AML-M2 Research Articles

Adult acute myeloid leukemia with TLS-ERG fusion gene positive: report of two cases and review of literature

Objective To investigate the clinical characteristics and prognosis of adult acute myeloid leukemia (AML) with TLS-ERG fusion gene positive. Methods Adult two AML patients with TLS-ERG fusion gene positive in the First Affiliated Hospital of Zhengzhou University in January 2017 and March 2018, respectively were enrolled, and their clinical characteristics and prognosis were analyzed. Results Both patients with TLS-ERG fusion gene positive were males. According to morphology classification, the first case was classified to AML-M5, and the other one was AML-M4. Both patients' immunophenotypic features showed CD56 expression, and CD25 was expressed in the first patient simultaneously. Both patients were treated with induction and consolidated intensive therapy based on Chinese guidelines for diagnosis and treatment of adult AML (not acute promyelocytic leukemia) and the guidelines for diagnosis and treatment of AML (relapse/refractory) in China. The first patient died 11 months after diagnosis, and the second patient remained in remission until the end of follow-up in February 2019 (12 months after diagnosis). Conclusion TLS-ERG fusion gene has low incidence rate, high recurrence rate, poor reinduction effect, poor prognosis and short survival time in adult AML. Key words: Leukemia, myeloid, acute; Translocation, genetic; TLS-ERG fusion gene

Induction of a Leukemic Bone Marrow Niche Mediated By TGFB1 of Leukemic Blasts in Pediatric Acute Mekaryoblastic Leukemia: Results of an in Vitro and In Vivo Analysis

Background: An increasing knowledge about the bone marrow niche demonstrates the high complexity of leukemogenesis. Mesenchymal stromal cells (MSC) are important members of the bone marrow niche and source of fibrosis. Further, the microenvironment seems to be regulated by megakaryocytes and platelets via cytokines, such as transforming growth factor beta 1 (TGFB1). Despite extensive research, the pathogenesis of the bone marrow niche in childhood leukemia and the therapeutic potential is still unclear. We focus on acute childhood megakaryoblastic leukemia (AMKL) as a disease model and include patients with (ML-DS) and without Down syndrome. Based on similar clinical progressions - myelofibrosis occurs as a side-effect of both leukemia subtypes; these two diseases suit to characterize the leukemic bone marrow niche. Methods: We performed a comprehensive characterisation of MSC from ML-DS (n=9), AMKL patients (n=5) and healthy donors (HD; n=6) via e.g. differentiation assays (adipogenic, osteogenic), gene expression profiles and western blot analysis. In addition, we established an in vivo model with humanized ossicles, representing a human bone marrow microenvironment (as described by Chen et al. 2012; Reinisch et al. 2015): We injected MSC mixed with pooled human umbilical vein endothelial cells (HUVEC) and Matrigel subcutaneously into NOD scid gamma (NSG) mice. After 8 weeks, the engrafted ossicles were injected with megakaryoblastic cells (CMK cell line); injected ossicles (n=16); uninjected ossicle (n=27), MSC from ML-DS (n=19 ossicles), AML M1 (n=15 ossicles) and HD (n=9 ossicles). After 4 more weeks, histopathology evaluation of fibrosis in the ossicles was performed in accordance with the European Consensus on Grading Bone Marrow criteria from an independent pathologist. Results: The detailed characterisation of MSC with ML-DS and AMKL demonstrated a high similarity to MSC of HD: morphology, osteogenic differentiation potential, colony forming unit-fibroblast assay, proliferation and gene expression profiles. However, two differences emerged in our analysis: MSC showed a decreased adipogenic differentiation potential in ML-DS and AMKL compared to HD (ML-DS vs. HD=0.26-fold, p<0.05; AMKL vs. HD=0.50-fold). Gene expression profiling identified an upregulation of IGF2BP3, an oncofetal RNA binding protein, in MSC of ML-DS compared to HD confirmed by qRT-PCR (2.6-fold, p<0.05). IGF2BP3 is known to be highly expressed in many cancers and seems to be associated with proliferation. The increased level of IGF2BP3 (protein: IF2B3) was confirmed at protein-level detected by western blot analysis (ML-DS vs HD: 37.3-fold, p<0.05 and AMKL-MSC vs HD: 13.1-fold, p<0.05). TGFB1 - known to be secreted by leukemic megakaryoblasts - induced a fibrotic state in MSC regardless of their origin indicated by decreased adipogenic differentiation potential (treated vs. untreated: ML-DS 0.22-fold; AMKL 0.08-fold; HD 0.06-fold, p<0.05) and increased expression of collagen genes (qRT-PCR; COL1A1: ML-DS=1.63-fold, AMKL=1.80-fold (p<0.01), HD=1.66-fold (p<0.05); COL3A1: ML-DS=1.31-fold, AMKL=1.52-fold (p<0.05), HD=1.24-fold). The humanized bone marrow niche in our mouse model demonstrated a development of myelofibrosis after injection of the megakaryoblastic cell line (CMK): Grade 1 or 2 in 81% of the ossicles. The induction was independent of the MSC entity (HD/ML-DS). Of note, a monocytic subpopulation, which engrafted unexpectedly in ossicle from HD-MSC (n=3 ossicle), did not induce fibrotic fibers. Conclusion: Our data impressively illustrate the mutual influence between MSC and leukemic blasts that leads to a fibrotic microenvironment. This correlation has been observed in vitro but also in a unique mouse model. The interaction of MSC and leukemic blasts seems to be the key factor for the development of the leukemic niche in AMKL mediated inter alia by the TGFB pathway. However, we could identify several disease specific characteristics of MSC. Our model offers a unique opportunity to fundamentally examine of the leukemic niche in order to subsequently evaluate the potential therapeutic use in further studies. Disclosures Reinhardt: Novartis: Other: Participation in Advisory Boards; CSL Behring: Research Funding; Jazz: Other: Participation in Advisory Boards, Research Funding; Roche: Research Funding.

