Infectious triggers and novel therapeutic opportunities in childhood B cell leukaemia.

B cell acute lymphoblastic leukaemia (B-ALL) is the most common form of childhood cancer. Although treatment has advanced remarkably in the past 50 years, it still fails in ~20% of patients. Recent studies revealed that more than 5% of healthy newborns carry preleukaemic clones that originate in utero, but only a small percentage of these carriers will progress to overt B-ALL. The drivers of progression are unclear, but B-ALL incidence seems to be increasing in parallel with the adoption of modern lifestyles. Emerging evidence shows that a major driver for the conversion from the preleukaemic state to the B-ALL state is exposure to immune stressors, such as infection. Here, we discuss our current understanding of the environmental triggers and genetic predispositions that may lead to B-ALL, highlighting lessons from epidemiology, the clinic and animal models, and identifying priority areas for future research.

Tracking preleukemic cells in vivo to reveal the sequence of molecular events in radiation leukemogenesis.

Epidemiological studies have demonstrated an increased leukemia incidence following ionizing radiation exposure, but to date, the target cells and underlying mechanisms of radiation leukemogenesis remain largely unidentified. We engineered a mouse model carrying a different fluorescent marker on each chromosome 2, located inside the minimum deleted region occurring after radiation exposure and recognized as the first leukemogenic event. Using this tailored model, we report that following radiation exposure, more than half of asymptomatic CBA Sfpi1 GFP/mCh mice presented with expanding clones of preleukemic hematopoietic cells harboring a hemizygous interstitial deletion of chromosome 2. Moreover, following isolation of preleukemic hematopoietic stem and progenitor cells irradiated in their native microenvironment, we identified the presence of Sfpi1 point mutations within a subpopulation of these preleukemic cells expanding rapidly (increasing from 6% to 55% in 21 days in peripheral blood in one case), hence identifying for the first time the presence of such cells within a living animal. Importantly, we also report a previously undescribed gender difference in the phenotype of the preleukemic cells and leukemia, suggesting a gender imbalance in the radiation-induced leukemic target cell. In conclusion, we provide novel insights into the sequence of molecular events occurring during the (radiation-induced) leukemic clonal evolution.

Preleukemia: one name, many meanings.

Definition of preleukemia has evolved. It was first used to describe the myelodysplastic syndrome (MDS) with a propensity to progress to acute myeloid leukemia (AML). Individuals with germline mutations of either RUNX1, CEBPA, or GATA2 can also be called as preleukemic because they have a markedly increased incidence of evolution into AML. Also, alkylating chemotherapy or radiation can cause MDS/preleukemia, which nearly always progress to AML. More recently, investigators noted that AML patients who achieved complete morphological remission after chemotherapy often have clonal hematopoiesis predominantly marked by either DNMT3A, TET2 or IDH1/2 mutations, which were also present at diagnosis of AML. This preleukemic clone represents involvement of an early hematopoietic stem cells, which is resistant to standard therapy. The same clonal hematopoietic mutations have been identified in older 'normal' individuals who have a modest increased risk of developing frank AML. These individuals have occasionally been said, probably inappropriately, to have a preleukemia clone. Our evolving understanding of the term preleukemia has occurred by advancing technology including studies of X chromosome inactivation, cytogenetics and more recently deep nucleotide sequencing.

Preleukemia: the normal side of cancer.

PURPOSE OF REVIEW: In the present review, we will define the preleukemic state. We aim at increasing awareness and research in the field of preleukemia that will nurture targeted therapy for the earlier steps of leukemia evolution. RECENT FINDINGS: Emerging evidence supports the role of hematopoietic stem/progenitor cells carrying recurrent leukemia-related mutations as the cell of origin of both myeloid and lymphoid malignancies. The preleukemic stem cells can maintain at least to some extent their functionality; however, they have increased fitness endowed by the preleukemic mutations that lead to clonal expansion. SUMMARY: The latent preleukemic period before overt leukemia presents can take years, and the majority of carriers will never develop leukemia in their lifetime. The preleukemic state is not rare, with greater than 1% of individuals having acquired one or more of the recognized preleukemic lesions. The high frequency of such abnormalities in the population may be the cost of growing old; however, another view could be that in order to survive to old age, the hematopoietic system must adapt to create robust hematopoietic stem/progenitor cells with an increased fitness and clonal expansion. Hence, leukemia does not necessarily start as a disease, but rather as a need, with the normally functioning preleukemic hematopoietic stem cells trying to maintain health for years but in time succumbing to their own acquired virtues.

Prominent megakaryocytic dysplasia after regression of transient abnormal myelopoiesis associated with GATA-1 mutation.

