Acute Myelomonocytic Leukemia Research Articles

Targeting Cell-Bound MUC1 on Myelomonocytic and Monocytic Leukemias and Leukemic Stem Cells: Therapeutic Implications

Monocytic neoplasms comprise a heterogeneous group of hematologic malignancies including chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5), and monocytic sarcoma. Monocytic or granulomonocytic hyperplasia is a finding frequently-if not invariably-shared by these different entities, as is a poor therapeutic outcome in the absence of hematopoietic stem cell transplantation. Cell surface molecules aberrantly expressed or overexpressed by leukemic cells represent potential disease-specific therapeutic targets. MUC1, a polymorphic type I high molecular weight glycoprotein represents such a molecule. MUC1 consists of an extracellular domain containing 20 to 125 tandem repeats of a 20 amino acid-long sequence, followed by a transmembrane domain and a short cytoplasmic tail leading to intracellular signaling. Cleavage of MUC1 yields two unequal chains: a large extracellular alpha subunit containing the tandem repeat array bound in a strong non-covalent interaction to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Essentially all anti-MUC1 antibodies reported to date target the highly immunogenic tandem repeat of the MUC1 alpha chain. Because the alpha chain binds the cell-bound domains of MUC1 only intermittently in an 'on-and-off' manner, agents directed against the alpha chain will not effectively target MUC1+ cells. In contrast, the MUC1 SEA domain represents a stable structure fixed to the cell surface at all times. We therefore generated mAbs that specifically recognize the cell-bound MUC1 SEA domain. One of them, a partially humanized murine mAb termed DMB-5F3 was used to examine the expression of MUC1 on AML cells by flow cytometry. A series of twenty-two AML samples (blood-derived n=12; bone marrow-derived n=10; AML0=2, AML1=2, AML2=10, AML4=1, AML5=5, AML6=2) collected either at the time of diagnosis or at relapse were analysed for MUC1 expression by flow cytometry. A murine mammary tumor cell line stably transfected with human MUC1 DNA served as control. Blasts cells from 5 AML samples highly expressed MUC1, and significantly, all were of monocytic or myelomonocytic lineage (AML4=1, AML5=4). Leukemic stem cells (CD34pos or CD34neg linneg) from the MUC1+ AMLs were examined and likewise found to express MUC1. In addition, AML cell lines MV411, MOLM14, and SHI-1 derived from monocytic leukemic lineage clearly expressed cell surface MUC1, while non- monocytic leukemic cell lines U937, K562, and HL60 had little or no expression. Normal monocytes and monocytes derived from patients with activated monocytosis were also found to express MUC1. Based on these findings we examined MUC1 expression in a series of myelomonocytic leukemia (CMML and JMML). In fifteen CMML samples examined (type 1 n=11, type 2 n=4) (blood n=7, BM n=7) 92%-100% (median 99.7%) of CD14+CD56+ CMML cells bound mAb DMB-5F3 to cell-surface MUC1. CD14+CD16+CD56+ blast cells from 2 pts with JMML were also found to express MUC1 (between 64% and 71 % positive). Based on these findings we conclude that expression of MUC1 is restricted to monocytic and myelomonocytic leukemias and that MUC1 represents an effective target for leukemic immunotherapy. Significantly, anti-MUC1 mAb also targets monocytic leukemic stem cells, reinforcing its therapeutic potential. The fact that the anti-MUC1 antibody DMB-5F3 can enter cells and thereby ferry Ab-bound toxin opens the way for us to demonstrate leukemic cell killing with anti-MUC1 mAb-immunotoxin conjugates. DisclosuresMoreau:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

P119: Cell aggregation increases drug resistance of acute myelomonocytic leukemia cells

