Acute Myelomonocytic Leukemia Research Articles

Post-transcriptional modulation of C/EBPα prompts monocytic differentiation and apoptosis in acute myelomonocytic leukaemia cells

CCAAT/enhancer binding protein alpha (C/EBPα) induction induces monocytic differentiation even in acute myeloid leukaemia (AML). In this study, the induction/activation of C/EBPα in myelomonocytic AML was investigated using a combination of all-trans retinoic acid (ATRA) and RAD001 (Everolimus), a mammalian target of rapamycin complex 1 (mTORC1) inhibitor. Combining these agents increased PU.1, C/EBPɛ and C/EBPα expression, increased the p42/p30 C/EBPα ratio, and decreased C/EBPα phosphorylation at serine 21, and was accompanied by growth inhibition, induction of CD11b expression and apoptosis in AML cell lines. Thus, agents that induce sufficient levels of C/EBPα expression might be useful in treating AML.

Establishment and characterization of a new human acute myelomonocytic leukemia cell line JIH-3

Here, a new acute myelomonocytic leukemia (AMML) cell line, JIH-3, is reported, and its biological characteristics are described. JIH-3 cells were maintained without any cytokines for 27 months. The JIH-3 cell line showed typical myelomonocytic morphological features. Additionally, it mainly expressed myeloid and monocytic markers (CD13, CD14, CD11b, CD15 and CD33), although it also expressed other antigens such as the markers of T and B lymphocytic lineage as well as stem cell, progenitor cell, and natural killer cell-related antigens (CD4, CD5, CD7, CD10, CD22, CD34, CD38, HLADR, CD16/CD56 and CD56); the expression of these markers, suggested that this cell line was in the early stage of myelomonocytic differentiation. Cytogenetic analysis initially showed a karyotype of 46, XY, del(7) (p1?3p2?2). During the passage period, the cells with this karyotype gradually decreased and were replaced by cells with a 45,XY,dic(4;7)(p11;p11),del(15)(q2?2) karyotype. Chromosome painting showed a deletion in the short arm of chromosome 7 for del(7)(p1?3p2?2) and der(4;7)(p11;p11). The latter had larger deleted segment than the former. Fluorescence in situ hybridization (FISH) revealed the dicentric nature of der(4;7), and Multiplex FISH (M-FISH) confirmed that der(4;7) was an unbalanced translocation. A deletion involving the 7p region on dic(4;7)(p11;p11) harbors many genes, including CDC2L5, C7ORF11, C7ORF10 and INHBA. Haploinsufficiency of the genes on 4p, 7p and 15q caused by deletions of 4p, 7p and 15q2?2 that resulted from dic(4;7)(p11;p11) and del(15)(q2?2) may play important roles in leukemogenesis and in the establishment of the JIH-3 cell line. JIH-3 cells did not express multidrug resistance (MDR)-related genes and apoptosis-related genes such as MDR1, multidrug resistance-related protein, lung resistance protein, BCL-2, Bax, GS-π or Fax, only P21 expression was detected, which probably leads the MDR indirectly through inhibition of the activities of cyclin-dependent kinase (CDK). JIH-3 cells had tumorigenic capacity in nude mice. In conclusion, JIH-3 is a new acute myelomonocytic leukemia cell line. It is from a well-characterized background and provides a new useful tool for the study of leukemia patients with a 7p deletion.

Myeloid Sarcoma and Acute Myelomonocytic Leukemia in an Adolescent with Tetrasomy 8: Staging with 18F-FDG PET/CT

Tetrasomy 8 is a relatively rare chromosomal abnormality that has been reported in only 33 cases in hematologic disorders. It is known for its association with aggressive acute myeloid leukemia (AML) and myeloid sarcoma and is considered a very poor prognostic factor. Myeloid sarcoma is a rare hematologic malignancy characterized by tumor masses consisting of immature myeloid cells, presenting at an extramedullary site. We present a case of a 17-year-old boy referred for an (18)F-FDG PET/CT for the evaluation of pleural masses and spinal bone lesions seen on CT, after presenting with a 4month history of chest pain. The PET/CT revealed extensive FDG-avid extramedullary disease in the soft tissues of the chest, abdomen, and pelvis, which were biopsy-proven to be myeloid sarcoma, as well as extensive intramedullary disease biopsy proven to be AML. This is the first report of the use of (18)F-FDG PET/CT to stage a subset of aggressive AML and myeloid sarcoma in a patient with an associated chromosomal abnormality (tetrasomy 8).

Multiple gingival metastasis of adenocarcinoma of the lung

Metastasis to the gingival soft tissues is an extremely rare phenomenon, and metastasis of multiple lesions (i.e. more than 2 lesions), is even more hard to find. In this study, we have reported a rare case of lung adenocarcinoma, which metastasized to the maxillary (2 lesions) and mandibular gingivae (1 lesion) in a 57 year old male patient. Metastasis was also seen to the vertebrae. The differential diagnosis consisted of acute myelomonocytic leukemia, chronic lymphocytic leukemia, haemangioma, pyogenic granuloma, giant cell granuloma, peripheral fibroma, primary gingival carcinoma and secondary metastasis. Contrast enhanced computed tomography of the lung showed a well-defined mass situated below the right hilum with lower lobe consolidation and pleural effusion on the right side with dorsal spinal metastasis. Excisional biopsy of the lesions was consistent with the diagnosis, and the immunohistochemical analysis was positive for cytokeratin 7, carcinoembryonic antigen (CEA), thyroid transforming factor 1 (TTF1), and negative for vimentin and cytokeratin 20 (CK20).

