Tessa L. Holyoake

Different subsets of primary chronic myeloid leukemia stem cells engraft immunodeficient mice and produce a model of the human disease.

Xenograft models of chronic phase human chronic myeloid leukemia (CML) have been difficult to develop because of the persistence of normal hematopoietic stem cells in most chronic phase CML patients and the lack of methods to selectively isolate the rarer CML stem cells. To circumvent this problem, we first identified nine patients samples in which the long-term culture-initiating cells were predominantly leukemic and then transplanted cells from these samples into sublethally irradiated NOD/SCID and NOD/SCID-β2microglobulin−/− mice. This resulted in the consistent and durable (>5 months) repopulation of both host genotypes with similar numbers of BCR-ABL+/Ph+ cells. The regenerated leukemic cells included an initial, transient population derived from CD34+CD38+ cells as well as more sustained populations derived from CD34+CD38− progenitors, indicative of a hierarchy of transplantable leukemic cells. Analysis of the phenotypes produced revealed a reduced output of B-lineage cells, enhanced myelopoiesis with excessive production of erythroid and megakaropoietic cells and the generation of primitive (CD34+) leukemic cells displaying an autocrine IL-3 and G-CSF phenotype, all characteristics of primary CML cells. These findings demonstrate the validity of this xenograft model of chronic phase human CML, which should enable future investigation of disease pathogenesis and new approaches to therapy.

A prospective study of real-time panfungal PCR for the early diagnosis of invasive fungal infection in haemato-oncology patients

Summary:A blinded prospective study was performed to determine whether screening of whole blood using a real-time, panfungal polymerase chain reaction (PCR) technique could predict the development of invasive fungal infection (IFI) in immunocompromised haemato-oncology patients. In all, 78 patients (125 treatment episodes) were screened twice weekly by real-time panfungal PCR using LightCycler™ technology. IFI was documented in 19 treatment episodes (five proven, three probable and 11 possible), and in 12, PCR was sequentially positive. PCR positivity occurred in: 4/5 proven; 2/3 probable; 6/11 possible; and 29/106 with no IFI. In 8/12 with IFI and sequentially positive PCR results, PCR positivity occurred before (median 19.5 days) and in 4/12 (median 10.5 days) after the initiation of empirical antifungal therapy. Based on sequential positive results for proven/probable IFI sensitivity, specificity, positive predictive value and negative predictive value were 75, 70, 15 and 98%, respectively. Real-time panfungal PCR is a sensitive tool for the early diagnosis of IFI in immunocompromised haemato-oncology patients. It may be most useful as a screening method in high-risk patients, either to direct early pre-emptive antifungal therapy or to determine when empirical antifungal therapy can be withheld in patients with antibiotic--resistant neutropenic fever. However, these strategies require further assessment in comparative clinical trials.

Nilotinib concentration in cell lines and primary CD34(+) chronic myeloid leukemia cells is not mediated by active uptake or efflux by major drug transporters.

Imatinib mesylate and nilotinib are highly effective at eradicating the majority of chronic myeloid leukemia (CML) cells; however, neither agent induces apoptosis of primitive CML CD34+ cells. One possible explanation is that CD34+ cells do not accumulate sufficient intracellular drug levels because of either inadequate active uptake or increased efflux. To determine the interaction of nilotinib with major clinically implicated drug transporters, we analyzed their interactions with MDR1 (ABCB1), MRP1 (ABCC1), ABCG2 (BCRP) and human organic cation transporter (hOCT)1 in CML cell lines and primitive (CD34+) primary CML cells. Nilotinib is neither dependent on active import by hOCT1, nor effluxed through the ATP-binding cassette transporters analyzed. Indeed, we found nilotinib to be an inhibitor of hOCT1, MDR1 and ABCG2. The efflux transporters MDR1, MRP1 and ABCG2 are expressed on CML CD34+ cells at 13.5, 108 and 291% of control, respectively, although hOCT1 expression was absent; however, inhibition of efflux transporter activity did not potentiate the effect of nilotinib on apoptosis, Bcr–Abl inhibition or CML CD34+ cell proliferation. Therefore, we have found no evidence for either active uptake of nilotinib through hOCT1 or efflux through MDR1, MRP1 or ABCG2, and it is therefore unlikely that these transporters will have any effect on the clinical response to this drug.