Acute myelomonocytic leukemia (FAB M4) is one of the most common forms of acute myeloid leukemia (AML). This AML form is characterized by rapid accumulation transformed myeloblasts and monoblasts in bone marrow, with the rapid suppression of normal hematopoiesis. Bone marrow microenvironment is one of the main factors determining drug resistance of leukemic cells. It is known that the adhesion of leukemic cells to mesenchymal stem cell and bone marrow extracellular matrix (laminin, collagen) enhances their drug resistance. However, it remains unknown whether the emergence of drug resistance when cell–cell contacts are formed only between leukemia cells, without the involvement of bone marrow stromal elements. We studied the role of cell aggregation in drug resistance of leukemic cells. We used the bone marrow mononuclear cells (BMMC) isolated from the patients with acute myelomonocytic leukemia. For the formation of multicellular aggregates, BMMC were cultivated in 96-well plates coated with 1.5% agarose. We showed that resistance of BMMC to bortezomib, doxorubicin and fludarabine in multicellular aggregates was increased. In three-dimensional multicellular aggregates of BMMC index IC50 for bortezomib, doxorubicin and fludarabine was 7 ± 1 ng/ml, 1 ± 0.4 mkM and 0.8 ± 0.05 mkM, respectively. In control condition, index IC50 bortezomib, doxorubicin and fludarabine was significantly lower, 2 ± 0.5 ng/ml, 0.3 ± 0.05 mkM and 0.07 ± 0.001 mkM, respectively. In multicellular aggregates of BMMC number of mitotic cells and expression of Ki-67 protein were not significantly different from the control. It has also been shown that cells in multicellular aggregates increased expression antiapoptotic protein Bcl-2. Suppression of BMMC aggregation by culturing the cells in medium containing 0.9% methylcellulose resulted in decreased IC50 index for bortezomib, doxorubicin and fludarabine, 2 ± 0.7 ng/ml, 0.12 ± 0.004 mkM and 0.04 ± 0.005 mkM, respectively. Expression of the Bcl-2 protein was also decreased. This work demonstrates the involvement of cell aggregation in the formation of drug resistance phenotype in leukemic cells. The work was supported by the Russian Foundation for Basic Research (Russia) (No. 14-04-32183 , 14- 04-32191 ), the scholarship of the President of the Russian Federation (Russia) (No. SP-6867.2013.4 , SP-1519.2015.4 ), and by the Government of the Russian Federation (Russia) (No. 14.Z50.31.0028 ).

Detection of Dual IDH1 and IDH2 Mutations by Targeted Next-Generation Sequencing in Acute Myeloid Leukemia and Myelodysplastic Syndromes

Studies in myeloid neoplasms have described recurrent IDH1 and IDH2 mutations as primarily mutually exclusive. Over a 6-month period of clinical testing with a targeted next-generation sequencing assay, we evaluated 92 patients with acute myeloid leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia and identified a subset of 21 patients (23%) who harbored mutations in either IDH1 or IDH2. Of the 21 patients with IDH mutations, 4 (19%) were found to have single nucleotide variants in both IDH1 and IDH2. An additional patient included in the study was found to have two different IDH2 mutations. The mutations were typically present at different variant allelic frequencies, with one predominating over the other, consistent with the presence of multiple subclones in a single patient. In one case, the variant allelic frequencies in both IDH1 and IDH2 were equally low in the setting of a high percentage of blasts, suggesting that the IDH mutations were unlikely to be present in the founding clone. Given these data, we conclude that dual IDH1/2 mutations likely were previously underestimated, a finding that may carry important treatment implications.

Abstract 5348: The extracellular domain of thrombomodulin induces erythroid differentiation and hampers proliferation of human leukemia cells in vitro and in vivo

Abstract Thrombomodulin (TM) is a glycosylated type I transmembrane molecule with multiple domains that include an NH2-terminal lectin-like region, six tandem epidermal growth factor (EGF)-like structures, an O-glycosylation site-rich domain, a transmembrane domain, and a cytoplasmic tail domain. TM is ubiquitously expressed on endothelial cells and acts as an anticoagulant via generation of activated protein C. Recombinant human soluble thrombomodulin (rTM) comprises the active, extracellular domain of TM, and inactivates coagulation. Use of rTM for the treatment of disseminated intravascular coagulation was approved in Japan in 2008. We previous showed that exposure of acute promyelocytic leukemia or acute myelomonocytic leukemia cells to rTM induced myeloid differentiation and growth arrest in vitro (Exp Hemat. 2012, Leuk Res. 2012). The present study found that exposure of K562 and KU812 cell lines that were derived from chronic myeloid leukemia cells to rTM increased expression of cell surface glycophorin CD235a in association with upregulation of GATA1 mRNA, indicating a differentiation of leukemia cells toward erythroid lineage. Interestingly, treatment of K562 bearing immunodeficient mice with rTM significantly decreased number of K562 cells infiltrating in liver and prolonged their survival compared to mice who received control diluent. These observations shed light on a novel function of TM in hematopoietic cells. Note: This abstract was not presented at the meeting. Citation Format: Takayuki Ikezoe, Jing Yang, Chie Nishioka, Goichi Honda, Akihito Yokoyama. The extracellular domain of thrombomodulin induces erythroid differentiation and hampers proliferation of human leukemia cells in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5348. doi:10.1158/1538-7445.AM2015-5348