About a quarter of children with Down syndrome and transient abnormal myelopoiesis (TAM) progress to acute megakaryoblastic leukemia (AMKL). We describe isolated dysmegakaryopoiesis despite complete resolution of TAM in an 18-month-old girl, who developed AMKL 6 months later.

Features of Japanese patients with myelodysplastic syndrome in an aging population of Sado Island.

Myelodysplastic syndrome (MDS) is relatively common in the elderly, and aging of populations is progressing in developed nations, notably so in Japan. The major age group in Japan and Sado Island are distributed between 30 and 60 and between 50 and 80, respectively. The aim of this study was to analyze the features of MDS in the population of Sado Island to anticipate the characteristics of the disease in the near future. One-hundred and fifty-three patients (71 male, 82 female, 19-94 years old, median 73 years old) with de novo MDS between 1985 and 2005 were retrospectively evaluated. All patients were reclassified according to WHO-2001 criteria. The predictive power of the international prognostic scoring system and the WHO classification-based prognostic scoring system were evaluated. The major causes of death were leukemic transformation (38%) in refractory anemia with an excess of blasts and infection (48%) for total MDS. Age was another independent prognostic factor. Elderly patients exhibited a significantly poorer prognosis mainly due to infections such as pneumonia. Although novel remedies for MDS and hyperferremia have recently been developed, prevention of infection remains important in MDS, particularly for older patients.

N-Ras(G12D) induces features of stepwise transformation in preleukemic human umbilical cord blood cultures expressing the AML1-ETO fusion gene.

AML1-ETO (AE) is a fusion product of t(8;21) observed in 40% French-American-British M2 type of acute myeloid leukemia (AML). Clinical data suggest that Ras mutation is a frequent cooperating event in t(8;21) AML. Whether constitutively active Ras promotes leukemogenesis on the t(8;21) background has not been demonstrated experimentally. Here, we retrovirally expressed N-Ras(G12D) in AE-expressing human hematopoietic cells to investigate cooperativity. The AE/N-Ras(G12D) cultures were cytokine-independent, enriched for CD34 positivity, and possessed increased colony-forming and replating abilities. N-Ras(G12D) expression led to Bcl-2 up-regulation and reduced apoptosis. Ectopic Bcl-2 expression also resulted in enhanced colony-forming and replating abilities but was insufficient to sustain cytokine independence. AE/N-Ras(G12D) cells were more sensitive to Bcl-2 inhibition with ABT-737 than parent AE cells. Enhanced engraftment of AE/N-Ras(G12D) cells was observed on intrafemoral injection into immunodeficient mice, presumably because of improved survival in the bone marrow microenvironment. N-Ras(G12D) promotes progression toward transformation in AE-expressing cells, partially through up-regulating Bcl-2.

Insights into the manifestations, outcomes, and mechanisms of leukemogenesis in Down syndrome.

Children with Down syndrome (DS) show a spectrum of clinical anomalies, including cognitive impairment, cardiac malformations, and craniofacial dysmorphy. Moreover, hematologists have also noted that these children commonly show macrocytosis, abnormal platelet counts, and an increased incidence of transient myeloproliferative disease (TMD), acute megakaryocytic leukemia (AMKL), and acute lymphoid leukemia (ALL). In this review, we summarize the clinical manifestations and characteristics of these leukemias, provide an update on therapeutic strategies and patient outcomes, and discuss the most recent advances in DS-leukemia research. With the increased knowledge of the way in which trisomy 21 affects hematopoiesis and the specific genetic mutations that are found in DS-associated leukemias, we are well on our way toward designing improved strategies for treating both myeloid and lymphoid malignancies in this high-risk population.

TEL-AML1 preleukemic activity requires the DNA binding domain of AML1 and the dimerization and corepressor binding domains of TEL.

The t(12;21)(p13;q22) translocation generates the TEL-AML1 (TEL, translocation-Ets-leukemia; AML1, acute myeloid leukemia-1) (ETV6-RUNX1) fusion product and is the most common chromosomal abnormality in pediatric leukemia. Our previous studies using a murine fetal liver transplantation model demonstrated that TEL-AML1 promotes the self-renewal of B-cell precursors in vitro and enhances the expansion of hematopoietic stem cells (HSCs) in vivo. This is consistent with the hypothesis that TEL-AML1 induces expansion of a preleukemic clone. Several studies have described domains within TEL-AML1 involved in the transcriptional regulation of specific target genes. However, it is unclear which of these domains is important for the activity of TEL-AML1 in preleukemic hematopoiesis. In order to examine this, we have generated a panel of deletion mutants and expressed them in HSCs. These experiments demonstrate that TEL-AML1 requires multiple domains from both TEL and AML1 to alter hematopoiesis. Furthermore, mutation of a single amino-acid residue within the runt homology domain of AML1, required for DNA binding, was sufficient to abrogate TEL-AML1 activity. These data suggest that TEL-AML1 acts as an aberrant transcription factor to perturb multiple pathways during hematopoiesis.

