Myeloblasts In Bone Marrow Research Articles

Very Short Telomeres As a Novel Mechanism Of Donor-Cell Derived Leukemia After Cord Blood Transplantation

Donor-cell derived leukemia (DCL) is a rare but serious complication of hematopoietic stem cell transplantation (HSCT), reported in 5% of all relapses following allogeneic HSCT. Only 12 cases of DCL after umbilical cord blood (UCB) HSCT are reported in the literature. Multiple mechanisms have been proposed for DCL, including occult donor leukemia, impaired immunity, drug toxicity, or a bone marrow “leukemogenic milieu”.We propose a novel mechanism of leukemogenesis, mediated by very short telomeres of donor cells with subsequent severe telomere attrition in vivo, genomic instability, and progression to complex cytogenetics acute myeloid leukemia (AML), based on study of a patient who underwent UCB HSCT for myelodysplasia (MDS). The patient was a 44-year-old woman, subsequently shown to have a T354M mutation in GATA2, who presented at age 19 years with multiple infections and MDS. At age 41, due to progression of MDS to AML, she received induction chemotherapy and underwent a single 4/6 HLA-matched UCB HSCT. She had delayed engraftment. achieving an absolute neutrophil count of 500/ul more than 100 days post-transplant. Chimerism studies demonstrated 100% donor cell at all time-points post-transplant. Two years and eight months after HSCT she presented with pancytopenia, circulating myeloblasts, and 50% myeloblasts in bone marrow, indicating recurrence of with AML. However, cytogenetics revealed complex abnormalities, t(2p;3q) and an interstitial deletion of 5q, male cells, indicating donor origin of AML.Telomere length of the transplanted cord blood cells measured by Q-PCR showed a severe decrease in length to 7.7 kb compared with an average length of 13 kb in control UCB (n=12). Moreover the telomere length decreased precipitously to 6.6 kb 2 years after transplantation and 5.6 kb at the time of diagnosis of AML. Single telomere length assay (STELA) was used to assess chromosome-specific telomere length. Very short telomeres (<3.0 kb) were present in the UCB before transplant that increased in proportion post-transplant before progression to AML. In contrast, very short telomeres were not present in control UCB STELA. Sequencing of cord blood-derived myeloid cells showed no mutation in TERT or TERC. Comparative genomic hybridization confirmed the complex cytogenetics.Telomere attrition occurs in the first year after HSCT, presumably due to proliferative stress on the stem cell compartment. In comparison to bone marrow, UCB have longer telomeres, but HSC number may be lower than in a BM inoculum, especially for adult recipients. In our case, initial telomere length of UCB was extremely short, for unclear reasons but possibly related to low cord blood cell viability. The long time that was needed for engraftment generated additional telomere attrition with accumulation of very short telomere and genomic instability leading to development of leukemia. Screening UCB for telomere length prior to HSCT might prove useful in identifying situations at risk for graft failure or malignant transformation. Disclosures:No relevant conflicts of interest to declare.

Abstract C257: Preclinical development of a potent dual PIM/FLT3 mutant kinase inhibitor for the treatment of AML and other hematological malignancies.

Abstract Despite an evolving understanding of acute myeloid leukemia (AML), a group of heterogeneous diseases with a common feature of abnormal levels of myeloblasts in bone marrow and in circulation, the current standard of care including chemotherapy and allogeneic hematopoietic stem cell transplantation results in less than 40 % cure rate with little progress over the last decades. One of the most extensively investigated approaches in targeted therapy of AML are FLT3 inhibitors that address the population of patients harboring FLT3 mutations that result in high relapse rate and decreased overall survival when compared to patients lacking such mutations. Therefore inhibition of the constitutively active mutants of FLT3 is a promising therapeutic approach. Unfortunately, the clinical development of selective or multitargeted FLT3 inhibitors turned out to result in rapid, but very transient responses followed by disease progression. One of the main reasons for development of resistance was selection of clones harboring both ITD and tyrosine kinase mutations (TKD). In parallel, the role of PIM kinases, especially PIM1 in FLT3 mediated leukemogenesis attracted attention of the pharmaceutical industry with currently two phase I programs targeting PIM kinases in hematological malignancies. PIM kinases are important downstream effectors of FLT3 signaling and play a crucial role in cell survival and inhibition of apoptosis upon expression. For that reason, PIM kinases represent an emerging therapeutic target class in AML with promising preliminary data from clinical trials. Due to the heterogeneous nature of AML, dual inhibition of FLT3 mutant kinase and PIM kinases could lead to improved efficacy and constitute a promising approach to overcome rapid resistance development to targeted therapies. Selvita has developed a potent and selective dual PIM/FLT3 mutant kinase inhibitor - SEL24-B489 which is highly active in in vitro and in vivo AML models. The compound shows high inhibitory activity against mutated FLT3 (FLT3-ITD and TKD mutations such as D835H, D835Y, N841I) and all three PIM kinase isoforms, comparable to activity of selective inhibitors of FLT3 (A220) and PIM (AZD1208). A head to head comparison of SEL24-B489 in cellular models reveals high activity across tested cellular AML models and biomarker inhibition in line with the expected kinase activity profile. Most importantly, SEL24-B489 showed strong synergistic effect in combination with the current standard of care in AML - cytarabine, and with other targeted inhibitors in advanced clinical development. The activity of B489 in vivo in xenograft models of AML and lymphomas after oral administration was higher than compared to selective PIM inhibitor and led to remissions in certain models. SEL24-B489 is a successful example of rational drug design and is currently in preclinical development. It represents a promising therapeutic approach which addresses alternative survival pathways downstream of FLT3 in addition to the activity on resistant FLT3 mutant kinases, which hopefully will translate into improved survival of AML patients in clinical trials. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C257. Citation Format: Wojciech Czardybon, Michal Galezowski, Pawel Guzik, Magdalena Zawadzka, Renata Windak, Izabela Dolata, Magdalena Salwińska, Radoslaw Obuchowicz, Krzysztof D. Brzózka. Preclinical development of a potent dual PIM/FLT3 mutant kinase inhibitor for the treatment of AML and other hematological malignancies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C257.