Prognostic factors of childhood and adolescent acute myeloid leukemia (AML) survival: Evidence from four decades of US population data

Growing insight into prognosis of pediatric acute myeloid leukemia (AML) survival has led to improved outcome over time and could be further enhanced through investigation using a large number of patients. To characterize the extent of the association of pediatric AML survival with its identified prognostic factors, we analyzed the United States population-based Surveillance Epidemiology and End Results (SEER) large dataset of 3442 pediatric AML patients diagnosed and followed between 1973 and 2011 using a Cox proportional hazards model stratified by year of diagnosis. Patients diagnosed between 10 and 19 years of age were at a higher risk of death compared to those diagnosed before age 10 (adjusted hazard ratio (aHR): 1.30, 95% confidence interval (CI): 1.17–1.44). African Americans (1.27, 1.09–1.48) and Hispanics (1.15, 1.00–1.32) had an elevated risk of mortality than Caucasians. Compared to the subtype acute promyelocytic leukemia, AML with minimal differentiation (2.44, 1.78–3.35); acute erythroid leukemia (2.34, 1.60–3.40); AML without maturation (1.87, 1.35–2.59); and most other AML subtypes had a higher risk of mortality, whereas AML with inv(16) had a substantially lower risk. Age at diagnosis, race-ethnicity, AML subtype, county level poverty and geographic region appeared as significant prognostic factors of pediatric AML survival in the US. Contrary to previous findings, the subtypes of AML with t(9;11)(p22;q23)MLLT3-MLL, AML without maturation and acute myelomonocytic leukemia emerged to be indicative of poor outcome.

The Fc-alpha receptor is a new target antigen for immunotherapy of myeloid leukemia.

Antibody-based immunotherapy of leukemia requires the targeting of specific antigens on the surface of blasts. The Fc gamma receptor (CD64) has been investigated in detail, and CD64-targeting immunotherapy has shown promising efficacy in the targeted ablation of acute myeloid leukemia (AML), acute myelomonocytic leukemia (AMML) and chronic myeloid leukemia cells (CML). Here we investigate for the first time the potential of FcαRI (CD89) as a new target antigen expressed by different myeloid leukemic cell populations. For specific targeting and killing, we generated a recombinant fusion protein comprising an anti-human CD89 single-chain Fragment variable and the well-characterized truncated version of the potent Pseudomonas aeruginosa exotoxin A (ETA'). Our novel therapeutic approach achieved in vitro EC50 values in range 0.2-3 nM depending on the applied stimuli, that is, interferon gamma or tumor necrosis factor alpha. We also observed a dose-dependent apoptosis-mediated cytotoxicity, which resulted in the elimination of up to 90% of the target cells within 72 hr. These findings were also confirmed ex vivo using leukemic primary cells from peripheral blood samples of three previously untreated patients. We conclude that CD89-specific targeting of leukemia cell lines can be achieved in vitro and that the efficient elimination of leukemic primary cells supports the potential of CD89-ETA' as a potent, novel immunotherapeutic agent.

Bilateral Adrenal Masses in a Patient in Remission From Acute Myelomonocytic Leukemia

CLINICAL PRESENTATION A 51-year-old man presented in the outpatient department with a 1-month history of persistent low-grade fever. He was diagnosed with acute myelomonocytic leukemia 4 years before and was treated with induction therapy of standard-dose cytarabine with daunorubicin, followed by consolidation therapy with cytarabine. Until this time, he had been in complete remission. Workup on admission revealed no pathogen detected in blood or sputum cultures. The early morning serum cortisol was 15.89 mg/dL, and urine vanillylmandelic acid level was normal. The bone marrow aspiration revealed normal cellularity and no residual blasts. The coronal computed tomography (CT) scan of abdomen and pelvis demonstrated bilateral adrenal masses, 10.8 cm in diameter on the right side (Figure 1A, black arrow) and 10.0 cm in diameter on the left side (Figure 1A, arrowhead). Celiac trunk nodal involvement was also found (Figure 1A, white arrow). 18Fluorine-fluorodeoxyglucose (18F-FDG) positron emission tomography/CT imaging showed “H” appearance of intense 18F-FDG uptake at bilateral adrenal glands (the maximum value of standardized uptake value [SUVmax] up to 30) and an ill-defined FDG-avid tumor (about 4.5 cm, SUVmax from 18.3 to 20.9) between bilateral lesions (Figure 1B). The core biopsy of the right adrenal mass disclosed features of diffuse large B-cell lymphoma, and he was treated with rituximab plus cyclophosphamide, liposomal doxorubicin, vincristine, and prednisone (R-CLOP). Follow-up CT scan obtained 3 months later showed nearly complete resolution of bilateral adrenal mass. Although secondary involvement of the adrenal gland occurs in up to 25% of patients with non-Hodgkin’s lymphoma, its clinical manifestation is usually ambiguous. Positron emission tomography/CT has potential advantages over traditional radiological examinations for diagnosis, staging of disease and detecting recurrences of malignancies. In this case, the “H” appearance of significantly higher 18F-FDG uptake helped to differentiate secondary adrenal involvement from a primary adrenal malignancy.