1100P - Interleukin-6 as a predictive marker for early response to induction chemotherapy in acute myeloblastic leukaemia

BackgroundThe early complete morphological remission (CR) after induction chemotherapy is a major independent prognostic factor for both achievements of CR and long-term outcome in acute myeloid leukemia. we studied the correlation between the serum IL-6 levels and the early morphological CR aiming to use serum level of IL-6 instead of invasive painful BM aspiration as a predictive factor for the early response to induction chemotherapy in AML patients. MethodsDuring the study period from February 2015 to May 2016, we investigate 36 patients. Eligibility criteria were the evidence of a diagnosis of AML (not AML-M3), both sexes, age of 18-60 years, Patients with a performance status of 0 to 2, and adequate organs functions. We excluded 6 ineligible patients from the study. All patients received induction chemotherapy of cytarabine and anthracycline in the standard “7 + 3” regimen. We did day 14 BM aspiration to detect the presence of early morphological complete remission (≤ 5% marrow blasts) and blood sampling for measurement of the interleukin-6 at presentation and day 14 of starting of induction chemotherapy. ResultsThis study included eligible 30 newly diagnosed AML patients with age ranged from 18-60 years (Mean ± SD, 39.53 ± 13.54). The range of marrow blasts at presentation was 20-93% with the Mean ± SD of 58.10% ± 23.10%. As regards the levels of IL-6 at presentation, the mean ± SD was 42.46±28.10 pg/ml and the range was 21.3 - 140.6. Day 14 BMA revealed 8 out of the 30 enrolled patients achieved complete morphological remission (≤ 5 % marrow blasts). There was a statistically significant decrease in IL-6 serum level in patients achieved morphological CR (mean + SD, 18.26 ± 5.75; range, 10.7 - 28.9pg/ml) than those did not (mean + SD, 26.07 ± 4.78; range, 21.3 - 32.7pg/ml) (P=0.017). At Day 14 of induction chemotherapy, there was a significant positive correlation between IL-6 and marrow blasts (r=0.745, P=0.000*). ConclusionsInterleukin-6 is suggested to be a good predictive marker for early response to induction chemotherapy in patients with acute myeloblastic leukemia and may be used instead of invasive day 14 BM aspiration as a marker of this response. Legal entity responsible for the studyFaculty of Medicine, Assiut University. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

