donor origin; both will proliferate in culture.