WEHI-3 cells inhibit adipocyte differentiation in 3T3-L1 cells

By investigating the anti-adipogenic effects of WEHI-3 cells – a murine acute myelomonocytic leukemia cell line – we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPARγ, C/EBPα, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway, β-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and β-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and β-catenin are involved in this process.

The antileukemia roles of PP242 alone or in combination with daunorubicin in acute leukemia.

PP242 is a novel dual mammalian target of rapamycin (mTOR) inhibitor that simultaneously inhibits mTORC1 and mTORC2, and its antileukemia effect has been sufficiently investigated here. The human acute leukemia cell lines and primary blasts were treated with PP242 alone or in combination with daunorubicin (DNR). Cell proliferation was examined using an MTT assay. The phosphorylation expression of the Akt/mTORC1/eIF4E signaling pathway was assessed by western blot analysis. The assembly of the eIF4F translation initiation complex was examined using a 7-methyl-guanosine cap affinity assay. PP242 significantly induced cytotoxicity in human acute leukemia cells, especially in combination with DNR. The phosphorylation levels of eIF4E (p-eIF4E) at Ser209 influence the antileukemia roles of PP242. As expected, the antiproliferative effects of PP242 on leukemia cells with low p-eIF4E expression, such as the acute promyelocytic leukemia NB4 cell line and AML-M3 primary blasts, were poor. Surprisingly, the effects of PP242 in leukemia cells with high p-eIF4E expression, such as the acute myelomonocytic leukemia THP-1 cell line and M4-M5 primary blasts, were also weak. In contrast, PP242 exerted a significant antiproliferative effect in the Ph+ acute lymphoblastic leukemia SUP-B15 cell line and the mantle cell lymphoma JEKO-1 cell line, which had intermediate p-eIF4E levels. PP242 inhibited the translation of the antiapoptotic protein Mcl-1 by downregulating the Akt/mTORC1/eIF4E signaling pathway. More importantly, DNR activated the Akt/mTORC1/eIF4E signaling pathway, whereas PP242 effectively eliminated this deleterious side effect of DNR and synergistically enhanced the anticancer ability of DNR treatment. PP242, especially in combination with DNR, exerts significant antileukemia effects.

Cytologic findings of hematologic malignancies in bronchoalveolar lavage fluid

Bronchoalveolar lavage (BAL) is often performed in leukemia and lymphoma patients with pulmonary infiltrates, mainly to rule out infection. However, malignant hematopoietic infiltrates are uncommon and a comprehensive cytologic study has not yet been performed. We retrospectively reviewed all BAL samples from our institution for the past 22years (November 1992-October 2014). There were 37 cases of hematologic malignancies identified on BAL specimens (21 female patients and 16 male patients, age 22-80 years). Eighteen patients (49%) had pneumonia-like symptoms at the time of initial diagnosis of their malignancy, including fever, dyspnea, respiratory distress/hypoxia, and cough. The biopsy-proven cases were 25 leukemia (12 acute myeloid leukemia, 6 acute promyelocytic leukemia, 2 acute monocytic leukemia, 2 acute myelomonocytic leukemia, 1 chronic myeloid leukemia in blast phase, 1 large granular leukemia, and 1 plasma cell leukemia), 11 lymphoma (8 diffuse large B-cell lymphoma, 1 mantle cell lymphoma, 1 natural killer/T-cell lymphoma, and 1 T-cell lymphoma), and 1 multiple myeloma. Chest X-ray findings included opacities, consolidation, and interstitial edema. Four patients had BAL specimens with concomitant microorganisms. Eighteen patients subsequently died (2 days to 4years), 15 were alive (3 weeks to 8 years of follow-up), and 4 were lost to follow-up. BAL is especially important in distinguishing inflammatory/infectious processes from neoplastic disorders because many patients with hematologic malignancies can have pneumonia-like symptoms as part of their initial disease presentation. Causative pathogens are identified in only a minority of malignant BAL specimens from these patients. Lung involvement in patients with hematologic malignancies carries a poor prognosis.