Acute myelomonocytic leukemia presenting with gingival enlargement as the only clinical manifestation

A 34 year old woman reported for evaluation of generalized gingival swelling pain and bleeding. The patient also reported menstrual irregularities. Based on the oral and systemic examinations and hematological investigations, a provisional diagnosis of ‘menstrual cycle associated gingivitis’ was made. The patient was referred for a gynecologic consultation. At the three-week dental recall, a worsening of gingival overgrowth with a necrotic component was noted. The hematologic investigations revealed markedly elevated leukocyte counts. The clinical and hematological findings led us to a diagnosis of leukemia. This report emphasizes the importance of the dentist as well as the physician (in this case, the gynecologist) in correlating the oral, systemic and hematological findings in the diagnosis and also of regular recall in the management of a case.

Runx1 Loss Minimally Impacts Long-Term Hematopoietic Stem Cells

RUNX1 encodes a DNA binding subunit of the core-binding transcription factors and is frequently mutated in acute leukemia, therapy-related leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia. Mutations in RUNX1 are thought to confer upon hematopoietic stem cells (HSCs) a pre-leukemic state, but the fundamental properties of Runx1 deficient pre-leukemic HSCs are not well defined. Here we show that Runx1 deficiency decreases both apoptosis and proliferation, but only minimally impacts the frequency of long term repopulating HSCs (LT-HSCs). It has been variously reported that Runx1 loss increases LT-HSC numbers, decreases LT-HSC numbers, or causes age-related HSC exhaustion. We attempt to resolve these discrepancies by showing that Runx1 deficiency alters the expression of several key HSC markers, and that the number of functional LT-HSCs varies depending on the criteria used to score them. Finally, we identify genes and pathways, including the cell cycle and p53 pathways that are dysregulated in Runx1 deficient HSCs.

Frequency and Prognosis of Clonal Chromosomal Abnormalities Following Stem Cell Transplantation (SCT) in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS)

Abstract 4897 Objective.To study the frequency, type and prognostic significance of clonal chromosomal abnormalities following SCT in patients with AML and MDS. Patients and methods.One hundred thirty patients were studied between 2000 and 2010. Karyotypes were analysed by G-banded chromosomes obtained from 24 hours bone marrow cultures, and were described according to ISCN 2009. Results.Clonal abnormalities were observed in 36/130 patients (28%) with a median follow-up was 11 months (range 3–131). Initial diagnosis (OMS 2008): AML with maduration (8 patients), acute erythroid leukaemia (4), acute monocytic leukaemia (3), AML with multilineage dysplasia (3), AML with myelodysplasia-related changes (3), AML with inv(16)(p13q22) (2), acute monoblastic leukaemia (2), AML with minimal differentation (2), AML without maduration (1), acute myelomonocytic leukaemia (1), refractory anaemia with excess blasts (4), chronic myelomonocytic leukaemia (2) and refractory cytopenia with multilineage dysplasia (1). Treatment before SCT: idarubicin, cytarabine, etoposide and mitoxantrone (25), idarubicin, cytarabine and etoposide (6), idarubicin, cytarabine, etoposide and gemtuzumab (2), FLAG-ida (2) and azacitidine (1). SCT type: autologous (23 patients), allogeneic of reduced intensity (7), allogenic (4), umbilical cord blood (1) and syngeneic transplant (1). Conditioning regimen: TBI and cyclophosphamide (24 cases), fludarabine and busulfan (7), busulfan and cyclophosphamide (4) and thymoglobulin, thiotepa, fludarabine and busulfan (1). The median time between SCT and the appearance of clonal abnormalities was 6.5 months (range 2–51). At the time of clonal abnormality detection, 32 patients were in cytological and/or clinical relapse and 4 in cytological remission. In 15 cases the initial clone reappeared, 2 showed the initial abnormalities with an acquired abnormality and 19 presented de novo clonal abnormalities (53%). The most frequent clonal chromosomal abnormalities observed were: complex karyotype (22%), estructural abnormalities with afection of chromosomes 1, 4, 6, 7, 10, 11, 12, 16 and 17 (22%), +21 (5%) and +11 (3%). The median survival from the appearance of clonal abnormalities was 2.5 months (range 1–100). There were no differences when we compared the survival of patients with de novo clonal abnormalities with that of patients with initial abnormalities (including those with acquired abnormality). Furtheremore there were no differences when we compared the median survival of relapsed patients with normal Karyotype with that of patients with clonal chromosomal abnormalities following SCT (2.5 months [range 1–25] and 2.5 months [range 1–100] respectively). At the time of the analysis only 1 patients are alive and in complete remission (100 months). Conclusions.1. The appearance of clonal abnormalities following SCT in patients with AML and MDS is frequent and the majority of cases are detected at the time of relapse. Half of cases presented de novo clonal abnormalities and the majority were not related with prior irradiation exposition, alkylating agents neither topoisomerase II inhibitors. The prognosis of patients with acquired clonal abnormalities after SCT is poor. Disclosures:No relevant conflicts of interest to declare.