Micronuclei‐associated MYC amplification in the form of double minute chromosomes in acute myeloid leukemia

A 41-year-old male was admitted to our hospital due to fever and purpura. Peripheral blood values were hemoglobin 89 g/L, platelets 16 × 109/L, and white blood cells 3.4 × 109/L with 46% myeloblasts. Bone marrow examination showed hypercellular marrow with 71.0% myeloblasts and 23.0% other myeloid cells. Some of myeloblasts in the peripheral blood and bone marrow had one or two micronuclei (Image 1A, B). Furthermore, many large granules, suggestive of pseudo-Chediak–Higashi granules, were found in the cytoplasm of bone marrow myeloblasts (Image 1C). Dysplastic changes including degranulation and abnormal lobulation were observed in differentiated myeloid cells. Myeloblasts stained positive for myeloperoxidase and chloroacetate esterase. These cells were also positive for CD13, CD33, and HLA-DR, but negative for CD34. G-banding analysis of the bone marrow cells showed 43,X,-Y,-5,add(7)(q22),-20,1∼40dmin[20] (Image 2A, B). Fluorescence in situ hybridization (FISH) with IGH/MYC/CEP 8 probe on metaphase spreads revealed multiple MYC signals on the double minute chromosomes, indicating marked amplification of the MYC gene (Image 2C). In addition, FISH on interphase nuclei confirmed that micronuclei were heavily labeled with the MYC probe in 2 of 100 cells (Image 2D). Taking these hematological and cytogenetic findings into consideration, we made a diagnosis of acute myeloid leukemia (AML) with myelodysplasia-related changes according to the World Health Organization classification, or AML M2 by the French–American–British classification. The patient received two courses of standard induction therapy, but did not achieve complete remission. He underwent allogeneic stem cell transplantation because of worst prognosis cytogenetics including monosomal karyotype 1. Micronuclei are defined as small nucleus-like structures that can be expelled from the nucleus. They usually contain amplified oncogenes, acentric chromosomal fragments, or whole damaged chromosomes 2. Double minute chromosomes (dmin) are small chromatin bodies that lack centromeres and frequently mediate oncogene amplification in human tumors, although they are rarely found in AML. These chromosomes are commonly correlated with amplification of MYC at 8q24 or less commonly MLL at 11q23 3-5. The presence of dmin as part of complex karyotypes in AML appears to bring about chemotherapy resistance and rapid progression of the disease. To our knowledge, the association between micronuclei in myeloblasts and dmin with MYC amplification has previously been demonstrated in only one study 2. In the present case, it is confirmed that dmin on metaphase cells and micronuclei on interphase cells also contained markedly amplified MYC. On the other hand, pseudo-Chediak–Higashi granules, which are formed by fusion of azurophilic granules and show myeloperoxidase activity, are occasionally found in AML with or without dmin 6. They were also detected in two of four AML cases with micronuclei and MYC amplification 2. Thus, simultaneous detection of cytoplasmic micronuclei and pseudo-Chediak–Higashi granules in myeloblasts may imply cytogenetic abnormalities including dmin, and eventually predict poor prognosis on initial diagnosis of AML.

Biology of Myelodysplasia Clonality: Clinical Implications of Whole-Genome Sequencing

Abstract SCI-32The discovery of acquired genetic mutations associated with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) has rapidly expanded with the use of next-generation sequencing technologies. The distinction between MDS and de novo or secondary AML (arising after MDS) and therapeutic recommendations are largely based on the presence of morphologic dysplasia and the percentage of bone marrow myeloblasts. Using whole genome sequencing to define the allele burden of hundreds of mutations, our group has shown that nearly all the bone marrow cells in seven patients with MDS and secondary AML were clonally derived (∼85%), regardless of the myeloblast count. We now have experimental evidence suggesting that the vast majority of mutations in these samples are randomly acquired during aging (passenger mutations) and carried forward in a cell when it is transformed. By using all the validated mutations in a sample (passengers and drivers) we found that five of the seven MDS samples harbored two or more malignant clones and that all seven contained a founding clone that gave rise to subsequent daughter clones. Thus, the MDS bone marrow samples are a mosaic of several tumors with unique sets of mutations each shaped by the acquisition of a series of mutations and clonal diversification. Each clone contained at least one coding gene mutation, and we identified 11 recurrently mutated genes in the seven samples, including recently described mutations in U2AF1, a spliceosome gene that is typically mutated in the MDS founding clone. In addition, the MDS founding clone always gave rise to the dominant secondary AML clone, suggesting that therapies targeting mutations in the founding clone may have benefit in treating both MDS and secondary AML. It is possible that therapeutic response and disease progression is driven not only by the presence of recurrent mutations, which have prognostic value, but also by the clone (i.e., founding and daughter) in which they arise. We are currently testing this hypothesis by monitoring the dynamic change in clone size, including driver mutation allele burdens, during treatment of patients with AML and MDS. Disclosures:No relevant conflicts of interest to declare.