A MOUSE MODEL OF ACUTE MEGAKARYOBLASTIC LEUKEMIA TO REVEAL NOVEL THERAPEUTIC VULNERABILITIES

Non-down syndrome pediatric acute megakaryoblastic leukemia (AMKL, AML M7; FAB classification) is an aggressive disease, and classical protocols used for the treatment of AML in children with AMKL have proven unsuccessful. This is largely due to current poor understanding of the molecular mechanisms by which oncogenics fusions – which are frequently occurring in pediatric but not adult AMKL - drive leukemogenesis and sustain cellular self-renewal. We recently developed a mouse model of AKML as a tool to identify and characterize novel therapeutic targets. AMKL was generated with a penetrance of 50-70% and affected mice displayed striking splenomegaly. Our model recapitulates many features of the human disease which is characterized by an accumulation of megakaryocytic blasts in the peripheral blood and bone marrow as well as infiltration into extramedullary tissues such as the spleen and liver. Blasts also express one or more megakaryocyte markers (CD61, CD41, CD42, factor VIII), and the morphology of bone marrow and spleen cells is consistent with megakaryoblasts and megakaryocytes. In addition, increased proportions of stem and progenitor cells (c-Kit+/Sca1+), progenitors (c-Kit+/Sca1-) and immature megakaryoblasts (c-Kit+/CD41+) were detected in the spleen and bone marrow of leukemic mice. Finally, genome-wide RNA sequencing of leukemic blasts revealed a megakaryocytic signature (Itga2b, Itgb3, Gp1ba, Vwf), while gene set enrichment analysis uncovered, among other pathways, increased Notch and Wnt/s-catenin signalling with an altered metabolic gene expression profile. To conclude, by taking advantage of our mouse model, we are currently investigating signalling pathways that are altered in leukemic megakaryoblasts to ultimately uncover unique therapeutic vulnerabilities specific to pediatric non-DS AMKL.

Auer Rods, Faggots and Phi Bodies and the Role in the DIC (Disseminated Intravascular Coagulopathy) of Acute Promyelocytic Leukaemia!) = AML M2 Microgranular Variety!

Auer rods are elongated, azurophilic plasmic inclusions in leukaemic blasts belonging to the myeloid series. Their presence confirms the diagnosis of acute myeloid leukaemia..

Clinical characteristics of myeloid sarcoma: analyses of 12 cases

Objective To investigate the clinical characteristics, curative effect and prognosis of myeloid sarcoma. Methods The clinical data of 12 patients with MS diagnosed at Xijing Hospital of Air Force Medical University from August 2008 to May 2018 were retrospectively analyzed. Their clinical manifestations, diagnosis, treatment and survival were analyzed. Results Twelve patients were 17 to 62 years old. The initial site included lymph node, external auditory canal, eye, buttock, lung, liver, pancreas, breast, skin, vertebra and its surroundings,and cervix. Among 11 patients with peripheral blood classification, bone marrow aspiration and bone marrow biopsy, 6 cases were isolated MS [one of which developed acute myeloid leukemia (AML)], 1 case was chronic myeloid leukemia in chronic phase, 2 cases were AML-M2, and 1 case was myelodysplastic syndrome (MDS), and 1 case was after aplastic anemia (AA) with no infiltration of bone marrow. Immunohistochemical results showed that LCA(+) (7/7), MPO(+) (12/12), CD43(+) (9/9), lysozyme(+) (5/7), CD3(-) (8/8), CD20(-) (9/9), CD34(+) (5/6), CD117(+) (7/7), and Ki-67(+) 30%-90%. Four patients were examined for bone marrow chromosomes, 2 patients with AML had t(8;21), 1 patient with MDS was 47, XX, +8, del(11)(q21), and 1 patient with CML was t(9;22). Two of the 12 patients were lost to follow-up. Among the 10 patients who were followed up, 6 died and 4 survived, and the median survival time was 21 months (2-27 months). Conclusions AA in stable phase with MS and CML in chronic phase with MS are rarely reported. The clinical manifestations of MS patients are varied, of which the common incidence sites are superficial lymph nodes, the infrequent sites are vertebra and its surrounding areas, and the rare sites are eye, pancreas, lung, liver, etc. The median survival time of MS patient is short and the curative effect is poor. Key words: Sarcoma, myeloid; Leukemia, myeloid, acute; Leukemia, myelogenous, chronic; Anemia, aplastic

Clinical Profile and Short Term Outcome of Adult Patients with Acute Myeloid Leukemia.