GATA2 deficiency.

GATA2 deficiency is a germline disease that causes a wide spectrum of phenotypes including viral and bacterial infections, cytopenias, myelodysplasia, myeloid leukemias, pulmonary alveolar proteinosis and lymphedema. The age of clinical presentation ranges from early childhood to late adulthood, with most occurring in adolescence to early adulthood. We review the expanding GATA2-deficient phenotype, molecular genetics of disease and developments in treatment. GATA2 mutations have been found in up to 10% of those with congenital neutropenia and/or aplastic anemia. Heterozygous mutations appear to cause haploinsufficiency due to either protein dysfunction or uniallelic reduced transcription. Disease-associated mutations in intronic regulatory elements or variations within the 5' leader exons indicate that regulation of GATA2 is critical. Those with GATA2 mutations are at high risk for myelodysplasia, cytogenetic abnormalities, acute myeloid leukemia or chronic myelomonocytic leukemia. Bone marrow transplantation has been successful for both hematopoietic and pulmonary alveolar proteinosis repair. GATA2 is a zinc finger transcription factor essential for embryonic and definitive hematopoiesis as well as lymphatic angiogenesis. GATA2 deficiency is caused by a variety of mutations in the GATA2 gene and can have variable presentation, onset and outcome. Patients are susceptible to mycobacterial, viral and fungal infections and can develop myelodysplasia, acute or chronic leukemias, lymphedema and pulmonary alveolar proteinosis. Hematopoietic stem cell transplantation reverses most of the clinical phenotype with good long-term outcomes.

Hematological Malignancies: Analysis of Myelogram Results over 21 Years in Lome Teaching Hospitals

Objectives: Determine the frequency of malignancies diagnosed in myelogram seen in hematology services of Campus and Sylvanus Olympio Teaching Hospitals in Lome from 1992 to 2013. Study the epidemiology profile of these diseases. Materials and methods: It was an analytical retrospective study of 469 cases of malignancies diagnosed in 1511 myelograms during 21 years in both teaching hospital of Lome. Results: Blood disorders represent 31.70% of the results of myelogram performed during 21 years in the Teaching Hospital of Lome. Each year, there is an average of 22.3 cases diagnosed hematological malignancies. The most hematological malignancies were Kahler's disease (28.57%), chronic myeloid leukemia (24.52%), chronic lymphocytic leukemia (22.17%), acute lymphoblastic leukemia (12.58%) and acute myeloid leukemia (8.10%). Other (4.05%) were represented by the essential hypereosinophilic syndrome (4 cases), hairy cell leukemia (4 cases), Sezary lymphoma (3 cases), essential thrombocythemia (3 cases), Waldenstrom's disease (2 cases), acute myelomonocytic leukemia (1 case), prolymphocytic leukemia (1 case) and plasma cell leukemia (1 case). By gender, the CLL is most predominant in women against CML in men. Hemopathies were observed at all ages of life. Conclusion: Hematological malignancies are diagnosed in the Teaching Hospital of Campus and Sylvanus Olympio in Lome with a high relatively frequency. This prevalence must be higher if all cases of malignancies diagnosed in Togo were recorded in a national cancer register. This study aims to knowledge of epidemiological aspects of hematological malignancies and improve the care of patients with hematological malignancies by the provision of adequate anti-mitotic drugs.

Azacitidine in outpatient treatment - single center experience.

Aim of the studyAzacitidine is a hypomethylating agent which is used in the treatment of myelodysplastic syndromes, acute myeloid leukemia and chronic myelomonocytic leukemia. Because of good tolerance to the drug, azacitidine can be administered both during hospitalization and in an outpatient setting.The aim of our retrospective analysis was to assess the efficacy of azacitidine treatment in patients with a myelodysplastic syndrome and with acute myeloid leukemia who had received treatment in hospital and in an ambulatory care setting. Offsets in the course of azacitidine administration and discontinuations of treatment have a negative impact on patients’ response to the therapy.Material and methodsThe study included 31 patients. Sixteen patients received azacitidine in an ambulatory care setting, 15 patients within their hospitalization.ResultsA hematologic response was achieved in 48% of the patients. Forty-one percent of the cycles were delayed. In an outpatient setting, 62% of the cycles were administered systematically, while during hospitalization the patients received 54% of cycles on time. Administrative problems caused the delay of 26% of the cycles.ConclusionsAzacitidine has a high tolerance level and a high safety profile which allows for its use in an outpatient care setting. Outpatient administration of azacitidine is feasible and safe without compromising efficacy.