G-CSF Induced Atypical Monocytic Proliferation Mimicking Acute Myelomonocytic Leukemia in Post-Induction Chemotherapy AML Patient

Abstract 4283 Introduction:Granulocyte colony-stimulating factor (G-CSF) is a major hematopoietic cytokine involved in control of neutrophil production, proliferation and survival.1 Nowadays, recombinant G-CSF is widely used in patients with hematopoietic malignanies receiving chemotherapy to increase leukocyte count.2 Studies have showed that G-CSF use in these settings is safe and does not increase relapse rate.3,4 However, A phenomena called transient atypical monocytosis, or pseudoleukemia, induced by G-CSF administration in AML patients with chemotherapy have been reported.5,6 This entity mimics recurrent/relapsed acute leukemia and causes confusion in pathologic diagnosis and potential problems in clinical management. The case presented in this report is an example of this entity. Pathology:A 55-year old male patient presented with leukocytosis and anemia. Peripheral blood smear examination revealed a blast population and monocytosis. Flow cytometry on the blood sample showed 37% blasts with expression of CD33, CD13, CD117, HLA-DR, CD34, CD14, CD15 and CD64. FISH study revealed a chromosomal abnormality of inversion 16. A diagnosis of acute myelomonocytic leukemia was established. The patient was treated with induction chemotherapy and was given Neupogen from day 6 through day 20. Bone marrow examination on the day 14th showed an aplastic marrow. However, examination of the patient's day 21 bone marrow revealed marked hypercellularity with sheets of large atypical monocytic cells and brisk mitotic figures. These cells showed round to ovoid nuclei, basophilic cytoplasm and prominent nucleoli, morphologically indistinguishable from blastic cells. Immunohistochemical studies confirmed that most of these atypical cells were lysozyme, CD163 and CD68 positive monocytes, some of them were positive for CD117. These findings raised concerns of recurrent/relapsed AML. However, FISH study was negative for inversion 16. These results indicate that the findings in the patient's day 21 marrow were most likely G-CSF-induced changes. The Neupogen was discontinued soon after. The patient was followed-up with periodic bone marrow examination and FISH study, which showed negative results. Currently, the patient is in complete remission. Discussion:We report one case of G-CSF induced atypical monocytic proliferation in an AML patient with induction chemotherapy. The findings could have created diagnostic problems if FISH study data had not available since those atypical cells were morphologically indistinguishable from blastic cells. Studies have shown that administration of recombinant G-CSF induces a rapid and sustained elevation in absolute peripheral neutrophil counts, and its use in leukemic patients during or after induction chemotherapy is safe and does not increase relapse rates.4 G-CSF shortens the transit time of developing granulocytes in bone marrow and accelerates the release of neutrophils that have undergone recent cell division.1 Our case of transient atypical monocytosis and a few similar cases reported in literatures showed an unusual response to G-CSF. This phenomenon was mostly seen in AML-M4, AML-M5 and AML-M3 cases7 and the mechanism is not entirely clear. A possible explanation is that G-CSF does not only act on neutrophils but also acts on progenitor hematopoietic cells including monocytes.8 One problem is that this entity can mimic recurrent AML causing confusion or potential misdiagnosis if clinical information or cytogenetic studies are not available. Therefore, it is important that both clinicians and pathologists be aware of this phenomenon in hematology/oncology settings. Appropriate discontinuation of G-CSF and following-up these patients with morphologic examination and cytogenetic studies is necessary. Meanwhile, a thorough understanding of exogenous G-CSF effects in cancer patients is essential to reduce any potential risks caused by its administration. Disclosures:No relevant conflicts of interest to declare.

Phase 1 Dose-Escalation Study of PF-04449913, An Oral Hedgehog (Hh) Inhibitor, in Patients with Select Hematologic Malignancies

