Analysis of genomic breakpoints in p190 and p210 BCR-ABL indicate distinct mechanisms of formation.

We sought to understand the genesis of the t(9;22) by characterizing genomic breakpoints in chronic myeloid leukemia (CML) and BCR-ABL-positive acute lymphoblastic leukemia (ALL). BCR-ABL breakpoints were identified in p190 ALL (n=25), p210 ALL (n=25) and p210 CML (n=32); reciprocal breakpoints were identified in 54 cases. No evidence for significant clustering and no association with sequence motifs was found except for a breakpoint deficit in repeat regions within BCR for p210 cases. Comparison of reciprocal breakpoints, however, showed differences in the patterns of deletion/insertions between p190 and p210. To explore the possibility that recombinase-activating gene (RAG) activity might be involved in ALL, we performed extra-chromosomal recombination assays for cases with breakpoints close to potential cryptic recombination signal sequence (cRSS) sites. Of 13 ALL cases tested, 1/10 with p190 and 1/3 with p210 precisely recapitulated the forward BCR-ABL breakpoint and 1/10 with p190 precisely recapitulated the reciprocal breakpoint. In contrast, neither of the p210 CMLs tested showed functional cRSSs. Thus, although the t(9;22) does not arise from aberrant variable (V), joining (J) and diversity (D) (V(D)J) recombination, our data suggest that in a subset of ALL cases RAG might create one of the initiating double-strand breaks.

Imatinib sensitivity as a consequence of a CSF1R-Y571D mutation and CSF1/CSF1R signaling abnormalities in the cell line GDM1.

Imatinib is usually a highly effective treatment for myeloproliferative neoplasms (MPNs) associated with ABL, PDGFRA or PDGFRB gene fusions; however, occasional imatinib-responsive patients have been reported without abnormalities of these genes. To identify novel imatinib-sensitive lesions, we screened 11 BCR-ABL-negative cell lines and identified GDM1, derived from a patient with an atypical MPN (aMPN), as being responsive to imatinib. Screening of genes encoding known imatinib targets revealed an exon 12 mutation in the colony-stimulating factor 1 receptor (CSF1R; c-FMS) with a predicted Y571D amino-acid substitution. CSF1R in GDM1 was constitutively phosphorylated, but rapidly dephosphorylated on exposure to imatinib. Y571D did not transform FDCP1 cells to growth factor independence, but resulted in a significantly increased colony growth compared with controls, constitutive CSF1R phosphorylation and elevated CSF1R signaling. We found that GDM1 expresses CSF1, and CSF1 neutralization partially inhibited proliferation, suggesting the importance of both autocrine and intrinsic mechanisms of CSF1R activation. An extensive screen of CSF1R in aMPNs and acute myeloid leukemia identified three additional novel missense variants. None of these variants were active in transformation assays and are therefore likely to be previously unreported rare polymorphisms or non-pathogenic passenger mutations.

Identification of a novel imatinib responsive KIF5B-PDGFRA fusion gene following screening for PDGFRA overexpression in patients with hypereosinophilia.

Idiopathic hypereosinophilic syndrome (IHES) is a disease that is difficult to classify, and diagnosis is one of exclusion. The identification of a cytogenetically invisible interstitial deletion resulting in the fusion of FIP1-Like-1 (FIP1L1) to platelet-derived growth factor receptor alpha (PDGFRA) has enabled many IHES cases to be reclassified as chronic eosinophilic leukemia. As it is likely that PDGFRA may fuse to other partner genes, we established a reverse transcriptase-PCR test to detect specific overexpression of the PDGFRA kinase domain as an indicator of the presence of a fusion gene. Overexpression was detected in 12/12 FIP1L1-PDGFRA-positive patients, plus 9/217 (4%) patients with hypereosinophilia who had tested negative for FIP1L1-PDGFRA. One of the positive cases was investigated in detail and found to have a complex karyotype involving chromosomes 3, 4 and 10. Amplification of the genomic breakpoint by bubble PCR revealed a novel fusion between KIF5B at 10p11 and PDGFRA at 4q12. Imatinib, a known inhibitor of PDGFRalpha, produced a complete cytogenetic response and disappearance of the KIF5B-PDGFRA fusion by PCR, from both genomic DNA and mRNA. This study demonstrates the utility of screening for PDGFRA kinase domain overexpression in patients with IHES and has identified a third PDGFRA fusion partner in chronic myeloproliferative disorders.