Acute myeloid leukemia has a poor outcome because of early deaths, high relapse rate and financial constraints. Our hospital provides care free of cost and this study assesses the short term outcome of acute myeloid leukemia in adults. The study was done from September 2013 to May 2015. All patients above 18years of age were included. Cytarabine infusion 100mg/m2 daily for 7days and Daunorubicin 60mg/m2 daily for 3days was used for induction chemotherapy followed by three cycles of high dose cytarabine as post-remission therapy. One hundred and two patients were included in the study. 48% were males. The median age was 41years. There was an intention to treat in 84 patients. 13 patients died before chemotherapy and 71 patients (57 non AML M3) received induction chemotherapy. 82% of them had a Eastern Cooperative Oncology Group performance score of ≤ 2. 28 (of 57 non AML M3) patients were alive after post-remission therapy (with 39% deaths during induction phase) and 15 of them were in remission after a median follow up of nine months. The overall event free survival at the end of the study was 22% (16 out of 71). Altogether, 63 out of 84 patients had died. Sepsis was considered as the cause of death in 46% of the patients, but the isolation of causative organism was limited (20%). The treatment outcomes of AML are poor at our centre and the current standard of care needs a significant improvement.

The frequency of nucleophosmin1 gene mutation in newly diagnosed patients of acute myeloid leukemia

Introduction: Nucleophosmin1 (NPM1) gene mutation is the most frequently occurring gene mutation in acute myeloid leukemia. This mutation is associated with good prognosis. AML patients should be screened for the NPM1 gene mutation to identify patients with good response to chemotherapy. Its association with FLT3-ITD mutation is related to poor outcome. B: To evaluate the frequency of NPM1 gene mutation in newly diagnosed patients of AML. Study design: Cross sectional study. Settings and duration: Armed Forces Institute of Pathology (AFIP), Rawalpindi from 1st September 2014 to 30th June 2015. Patients and duration: Patients of age between 5 to 85years and both gender diagnosed with AML at AFIP were included. After informed consent 2ml bone marrow samples were collected from patients. RNA was extracted and complimentary DNA was synthesized using reverse transcriptase. Polymerase chain reaction (PCR) was done using Amplification refractory mutation system methodology. PCR products were visualized by electrophoresis on Poly Acryl Amide Gel. Results: Out of 95 AML patients34 (35.7%) were positive for NPM1 gene mutation with a median age of 47years. 80 were males while 15 were females. Out of 34 NPM1 positive patients 14 (26.9%) were AML M2 subtype, 13 patients (38.2%) were AML M4 , 03 (8.82%) were AML 3 while there were 4(11.7%) cases of AML M5. Conclusion: NPM1 gene mutation is quite frequent in our patients of AML and its identification will help identify patients with good response to chemotherapy.