Abstract 424 Background:Hh signaling is activated in a variety of human cancers and is required for maintenance of leukemic stem cell (LSC) populations in several hematologic malignancies. PF-04449913 selectively inhibits Hh signaling by binding Smoothened, a membrane protein that regulates the Hh pathway. PF-04449913 significantly reduced LSC burden following xenotransplantation of patient-derived CD34+ imatinib-resistant chronic myeloid leukemia (CML) cells in preclinical models. Methods:This first-in-patient Phase 1a dose-escalation study is assessing first-cycle dose-limiting toxicities (DLTs) and the recommended Phase 2 dose (RP2D) of PF-04449913 in patients with select hematologic malignancies (primary endpoint). Secondary endpoints include safety, pharmacokinetics (PK), pharmacodynamics, and preliminary signs of efficacy as defined by disease-specific guidelines. Patients had refractory, resistant, or intolerant select hematologic malignancies and could be previously untreated but not candidates for standard therapies: CML including T315I mutations (any phase), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myelofibrosis (MF), or chronic myelomonocytic leukemia (CMML). Cohorts of ≥3 patients received PF-04449913 alone, administered continuously in 28-day cycles, starting at a dose of 5 mg orally once daily. Results:Thirty-two patients have been enrolled at doses up to 270 mg: 18 males/14 females; AML, 18; MF, 6; CML, 5; MDS, 3; with a median age of 68.5 (35–79) years. ECOG PS was 0/1/2: n=11/16/5. Treatment duration ranged from 1 to 387 days (AML: 1–266; CML: 1–281; MDS: 2–335; MF: 44–387 days). One patient discontinued the study due to a treatment-related adverse event (AE) after 137 days of therapy at the 10 mg dose level (grade [G] 3 hemorrhagic gastritis in the setting of chronic proton pump inhibitor administration prior to and during the study). One AML patient evolved from CMML on 80 mg had a DLT comprising G3 hypoxia and G3 pleural effusion. The majority of AEs were of G1/2 severity; the most frequent treatment-related AEs included dysguesia (16%), alopecia (6%), arthralgia (6%), decreased appetite (6%), nausea (6%), and vomiting (6%). Preliminary indications of efficacy were observed across all hematologic diseases studied. One patient with AML (evolved from CMML and with a concurrent diagnosis of systemic mastocytosis) achieved a complete remission with incomplete blood count recovery; bone marrow blast count decreased from 92% to 1%. Five AML patients had a ≥50% reduction in bone marrow blast counts (20% to 10%, 70% to 20%, 44% to 8%, 14% to 7%, 40% to 10%). One patient with low-risk MDS, currently remaining on study after 335 days, achieved significant reduction in spleen size and a hematologic improvement in platelets (from 98.5 to 369 ×109/L) and neutrophils (ANC from 410 to 5490), and is no longer granulocyte colony-stimulating factor (G-CSF) dependent. Five patients with MF attained stable disease; an additional MF patient, currently remaining on study after 385 days, achieved clinical improvement with a >50% reduction in extramedullary disease (spleen size decreased from 10 cm to 3.5 cm sustained over 8 weeks). One patient with T3151 lymphoid blast crisis CML on study for 115 days achieved a major cytogenetic response with loss of their T3151 mutation. PF-04449913 PK data (5–180 mg) indicated a dose-proportional profile, with a median time to maximum concentration (Tmax) of 1–2 hours, a mean half-life ranging from 17 to 35 hours, and a large volume of distribution (mean range 250–480 L). Following daily dosing, steady state was achieved by Day 8 and the median accumulation ratio ranged from 1.3 to 2.9. Conclusions:PF-04449913 was safe and well tolerated, with early signs of efficacy observed in all hematologic diseases studied. Several patients with aggressive malignancies remained on trial for prolonged durations with improved quality of life; some exhibited cellular differentiation as determined by flow cytometry. On-target AEs (e.g. dysgeusia and alopecia) were observed at multiple dose levels. Pharmacokinetics were linear, predictable, and compatible with once-daily dosing. On the basis of these encouraging results, a Phase 1b study of PF-04449913 in combination with bosutinib and dasatinib is planned in patients with CML. Disclosures:Jamieson:Pfizer Oncology: Research Funding; Celgene: Research Funding; Novartis: Honoraria; Wintherix: Equity Ownership. Cortes:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Pfizer Oncology: Research Funding; Novartis: Research Funding; BMS: Research Funding; Infinity: Research Funding. Oehler:Pfizer Oncology: Research Funding. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Zhang:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Shaik:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Courtney:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Levin:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Martinelli:Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

Jumping Translocation of Homogeneously Staining Region Hsr(11q) in An Erythroid Leukemia: Identification of Amplified Regions by Fluorescence Hybridization, M-FISH and M-Banding.