TEL-AML1 promotes development of specific hematopoietic lineages consistent with preleukemic activity.

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality yet identified in any pediatric leukemia and gives rise to the TEL-AML1 fusion product. To investigate the effects of TEL-AML1 on hematopoiesis, fetal liver hematopoietic progenitor cells (HPCs) were transduced with retroviral vectors expressing this fusion protein. We show that TEL-AML1 dramatically alters differentiation of HPCs in vitro, preferentially promoting B-lymphocyte development, enhancing self-renewal of B-cell precursors, and leading to the establishment of long-term growth factor-dependent pre-B-cell lines. However, it had no effect on myeloid development in vitro. Further experiments were performed to determine whether TEL-AML1 also demonstrates lineage-specific activity in vivo. TEL-AML1-expressing HPCs displayed a competitive advantage in reconstituting both B-cell and myeloid lineages in vivo but had no effect on reconstitution of the T-cell lineage. Despite promoting these alterations in hematopoiesis, TEL-AML1 did not induce leukemia in transplanted mice. Our study provides a unique insight into the role of TEL-AML1 in leukemia predisposition and a potential model to study the mechanism of leukemogenesis associated with this fusion.

Lack of circulating megakaryoblasts in newborn peripheral blood: development and validation of a sensitive flow cytometric detection method.

It is currently thought that approximately 1% of children with Down syndrome will develop a "premalignant" syndrome known as transient myeloproliferative disorder (TMD). Prospective, population-based studies of the incidence of TMD in Down syndrome infants is lacking. Although most cases of TMD resolve by 1 year of age, data suggest that 10% to 20% of Down syndrome patients with TMD develop AML-M7 (megakaryoblastic leukemia). To identify the true incidence of TMD in the Down syndrome population, a sensitive, rapid, and cost-effective method of quantifying circulating megakaryoblasts in large numbers of patients was needed. In this pilot study, the authors tested the hypothesis that there are fewer than 1% megakaryoblasts of nucleated cells circulating in the blood of normosomic infants. Four-antigen flow cytometry was used to establish the percentage of megakaryoblasts present in each of 100 cord blood samples collected blindly from "normosomic" live births. There was a mean percentage of 0.017% megakaryoblasts in 100 cord blood samples from normosomic infants. Flow cytometry proved to be a sensitive, rapid, and reproducible method for the quantification of megakaryoblasts. Less than 1% of circulating nucleated cells in the blood of newborn infants are megakaryoblasts, providing a comparison population for the authors' larger proposed incidence study.

Protein kinase C mediates mutant N-Ras-induced developmental abnormalities in normal human erythroid cells.

RAS mutations are one of the most frequent molecular abnormalities associated with myeloid leukemia and preleukemia, yet there is a poor understanding of how they contribute to the pathogenesis of these conditions. Here, we describe the consequences of ectopic mutant N-Ras (N-Ras*) expression on normal human erythropoiesis. We show that during early (erythropoietin [EPO]-independent) erythropoiesis, N-Ras* promoted the amplification of a phenotypically primitive but functionally defective subpopulation of CD34(+) erythroblasts. N-Ras* also up-regulated the expression of megakaryocyte antigens on human erythroblasts. Although early erythroblasts expressing N-Ras* were able to respond to erythropoietin and generate mature progeny, this occurred with greatly reduced efficiency, probably explaining the poor colony growth characteristics of these cells. We further report that this oncogene promoted the expression and activation of protein kinase C (PKC) and that the effects of N-Ras* on erythropoiesis could be abrogated or attenuated by inhibition of PKC. Similarly, the effects of this oncogene could be partially mimicked by treatment with PKC agonist. Together, these data suggest that expression of N-Ras* is able to subvert the normal developmental cues that regulate erythropoiesis by activating PKC. This gives rise to phenotypic and functional abnormalities commonly observed in preleukemia, suggesting a direct link between RAS mutations and the pathogenesis of preleukemia.

[Preleukemic states: an experimental murine model for myelodysplastic human syndromes]. / Les états préleucémiques: d'un modèle expérimental murin aux syndromes myélodysplasiques humains.

Using a model of experimental leukemia in mice, we have demonstrated that tumor development depends upon interactions between preleukemic cells and their microenvironment whose functions are altered. Cytokine injections inhibit tumor development by inducing a functional restoration of this environment. Human myelodysplastic syndromes (MDS) are marrow pathologies considered as preleukemic stages. As in murine leukemias, it is possible that marrow environment could play a key role in their evolution. We currently establish a model of human hematopoiesis in NOD/SCID mice grafted with human bone fragments. We hope that this model would allow to analyse the role of the marrow stromal cells in MDS and to establish treatments restoring their functions.