Sorafenib treatment of FLT3-ITD+ acute myeloid leukemia: favorable initial outcome and mechanisms of subsequent nonresponsiveness associated with the emergence of a D835 mutation

Internal tandem duplication (ITD) of the fms-related tyrosine kinase-3 (FLT3) gene occurs in 30% of acute myeloid leukemias (AMLs) and confers a poor prognosis. Thirteen relapsed or chemo-refractory FLT3-ITD(+) AML patients were treated with sorafenib (200-400 mg twice daily). Twelve patients showed clearance or near clearance of bone marrow myeloblasts after 27 (range 21-84) days with evidence of differentiation of leukemia cells. The sorafenib response was lost in most patients after 72 (range 54-287) days but the FLT3 and downstream effectors remained suppressed. Gene expression profiling showed that leukemia cells that have become sorafenib resistant expressed several genes including ALDH1A1, JAK3, and MMP15, whose functions were unknown in AML. Nonobese diabetic/severe combined immunodeficiency mice transplanted with leukemia cells from patients before and during sorafenib resistance recapitulated the clinical results. Both ITD and tyrosine kinase domain mutations at D835 were identified in leukemia initiating cells (LICs) from samples before sorafenib treatment. LICs bearing the D835 mutant have expanded during sorafenib treatment and dominated during the subsequent clinical resistance. These results suggest that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations, and might provide important leads to further improvement of treatment outcome with FLT3 inhibitors.

Therapy of Refractory AML with Del(5q) with Low-Dose Lenalidomide

Abstract 4286AML M-4 with >30% bone marrow myeloblasts was detected in 63 y old woman 2 mo after a diagnosis of chronic myelomonocytic leukemia (CMMLgrade 2). Cytogenetics showed 46, XX, del(5)(q12 q14) (N=4)/46, XX (N=18). Immune-phenotyping showed CD34+ CD117+ CD13+ CD33+ CD38+ HLADR+ CD2+. Leukemia cells were negative for inv (16), BCR/ABL, NPM, TEL/AML1, MLL/AF4, PML/RARA and AML1/ETO. She was RBC-transfusion-dependent. Therapy with cytarabine and daunorubicin and later with FLAG was ineffective and bone marrow myeloblasts increased to >70% with severe granulocytopenia and infection including pulmonary aspergillosis. She then received lenalidomide, 10 mg/d for 21 d every 4 w Bone marrow myeloblasts decreased to about 20% and granulocytopenia resolved. RBC-transfusion–dependence decreased. Cytogenetic and FISH-testing showed no detectable cells with del(5). This has persisted for 5 mo. Disclosures:Off Label Use: Lenalidomide for the treatment of AML with 5q- deletion. [Display omitted]

Transient myeloproliferative disorder in a newborn with down syndrome treated with rasburicase for the risk of development of tumor lysis syndrome: A case report

IntroductionTransient myeloproliferative disorder is a hematologic abnormality characterized by an uncontrolled proliferation of myeloblasts in peripheral blood and bone marrow that primarily affects newborns and babies with Down syndrome. Tumor lysis syndrome is rarely associated with transient myeloproliferative disorder.Case presentationTransient myeloproliferative disorder was diagnosed in a seven-day-old baby girl with Down syndrome, who was referred to our department due to hyperleukocytosis. Our patient developed tumor lysis syndrome, successfully treated with rasburicase, as a complication of transient myeloproliferative disorder resulting from rapid degradation of myeloid blasts after initiation of effective chemotherapy.ConclusionsTumor lysis syndrome is rarely reported as a complication of transient myeloproliferative disorder. To the best of our knowledge, this is the first case of a newborn with Down syndrome and transient myeloproliferative disorder treated with rasburicase for developing tumor lysis syndrome.

Angiotensin‐converting enzyme is required for normal myelopoiesis

Inhibition of angiotensin-converting enzyme (ACE) induces anemia in humans and mice, but it is unclear whether ACE is involved in other aspects of hematopoiesis. Here, we systemically evaluated ACE-knockout (KO) mice and found myelopoietic abnormalities characterized by increased bone marrow myeloblasts and myelocytes, as well as extramedullary myelopoiesis. Peritoneal macrophages from ACE-KO mice were deficient in the production of effector molecules, such as tumor necrosis factor-α, interleukin-12p40, and CD86 when stimulated with lipopolysaccharide and interferon-γ. ACE-KO mice were more susceptible to Staphylococcus aureus infection. Further studies using total or fractionated bone marrows revealed that ACE regulates myeloid proliferation, differentiation, and functional maturation via angiotensin II and substance P and through the angiotensin II receptor type 1 and substance P neurokinin 1 receptors. Angiotensin II was correlated with CCAAT-enhancer-binding protein-α up-regulation during myelopoiesis. Angiotensin II supplementation of ACE-KO mice rescued macrophage functional maturation. These results demonstrate a previous unrecognized significant role for ACE in myelopoiesis and imply new perspectives for manipulating myeloid cell expansion and maturation.

Prognosis In Patients with MDS or AML and Bone Marrow Blasts Between 10% and 30% Is Not Associated with Blast Count but Depends on Cytogenetic and Molecular Genetic Parameters