Abstract 4890Gene amplification is a mechanism whereby a tumor cell can increase the copy number of specific gene sequences and gain a proliferative advantage. Although amplifications are common in solid tumors, they are relatively rare in hematological neoplasms. We report here on a patient with an acute erythroid leukemia and a jumping translocation of a hsr(11q) in a complex karyotype, without MLL amplification.The patient was 40 years old when he was admitted for asthenia, peripheral blood showing pancytopenia (Hb=111G/L; neutrophils= 0.23 G/L; platelets 92 G/L), without circulating blast cells. Bone marrow was hypercellular with the presence of more than 50% of erythroid precursors and more than 20% of myeloid blast cells in non-erythroid cell population, leading to the diagnosis of erythroblastic acute leukemia. A marked dysgranulopoiesis was also detected.Conventional cytogenetics showed a complex karyotype with a del(5q), a unbalanced t(7;17) leading to partial 7q deletion and homogeneously staining regions (hsr). Fluorescence in situ hybridization confirmed the del(5q), and showed that TP53 was not deleted in the t(7;17). Multi-FISH (M-FISH) pointed out that the hsr consisted of chromosome 11 and was implicated in 3 different translocations with 3 different partner chromosomes in 3 different clones. To further characterize the amplicon and determine which bands were implicated, we used a chromosome 11 m-band probe. It revealed that the bands implicated are the same on the der(3), der(12) and der(20) and are localized between 11q23.3 and 11q25. A series of BAC probes showed that different genes, present in these regions, were amplified: ETS1, FLI1, KCNJ5, NFRKB, SNX19, HNT, OPCM, but not MLL.Amplifications of chromosome 11q usually includes MLL, but more telomeric amplicons have also been reported in AML and myelodysplatic syndromes (MDS). A very close 11q amplification was identified in 3 AML/MDS cases (Crossen et al, 1999; Tyybäkinoja et al, 2006). Also, the ETS1 oncogene was found to be rearranged and 30-fold amplified in a case of acute myelomonocytic leukaemia, in which an hsr occurred on 11q23 (Rovigatti et al, 1986).The role of chromosomal amplifications in leukemia is unclear, but it has been suggested that they are associated with rapid disease progression and short survival. Jumping translocations are an unusual phenomenon and have been rarely reported in hematological malignancies. Whether jumping translocations play a role in tumor genesis or confer a selective growth advantage on tumor cells is unknown. The combination of hsr and jumping translocations as described here are very rare in hematological malignancies. The cases of the literature (Yoshida et al, 1999) and the one presented here suggest that the 11q24-q25 region may harbor new candidate oncogenes, together with unusual chromosomal mechanisms. Disclosures:No relevant conflicts of interest to declare.

Pretreatment of Low-Dose Decitabine to Leukemia Cells Markedly Enhances the Cytotoxicity of Gemtuzumab Ozogamicin

Abstract 5000Prognosis of relapsed/refractory acute myeloid leukemia (AML) patients has been poor due to its drug resistance. Although gemtuzumab ozogamicin (GO) targets CD33 antigen, which is overexpressed on most AML cells, the efficacy of GO alone for relapsed/refractory patients remains to be unsatisfied. Therefore we aimed to improve GO effects by combination with the other drugs.GO needs target cells intact to complete several steps for its cytotoxicity: GO binding to CD33 antigen, internalization of GO with CD33 antigen, fusion of the endosome to lysosome, digestion of the linker by lysozyme and release of calicheamicin, and intercalation of activated calicheamicin to DNA. GO could exert its cytotoxicity only when these cellular steps of target cells are intact. This paradoxical mechanism prompted us to study decitabine (DAC), azacitidine (AZA) and, valproic acid (VPA), which modify epigenetic alterations in leukemia cells with modest cytotoxicity. Low-dose DAC, AZA, or VPA have been reported to differentiate AML cells, with the increased expression of myeloperoxidase and lysozyme. Differentiation of AML cells by some agents, for example G-CSF, has been reported to reduce the expression of P-glycoprotein. Thus, we hypothesized that DAC, AZA or VPA could activate the cellular functions of target cells and enhance the cytotoxicity of GO.As refractory leukemic cells, we used SKK-1 and SKM-1 cells derived from patients with leukemia progressed from myelodysplastic syndrome, and SKNO-1, KO52, and Kasumi3 cells with complex karyotypes. We also used an acute promyelocytic leukemia cell line (NB4) and an acute myelomonocytic leukemia cell line (U937).Low-dose of DAC (100nM), AZA (2μM), or VPA (1mM) showed negligible cytotoxic effects. Simultaneous administration of GO (2.5 μg/ml) and DAC did not enhance cytotoxicity of GO. In contrast, GO cytotoxicity was enhanced only when cells were pretreated with DAC prior to GO. Pretreatment with DAC for 48 hours showed maximum enhancing effects. AZA also required pretreatment for enhancing effect. It was suggested that DAC and AZA induced some cellular response which enhance the cytotoxic steps of GO during pretreatment. VPA enhanced the cytotoxicity added either simultaneously or antecedently. The enhancing effect varied in each cell line. Pretreatment with DAC showed the most prominent enhancing effects among the three agents.DAC treatment changed neither the expression of CD33 antigens nor internalization of GO. DAC enhanced GO cytotoxicity partly by both promoting intercalation of calicheamicin to DNA and suppressing the activity of a drug-efflux pump, MRP-1.DAC-enhanced GO cytotoxicity in vitro was also observed in primary leukemic blasts of four (29%) among 14 relapsed/refractory AML patients.A large phase III study about GO, S0106 trial conducted by SWOG, revealed no significant improvement either in CR rate or in LFS for de novo AML patients, whereas significant increase of early complication death, in concurrent use of chemotherapy and GO. The result of SWOG trial and the mechanism of GO cytotoxicity might suggest that concurrent use of strongly-cytotoxic agents is not the best way of combination for GO. In contrast to SWOG trial, the present study targeted relapsed/refractory AML cells, employed pretreatment of low-dose DAC, and demonstrated preclinical possibility that the combination of DAC and GO would be useful for relapsed/refractory AML. Disclosures:No relevant conflicts of interest to declare.

