Engraftment of NOD/SCID/gammac(null) mice with multilineage neoplastic cells from patients with juvenile myelomonocytic leukaemia

Several lines of evidence indicate the clonal nature of juvenile myelomonocytic leukaemia (JMML), involving myeloid, erythroid, megakaryocyte and B-lymphoid lineages. However, it is unclear whether the T-lymphocyte lineage is involved. We demonstrated that cells from six patients with JMML repopulated in non-obese diabetic/severe combined immunodeficient/gammac(null) mice and differentiated into granulocytes, monocytes, erythrocytes, B lymphocytes, T lymphocytes and natural killer cells. The percentage of human CD45 antigen-positive cells ranged from 41% to 73% in the murine bone marrow 12 weeks after transplantation. To examine the involvement of lymphocyte subpopulations, we purified human CD3(+), CD19(+) and CD56(+) cells from murine bone marrow cells transplanted from a patient with monosomy 7. Fluorescence in situ hybridization (FISH) showed the clonal marker in 96-100% of purified CD3(+), CD19(+) and CD56(+) subpopulations. These findings support the concept that JMML originates in transplantable multilineage haematopoietic stem cells. This novel murine xenotransplant model should be useful for investigating the nature of stem cells and testing new therapies for patients with JMML.     

Revised guidelines for the diagnosis and management of hairy cell leukaemia and hairy cell leukaemia variant*

The British Committee for Standards in Haematology first produced guidelines for the diagnosis and management of hairy cell leukaemia and hairy cell leukaemia variant in 2000. This revision updates those guidelines and covers the areas of diagnosis, treatment and assessment of response to therapy.     

How I manage patients with hairy cell leukaemia

Patients with hairy cell leukaemia (HCL ) have highly favourable outcomes after purine analogue therapy. However, most patients subsequently relapse and require re‐treatment. A minority of patients develop purine analogue‐refractory disease. Targeted therapies have improved outcomes for such patients. Recently, the BRAF V600E mutation was identified in most patients with classical HCL , resulting in constitutive mitogen‐activated protein kinase pathway activation; impressive responses are achieved in heavily pre‐treated patients with BRAF inhibition. The CD 22‐targeted immunoconjugate moxetumomab pasudotox and BTK inhibitor ibrutinib also achieve responses in relapsed and refractory patients. HCL variant and the IGHV 4‐34 molecular variant of HCL lack BRAF mutation and have inferior outcomes with standard purine analogue therapy. The addition of rituximab to purine analogues achieves very high rates of minimal residual disease‐negative complete remission and improves outcomes for patients with HCL variant. Given the rarity of HCL , optimal integration of novel therapies into treatment algorithms will require well‐designed, collaborative studies.     

Efficient assay for evaluating human thrombopoiesis using NOD/SCID mice transplanted with cord blood CD34+ cells

A suitable model for the preclinical study of human platelet production in vivo has not been available. NOD/SCID mice were characterized as representing an efficient engraftment model for human hematopoietic stem cells, which resulted in the production of human platelets. Here, we evaluated in vivo human thrombopoiesis and ex vivo human platelet functions in NOD/SCID mice transplanted with human cord blood (CB) CD34(+) cells. Human platelets and human CD45(+) cells appeared in peripheral blood of NOD/SCID mice from 4 wk after transplantation. Human platelets produced in these mice showed CD62P expression and the activation of GPIIb/IIIa on human platelets on stimulation with an agonist. PEG-rHuMGDF (0, 0.5 and 5 microg/kg/d s.c.) was injected for 14 d into mice that had been confirmed to produce human platelets stably. The number of human platelets increased about twofold at 0.5 microg/kg/d and about fivefold at 5 microg/kg/d after 14 d. Withdrawal of PEG-rHuMGDF administration caused the human platelet count to return to the pretreatment level. Further, re-administration of PEG-rHuMGDF induced a similar human thrombopoietic response as it did on initial administration. These results suggest that NOD/SCID mice engrafted with human CB CD34(+) cells will be useful for the study of human platelet production in vivo.     