AbstractAbstract 2703 Introduction:Since the WHO classification in 2001, myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are separated by 20% of blasts in the bone marrow (BM). However, whether this arbitrary threshold can be justified from biologic or genetic aspects is in continuous debate. Design of study: We analyzed clinical outcomes and cytogenetic/molecular genetic features in 292 pts with 10–30% of BM blasts as assessed by cytomorphology. According to WHO criteria (2008), 111 pts were diagnosed as MDS with 10–19% of myeloblasts, while 181 had a diagnosis of AML with 20–30% of myeloblasts (“AML with recurrent genetic abnormalities” were excluded). Patients and Methods:A total of 292 pts (f/115; m/177; median, 68.4 yrs; 20.4–88.3 yrs) were investigated by MGG staining of BM, chromosomal banding analysis, FISH, and PCR for the FLT3-ITD, NPM1, and MLL-PTD. Results:Median age did not differ significantly between the MDS and AML cohorts (68.8 yrs versus 68.2 yrs; n.s.; T-test). Aberrant karyotypes (MDS: 45/111; 40.5%; vs. AML: 79/181; 43.6%) and cytogenetic risk groups (classified according to MRC cytogenetic criteria; Grimwade, 2010) were similar in MDS and AML cohorts: intermediate karyotypes: MDS: n=86/111; 77.5%; vs. AML: 140/181; 77.3%; unfavorable karyotypes: MDS: 25/111; 22.5%; vs. AML: 41/181; 22.7% (Fisher's exact test; n.s.). Subsequently, the different molecular markers were compared between the different categories. No significant differences were detected between MDS and AML cohorts regarding the NPM1mut (total cohort: n=55/292; 18.8%; MDS: 23/111; 20.7%; vs. AML: 32/181; 17.7%; n.s.), FLT3-ITD (total: 12/292; 4.1%; MDS: 3/111; 2.7%; vs. AML: 9/181; 5.0%; n.s.), and MLL-PTD (total: 18/292; 6.2%; MDS: 5/111; 4.5%; vs. AML: 13/181; 7.2%; n.s.). Overall survival (OS) was first compared between the two above defined morphological subgroups according to thresholds of 20% of blasts. No significant differences in the median OS were observed (MDS: 27.3 mo. vs. AML: 43.7 mo.; p=0.687). This was followed by analysis of OS according to 4 blast categories: 1) 10–14% of blasts: n=61, median OS: 34.9 mo.; 2) 15–19%: n=50: 21.5 mo.; 3) 20–24%: n=98: 43.7 mo.; and 4) 25–30%: n=83: median not reached). When comparison was performed between all of the subgroups, no significant differences were detected (p=0.422). In contrast, when the total cohort was subdivided according to cytogenetics, pts with unfavorable karyotypes (KTs) (n=66/292; 22.6%; median OS, 14.1 mo.) had significantly worse outcomes than those with intermediate karyotypes (226/292; 77.4%; 43.7 mo.; p<0.001). The cytogenetic risk group had a significant prognostic impact also when MDS (unfav. KTs: med. OS, 13.1 mo.; intermed. KTs: 34.9 mo.; p=0.002) and AML (unfav. KTs: med. OS, 19.0 mo.; intermed. KTs: 43.7 mo.; p=0.046) were separately investigated. Focusing again on the total cohort, an NPM1 mutated status conferred a better prognosis when compared to NPM1 wildtype pts (median not reached versus 27.3 mo.; p=0.031), while FLT3-ITD and MLL-PTD had no significant influence on outcomes (FLT3-ITD positive: 14.7 mo. vs. FLT3 wildtype: 34.9 mo.; p=0.896; MLL-PTD positive: median not reached; MLL-PTD negative: 34.4 mo.; p=0.496). Furthermore, OS was better in the NPM1mut/FLT3wt subgroup (n=46; median OS, not reached) when compared to all other molecular subgroups (n=246; median OS, 27.3 mo.; p=0.016). In univariate analysis for the total cohort, age (p=0.001), cytogenetic risk group (p=0.001), the NPM1mut/FLT3wt status (p=0.022) were prognostically relevant, whereas the blast percentages (10-19% vs. 20–30%; p=0.687), WBC (p=0.496), hemoglobin (p=0.071), presence of FLT3-ITD (p=0.896), and MLL-PTD (p=0.499) had no relevance. In multivariate analysis, only age (p=0.008) and the cytogenetic risk group (p=0.004) were significantly associated with OS. Conclusions:1) The separation of patients demonstrating 10–30% of bone marrow myeloblasts into MDS and AML does not distinguish between groups with different prognosis. 2) Cytogenetic and molecular genetic risk factors occur with comparable frequencies in MDS and AML subsets. 3) Karyotype and NPM1 mutation status are powerful parameters for risk categorization and treatment stratification in both subgroups with borderline blasts. Disclosures:No relevant conflicts of interest to declare.

Functional Characterization of FLT3 Receptor Signaling Deregulation in Acute Myeloid Leukemia by Single Cell Network Profiling (SCNP)

BackgroundMolecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative.Methodology/Principal FindingsUsing single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management.Conclusions/SignificanceThese studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations.