Disruption and aberrant expression of HMGA2 as a consequence of diverse chromosomal translocations in myeloid malignancies.

Chromosomal translocations that target HMGA2 at chromosome band 12q14 are seen in a variety of malignancies, notably lipoma, pleomorphic salivary adenoma and uterine leiomyoma. Although some HMGA2 fusion genes have been reported, several lines of evidence suggest that the critical pathogenic event is the expression of truncated HMGA2 isoforms. We report here the involvement of HMGA2 in six patients with myeloid neoplasia, dysplastic features and translocations or an inversion involving chromosome bands 12q13-15 and either 7p12, 8q22, 11q23, 12p11, 14q31 or 20q11. Breaks within or very close to HMGA2 were found in all six cases by molecular cytogenetic analysis, leading to overexpression of this gene as assessed by RT-PCR. Truncated transcripts consisting of HMGA2 exons 1-2 or exons 1-3 spliced to intron-derived sequences were identified in two patients, but were not seen in controls. These findings suggest that abnormalities of HMGA2 play an important and previously unsuspected role in myelodysplasia.

Contact-mediated inhibition of human haematopoietic progenitor cell proliferation may be conferred by stem cell antigen, CD34.

INTRODUCTION: The function of CD34, a transmembrane sialomucin expressed by human haematopoietic progenitor cells, is poorly understood. Its structure suggests it may act as a cell adhesion and signalling molecule. MATERIALS AND METHODS: KGIa cells and primary CD34-positive marrow cells were tested for their ability to aggregate in the presence of the anti-CD34 antibody QBEND10; CFU-GM colonies were grown using standard methods and tested for their content of colony-forming cells by replating; 'haematons' were isolated from marrow by filtration; the phosphorylation of CD34 was investigated by immunoprecipitation and Western blotting DISCUSSION: CD34-positive cells in human bone marrow, like KG1a cells, aggregate when incubated with QBEND10. Staining aggregates with anti-CD34-FITC revealed that aggregation involved co-localisation of CD34 at intercellular binding sites. We examined myeloid colonies (CFU-GM) grown from normal human bone marrow cells, and multicellular aggregates ('haematons') separated from freshly aspirated marrow by filtration, and found CD34-positive cells bound together with co-localisation of the CD34 at the binding sites. This finding shows that CD34-positive cell-cell adhesion occurs physiologically in vitro and in vivo. QBEND10-induced aggregation of KG1a and CD34-positive cells was enhanced by staurosporine (a protein kinase C inhibitor) and inhibited by genistein (a protein tyrosine kinase inhibitor). Moreover, aggregated cells had increased phosphorylation of tyrosine on CD34 and translocation of protein kinase C (PKC) to the cytoplasm, compared with non-aggregated cells. We used the ability of primary colonies to produce secondary colonies on replating as a functional parameter and found that the replating ability of the colonies was increased by treatment with genistein (P=0.003). In addition, the ability of individual samples of primary CD34-positive cells to undergo QBEND10-induced aggregation and the ability of CD34-positive cell-derived colonies to produce secondary clones on replating were inversely related (r=0.86). CONCLUSION: Our results suggest that homotypic aggregation of haematopoietic progenitor cells may be an important mechanism for preventing inappropriate proliferation in vivo. Thus, regulation of expression of the CD34 molecule may play an important role in maintaining the normal level of haematopoietic activity by contact-mediated inhibition of progenitor cell proliferation.

Cell biology of CML cells.