NADPH Oxidase Regulates Cytarabine-Induced Apoptotic Death in Acute Myeloid Leukemia Cells

Abstract 4258 Background:Cellular metabolism and oxidative stress are important in the biology and pathophysiology of malignancies. Both increased reactive oxygen species [ROS] levels and induction of anti-oxidative pathways have been described in several malignancies, and may be modulate tumor biology and susceptibility to chemotherapy. Limited studies point to metabolic pathways, including ROS production, influencing pathogenesis and chemo-sensitivity of leukemia. NADPH oxidase is a critical enzyme in antimicrobial host defense and its activation results in ROS generation in myeloid leukemia cells. Our prior studies show that NADPH oxidase can activate Nrf2, a transcriptional factor that induces anti-oxidant and cytoprotective pathways. However the role of NADPH oxidase in chemotherapy-mediated apoptosis induction in leukemic cells is not well-known. Hypothesis:NADPH oxidase-derived ROS will increase sensitivity of AML cells to chemotherapy, whereas Nrf2 will be associated with chemotherapy resistance Methods:We evaluated the role of NADPH oxidase and Nrf2 in regulating cytarabine-induced cell death in wild-type [WT] and engineered PLB-985 cells, a human acute myelomonocytic leukemia cell line derivative. NADPH oxidase-deficient PLB-985 cells were generated by recombination with mutant gp91phox, a necessary component of NADPH oxidase. Nrf2-deficient cells were generated by shRNA (Nrf2shRNA) and depletion (>70%) of Nrf2 mRNA was confirmed by quantitative-PCR. WT and engineered PLB-985 cells were treated with cytarabine (12.5 to 750ng/ml for 24 – 48 hours) and cell death was determined by trypan blue exclusion and Annexin V/7-AAD staining. Results:NADPH oxidase-deficient PLB-985 cells were significantly more resistant to cytarabine compared to WT cells. Cytarabine (500 ng/ml for 48h) induced apoptotic cell death in 25% of NADPH oxidase-deficient vs. 53% of WT PLB-985 cells. Additional dose-response studies confirmed a significant effect of NADPH oxidase in potentiating cytarabine-induced cell death. Nrf2shRNA PLB-985 cells had either similar or modestly increased susceptibility to cytarabine-induced cell death compared to WT PLB-985 cells with empty vectors. NADPH-deficient/Nrf2shRNA PLB-985 cells had similar susceptibility to cytarabine as NADPH-deficient cells with empty vector. Conclusions:Our results show that NADPH oxidase potentiates apoptotic cell death by cytarabine in a myelomonocytic leukemia cell line. However, we did not observe a consistent effect of Nrf2 depletion on apoptotic cell death by cytarabine. These studies suggest that modulation of redox-stress may be a potential therapeutic approach in AML that merits further study. Disclosures:No relevant conflicts of interest to declare.

Histological and immunohistochemical features of gingival enlargement in a patient with AML

Here, we discuss the pathophysiology of leukemia-associated gingival enlargement based on a case of acute myelomonocytic leukemia (AML-M4) with typical gingival enlargement. Uniquely, this patient was well enough to allow full periodontal examination and incisional gingival biopsy to be performed both before and after chemotherapy. The patient was a 39-year-old Japanese woman with AML-M4 showing gingival enlargement. Histological and immunohistochemical features of gingiva and bacterial counts in the periodontal pockets were examined before and after chemotherapy. The results were as follows: (1) infiltration of myelomonocytic blasts in enlarged gingiva; (2) resolution of gingival enlargement with complete remission of AML by anticancer chemotherapy; and (3) the numbers of bacteria in the periodontal pockets were not high and were not altered before or after chemotherapy. In patients with AML-M4, remarkable mucosal enlargement is not generally observed in the body except in the gingiva. We hypothesized that antigens derived from periodontal bacteria, even if they are not present in large numbers, could act as chemoattractants for myelomonocytic leukemic cells.

Inversion and deletion of 16q22 defined by array CGH, FISH, and RT-PCR in a patient with AML