Hemophagocytic lymphohistiocytosis associated with myelodysplastic syndromes

Hemophagocytic lymphohistiocytosis/hemophagocytic syndrome (HLH/HPS) is a reactive disorder of the mononuclear phagocytic system, characterized by generalized histiocytic proliferation with marked hemophagocytosis in the bone marrow [1]. It comprises two different categories: a primary (genetic) and a secondary (acquired) form. Malignant neoplasm-associated HPS (MAHS) is categorized as a secondary HLH. MAHS is mainly associated with lymphoma and rarely with other carcinomas [2]. To our knowledge, HLH associated with myelodysplastic syndromes (MDS) has not been described, although MDS with hemophagocytosis has been observed. Here, we describe a case of HLH associated with MDS, which presented with abundant CD8 T cells in the bone marrow and elevated plasma-soluble interleukin 2 receptor (sIL-2R). A 60-year-old Japanese man was admitted with pancytopenia, epitaxis, fever and general fatigue. A hematological examination showed a hemoglobin concentration of 6.5 g/dL, a platelet count of 14,000/lL and a leukocyte count of 3,200/lL with 42.8% atypical lymphocytes (Fig. 1a). Blood biochemistry showed increased levels of lactate dehydrogenase (LDH) (331 IU/L), ferritin (649 ng/mL) and sIL-2R (4,054 U/mL). NK cytolytic activity was measured by the standard 51-chromium (Cr) release assay. NK cytolytic activity was not reduced at both the E/T ratios of 10:1 (13.1%, normal range 8.9–29.5%) and 20:1 (22.5%, normal range 17.1–48.7%). Serological studies for hepatitis B, hepatitis C and HTLV-1 were negative. IgM antibodies to parvovirus were negative. Serological test for EBV revealed that anti-viral capsid antigen IgM was negative in the presence of anti-viral capsid antigen IgG (1609) and anti-EBV nuclear antigen antibody (409). The number of EBV DNA copies in serum was under 2.0 9 10 copies/mL (normal \200 copies/mL). Computed tomography (CT) revealed hepato-splenomegaly, ascites and bilateral pleural effusion. Bone marrow aspirate showed dyserythropoietic changes (abnormally lobulated nuclei) (Fig. 1c) and dysplastic megakaryocytes (micromegakaryocytes) (Fig. 1d). Erythrocytic precursors were 10.4% and bone marrow myeloblasts were 2.0% (Fig. 1e). Chromosome analysis showed severe complex karyotype: 54–57XY, ?1[2],?3[2],?4[4],?6[4],?8[4],add(9)(p22)[2],?11[3],add (15)(p11.2)[4],add(16)(q24)[4],add(19)(p13.1)[4],add(20) (p13)[4],?21[2],2–5mar[cp4]/46,XY[8]. A diagnosis of refractory anemia with excess blasts-1 (WHO classification) was made. In addition, bone marrow examination revealed increased numbers of atypical lymphocytes (Fig. 1b) and multiple sites of active hemophagocytosis (Fig. 2a–c). Atypical lymphocytes in bone marrow were positive for CD2, CD3 (surface, cytoplasmic), CD5, CD7, CD8, HLA-DR, TCR-ab, and negative for CD4. No monoclonal rearrangement of TCR beta, gamma and delta chain genes was detected by Southern blotting. These findings suggested that the atypical lymphocytes were reactive CD8 T cells. Combining fever, hemophagocytosis, splenomegaly, increased levels of ferritin and sIL-2R, a diagnosis of HLH was established. Although steroid therapy was administered for HLH, pancytopenia was not improved. Two months after the initial diagnosis, the levels of LDH and ferritin were further elevated (LDH 1,474 U/mL, ferritin 4,347 ng/mL). Bone marrow examination T. Tsuji (&) H. Yamasaki H. Tsuda Division of Hematology and Oncology, Kumamoto City Hospital, Kotoh 1-1-60, Kumamoto 862-8505, Japan e-mail: tsuji.takahiro@city.kumamoto.lg.jp

Childhood rhabdomyosarcoma metastatic to bone marrow presenting with disseminated intravascular coagulation and acute tumour lysis syndrome: review of the literature apropos of two cases

The paper presents diagnostic and therapeutic difficulties in two adolescents with widespread rhabdomyosarcoma (RMS) presenting with severe haemorrhages resulting from disseminated intravascular coagulation (DIC) and with laboratory features of acute tumour lysis syndrome (ATLS). Other published cases of childhood RMS with DIC at admission have been listed and reviewed. It has been concluded that the clinical picture of a widespread RMS in children may resemble acute hematologic malignancy and pose a big diagnostic problem. That is why the presence of small blue round cells morphologically similar to lymphoblasts and/or myeloblasts in bone marrow (BM), lacking hematopoietic makers, should prompt the pathologist to consider possible diagnosis of RMS. Inclusion of desmin, MyoD1 and myogenin Myf4 to the immunohistochemical panel is obligatory in such cases. When the representative histopathological tumour specimens are difficult to obtain, the flow cytometric immunophenotyping of BM metastases could help the standard morphological/immunohistological diagnostic procedures and advance the diagnosis. Recently, the flow cytometric CD45− CD56+ immunophenotype together with Myf4 transcript has been assigned to RMS cells infiltrating BM. In children with disseminated RMS complicated with DIC rapid polychemotherapy aimed at diminishing the malignancy-triggered procoagulant activity should be initiated. However, in cases with concomitant ATLS the initial doses of chemotherapy should be reduced and the metabolic disorders and renal function monitored. The prognosis in children with RMS metastatic to BM with signs of DIC or ATLS at admission depends on the response to chemotherapy, however generally it is highly disappointing.

Non-p53 Dependent, Leukemia Initiating-Cell Selective, Therapy.