At the cellular level, expansion of haemopoiesis in chronic myeloid leukaemia (CML) must involve some imbalance in cell production along the myeloid maturation pathway. The relevant kinetic parameters are cell loss by apoptosis and differentiation and cell gain by proliferation (self-renewal). In spite of the predominance of the BCR-ABL-positive leukaemic cells, some BCR-ABL-negative, presumably normal, progenitor cells remain for long periods in chronic phase CML. Thus, understanding the kinetics of CML and normal progenitor cells may lead to therapeutic strategies capable of reducing malignant cell growth and reactivating normal haemopoiesis.

Abnormal patterns of immunoglobulin heavy chain gene DNA fingerprinting during chronic phase chronic myeloid leukemia.

Using IgH DNA fingerprinting we have previously demonstrated clonal immunoglobulin heavy chain (IgH) gene rearrangements during chronic phase (CP) chronic myeloid leukemia (CML) in patients destined to develop lymphoid blast crisis (L-BC). In view of this we decided to follow a cohort of CP CML patients to determine the frequency with which abnormal IgH fingerprints are found and their relationship, if any, to treatment regimen. Thirty three, initially CP, CML patients were studied on 111 occasions over a 16 month period using consensus PCR amplification of the third complementarity determining region (CDR3) of the IgH gene and high resolution polyacrylamide gel electrophoresis (IgH DNA fingerprinting). Of these 33 patients, thirteen received interferon-alpha (IFN) containing regimens and 15 non-IFN containing regimens throughout the study period. Five patients received variable therapy. During the period of observation 7 patients experienced disease progression: 5 accelerated phase, I L-BC and I myeloid blast crisis (M-BC). Abnormal IgH fingerprints were seen in 29 of the 111 (26%) specimens analysed. The 28 patients who received uniform therapy (IFN or non-IFN) over the 16 months were classified as "normal" (n = 18, normal pattern on all occasions) or "abnormal" (n = 10, abnormal on 1 or more occasions). Analysis by patient group (normal vs abnormal) showed that fingerprint abnormalities were associated with an elevated peripheral blood lymphocyte count (p = 0.0001) but not with changes in the total white cell count. Comparison of the IFN vs. non-IFN groups showed the former all had normal patterns whereas 10 of 15 non-IFN therapy patients were abnormal (p = 0.00023). The peripheral blood lymphocyte counts in the normal vs abnormal patients within the non-IFN group were not significantly different. The patient who developed L-BC demonstrated a persistent IgH fingerprint pattern abnormality from 7 months prior to the diagnosis of L-BC. The M-BC patient had a normal pattern at all times. We conclude that: (1) abnormal IgH fingerprints are found in a significant number of CP CML patients; (2) in this cohort the use of IFN was associated with normal CP CML IgH fingerprints, and (3) detection of abnormal IgH fingerprints may be highly predictive for the lineage of impending blast crisis.

A two-color BCR-ABL probe that greatly reduces the false positive and false negative rates for fluorescence in situ hybridization in chronic myeloid leukemia.

The t(9;22) translocation resulting in the fusion of BCR and ABL genes is pathognomonic in chronic myeloid leukemia (CML) and may be investigated at the molecular level using fluorescence in situ hybridization (FISH). Two-color BCR-ABL probes visualizing one fusion signal (1F FISH) have high false positive rates (FPR) and false negative rates (FNR). The FPR is a result of the random spatial association of probe signals within normal interphase cells so that some cells appear to contain the BCR-ABL fusion gene. The FNR of 1F FISH probes depends on the distance between the BCR and ABL probes hybridized to the BCR-ABL fusion gene (< or =368 kb); the "gap" between the signals causing the cell to be interpreted as normal. To overcome these difficulties, a two-color probe was used, employing four yeast artificial chromosome (YAC) sequences that span the breakpoint regions of the BCR and ABL genes and that visualize the two fusion signals BCR-ABL and ABL-BCR in CML cells (2F FISH). The FNR for the 2F FISH probes was assessed on clonal Ph+ granulocyte-macrophage-colony-forming cell (CFU-GM) derived colonies and was reduced to 0.4% (2/450), compared with an FNR of 13.5% (111/823) with 1F FISH. The FPR in normal mononuclear cells for the 2F FISH was 0. 19 +/- 0.12% (3/1,700), whereas the FPR using 1F FISH was 4.5 +/- 2.3% (63/1,294). The 2F FISH can thus be used to evaluate very small frequencies of BCR-ABL-positive and -negative interphase cells and may be of use in the clinical monitoring of CML.