Acute myelomonocytic leukemia with eosinophilia is commonly associated with pericentric inversions of chromosome 16, involving the core binding factor beta gene (CBFB) on 16q22 and the myosin heavy chain gene (MYH11) on 16p13. The inv(16)(p13q22) results in a fusion gene comprising the 5'CBFB gene and the 3'MYH11 gene on the short arm of chromosome 16. The fusion gene interferes with the normal transcription of the CBFA/CBFB heterodimer and disrupts myeloid differentiation. The inv(16) is associated with a good prognosis. The inv(16) with deletion of the 3'CBFB region of the gene is a very rare occurrence. Although the number of cases is small, inv(16) with a deleted 3'CBFB seems to be associated with a poorer prognosis than that generally associated with inv(16). Our patient was a 30-year-old man with newly diagnosed acute myeloid leukemia who was found to have a CBFB-MYH11 fusion by reverse transcriptase-polymerase chain reaction. The high blast count and lack of differentiation were not typical for this entity and suggested clonal progression. The initial karyotype by conventional cytogenetic analysis, in all metaphases examined, was 46,XY,del(7)(q32),del(16)(q22). Fluorescence in situ hybridization analysis with a dual-color, break-apart probe corresponding to the CBFB gene locus (Abbott, Des Plaines, IL) showed a derivative chromosome 16 resulting from an inversion of the CBFB gene with a deletion of the 3'CBFB probe region. Oligonucleotide array comparative genetic hybridization analysis was performed on this patient's diagnostic bone marrow DNA referenced to a normal male control DNA by using the DNAarray Heme Profile (CombiMatrix Diagnostics, Irvine, CA) microarray. This analysis showed a 1.2 Mb loss of 16q22.1, which did not include loss of the 3'CBFB gene locus, but rather sequences distal to this locus. The DNAarray Heme Profile results illustrate the importance of microarray in the correct identification of abnormalities that will affect prognosis.

A rare case of supraspinatus tendon rupture

Dear Editor, A 66-year-old man was treated for acute myelomonocytic leukemia with mitoxantrone, cytarabine, and etoposide. As it is our practice, we gave him 500 mg ciprofloxacin every 12 h per os for antibacterial prophylaxis. Two weeks later, he complained of pain in both shoulders which worsened until he could no longer raise his arms. He also had pain in his hips and knees, but the Achilles tendons were not involved. There were no signs of inflammation, but palpation of the insertion point of the supraspinatus tendon proved painful. Ultrasound examination showed a bilateral partial rupture of the supraspinatus tendon and subscapularis tendinopathy (Fig. 1). We suspected ciprofloxacin toxicity so we stopped the drug and he was given analgesics and physiotherapy. The pain resolved completely within a few days; however, full functional recovery took several months. We did not re-challenge with ciprofloxacin as the side effects had been so severe. Fig. 1 Ultrasound of the left supraspinatus tendon. The ultrasound shows a partial longitudinal rupture of the supraspinatus tendon (white arrows) Antibiotic prophylaxis reduces infections and enhances survival when given to high-risk patients receiving chemotherapy for acute leukemia or stem cell transplant. Current guidelines recommend prophylaxis be given from the start of chemotherapy until bone marrow recovery when the expected duration of neutropenia is more than 7 days [1, 2]. Fluoroquinolones, which provide a broad antimicrobial spectrum, are generally well tolerated and have a good safety profile. However, they can cause tendinitis and other forms of tendon damage in 10–15 per 100,000 prescriptions, a rate that increases with older age, renal dysfunction, and corticosteroid use. The onset of symptoms is typically within 2 weeks after starting treatment, but symptoms can occur within 2 h after the first dose until 6 months after cessation of treatment. The Achilles tendon is affected in 90% cases, almost half involving both ankles and the tendon ruptures in approximately 50% of the cases. Other sites of tendon injury have been reported but are rare. Recovery usually takes 1–2 months requiring rest and immobilization and surgery is seldom needed [3, 4]. How fluoroquinolones induce tendon injury is not understood. Our patient's age appears to have been the only risk factor as he had not used corticosteroids and did not have renal dysfunction. In July 2008, the FDA added a black box warning to alert doctors and patients of the raised risk of tendinitis and tendon rupture associated with fluoroquinolones [5]. As clinicians will be treating an increasing number of elderly patients for hematological malignancies, it is very important for them to recognize these symptoms and switch to an alternative treatment before tendon rupture occurs.

Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia

Abnormal epigenetic regulation has been implicated in oncogenesis. We report here the identification of somatic mutations by exome sequencing in acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5). We discovered mutations in DNMT3A (encoding DNA methyltransferase 3A) in 23 of 112 (20.5%) cases. The DNMT3A mutants showed reduced enzymatic activity or aberrant affinity to histone H3 in vitro. Notably, there were alterations of DNA methylation patterns and/or gene expression profiles (such as HOXB genes) in samples with DNMT3A mutations as compared with those without such changes. Leukemias with DNMT3A mutations constituted a group of poor prognosis with elderly disease onset and of promonocytic as well as monocytic predominance among AML-M5 individuals. Screening other leukemia subtypes showed Arg882 alterations in 13.6% of acute myelomonocytic leukemia (AML-M4) cases. Our work suggests a contribution of aberrant DNA methyltransferase activity to the pathogenesis of acute monocytic leukemia and provides a useful new biomarker for relevant cases.