Abstract 2077Poster Board II-54Conventional drug therapy for AML is limited by toxic effects on normal hematopoietic stem cells (nHSC), and dependence on p53/apoptosis pathways that are impaired in malignancy. In hematopoiesis, key transcription factors (TF) determine cell-fate. Here, a difference in nHSC versus leukemia initiating-cell (LIC) TF expression is used to overcome the above limitations.The DNA methylating enzyme DNA methyl-transferase 1 (DNMT1) is also a component of multi-protein histone methyl-transferase complexes. Accordingly, shRNA mediated depletion of DNMT1 in hematopoietic cells hypomethylated DNA and decreased global H3K27 and H3K9 trimethylation (histone marks associated with transcription repression) by >70%. These epigenetic modifications were reproduced using a clinically relevant method: the cytosine analogue decitabine, added to normal human CD34+ hematopoietic precursor cells at 0.2–0.5uM 2–3X/week, depleted DNMT1, H3K27 and H3K9 trimethylation by >70% and significantly hypomethylated DNA (Illumina CpG Microarray). These decitabine levels did not cause measurable DNA damage (H2AX phosphorylation and Fast Micromethod) or apoptosis (Annexin staining and caspase 3 activity). Therefore, at low levels, decitabine can produce broad chromatin changes that increase TF access to target genes, without causing measurable DNA damage or apoptosis.The gene-expression/cell-fate consequences of opening chromatin with decitabine likely depend on the pre-existing TF expression pattern. HOXB4 (stem cell TF), CEBPa (lineage-specifying TF), and CEBPe (late differentiation TF) levels were measured by RQ-PCR in CD34+ cells from AML (n=3) versus normal bone marrow (n=3). AML CD34+ expressed >50-fold higher CEBPa, but HOXB4 and CEBPe levels comparable to normal CD34+ cells, a pattern confirmed in microarray gene expression analysis (CD34+ and myeloblasts, AML n=321, normal n=51 (GEO)). Repression of late differentiation TF likely involves chromatin-modification, regardless of underlying cause. Therefore, depleting DNMT1 to open chromatin in AML cells expressing high lineage-specifying TF could resume differentiation and terminate AML self-renewal, while nHSC, with high stem cell TF and little lineage-specifying TF, should continue to self-renew. nHSC and human MLL-AF9 AML cells were treated identically with decitabine for 7 days, then 300,000 each viable MLL-AF9 and nHSC were combined and transplanted into NSG mice (n = 8). Mice that received PBS treated cells died by week 5 (>90% human myeloblasts in bone marrow). Mice that received decitabine treated cells remained healthy until sacrifice for analysis at week 12 (log-rank p = 0.02, no detectable leukemia, >80% normal human hematopoietic cell marrow engraftment). Direct treatment of mice with established MLL-AF9 leukemia with very low dose decitabine 1mg/m2 3X/week extended survival by >20% (log-rank p = 0.04).Decitabine 0.5uM 2X/week induced morphologic differentiation, but not early apoptosis, in primary patient samples (n=15) and leukemia cell-lines (n=4). Cell-cycle exit by differentiation versus apoptosis may utilize different cyclin dependent kinase inhibitors (CDKN). The THP1 AML cell line contains a homozygous frame-shift mutation in TP53 (p.R174fs*3) and no detectable p53 RNA/protein. THP1 cells were treated with equimolar Ara-C or decitabine. Ara-C weakly upregulated CDKN1A (p21) but not CDKN2B (p15), and produced a transient decrease in cell-counts (D3-5) with recovery and growth similar to control by D7. Decitabine strongly upregulated p15, weakly upregulated p21, and produced gradual but complete and durable abrogation of cell growth by D7.A 66y patient with transfusion dependent RCMD with 5q-, 15q- and severe comorbidities was treated with metronomic (instead of cycled) very low dose SQ decitabine (0.2mg/kg [7.5mg/m2] 2X/week) to avoid cytotoxicity and sustain differentiation modification. Platelets increased by week 4, hematologic remission occurred by week 8 and cytogenetic remission by week 14 (without significant side-effects).Rationalizing dose and schedule of decitabine exploits a difference in nHSC and LIC TF expression to selectively terminate LIC self-renewal by a non-p53 dependent differentiation pathway. This approach, distinct from conventional apoptosis-based therapy, could have a very favorable safety profile, with efficacy in MDS/AML with complex cytogenetic abnormalities. Disclosures:Off Label Use: Decitabine, to treat myelodysplastic syndrome using a novel dose and schedule. Advani:Cephalon: Research Funding. Saunthararajah:HemaQuest: Consultancy.

Correlation between flow cytometric identification of CD33-positive cells and morphological evaluation of myeloblasts in bone marrow of patients with acute myeloblastic leukemia

Determination of the percentage of myeloblasts in bone marrow is important for the evaluation of acute myeloblastic leukemia (AML) and related disorders. Using flow cytometry with a CD45-blast gate (FCM/CD45), 226 bone marrow aspiration samples serially collected from 71 patients with de novo AML were analyzed. Bone marrow smears were evaluated independently by pathologists who did not know the corresponding flow cytometric data in advance. Patients received remission induction followed by consolidation. The CD33+ cell percentages evaluated by FCM/CD45 were strongly correlated to the myeloblast percentages determined by microscopic examination (r=0·8360, p<0·001). When only samples containing leukemic cells demonstrated by chromosomal or fluorescence in situ hybridization (FISH) analysis after induction were evaluated, positive correlations were found between CD33+ cell percentages determined by FCM/CD45 and myeloblast percentages determined by morphology (r=0·672, p<0·001). The identification of CD33+ cells by FCM/CD45 is useful for the evaluation of bone marrow myeloblasts in AML.

The Reduction of Leukemic Blasts in Bone Marrow Aspirate on Day 6 of Remission Induction Treatment Is Predictive for Complete Remission Rate and Survival in Adult Acute Myeloid Leukemia; the Results of Multicenter, Prospective Polish Adult Leukemia Group Trial.

The goal of this study was to prove the significance of an early evaluation of leukemic blast reduction based on cytological examination of bone marrow aspirates obtained on day 6 of remission induction treatment. Patients. 90 adult AML patients aged 18–60 (median 47) registered to the PALG prospective trial evaluating the efficacy of 3 remission induction protocols:A.DAF: daunorubicine (DNR) 60 mg/m2/d iv, d 1–3; cytarabine (AraC) 200 mg/m2/d ci, d 1–7, and fludarabine 25 mg/m2 2h inf. iv d 1–5),B.DAC (like DAF but cladribin is used at 5 mg/m2 instead of fludarabine) andC.the standard DA 3+7 regimen.Bone marrow aspirates were performed before the treatment and on the 6-th day of the first remission induction course, and the MGG stained marrow smears were evaluated at each centre by two experienced hematologists.Results. Based on the proportion of blasts in bone marrow specimen on day 6 patients were arranged into 2 groups:1.“responders”with ≤5% of blasts (n=59) and2.“non responders” with >5% of myeloblasts in bone marrow (n=31).The complete remission rate (CR) in the first group equalling 89% (51/59) was significantly higher in comparison to that obtained in the second group 29%(9/31), p=0,008. The probability of overall survival (OS) was also higher in the group of “responders” if compared to “non responders”; 65% versus 45% respectively, p=0,009. In multivariate analysis including bone marrow examination, cytogenetic risk group, age, leukocyte count at diagnosis and the induction arm only the persistence of leukemic blasts >5% in bone marrow specimen on 6-th day of induction was associated with higher risk of not achieving CR (HR = 52,6; p=0,00002), and with shorter OS (HR=3,13; p=0,02). Conclusion. This prospective, multicenter study demonstrates that a simple and commonly accessible evaluation of the leukemic blasts reduction in bone marrow smear on the 6-th day of remission induction treatment is a reliable and independent predictor for achieving CR and for overall survival. This offers a decision point for an early modification of the treatment strategy.