Treatment with interferon-alpha preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors (CFU-GM) from patients with chronic myeloid leukemia but spares normal CFU-GM.

The biological target for interferon (IFN)-alpha in chronic myeloid leukemia (CML) is unknown, but one possibility is that amplification of granulocyte-macrophage colony-forming cells (CFU-GM) is reduced. Replating CFU-GM colonies and observing secondary colony formation provides a measure of CFU-GM amplification. Amplification of CML, but not normal, CFU-GM in vitro was significantly inhibited by IFN-alpha (P = 0.02). In 5 out of 15 CML cases studied by fluorescence in situ hybridization, in vitro treatment with IFN-alpha increased the proportion of CFU-GM, which lacked BCR-ABL. The ability of patients' CFU-GM to amplify, and suppression of this ability by IFN-alpha, predicted responsiveness to IFN-alpha therapy in 86% of cases. Investigation of patients on treatment with IFN-alpha showed a threefold reduction in CFU-GM amplification in responders (P = 0.03) but no significant change in nonresponders (P = 0.8). We conclude that IFN-alpha preferentially suppresses amplification of CML CFU-GM to varying degrees. The differing in vitro sensitivities to IFN-alpha and growth kinetics of individual patients' cells could help differentiate those who will or will not benefit from treatment with IFN-alpha.

BCR/ABL-negative progenitors are enriched in the adherent fraction of CD34+ cells circulating in the blood of chronic phase chronic myeloid leukemia patients.

Philadelphia chromosome-positive (Ph+) hemopoietic cells predominate in patients with chronic myeloid leukemia (CML) in chronic phase, but some Ph presumably normal stem cells persist in most patients. Ph cells are relatively frequent, compared to mature cell populations, in primitive hemopoietic cell populations from CML patients. We have purified CD34+ cells from chronic phase CML blood and separated them into two fractions on the basis of adherence or non-adherence to tissue culture plastic. We also separated CD34+ CML cell populations into HLA-DR(hi) and HLA-DR(lo) fractions and CD38(hi) and CD38(lo) fractions by flow cytometry. The CD34+ cells that adhered to plastic were predominantly CD33-, CD38- and HLA(-)-DR; cells with these phenotypic properties were significantly rarer in the CD34+ non-adherent cell population (P = 0.008-0.02). Expression of p210 BCR/ABL mRNA by adherent, non-adherent, HLA-DR(hi) and HLA-DR(lo)CD34+ cell subpopulations was demonstrated by RT-PCR. Using fluorescence in situ hybridization (FISH) in conjunction with BCR and ABL probes we detected Ph+ and Ph- cells in both adherent and non-adherent CD34+ cell fractions of 15/15 patients studied and in the HLA-DR(lo) or CD38(lo) sorted CD34+ cell fractions. The concentration of Ph- cells in the adherent CD34+ cell fraction was three-fold higher than in the non-adherent fraction (P = 0.001). Ph- adherent cells were detected in untreated CML patients and as late as 6 years after diagnosis of CML in patients treated with hydroxyurea (HU) or interferon-alpha (IFN-alpha). We conclude that whilst appreciable numbers of Ph- primitive hemopoietic progenitors are present in the circulation in untreated patients and also in treated patients in late chronic phase, the majority of cells expressing CD34 but not CD33, CD38 or HLA-DR antigens, are part of the CML clone.

Stromal cells negatively regulate primitive haemopoietic progenitor cell activation via a phosphatidylinositol-anchored cell adhesion/signalling mechanism.