Inhibitory effect of simvastatin on the proliferation of human myeloid leukaemia cells in severe combined immunodeficient (SCID) mice

SCID mice were inoculated intravenously with cells from the human HL60 myeloblastic leukaemia cell line and then treated with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, simvastatin, by subcutaneous continuous infusion. The effect of the drug was measured by subsequent colony formation of surviving HL60 cells in vitro and flow cytometry. The number of clonogenic HL60 cells was reduced in the bone marrow of mice that received simvastatin compared with control mice by 65% and 68% in two separate experiments. The number of clonogenic, normal, murine, bone marrow progenitor cells concomitantly exposed to simvastatin in vivo , was not affected in either experiment. Flow cytometric analysis of bone marrow and spleen cells confirmed these results by showing that simvastatin had reduced the percentage of human leukaemia cells in these tissues by 70% and 88% respectively.   The data show that the reported selective effect of simvastatin against acute myeloid leukaemia cells in vitro, can be extended to this in vivo model. HL60 bears an N-ras mutation. In further in vitro studies, ketoconazole, an inhibitor of cholesterol biosynthesis post farnesyl pyrophosphate synthesis, had a similar effect to simvastatin on HL60 colony development. Furthermore, the clonogenicity of a population of N-ras mutated, primary acute myeloid leukaemia (AML) cells was no more sensitive to simvastatin than a population without the mutation. The data suggest that the inhibition of AML cell proliferation by simvastatin may be independent of the RAS signalling pathway.     

A morphometric study of bone marrow angiogenesis in hairy cell leukaemia with clinicopathological correlations

Bone marrow angiogenesis has recently been implicated in the pathophysiology and course of various haematological malignancies. Little is known, however, about the significance of this phenomenon in hairy cell leukaemia (HCL). We evaluated various morphometric characteristics of microvessels, highlighted by means of anti-CD34 immunohistochemistry, in the bone marrow of 44 patients with typical HCL, before and after treatment with interferon-alpha (IFN-alpha). Overall, bone marrow from 103 HCL patients and 20 controls was examined. Microvessel density (MVD) and several size- and shape-related parameters were quantified in the region of most intense vascularization using image analysis. MVD, size-related parameters and the percentage of branching microvessels were higher in HCL than in controls. Likewise, perimeter counts were higher in partial/non-responders than in complete responders. Achievement of complete response was accompanied by smaller calibre microvessels. IFN-alpha induced a decrease in MVD and branching values in cases with diffuse marrow involvement. In univariate analysis, progression-free survival was adversely affected by MVD, branching and major axis length. Multivariate analysis indicated that MVD/branching independently affected progression-free survival and the likelihood of complete response. Our data suggest that the generation of bone marrow microvessels indicated an increased risk of progression and IFN-alpha treatment failure in HCL. Furthermore, the prognostic significance of angiogenesis requires the concomitant assessment of MVD and the complexity of the microvascular network.     

Severe decrease in peripheral blood dendritic cells in hairy cell leukaemia

Clinical studies in hairy cell leukaemia (HCL) have linked the frequent occurrence of infections due to intracellular pathogens and a profound monocytopenia. More recently, dendritic cells (DC), a subset of which are related to monocytes, were shown to be the professional antigen-presenting cells which stimulate the adaptive immune response. Using membrane markers and flow cytometry, we determined in peripheral blood whether various DC subsets and monocytes were impaired in HCL. Lymphoid and myeloid DC were virtually absent in five HCL patients with active disease. After treatment, both DC and monocytes recovered slowly. The decrease in DC suggests that defective antigen presentation could affect susceptibility to intracellular pathogens in HCL.     

Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haemopoietic cells in NOD/SCID mice transplanted with human cord blood

Time course studies of sublethally irradiated non-obese mice with severe combined immunodeficiency (NOD/SCID mice) transplanted intravenously with 10⁷ human cord blood cells showed a rapid and parallel regeneration of human erythroid, granulopoietic, megakaryopoietic and B-lymphoid progenitors, as well as more primitive subpopulations of CD34⁺ cells (defined by their multi-lineage in vitro colony-forming ability, coexpression of Thy-1, or functional activity in long-term culture-initiating cell [LTC-IC] assays), in the marrow, spleen and blood. Maximum numbers of human cells were reached within 6 weeks and were then sustained for another 18–20 weeks. ³H-thymidine suicide studies showed all types of in vitro clonogenic human progenitors tested and the human LTC-IC to be proliferating in vitro throughout this period. A 2-week course of injections of human Steel factor, interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin given just prior to assessment of the mice had no effect on any of these human engraftment parameters. 4–6 weeks post-transplant, the marrow of primary NOD/SCID recipients contained human cells that were able to regenerate lymphopoiesis and/or myelopoiesis in secondary irradiated NOD/SCID mice. These findings establish a baseline for the kinetics of engraftment, multi-lineage differentiation and self-renewal of human cord blood stem cells in this xenogeneic transplant model and thus set the stage for future studies of their regulation in vivo .     

In utero transplantation of human fetal haemopoietic cells in NOD/SCID mice

We have previously demonstrated that high levels of allogeneic, donor-derived mouse haemopoietic progenitor cells engraft following in utero transplantation in NOD/SCID mice. To evaluate whether the fetal NOD/SCID haemopoietic microenvironment supports the growth and development of human fetal haemopoietic progenitor cells, we injected fetal liver mononuclear cells (FL) or fetal bone marrow (FBM) derived CD34⁺ cells into NOD/SCID mice on day 13/14 of gestation. At 8 weeks of age 12% of FBM recipients and 10% of FL recipients were found to have been successfully engrafted with CD45⁺ human cells. CD45⁺ cells were present in the BM of all chimaeric animals; 5/6 recipients showed engraftment of the spleen, and 4/6 recipients had circulating human cells in the peripheral blood (PB). The highest levels of donor cells were found in the BM, with up to 15% of the nucleated cells expressing human specific antigens. Multilineage human haemopoietic engraftment, including B cells (CD19), myelomonocytic cells (CD13/33) and haemopoietic progenitor cells (CD34), was detected in the BM of chimaeric mice. In contrast, no human CD3⁺ cells were detected in any of the tissues evaluated. When the absolute number of engrafted human cells in the PB, BM and spleens of chimaeric mice was determined, a mean 16-fold expansion of human donor cells was observed. Although multilineage engraftment occurs in these fetal recipients, both the frequency and the levels of engraftment are lower than those previously reported when human cells are transplanted into adult NOD/SCID recipients.     

VH gene expression in hairy cell leukaemia

Hairy cells are characterized by their typical morphology and expression of specific surface antigens. Although their B-cell origin is now confirmed, their exact position in B-cell development remains unclear. To better define the origin of hairy cells, we analysed the immunophenotype and the Ig VH nucleotide sequence of seven cases of hairy cell leukaemia (HCL). Six of them were typical HCL and the remaining case corresponded to a variant HCL. Analysis of sequenced VH genes revealed that the VH1 family was used in one case, VH2 in one, VH3 in two, VH4 in two and VH5 in one. No preferential usage of VH genes was observed in this small series. In five cases high rates of somatic mutations were observed, with a predominance of mutations and replacements in CDR regions for three, indicating that these cells originate from cells that have been exposed to the hypermutation mechanism. The distribution of mutations in our small series provides some evidence of a selective mutational process.     