LEUKEMIA MEGAKARIOBLASTIK AKUT PADA SEORANG ANAK

Acute Megakaryoblastic Leukemia (FAB AML M7) occurs in all age groups with two peaks in distribution. The one is in adults and theother in children 1 to 3 years of age especially in those with Down's syndrome. The diagnosis of AML M7 requires more than 30% of thenucleated bone marrow cells being megakaryoblasts. The AML M7 was under diagnosed before the availability of monoclonal antibodies.The more common types of AML MO-M6 have to be excluded by morphological and cytochemical analysis whereas immunology is neededto exclude ALL. The megakaryocytic nature of the leukemia has to be proven by ultrastructural demonstration of platelet peroxidase or byimmunological demonstration of CD61, CD42, CD41 on the surface of the leukemic blasts. Megakaryocytic/megakaryoblastic leukemiasshow a wide morphologic spectrum. Cytoplasmic blebs and protrusions are the most prominent feature of many cases. The nuclei ofthese cells are round with more finely reticulated chromatin and with prominent nucleoli. The megakaryoblastic nature of these cells canbe suggested by morphology. Cytochemistry is of limited diagnostic value in megakaryoblastic leukemias. Usually it is used to excludethe more common types of leukemia. An eighteen months girl was admitted to hospital with anemia and hepatosplenomegaly. There isdismorphic - hypertelorism face and enlargement of neck lymph nodes. The laboratory examination found anemia, hyperleukocytosis with75 % blast cells. Morphologically the blast cells show prominent blebs and cytoplasmic budding resemble features of budding platelets.The cytochemistry staining for granulocyte and erythrocyte lineages were negative. The expressions of lymphoid and myeloid lineagesmarkers by immunoflowcytometry method were also negative. Cytogenetic examination was followed. The physical and laboratoryexamination result conclude a child with Acute Megakaryoblastic Leukemia. Cytogenetic examination was followed

Abstract 515: Alterations in phosphatidylinositol 3-phosphate (PI3P) pathway and cAMP pathway confirm poor prognosis and reduced overall survival (OS) in a series of 209 acute myeloid leukemia patients

Abstract Introduction PI3P is a molecule that regulate cell growth and mediates cell proliferation via PI3K/AKT/mTOR in response to various growth signals. Abnormal activation of genes in its pathway is associated to oncogenic activity and poor Overall Survival (OS). AMPK plays a role as a regulator of cellular energy homeostasis. Aims The aim of the this study is to define the role of PI3P pathways and AMPK pathway in AML. Methods In this work we analyzed 208 consecutive newly diagnosed non M3 AML patients, screened for TP53, FLT3, NMP1, IDH1, IDH2, and DNMT3A mutations. Remission status was assessed with bone marrow biopsy. We performed Microarray-based Comparative Genomic Hybridization with Affymetrix SNP array 6.0 or Cytoscan HD in all the patients; we performed Whole Exome Sequencing (WES)in 80/208 patients. Survival data were collected prospectively, with a median follow-up of 18 months. Survival analysis was performed with Kaplan Meyer method using log rank test. Univariate and multivariable regression and Cox Hazard Ratio(HR) model was performed. Correlation between variables was assessed with Fisher’s exact test. Results We selected genes in pathways basing on literature and GO data. Alterations in these pathways involved 103/209 patients (48%). We analyzed the gene in two different pathways. PI3K/AKT/mTOR pathway includes the following genes: pik3ca, cdkn1a, akt1, akt3, mtor and pten, pdk1,pik3r1 and irs1. The second one is AMPK pathway and it include: sesn, prkaa1, prkab1, prkag1, prkag3. Alterations in PI3K/AKT/mTOR pathway confer worst OS (p = .035) when compared with unaltered patient, but events in these pathways did not affect therapy response. Alterations in AMPK pathway confer worst OS (p<.001); Alteration of regulators in cAMP were related to lower CR% after induction in univariate (p<.001) and multivariable analysis with age, karyotype, secondary AML, TP53 mutation (p=0.009). AMPc pathway alteration was significantly associated with complex karyotype and TP53 mutation (p<.001). WES in a sub-cohort of patients did not found any significant mutation in genes we analyzed, according to literature. Conclusions Our work investigates the role of PI3P and cAMP pathways in AML. Surprisingly, it showed that alterations in these pathways are associated with poor prognosis. Significantly, alterations in cAMP pathways were associated with therapy resistance. Acknowledgement: ELN, AIL, AIRC, PRIN, Progetto Regione-Università 2010-12, FP7 NGS-PTL project, HARMONY Citation Format: Mariachiara Abbenante, Mariachiara Fontana, Giovanni Marconi, Giorgia Simonetti, Antonella Padella, Elena Tenti, Eugenia Franchini, Anna Ferrari, Sarah Parisi, Emanuela Ottaviani, Nicoletta Testoni, Viviana Guadagnuolo, Chiara Sartor, Silvia Lo Monaco, Cristina Papayannidis, Giovanni Martinelli. Alterations in phosphatidylinositol 3-phosphate (PI3P) pathway and cAMP pathway confirm poor prognosis and reduced overall survival (OS) in a series of 209 acute myeloid leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 515. doi:10.1158/1538-7445.AM2017-515

Prognostic significance of alterations of pathways regulating autophagy in acute myeloid leukemia.