BCR-ABL activity and its response to drugs can be determined in CD34+ CML stem cells by CrkL phosphorylation status using flow cytometry.

In chronic myeloid leukaemia, CD34+ stem/progenitor cells appear resistant to imatinib mesylate (IM) in vitro and in vivo. To investigate the underlying mechanism(s) of IM resistance, it is essential to quantify Bcr-Abl kinase status at the stem cell level. We developed a flow cytometry method to measure CrkL phosphorylation (P-CrkL) in samples with <104 cells. The method was first validated in wild-type (K562) and mutant (BAF3) BCR-ABL+ as well as BCR-ABL− (HL60) cell lines. In response to increasing IM concentration, there was a linear reduction in P-CrkL, which was Bcr-Abl specific and correlated with known resistance. The results were comparable to those from Western blotting. The method also proved to be reproducible with small samples of normal and Ph+ CD34+ cells and was able to discriminate between Ph−, sensitive and resistant Ph+ cells. This assay should now enable investigators to unravel the mechanism(s) of IM resistance in stem cells.

Lonafarnib reduces the resistance of primitive quiescent CML cells to imatinib mesylate in vitro.

Recent studies indicate that a rare population of primitive quiescent BCR-ABL+ cells are innately insensitive to imatinib mesylate (IM) and persist after IM therapy of patients with chronic myeloid leukemia (CML). New approaches to the eradication of these cells are therefore likely to be crucial to the development of curative therapies for CML. We have now found that Ara-C, LY294002 (a PI-3 (phosphatidylinositol-3′ kinase) kinase inhibitor), 17AAG (a heat-shock protein (HSP)-90 antagonist) and lonafarnib (a farnesyltransfease inhibitor) all enhance the toxicity of IM on K562 cells and on the total CD34+ leukemic cell population from chronic phase CML patients. However, for quiescent CD34+ leukemic cells, this was achieved only by concomitant exposure of the cells to lonafarnib. Ara-C or LY294002 alone blocked the proliferation of these cells but did not kill them, and Ara-C, LY294002 or 17AAG in combination with IM enhanced the cytostatic effect of IM but did not prevent the subsequent regrowth of the surviving leukemic cells. These studies demonstrate the importance of in vitro testing of novel agents on the subset of primary leukemic cells most likely to determine long-term treatment outcomes in vivo.

No strong relationship between mannan binding lectin or plasma ficolins and chemotherapy-related infections.

Chemotherapy causes neutropenia and an increased susceptibility to infection. Recent reports indicate that mannan‐binding lectin (MBL) insufficiency is associated with an increased duration of febrile neutropenia and incidence of serious infections following chemotherapy for haematological malignancies. We aimed to confirm or refute this finding and to extend the investigation to the plasma ficolins, P35 (L‐ficolin) and the Hakata antigen (H‐ficolin). MBL, L‐ficolin and H‐ficolin were measured in 128 patients with haematological malignancies treated by chemotherapy alone or combined with bone marrow transplantation. Protein concentrations were related to clinical data retrieved from medical records. MBL concentrations were elevated compared with healthy controls in patients who received chemotherapy, while L‐ficolin concentrations were decreased and H‐ficolin levels were unchanged. There was no correlation between MBL, L‐ficolin or H‐ficolin concentration and febrile neutropenia expressed as the proportion of neutropenic periods in which patients experienced fever, and there was no relation between abnormally low (deficiency) levels of MBL, L‐ficolin or H‐ficolin and febrile neutropenia so expressed. Patients with MBLu2003≤u20030·1u2003µg/ml had significantly more major infections than no infections within the follow‐up period (Pu2003<u20030·05), but overall most patients had signs or symptoms of minor infections irrespective of MBL concentration. Neither L‐ficolin nor H‐ficolin deficiencies were associated with infections individually, in combination or in combination with MBL deficiency. MBL, L‐ficolin and H‐ficolin, independently or in combination, did not have a major influence on susceptibility to infection in these patients rendered neutropenic by chemotherapy. These results cast doubt on the potential value of MBL replacement therapy in this clinical context.