Long-term follow-up of patients with hairy cell leukaemia after treatment with pentostatin or cladribine

We report the long-term follow-up results on two groups of patients with hairy cell leukaemia (HCL) treated with either pentostatin (deoxycoformycin) or cladribine (2-chlorodeoxyadenosine). 165 HCL patients received treatment with pentostatin (between 1986 and 1994), and 45 were treated with cladribine (between 1992 and 1997). Age and sex characteristics were similar in the two groups. 38 patients in the pentostatin group and 12 in the cladribine group were previously untreated. 22 patients in the cladribine group had received prior treatment with pentostatin; four were resistant, 17 had relapsed following partial (four) or complete (13) responses, and one was not evaluable for response. The response rates were the same in the two groups: 82% complete response (CR), 15% partial response (PR) for pentostatin and 84% CR, 16% PR for cladribine. Relapse rates were 24% for pentostatin and 29% for cladribine after median follow-up of 71 and 45 months respectively. At 45 months, however, the relapse rate for pentostatin was only 9.7%. We found a statistically significant difference in the disease-free interval (DFI) between the two groups suggesting that patients may relapse more quickly after cladribine. The majority of relapsed patients achieved second remissions following further therapy with either pentostatin or cladribine, with no evidence of cross resistance between the two agents. The 5-year survival for all patients was 97% and treatment- related toxicity was low. We conclude that both pentostatin and cladribine induce durable remissions in the majority of HCL patients. Longer follow-up is required to establish whether some patients are cured as there is no plateau in DFI, and which of these two agents may be the treatment of choice.     

Characterisation of a new cell line (CJ18) from a patient with ‘hairy’ cell leukaemia

A cell line (CJ18) has been established from the peripheral blood of a patient with hairy cell leukaemia (HCL) in a leukaemic phase. They grow slowly in large clumps with a doubling time of 3–4 d. 8% show positivity for surface immunoglobulin G and a small percentage (5%) are positive for intracytoplasmic immunoglobulin. They are B1, Ia1 positive and CALL, TdT and OKM1 negative. Although they are Epstein Barr Virus Nuclear Antigen (EBNA) positive they have several features not found in other EBNA positive B lymphoblastoid cell lines which suggest they may be derived from the patient's leukaemic hairy cells. They are strongly positive for tartrate resistant acid phosphatase, esterase positive, contain numerous lysosomes and are able to phagocytose sheep red blood cells after exposure to tetradecanoyl-12, 13-phorbol acetate (TPA). Following exposure to retinoic acid and TPA they adhere to plastic with numerous slender processes, a feature seen in HCL cells.     

Primary splenic hairy cell leukaemia: a case report and review of the literature

Hairy cell leukaemia affecting primarily the spleen is a very rare feature of this disease at presentation. Splenectomy in such cases would seem to provide a cure. We report a case of primary splenic hairy cell leukaemia in which clinical and haematological remission were achieved after splenectomy, and we review the literature.     

Treatment outcomes and secondary cancer incidence in young patients with hairy cell leukaemia

Repeated therapy of hairy cell leukaemia (HCL) with treatments that have potential long‐term toxicities has raised concerns regarding increased risk for younger patients. We compared clinical outcomes and disease complications in 63 patients with HCL aged ≤40 years at diagnosis with 268 patients >40 years treated at Memorial Sloan Kettering Cancer Center. The rate of complete remission following initial therapy was 87% and 83% (P = 0·71) and estimated 10‐year overall survival was 100% and 82% (P = 0·25) in younger and older patients, respectively. Younger patients required therapy earlier and had a significantly shorter time between first and second therapy (median: 63 months vs. 145 months) (P = 0·008). Younger patients required significantly more lines of therapy during follow‐up. The 10‐year cumulative incidence of secondary malignancies in young and old patients was 0·205 and 0·287, respectively (P = 0·22). The incidence of secondary cancers in patients aged >40 years at diagnosis increased with the number of treatments for HCL (P = 0·018). These results highlight that young patients with HCL have shorter responses to treatment and require more lines of therapy to maintain disease control, while attaining similar long‐term survival. This has implications in the design of future clinical trials given our findings that secondary malignancies increase with more chemotherapy exposure.     