7038 Background: Nowadays, science is debating if autophagy in cancer can lead to therapy resistance or it can favor apoptosis. Autophagy pathways are involved pro-apoptotic mechanism, or they can improve stresses survival eliminating damaged mitochondria and proteins. Levels and activity of pro-apoptotic and anti-apoptotic proteins (eg. bcl-2 and p53), high levels of cAMP, and a pink/park complex could play as fulcrum on this lever. Our study aims to define the role of autophagy in AML. Methods: We analyzed 148 consecutive non M3 AML with Affymetrix SNP array. We screened all patients for TP53, FLT3, NMP1 mutations. Patients was treated with intensive induction chemotherapy regimens. Survival data were collected prospectively, with a median follow-up of 18 months. Results: Autophagy alteration (gene group 1: ULK1 CHR11; ULK1 CHR17; BECN1; ATG14; AMBRA1; UVRAG; ATG9A; ATG9B; PIK3C3; PIK3R4) was related to lower Complete Remission rate (CR%) after induction in univariate (p < .001) and multivariable regression model with age, karyotype, secondary AML, TP53 mutation (p = .014); autophagy alteration shown to confer worst Overal Survival (OS) (p < .001) and was significantly associated with complex karyotype and TP53 mutation (p < .001). We detected significant differences in term of survival independently both in gain and loss in group 1 genes (p < .001). Alterations in genes in cAMP pathway (group 2: SESN1; PRKAA1 CHR 3; PRKAB1: PRKAA1 CHR 1: PRKAG1 CHR11; PRKAG1 CHR 7; PRKAG3; PRKAB1) and in genes that could be related to a switch from a physiological role of autophagy to a resiliency mechanism (group 3: CCND1; BCL2; PINK1; PARK2; TP53; MDM1; MDM4) showed to confer worst OS (p < .001 in both groups); Alteration in group 2 and group 3 were related to lower CR% after induction (p < .001 in both groups). Whole Exome Sequencing on 56 patients in our set did not found any significant mutation in genes we analyzed with the exception of TP53. Conclusions: Alterations in autophagy regulator genes are associated with poor prognosis and therapy resistance. A loss in autophagy could block apoptosis, a gain could confer cell resiliency. Acknowledgements: ELN, AIL, AIRC, Progetto Regione-Università 2010-12,FP7 NGS-PTL, HARMONY

Loss of CD45 expression at relapse of acute myeloid leukemia.

A 49-year-old female was initially diagnosed with acute myeloid leukemia (AML) M4 with a CD45＋CD13＋CD33＋CD34-HLA-DR＋ immunophenotype. She underwent allogeneic bone marrow transplantation, but the disease recurred. The bone marrow was infiltrated with 87.0% blasts negative for myeloperoxidase (MPO) staining. Immunophenotyping by flow cytometry identified the presence of a CD45-negative blast population. These blasts exhibited a CD13＋CD33＋CD19-CD10-CD34-HLA-DR- immunophenotype. The lack of CD45 expression is often observed in B-cell acute lymphoblastic leukemia, whereas CD45-negative AML is extremely rare; only one older male with AML-M0 has been reported. In the present case, the CD45-negative blasts had an MPO-CD13＋CD33＋ phenotype, which is similar to AML-M0.