[The characteristics of the pre- and leukemic stages of chronic myeloproliferative diseases in persons suffering as a result of the Chernobyl catastrophe]. / Kharakteristika pre- i leikemicheskoi stadii khronicheskikh mieloproliferativnykh zabolevanii u lits, postradavshikh vsledstvie chernobyl'skoi katastrofy.

Clinical, morphological and cytogenetic investigations were done in those patients with leukocytosis having become victims of the Chernobyl catastrophe. Of these (n = 10), six patients demonstrated chromosomal abnormalities. In the study made at a later date in six patients with cytogenetic abnormalities, five patients were found to have chronic myeloproliferative disorders, with four cases presenting with chronic myeloid leukemia and one patient having osteomyelofibrosis.

[Leukemia and pre-leukemic conditions occurring after treatment of breast cancer]. / Leucémies et états préleucémiques survenus après traitement d'un cancer du sein.

OBJECTIVE: The purpose of this study was to determine the clinical and prognostic features of leukemias and preleukemic states, whatever the mode of development, observed in patients after treatment of breast cancer. PATIENTS AND METHODS: A retrospective multicentric analysis was made of 121 patients treated for breast cancer and who later developed leukemia or a preleukemic state. Initially, 44 patients had undergone mastectomy, 72 had conservative surgery and 119 had locoregional irradiation. At least one chemotherapy session was performed in 90 patients and 48 had received tamoxifen. The risk of relapse of breast cancer was high, moderate or low for 44, 46 and 24 patients respectively (data not available for 7 patients). RESULTS: By class, the hematology diseases found were: myelodysplasia (n = 9), refractory anemia with blast excess (n = 7), acute lymphoblastic leukemia (n = 6), acute myoblastic leukemia (n = 93 including a majority of type 2 and type 4). For acute myeloblastic leukemia, mean delay to onset was 65 and 37 months respectively without and after chemotherapy. The prognosis of these cases of leukemia and preleukemic states was poor with an overall death rate of 86%. CONCLUSION: In light of the recent development of indications for adjuvant chemotherapy even for subgroups of patients at moderate risk, it is important to more precisely assess the absolute benefit in terms of survival compared with the risk of severe complications, particular secondary leukemia. In the future, a systematic registry and a case-control study are required.

Possible retroviral origin of prion disease: could prion disease be reconsidered as a preleukemia syndrome?

A retroviral etiology might explain why amyloid plaque and/or spongiosis are or are not associated with neuronal death in prion diseases. While retroviral genes themselves may be responsible for neuronal death, a retrovirus may also cause mutations in cellular genes. Hence, the prion gene may be altered by a retrovirus in the same way as a cellular proto-oncogene is altered to produce an oncogene, either by transduction or by integration of the provirus in its vicinity. In both cases, the resulting abnormal prion protein, acting as a catalyst, may induce the formation of amyloid plaques. In addition, a wild type retrovirus may recombine to the vesicular stomatitis virus (VSV) to give rise to a pseudotyped retrovirus able to induce spongiosis. It is reported here that in scrapie, a blood monocytoid cell proliferates in vitro. If confirmed in other species, this raises the question of the potential link between prion disease and leukemia. Indeed neurovirulent strains of murine leukemia virus, a slow acting retrovirus, are known to induce spongiform encephalopathies. A preliminary attempt to purify reverse transcriptase by chromatography, using the classical protocol, failed because of the presence of a prion-like protein secreted by the blood mononuclear cells which stuck to the phosphocellulose column. Therefore, if a retrovirus is present in prion diseases, it would be evidenced only in animals developing the disease in the absence of prion protein. From this point of view, mice obtained in 1997 by the group of D. Dormont in France, offer a unique opportunity to test the retroviral hypothesis.

Pancytopenic prodrome (pre-ALL) of acute lymphoblastic leukemia in adults: possible pathogenesis.

We report two cases of adult acute lymphoblastic leukemia presenting with preleukemic phase of pancytopenia with a few abnormal lymphoid cells in bone marrow aspirates. The initial diagnosis of each case was suspicious aplastic anemia and hypoplastic anemia. Both cases progressed to overt acute lymphoblastic leukemia within 1 year. We suggest that initial pancytopenic phase (pre-ALL) may precede the diagnosis of acute lymphoblastic leukemia in adults and differential diagnosis from myelodysplastic syndrome and primary aplastic anemia will be needed. We also suggest that primary bone marrow lymphoma and "primary unknown metastatic lymphoma of bone marrow" may be possible as the pathogenesis in a case like ours.