DEK-CAN mRNAにより微小残存病変を追跡できた6;9転座型急性骨髄単球性白血病 (M4)

DEK-CAN mRNAにより微小残存病変を追跡できた6;9転座型急性骨髄単球性白血病 (M4)

染色体異常を有するsideroblastic anemiaで発症し, acute myelomonocytic leukemiaに移行した1症例

染色体異常を有するsideroblastic anemiaで発症し, acute myelomonocytic leukemiaに移行した1症例

Recurrent Mutations Found In Acute Monocytic Leukemia by Exomes Sequencing

AbstractAbstract 2490Acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5), is a distinct group with characteristic clinical features and has been reported to have a poor prognosis. A subset of AML-M5 is associated with various chromosomal translocations involving the MLL locus at 11q23, while mutations are also reported in genes such as NPM1, FLT3, NRAS at different frequencies in this disease. However, these genetic changes occur only in a part of AML-M5 patients and some of them lack specificity due to the presence in other AML subtypes. To address the important genetic and molecular factors for the pathogenesis of M5 in a comprehensive manner, we sequenced exomes from nine AML-M5 initial bone marrow (BM) samples and matched control samples. We identified 64 somatic mutations within the coding sequences of 61 genes including 57 point mutations and 7 insertions or deletions (indels), among which 17 genes had at least 2 cases of mutations in 100 validated AML-M5 BM samples. We tested a part of these gene mutations in different subtypes of AML and found that some mutations are restricted to AML-M5 and AML-M4 (acute myelomonocytic leukemia) with more than 15% and 10% frequency, respectively. Thus, by systemic sequencing of exomes from a group of AML-M5 cases, we discovered recurring mutations that may play an essential role in the pathogenesis of AML with monocytic features.The prognostic significance of the mutations will be present in details. Disclosures:No relevant conflicts of interest to declare.

5-Azacitidine In Patients with Myelodysplasia and Acute Myeloid Leukemia: a Single Centre Experience

AbstractAbstract 4969 IntroductionHypomethylating agents have recently been shown to prolong overall survival and improve quality of life in patients with INT-2 and high IPSS risk myelodysplasia (MDS) and low bone marrow blast count acute myeloid leukemia (AML). Patients and MethodsSince September 2008 we have been treating 26 patients affected by acute myeloid leukemia (13 patients), MDS (11 patients) or chronic myelomonocytic leukemia (2 patients) with 5-azacitidine.According to recent guidelines, most of myelodysplastic patients eligible for treatment belonged to the IPSS INT-2 or high risk groups. Patients with acute myeloid leukemia had a medullary blast count of 20–30%, except for four cases. Patients with chronic myelomonocytic leukemia had a medullary blast count > 10% and < 20%. The median age of patients when treatment was started was 69,5 years (range: 51–82). Azacitidine was administered subcutaneously (75 mg/m2/d) for 7 days of every 28-day cycle until loss of response or disease progression. Patients received a median number of 5,5 cycles of therapy (range 1–24). We evaluated overall improvement (CR + PR+ HI), the best response obtained and adverse events in the overall study population, according to International Working Group MDS and LMA criteria. In the subgroup of patients who received at least 6 cycles of therapy (10 patients) we also evaluated overall survival (OS) and progression free survival (PFS). ResultsThe results of 5-azacitidine therapy in our cohort of patients are described in Table 1.In the AML cohort, after a median number of 4 cycles (range 1–10), we observed a hematological improvement (HI) in 15% of the patients, a stable disease (SD) in 31% of patients and a lack of response in 38% of patients. In the MDS cohort, after a median number of 7 cycles (range 2–24), we observed a complete response (CR) (including a cytogenetic response) in 36.5% of patients, a partial response (PR) in 9% of patients, a hematological improvement in 45.5% of patients and a stable disease in 18% of patients. The overall improvement (CR + PR + HI) was 15% in the AML cohort and 91% in the MDS cohort. In the LMMC cohort, after a median number of 2.5 cycles (range 1–4), we observed a hematological improvement in 50% of the patients.In the subgroup of patients who received at least six cycles of therapy, the overall survival was 11.5 months and the progression free survival was 9 months. In the overall study population, two patients (8%) discontinued treatment as a result of adverse events. In four non-responder patients (15%) death was due to a rapid progression of disease. DiscussionIn this analysis we have reported good response rates in MDS patients, with 36.5% of complete response (including a complete cytogenetic response). No CR or PR were observed in AML patients; however, in this subgroup of patients the median follow-up was short (median number of cycles of therapy: four). Furthermore, in these patients even a stable disease with no need of hospitalization and a good quality of life can be considered an important result. Due to the short period of follow-up, 57.5% of our patients received fewer than 6 cycles of 5-azacitidine and their therapy is still ongoing. ConclusionsThe limited number of cases and the short period of follow-up did not allow us to evaluate overall survival and progression free survival in the overall study population. We will update these data as time progresses. We reported these data in the subgroup of patients who received at least six cycles of therapy. According to current evidence, we observed that 5-azacitidine plays an important role in the treatment of patients with MDS (both with high- and low-IPSS risk) and low bone marrow blast counts AML. In these subgroups of patients, 5-azacitidine prolongs survival and is well tolerated. In our limited experience, this drug had no efficacy when a higher degree of bone marrow blasts (> 30%) was present.Further trials should assess the number of cycles required for treatment, the role of hypometilating agents in low-risk MDS and in patients with AML and a bone marrow blasts counts > 30%. Disclosures:No relevant conflict of interest to declare. [Display omitted] Disclosures:No relevant conflicts of interest to declare.