Omacetaxine may have a role in chronic myeloid leukaemia eradication through downregulation of Mcl-1 and induction of apoptosis in stem/progenitor cells

Chronic myeloid leukaemia (CML) is maintained by a rare population of tyrosine kinase inhibitor (TKI)-insensitive malignant stem cells. Our long-term aim is to find a BcrAbl-independent drug that can be combined with a TKI to improve overall disease response in chronic-phase CML. Omacetaxine mepesuccinate, a first in class cetaxine, has been evaluated by clinical trials in TKI-insensitive/resistant CML. Omacetaxine inhibits synthesis of anti-apoptotic proteins of the Bcl-2 family, including (myeloid cell leukaemia) Mcl-1, leading to cell death. Omacetaxine effectively induced apoptosis in primary CML stem cells (CD34+38lo) by downregulation of Mcl-1 protein. In contrast to our previous findings with TKIs, omacetaxine did not accumulate undivided cells in vitro. Furthermore, the functionality of surviving stem cells following omacetaxine exposure was significantly reduced in a dose-dependant manner, as determined by colony forming cell and the more stringent long-term culture initiating cell colony assays. This stem cell-directed activity was not limited to CML stem cells as both normal and non-CML CD34+ cells were sensitive to inhibition. Thus, although omacetaxine is not leukaemia stem cell specific, its ability to induce apoptosis of leukaemic stem cells distinguishes it from TKIs and creates the potential for a curative strategy for persistent disease.

Transcriptional Analysis of Quiescent and Proliferating CD34+ Human Hemopoietic Cells from Normal and Chronic Myeloid Leukemia Sources

Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long‐term engraftment potential predominantly resides in the quiescent HSC subfraction, and G‐CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML‐derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR‐ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization.

CD34 + Positive Haemopoietic Cells: Biology and Clinical Applications

CD34+ is a heavily glycosylated surface antigen which is preferentially expressed on haemopoietic stem/progenitor cells. No definitive function has been attributed to CD34+, but it appears to play a role in cell to cell adhesion and may be involved in signal transduction to regulate the expression of other haemopoiesis-associated genes. A number of monoclonal antibodies to CD34+ have been raised and these have allowed the identification and characterization of a whole range of haemopoietic progenitor cells. CD34+ is expressed most strongly on the most primitive cells and is progressively lost as cells differentiate. The restricted expression of CD34+ to haemopoietic stem/progenitor cells has been exploited for transplantation studies. Several techniques have been developed to select cells expressing CD34+ from haemopoietic tissues. Successful sustained engraftment can be achieved using such positively selected cells. Alternatively, CD34+ cells may be expanded in vitro by incubation with synergistic cytokine combinations before being re-infused. An exciting new development has been the use of purified populations of CD34+ cells as the targets for gene marking and gene therapy protocols.

Telomere shortening correlates with prognostic score at diagnosis and proceeds rapidly during progression of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is associated increased stem cell turnover. We have previously shown that short telomeres in chronic phase (CP) predict for early progression to blast phase (BP). Poor prognostic score patients may therefore exhibit increased telomere loss at diagnosis and/or a greater than normal rate of loss during the disease course. We prospectively studied newly diagnosed CML patients for degree of telomere loss; measured telomere length in CML patients at all stages of disease; and performed follow-up sampling according to cytogenetic response to imatinib mesylate. Using flow-FISH, telomere length in peripheral blood leucocytes (PBL) from 32 consecutive newly diagnosed patients was measured (with ex-vivo expanded T-cells as an internal BCR-ABL negative control), in addition to 65 samples from all CML stages and 7 paired CP/BP samples. Fifty-five normal individuals served as a control population. Patients who attained either a complete cytogenetic response (CCR, 0% Ph+, n = 10) or no CR (100% Ph+, n = 11) underwent follow-up measurement. All statistical tests were two sided. Telomeres in accelerated phase (AP) and BP patients were significantly shorter than in CP, and mean telomere shortening was significantly greater in high-risk score than low-risk patients (P < 0.05) at diagnosis. The rate of shortening during disease progression was 10-20 times the rate observed in normal granulocytes. BP samples had undergone at least 30-60 additional divisions from baseline Ph- telomere length. Our findings show that telomere shortening in CML is greatest in high-risk score patients at diagnosis, and occurs rapidly during disease progression.