Cardiopulmonary Failure Requiring ECMO Bypass Resulting from Leukemia Cell Lysis in a Patient with Childhood Acute Myelomonocytic Leukemia.

Background. Childhood AML patients are at increased risk for early fatal pulmonary complications. Pulmonary leukostasis and systemic inflammatory response syndrome (SIRS) following leukemia cell lysis are the likely etiologies. Observation. Soon after initiation of AML chemotherapy, an 18-month-old female who met SIRS criteria sustained cardiopulmonary failure requiring ECMO support. Upon recovery, the patient went on to complete therapy and remains in remission without permanent neurologic or cardiac sequelae. Conclusion. Cytokine release syndrome from rapid cell lysis was the likely cause as infectious workup failed to reveal a definitive etiology and drug hypersensitivity testing to the chemotherapy agents was negative.

Alkaline phosphatase is a useful cytochemical marker for the diagnosis of acute myelomonocytic and monocytic leukemia in the dog.

Immunophenotyping has replaced cytochemical staining as the preferred technique for classifying acute leukemia. However, some acute myeloid leukemias (AML) lack lineage-associated markers. In our experience, alkaline phosphatase (ALP) is expressed in immature canine monocytes. We hypothesized that ALP is a useful marker for monocytic AML. The objective was to compare ALP expression in neoplastic cells from dogs with lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoid leukemia (ALL), and AML. Alkaline phosphatase results were retrieved from medical records of dogs with acute leukemia. Smears from dogs with lymphoma or leukemia were also prospectively stained for ALP activity. CLL was based on persistent lymphocytosis (10×10(9) /L) and acute leukemia on ≥20% blasts in blood or bone marrow. ALL was classified based on positive phenotyping for T- or B-lymphocyte antigens, and AML on positive phenotyping for CD11b, CD11c or CD14, or cytochemical staining for chloroacetate esterase, Sudan Black B, or myeloperoxidase. There was no ALP activity in all 49 lymphomas and 7 CLLs. Weak ALP activity was seen in 31% of 14 ALL (all T-ALL). ALP activity was seen in all 20 AML (P<.001 vs ALL) with strong activity in 64% (vs 25% ALL) in most neoplastic cells (median 75% vs 9% ALL, P=.020). Of AML, 80% were CD34+ (vs 39% ALL, P=.027) and 100% were MHCII- (vs 43% ALL, P=.002). ALP activity may be useful for AML confirmation in dogs, particularly if neoplastic cells only express CD34+ on immunophenotyping.

Specific Patterns of Molecular Mutations Determine the Morphologic Differentiation Stages in Acute Myeloid Leukemia (AML)