Evidence for a paracrine pathway of B-cell stimulation in hairy cell leukaemia

It is a well-known phenomenon that the growth of malignant B-lymphocytes, i.e. hairy cells, is regulated by cytokines. Several investigators have suggested that the stimulating cytokines are produced by the malignant B cells themselves, indicating an autocrine growth regulation. In this paper we demonstrate that T-lymphocyte clones produce soluble mediators which stimulate the growth of malignant B lymphocytes. The incidence of the growth-stimulating T-cell clones derived from peripheral blood is identical in patients with hairy cell leukaemia (HCL) and healthy controls. About 50% of the clones stimulate the growth of hairy cells, but not the growth of purified B lymphocytes of healthy donors. The stimulating activity of a single clone varies when tested on different hairy cells. Interferon alpha (IFNa), but not antibodies against tumour necrosis factor alpha (TNFQ) or interleukin-2 (IL-2), completely inhibit the growth-stimulating activity. Our results indicate that a paracrine growth regulation has to be considered in addition to the postulated autocrine loop in the growth regulation of malignant B cells.     

2-Chlorodeoxyadenosine in the treatment of hairy cell leukaemia: differences in response in patients with and without abdominal lymphadenopathy

We treated 26 patients with hairy cell leukaemia (HCL) with 2-chlorodeoxyadenosine, including nine with abdominal lymphadenopathy and of whom two had HCL-variant; 18 were previously treated. The overall response in 23 evaluable HCL patients was 100% with 87% complete remission (CR). The CR rate was 57% in patients with abdominal lymphadenopathy. Neither of the patients with HCL-variant achieved CR. Of four patients who had become refractory to 2'-deoxycoformycin, two achieved CR and one partial remission (PR). The lower response in HCL with abdominal lymphadenopathy supports the view that this represents a more resistant form of the disease.     

Glucose Homeostasis in Pre-diabetic NOD and Lymphocyte-Deficient NOD/SCID Mice During Gestation

BACKGROUND: Unlike other strains, spontaneously type 1 non-obese diabetic (NOD) mice experience transient hyperinsulinemia after weaning. The same applies for NOD/SCID mice, which lack functional lymphocytes, and unlike NOD mice, do not develop insulitis and diabetes like NOD mice. AIMS: Given that β-cell stimulation is a natural feature of gestation, we hypothesized that glucose homeostasis is disturbed in gestate pre-diabetic NOD and non-diabetic NOD/SCID mice, which may accelerate the onset of diabetes and increase diabetes prevalence. METHODS: During gestation and postpartum, mice were analyzed under basal feed conditions, and following glucose injection (1 g/kg, i.p.) after overnight fast, using glucose tolerance test (GTT). Glycemia, corticosteronemia, blood and pancreatic insulin, glucagon levels, islet size, and islet morphology were evaluated. Glycemia and mortality were assessed after successive gestations in NOD mice mated for the first time at 2 different ages. RESULTS: 1. Basal glucagonemia rose markedly in first-gestation fed NOD mice. 2. β-cell hyperactivity was present earlier in first-gestation non-diabetic fasted NOD and NOD/SCID mice than in age-matched C57BL/6 mice, assessed by increased insulin/glucose ratio after GTT. 3. Overnight fasting increased corticosteronemia rapidly and sharply in pre-diabetic gestate NOD and NOD/SCID mice. 4. Islet size increased in non-diabetic gestate NOD mice compared with C57BL/6 mice. 5. Successive gestations accelerated diabetes onset, and contributed to increased mortality in NOD mice. CONCLUSIONS: First-gestation pre-diabetic NOD and non-diabetic NOD/SCID mice exhibited β-cell hyperactivity and deregulation of glucagon and/or corticosterone secretion. This amplified normally occurring insulin resistance, further exhausted maternal β-cells, and accelerated diabetes in NOD mice.