Acute Promyelocytic Leukemia with FLT3/ITD and RUNX1 Mutation Presenting As Extramedullary Soft Tissue Mass

Background: APL formerly known as AML M3 is a variant of myeloid malignancies characterized by recurrent cytogenetic abnormality of PML/RARA translocation, t(15;17). Myeloid sarcoma, also called granulocytic sarcoma or chloroma, is a very rare presentation of extramedullary APL. In rare cases seen, it presents mainly as CNS disease in relapsed or refractory patients. Extramedullary APL on initial presentation is even rarer. As a result no significant information exists on the prognosis of such patients.Case report: A 50-year-old Caucasian female with no past medical history, presented with 6-month duration of chest pain, which was treated as costochondritis without any improvement. Three months later, a soft tissue mass was identified on her sternum. Complete blood count revealed pancytopenia with white blood cell 3.24 x 103/uL, hemoglobin 10.0 g/dL, platelets 58x 103/uL, absolute neutrophil count 1.13 x103/uL, and monocytes 0.13 x103/uL which indicated "low risk disease". A PET/CT scan revealed a 4.6 cm x 4 cm sternal soft tissue mass. The biopsy of the mass confirmed myeloid sarcoma. Bone marrow biopsy showed acute promyelocytic leukemia, approximately 20% blasts in a hypercellular marrow with 70-80% cellularity. Cytogenesis test was negative. FISH analysis subsequently showed a partial RARA transcript inserted at the PML locus suggestive of a cryptic PML/RARA. This was confirmed by PCR for the fusion transcript PML/RARA from the bone marrow. Next generation genome sequencing identified FLT3/ITD and RUNX1 mutation. The patient was treated as a high risk disease due to the presence of chloroma and immediately started on treatment with Daunorubicin, Arsenic Trioxide (ATO), and All-Trans-Retinoic Acid (ATRA).Conclusion: We present an extremely rare case of APL initially presenting with concomitant extramedullary sternal soft tissue mass with cryptic t(15;17) and somatic mutations in FLT/ITD as well as RUNX1 mutations. To our knowledge this is the first reported case of extramedullary APL with FLT/ITD and RUNX1 mutations. Early recognition of this rare presentation with timely chemotherapy may improve the outcomes of these patients. DisclosuresNo relevant conflicts of interest to declare.

Strong Correlation between the Expression Levels of HDAC4 and SIRT6 in Hematological Malignancies of the Adults.

Histone deacetylase enzymes, confirmed to have important role in the pathogenesis of leukemia, are promising targets of epigenetic treatment. However, in acute myeloid leukemia, our knowledge on their expression levels is limited, and controversial data have been published about their potential oncogenic or tumorsuppressor properties in solid tumors. In our study, the expression levels of HDAC4 and SIRT6 were evaluated via Western blot analysis in 45 bone marrow samples (2 uninfiltrated and 43 concerned by different kinds of hematological malignancies), including 32 specimens obtained from patients with newly diagnosed AML. Significantly higher HDAC4 level was detected in case of FLT3-ITD mutation compared to the group of patients without carrying this mutation (p<0.05). Compared to the non-infiltrated samples, the expression level of HDAC4 in AML M5 patients has been proved to be significantly higher (p<0.05). Decreasing expression levels of both HDAC4 and SIRT6 were observed during the induction treatment of FAB M5 type AML. Strong correlation has been proved between the expression levels of HDAC4 and SIRT6 (r=0.722 in full cohort and r=0.794 in AML), that confirms the recently suggested cooperation between NAD+-independent and NAD+-dependent HDAC enzymes in leukemia.

急性骨髄性白血病（AML M0）に先行した骨髄肉腫の1例

急性骨髄性白血病（AML M0）に先行した骨髄肉腫の1例

Abstract 3561: Role of bcl-2 family proteins in phorbol ester-induced apoptosis of acute myeloid leukemia cells