Management of RBC-Transfusion Dependence

Strategies for the management of anemia in patients with myelodysplastic syndrome (MDS) have evolved following the U.S. Food and Drug Administration (FDA) approval of three new therapeutics from one of symptom amelioration with red blood cell (RBC) transfusions to one of active treatment. Most patients develop transfusion-dependent anemia over the course of their disease, however, and its adverse consequence on the natural history of disease has only recently been appreciated. Although severe anemia contributes to symptoms of fatigue and reduced quality of life, transfusion dependence increases the risk of organ complications from iron overload coupled with an increased risk of leukemia transformation. Among World Health Organization categories without elevation in bone marrow myeloblasts, an incremental rise in RBC transfusion burden is associated with a proportionate reduction in both overall survival and leukemia-free survival, implying that anemia severity is an important variable limiting the otherwise favorable natural history of patients with lower risk disease. Moreover, therapeutic strategies that successfully restore effective erythropoiesis, such as erythropoetic stimulating agents, immunomodulatory agents, immunosuppressive therapies, or hypomethylating agents, may favorably affect the natural history of this disease, creating perhaps a new urgency for the initiation of erythropoietic promoters that have durable clinical benefit. Selection of primary therapy for the management of anemia should consider four response determinants: age, RBC transfusion burden and duration, endogenous erythropoietin production, and karyotype.

Phase-1 Study of ZIO-101: A New Organic Arsenic Active in Acute Myelogenous Leukemia (AML) and Multiple Myeloma (MM).

Background: Although arsenic trioxide (As2O3) is active in vitro against diverse hematological cancers, clinical data show activity only in acute progranulocytic leukemia (APL); there is little if any single-agent activity in multiple myeloma and other hematological cancers. This discordance may be because relatively little As2O3 can be given consequent to its substantial toxicity (especially QTc-prolongation) or because the mode of action (MOA) in APL (differentiation) is inoperative in other hematological cancers (or both). ZIO-101 (S-dimethylarsino-glutathione), a new organic arsenic, is active against diverse cancers in vitro and in animal models including AML and MM. Cell-killing by ZIO-101 is mediated by mitochondrial-disruption and apoptosis-induction rather than the differentiation MOA of As2O3. ZIO-101 can be given at doses ≥ 50-fold higher than As2O3 and achieves 5–10-fold higher intracellular concentrations at equimolar extracellular As concentrations. Gene-expression profiling data suggest different cellular responses to ZIO-101 and As2O3. These features make ZIO-101 attractive for evaluation in AML and MM.Methods: 2 phase-1 studies evaluating safety, activity and pharmacokinetic (PK) profile of ZIO-101 in 21 subjects with advanced AML (N=8) or MM (N=13). Median age is 58 y (range, 41–85 y). Median N of prior therapies is 5 (range, 2–12) including 4 subjects failing prior As2O3 therapy. Starting dose was 78 mg/me2/d IV for 5 consecutive d every 4 w.Results: Subjects received a median of 2 cycles (range, 1–6). Therapy was well-tolerated; adverse events ≥ grade-2 occurring in > 25% of subjects included neutropenia, hypokalemia and hyperglycemia. There was no clinically-important renal, liver or heart toxicity nor QTc-prolongation. Maximum tolerated dose (MTD) was 420 mg/me2/d. Pharmacokinetic (PK) studies at this dose showed a tmax=1 h (SD±0.9), Cmax=1.06 μg/mL (SD±0.07 μg/mL), t1/2=17.8 h (SD±1.4 h) and AUC0-∞=25.9 μg·h/mL (SD±0.8 μg·h/mL). 4 subjects with AML had stable disease (SD) after 1 cycle and received 2–4 more cycles before progressing. Blood myeloblasts decreased substantially in 1 subject and completely resolved in 2. Bone marrow myeloblasts decreased in 1. 1 subject with prior myelodysplastic syndrome (MDS) stopped requiring frequent RBC transfusions. 1 subject with rapidly-progressing As2O3- and bortezumib-resistant MM has stable disease (SD) >6 mo.Conclusions: These early data suggest activity of ZIO-101 in advanced AML and MM. The MTD is 420 mg/me2/d, ≥ 50-fold higher than As2O3. Plasma levels exceed the IC50 for AML and MM cell in vitro and animal models. Clinically-important QTc-prolongation was not seen. Some subjects failing As2O3 responded to ZIO-101 indicating efficacy of a higher dose, different MOA or both. Because of these favorable results phase-2 studies in hematological cancers are in progress.