We have tested the effect of stromal cells on the proliferation in long- and short-term cultures of primitive (Thy-1+, CD34+, CD33-, CD38- , HLA-DR , adherent in vitro and quiescent in vivo) progenitors in normal human bone marrow. These primitive cells produce granulocyte-macrophage colony-forming cells (CFU-GM) that are measured in secondary clonogenic assays. Addition of stromal cells to normal adherent haemopoietic progenitor cells reduced CFU-GM production by 80% (P =0.0002) after 1 week of incubation. In long-term culture (LTC), in the presence of stroma. the normal adherent cells did not produce significant numbers of CFU-GM until 3-4 weeks later which suggests that stromal cells reduce the probability of quiescent cell activation. This effect could not be attributed to soluble inhibitory factors and was specific to stroma grown with, rather than without, methylprednisolone. It was blocked by heparanase (H'ase) II treatment of stromal cells, by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment of progenitor cells, by antibody blocking of beta1 integrin molecules or by exposure to glucose/N-acetyl-D-glucosamine/alpha-methyl-D-mannoside, but not by exposure to galactose or fructose. Moreover, these interventions enabled the progenitor cells to respond to stimulatory factors in the culture supernatant. We interpret these results as support for a model involving primitive progenitor cell binding to stroma by PI-CAM/HS, beta1 integrin activation via lectin-like interactions and the transduction of signals which reduce the ability of primitive cells to respond to ambient stimulators. This model provides a mechanism for the maintenance of the quiescent state of stem cells by adhesion to stromal cells.

Phenotype and progeny of primitive adherent human hematopoietic progenitors.

Hematopoietic progenitor cells can be classified as plastic- and stroma-adherent (P+S+), stroma-adherent (P-S+) and non-adherent (P-S-). Both P+S+ and P-S+ populations are detected in delta (delta) culture systems where they produce non-adherent (P-S-) granulocyte-macrophage colony-forming cells (CFU-GM) and erythroid burst-forming units (BFU-E). Here we demonstrate that the plastic-adherent progenitor cells (P delta cells) comprise 5-10 percent of the CD34+, population in adult human marrow. Moreover, they do not express CD3 or CD22 and 88 percent of them are CD38-, 88 percent are CD33- and 74 percent are HLA-DR-. Production of CFU-GM by purified plastic-adherent CD34+, adherent cells was 60 percent of the number produced by recombined CD34+, and CD34- fractions. We have shown also that the plastic-adherent P+S+ cells are the precursors of the stroma-adherent P-S+ cells (S delta cells), day 21 cobblestone-area forming cells (CAFC) and cells capable of sustained hematopoiesis in a modified long-term bone marrow culture system. These observations support the primitive nature of P delta cells and establish a phenotypic sequence of plastic and stroma adherence through stroma adherence to non-adherence in hematopoietic cell development. To further investigate the relationship between P delta cells, S delta cells and long-term culture-initiating cells (LTCIC), we cultured whole mononuclear cell tractions and plastic-adherent cell-depleted mononuclear cell fractions in long-term culture and in the S delta assay. The results indicated the P delta cells were inhibited in the presence of stromal cells.

Apoptosis in chronic myeloid leukaemia: normal responses by progenitor cells to growth factor deprivation, X-irradiation and glucocorticoids.

Inhibition of apoptosis (genetically programmed active cell death) by p210 BCR-ABL expression is a mechanism that might contribute to clonal expansion in chronic myeloid leukaemia (CML). Since cell death following exposure to ionizing radiation and many chemotherapeutic agents can occur by the apoptotic pathway, inhibition of apoptosis would be expected to confer a relative resistance to these treatments. Similarly, cells deprived of growth factors in vitro die by apoptosis, and inhibition of apoptosis would therefore be expected to allow cells to survive better in growth factor-deprived conditions. We found that the survival of normal and CML myeloid progenitors was the same after in vitro incubation in deprived conditions and after treatment with X-irradiation or glucocorticoids. We also found that mature cells in colonies produced by CML progenitors (CFU-GM) did not survive better than those produced by normal progenitor cells. Flow cytometric analysis of propidium iodide-stained cells provided a direct indication that the degree of apoptosis may correspond to the degree of deprivation. These results suggest that inhibition of apoptosis may not be the primary mechanism whereby BCR-ABL influences the expansion of the malignant clone in CML.