Background: The FAB classification of AML was based on the differentiation state and the involved cell lineages and recognized some close associations between genetic abnormalities and cytomorphology: CBFB-MYH11 and AML with abnormal eosinophils, PML-RARA and acute promyelocytic leukemia; RUNX1-RUNX1T1 and AML with maturation (FAB subtype M2). Associations with molecular mutations have not been examined in detail yet.Aim: Evaluate the impact of molecular mutations on the AML differentiation state.Material and Methods: The cohort comprized 3,703 patients with de novo AML (median age 67 yrs; range: 18.1-100.5 yrs). All cases were analyzed by cytomorphology and molecular mutation screening for 12 genes by next generation or Sanger sequencing in our laboratory. Based on cytomorphology and immunophenotyping we categorized the cohort into 11 subgroups: 1. AML with minimal differentiation (according to the FAB classification: M0; n=192), 2. AML without maturation (M1; n=1,015), 3. AML with maturation (M2; n=1,324), 4. acute promyelocytic leukemia (APL) with PML-RARA (M3; n=124), 5. the microgranular variant of APL (M3v, n=110), 6. acute myelomonocytic leukemia (M4; n=604), 7. acute myelomonocytic leukemia with abnormal eosinophils and CBFB-MYH11 (M4eo; n=81), 8. acute monoblastic leukemia (M5a; n=86), 9. acute monocytic leukemia (M5b; n=63), and 10. acute erythroid leukemia (M6; n=101). Cases with acute megakaryoblastic leukemia (M7; n=3) were not considered for statistical evaluation. All 3,703 cases were analyzed for FLT3-ITD and FLT3-TKD. All cases except M3/M3v and M4eo (n=3,487) were screened for NPM1 und MLL-PTD. Data on the mutation status of the following genes was available: FLT3-ITD (n=3,703), FLT3-TKD (n=3,703), CEBPA (n=2,684), RUNX1 (n=2,320), WT1 (n=2,224), ASXL1 (n=1,591), DNMT3A (n=1,294), NRAS (n=1,232), TP53 (n=1,046) and TET2 (n=974). Statistical significance of associations between the mutation frequencies and AML subtypes were assessed by Fisher’s exact test and corrected according to Benjamini-Hochberg (1995) to compensate for multiple testing.Results: The mutation rates of the analyzed genes are in line with published data.Total cohort: Significant associations were observed between the mutation frequencies of distinct genes and the differentiation stage of the AML. Figure 1 depicts genes showing significantly different mutation rates within distinct AML subtypes compared to the rate of the total cohort. In detail, mutations in RUNX1 were most frequently observed in M0 (43.6%) and M2 (24.0%) and were nearly absent in M5a (3.5%) and M5b (0%). The highest TP53 mutation rates were observed in M6 (35.3%) and M2 (15.0%), the lowest in M4 (3.0%) and M1 (5.7%). NPM1 mutations were significantly more frequent in M5b (69.8%), M4 (56.3%), M5a (47.7%) and M1 (42.1%) than in M2 (24.2%), M6 (20.8%), M0 (3.1%), and absent in M3, M3v, and M4eo, respectively.NPM1 mutated subcohort (n=1,200): The overall DNMT3A mutation rate in this subcohort was 49.1%. It was significantly higher in M5b (75.0%) and M4 (61.6%) and significantly lower in M1 (40.6%), M5a (22.2%), and M6 (13.3%). Contrary, RUNX1 (overall frequency in NPM1mut cases 1.2%) and ASXL1 (overall 2.5%) mutations were more frequent in M5a (8.3% and 34.8%, respectively), while FLT3-ITD was more frequent in M1 (52.5%), and WT1 mutations more frequent in M6 (26.7%).CEBPA double mutated subcohort (n=108): ASXL1 and RUNX1 mutations were 5- and 7-fold more frequent in M2 than in M1, but did not reach significance.Conclusions:Specific patterns of molecular mutations are linked to distinct AML differentiation stages: 1) RUNX1 and ASXL1 with M0 2) NPM1, ASXL1 and TET2 with M5a, and 3) NPM1 and DNMT3A with M5b. 4) The differentiation stage of NPM1 mutated AML is driven by the presence of additional mutations: DNMT3A mutations lead to a (myelo-)monocytic differentiation, while mutations in RUNX1 or ASXL1 lead to a monoblastic phenotype. 5) FLT3-ITD is most frequent in NPM1 mutated AML without maturation and WT1 mutations are most frequent in acute erythroid leukemia bearing NPM1mut. [Display omitted] DisclosuresRose:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Leukocytapheresis for the treatment of hyperleukocytosis secondary to acute leukemia.

Patients presenting with new or recurrent acute leukemia, particularly of the myeloid lineage, with WBC counts exceeding 100 × 10(9)/L are often considered for leukocytapheresis, especially if they are experiencing symptoms of leukostasis. These symptoms are thought to occur because of blast aggregates and WBC thrombi in the circulation, which reduce blood flow. Leukostasis may cause various complications, including hyperviscosity syndrome, vascular occlusion resulting in intracranial hemorrhages and respiratory failure, and perivascular leukemic infiltrates. Leukostasis occurs more commonly with a high WBC count and with leukemias of monocytoid lineage such as acute myelomonocytic leukemia, which is a reflection of the nature of the leukemic blasts. Leukocytapheresis is used in an effort to quickly decrease a patient's circulating blast count, which can both prevent the development of leukostasis and provide symptomatic relief of leukostasis. However, the impact of leukocytapheresis on early- and long-term mortality is controversial, with several studies producing conflicting results. In this chapter, the pathophysiology of leukostasis, performance of leukocytapheresis, and efficacy of this treatment are reviewed.