A phase 1 trial of a novel organic arsenic S-dimethylarsino-glutathione (ZIO-101) in hematological malignancies

16503 Background: Inorganic Arsenicals (IA, e.g. AS2O3) kill many human leukemia cell lines and have efficacy in APL. Evaluation of efficacy against other types of leukemia is limited by the substantial toxicity of IA. Organic Arsenicals (OA) are less toxic than IA. ZIO-101 (S-dimethylarsino-glutathione) is an OA with activity against diverse cancers in in vitro tests and animals mediated through mitochondrial damage and apoptosis induction. ZIO-101 may induce apoptosis by different mechanisms than As2O3 and can kill As2O3-resistant cancer cells. Compared to As2O3, the LD50 of ZIO-101 in mice is 50X higher and at equimolar extracellular arsenic concentrations ZIO-101 produces 15× higher intracellular arsenic concentration. These features make ZIO-101 attractive for evaluation in leukemia. Methods: A phase-1 study evaluating the safety and pharmacokinetic (PK) profile of ZIO-101 in subjects with advanced hematological malignancies is underway. 8 patients (PATS) with acute myelogenous leukemia (AML) (median 3 prior treatments) received ZIO-101 at doses of 78, 109 or 153 (n = 4, 3, 1) mg/m2/d IV for 5 d every mo. Results: Therapy with ZIO-101 has been safe and well-tolerated. Toxicities ≥ grade-2 included hyperglycemia (N = 3) and decreased albumin (N = 3) and Ca (N = 3) and 1 subject each had ≥ grade 2 elevations in LFT, or decreases in K, or PO4, febrile neutropenia and rash. Preexisting anemia and thrombocytopenia increased by 1 grade in 4 and 3 PATS each. Grade ≥3 neutropenia occurred in 2 PATS. No significant renal, liver or heart toxicity occurred, 1 patient with prior 10AV block had transient prolongation of QT interval non-recurring on rechallenge. PK studies at 109 mg/m2/d showed a tmax = 1.5 h (SD ± 0.7 h), Cmax = 402 ng/mL (±33 ng/mL), t1/2 = 14.4 h (±2.9 h) and AUC0-∞ = 8.93 μg · min/mL (±1.82 μgzmdmin/mL). 4 PATS progressed after 1 cycle, 3 had stable disease and received a 2nd cycle: 1 progressed and 2 remain on study. 1 patient is too early to evaluate. Blood myeloblasts decreased (9 to 1.1 × 10e9/L at day 13) in 1 subject and completely resolved in 2 (pre-therapy: 100 and 336 × 10e9/L). Bone marrow myeloblasts were unchanged. Conclusions: Administration of ZIO-101 to PATS with advanced AML was safe and dose-escalation continues and a modest antileukemic effect has been observed. [Table: see text]

A Phase I Study of Temozolomide (Temodar®) in Pediatric Patients with Relapsed or Refractory Leukemia—A Children's Oncology Group Study.

A phase I study of oral temozolomide, administered daily for 5 days every 28 days, was performed in children with relapsed or primary refractory leukemias. The starting dose was 200 mg/m2/day with subsequent dose escalation to 260 mg/m2/day. Ten patients (5 male, 5 female), median age 9 years (range: 1–18 years), with leukemia (6 with acute lymphocytic leukemia [ALL], 4 with acute myelogenous leukemia [AML]) received a total of 14 cycles of temozolomide. Serum pharmacokinetic (PK) samples were obtained in 6 patients using a limited pharmacokinetic sampling strategy. Samples were also obtained from 8 patients for correlative biology studies, including analysis of methyl-guanine methyl transferase (MGMT) activity and microsatellite instability (MSI). MGMT activity was measured in 7 patients; all 3 patients with ALL tested for MGMT had elevated MGMT activity, with a median MGMT activity of 1240 fmol/mg protein (range: 356–1756). In contrast, 3 out of 4 patients with AML had MGMT activity below the limits of detection. Eight patients were tested for microsatellite instability by multiplex PCR using a panel of 13 MSI loci. Two patients (one with ALL and one with AML) demonstrated probable MSI instability. Temozolomide has been well tolerated, with no patients developing dose-limiting toxicity to date. One patient with AML, undetectable MGMT activity and no microsatellite instability had a partial response (PR) lasting 4 cycles (<10% myeloblasts in bone marrow). Further analysis is underway to determine the frequency of elevated MGMT activity in ALL and AML patients at diagnosis and following relapse.

A Comparative Review of Classification Systems in Myelodysplastic Syndromes (MDS)

Reliable classification and prognostic scoring systems for myelodysplastic syndromes (MDS) are needed to facilitate medically appropriate treatment and management decisions. The French-American-British (FAB) classification scheme for MDS was described in 1982 and has become the reference standard for subsequent MDS classification schemes. The FAB classification system divides MDS into five subgroups, based mainly on morphologic criteria and the percentage of myeloblasts in bone marrow (BM). More recently, the increasing availability of BM cytogenetics, immunologic markers, and molecular genetics has provided important information for staging, prognosis, and treatment of MDS. A World Health Organization panel incorporated this new diagnostic information into a revised classification system that modified the FAB criteria while retaining most of its basic features. The major changes included the creation of additional categories (eg, 5q- syndrome), distinction of unilineage from multilineage dysplasias in the refractory anemias, and subdivision of the heterogeneous refractory anemias with excess blasts into two categories based on BM blast percentage. Additionally, some MDS subtypes were removed or merged with other myeloid disorders into newly created categories. In independent validations, the World Health Organization revisions were shown to provide more-homogeneous subgroups of patients and greater prognostic power compared with the FAB system, although controversies remain. The International Prognostic Scoring System combines blast percentage, karyotype, and number of cytopenias to generate a scoring system that reliably estimates survival and risk of transformation to acute myeloid leukemia for patients with MDS. This universally accepted scoring system is often combined with FAB or World Health Organization morphologic criteria to provide a more complete clinical picture and the most accurate prognostic assessment possible. As more is learned about the pathogenesis of MDS at the molecular level, it is anticipated that these classification and scoring systems will continue to evolve to incorporate the new information.