Azacitidine access program for Belgian patients with myelodysplastic syndromes, acute myeloid leukemia or chronic myelomonocytic leukemia

Objective:Azacitidine (Vidaza) is approved in Europe for treatment of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) with 20–30% bone marrow (BM) blasts, and chronic myelomonocytic leukemia (CMML) with 10–29% BM blasts and no myeloproliferative syndrome (i.e. <13.000/μL white blood cells). In Belgium, the azacitidine reimbursement process can take several months, and is often delayed at submission for medical assessment by the Belgian National Institute for Health and Disability Insurance of incomplete patient dossiers, due to disease complexity and classification, and administrative burden. We describe the Vidaza Access Program and its application to an initial 175 patients. Individual medical dossiers were reviewed for completeness to facilitate patient access to treatment in Belgium.Methods:A standardized anonymized patient information form is completed by the physician and sent for review to the Belgian Celgene Medical Department. The form is reviewed within three working days and, for complete dossiers, Celgene grants a financial guarantee for treatment with azacitidine. The patient can then be treated without the hospital being subjected to financial risk.Results:Between January 2013 and June 2014, 63 physicians (53 Belgian hospitals) recruited 175 patients. In total, 163 patient dossiers were approved by Celgene (120 MDS, 36 AML, and 7 CMML), of which 104 dossiers were also approved by the review committee and 49 have been waiting for a final decision for a median of 6 months; no information is currently available for the remaining 10. No dossiers approved by Celgene have been rejected by the review committee.Conclusions:The Celgene Vidaza Access Program offers support to healthcare professionals in the appropriate use of azacitidine. By facilitating the assessment of patient dossiers and providing a financial guarantee for prescribers and hospitals, treatment can be initiated more rapidly and patients may better benefit from azacitidine treatment.

Leukemia-induced Gingival Overgrowth as an Early Manifestation in Pregnancy: A Case Report

Leukemia is a neoplastic disease with early oral and periodontal manifestations such as ulceration, infection, bleeding and gingival hyperplasia. This paper describes a 39-year-old pregnant woman with a diagnosis of acute myelomonocytic leukemia (AML), with gingival enlargement in the upper and lower jaws. A gingival biopsy was performed, followed by a complete blood count and peripheral blood smear. From a histopathological view, infiltration of the neoplastic (myelomonocytoblastic) cells was observed and many immature lymphoid cells were revealed by the hematologic tests. The interesting clinical finding about this case was the absence of spontaneous bleeding and profuse bleeding on probing. This case is reported to emphasize the leukemia-induced gingival enlargement in pregnancy and the early diagnosis of AML by dentists, which results in immediate treatment and management of the patient.

Abstract 2306: Pre-clinical development of 4′-thio-2′-deoxycytidine (TdCyd) as a DNA-demethylating agent for use in treating solid tissue tumors

Abstract Targeting cancer epigenetic control of cell growth via DNA methylation has been successful in treating hematologic diseases, such as Decitabine (DAC) and Azacitidine for Myelodysplastic Syndrome including Acute Myelomonocytic Leukemia. This success has not extended to solid tissue tumors. The Division of Cancer Treatment and Diagnositcs of NCI has initiated pre-clinical development of TdCyd as an agent for treating solid tumors after promising early results in a lung adenocarcinoma xenograft model (NCI-H23). IP dosing at 5MKG (0.56 MTD) in nu/nu mice on a Q5D x 3 cycle schedule resulted in tumor stasis with no accompanying weight loss in the mice. A DAC-treated control arm treated at MTD resulted in tumor growth delay but not stasis, and a 10% weight loss was noted. Intratumoral levels of DNMT1 were reduced to undetectable levels in xenografts post administration of TdCyd by ELISA and Western Blot assays, but were unaffected by DAC treatment. Mass Spectrometry analysis demonstrated incorporation of both TdCyd and thiothymidine (TdThd) into H23 DNA. In vitro experiments on a selected panel of cancer cell lines confirmed the conversion of TdCyd to the triphosphate and re-expression of tumor suppressor proteins p15 and p16. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Robert J. Kinders, Melinda Hollingshead, Jaideep Thottassery, William B. Parker, Thomas D. Pfister, Lawrence W. Anderson, Joseph E. Tomaszewski, Jerry M. Collins, James H. Doroshow. Pre-clinical development of 4′-thio-2′-deoxycytidine (TdCyd) as a DNA-demethylating agent for use in treating solid tissue tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2306. doi:10.1158/1538-7445.AM2014-2306