Abstract Human acute myeloid cell lines undergo genetic re-programming in response to differentiation inducers. The M3 AML cell line, HL-60, has served as a model system for studying phorbol ester-induced myeloid differentiation into macrophage-like cells. Macrophage differentiation is preceded by cell cycle arrest and followed by apoptosis. Therefore, the system elucidates the changes in the leukemia cell gene expression program necessary to override the genetic alterations driving continuous cell division, preventing myeloid differentiation, and blocking programmed cell death. We hypothesize that defining the evolving transcriptome as AML cells reverse their proliferative, undifferentiated, and pro-survival phenotype will reveal genes regulating these processes and possibly identify new targets for AML treatment. Whole genome exon DNA microarray analyses identifies 1260 genes whose RNA expression levels change significantly during the reprogramming. Among these genes are members of the BCL-2 family that encode both pro- and anti-apoptotic proteins (BBC3, PMAIP1, BID and BCL2, MCL1, BCL2A1, respectively). Myeloid cell leukemia 1 (MCL1) is a BCL-2 family protein defined as an anti-apoptotic protein, however MCL1 increases in expression following phorbol ester treatment in HL-60 cells, as well as other AML lines including THP-1, Kasumi-1, and KG-1. The MCL1 gene can be alternatively spliced to generate a transcript encoding a BH3-only variant (MCL1S) known to exhibit pro-apoptotic function. Western blotting demonstrates the accumulation of MCL1S protein prior to the initiation of apoptosis in phorbol ester treated cells. Here we present evidence for splice variant switching and explore the possibility that reduction in the pro-survival form, MCL1L, and accumulation of MCL1S mediates the p53-independent initiation of apoptosis. Currently, we are evaluating the effects on the leukemic phenotype and apoptotic response to phorbol ester in HL-60 cell lines overexpressing MCL1L, MCL1S, and PMAIP1, as well as HL-60 lines containing CRISPR-Cas9 mediated knockouts of MCL1 and PMAIP1. Citation Format: Cassandra Holbert, Jeffrey Forrester, Michael Roberts. Role of bcl-2 family proteins in phorbol ester-induced apoptosis of acute myeloid leukemia cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3561.

Expression of T-cell immunoglobulin mucin-3 gene in acute leukemia and its clinical significance

Objective To investigate the expression of T-cell immunoglobulin mucin-3 (Tim-3) gene in acute leukemia (AL) patients and cell lines, and to find its clinical significance. Methods 8 kinds of AL cell lines, 65 initial AL patients and 15 healthy donors were included. DNA was extracted from the separation of mononuclear cells, and Tim-3 mRNA expression was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). The expression of Tim-3 in different cell lines and the relationships between Tim-3 expression and characteristics of patients were analyzed. Results The expression levels of Tim-3 in NB4, Kasumi-1, HL60, SHI-1, THP-1, Jurkat, CCRF-CEM and Mutz-1 cell lines were (4.51±0.62) × 10-5, (79.22± 2.91) × 10-5, (41.14±3.01) × 10-5, (27.70±3.50) × 10-5, (60.47±4.97) × 10-5, (10.44±1.77) × 10-5, (42.80±2.95) × 10-5 and (11.68±1.96) × 10-5, respectively. The expression levels of Tim-3 mRNA were higher in non-M3 cell lines (Kasumi-1, HL60, Jurkat SHI-1, THP-1, CCRF-CEM, Mutz-1) than that in M3 cell line (NB4) (all P 0.05). Tim-3 expression level was significantly increased in AML-M4 patients compared with in other non-M3 AML patients according to FAB typing (P 0.05). Conclusion Tim-3 mRNA expression level is high in non-M3 AL and cell lines , which may have no significant correlation with clinical characteristics including sex, age and bone marrow blast percentage. Key words: T-cell immunoglobulin mucin-3; Leukemia, acute; Real-time quantitative polymerase chain reaction; Clinical characteristics

De Novo Philadelphia Positive Acute Myeloid Leukemia with Extensive Basophilia: A Diagnostic Dilemma.

Basophilia in peripheral blood as well as bone marrow is an unusual finding, seen in certain reactive and neoplastic conditions. Amongst the malignant hematological diseases, it is a diagnostic hall mark of chronic myeloproliferative disorders, particularly, chronic myeloid leukemia. Basophilia may also be seen in cases acute myeloid leukemia, particularly FAB AML M2 and M4. Here we document an interesting case of de novo acute myeloid leukemia which had extensive peripheral blood and bone marrow basophilia. Molecular analysis revealed p190, bcr-abl fusion transcript. A short clinical course, absence of organomegaly and features suggestive of an underlying myeloproliferative disorder, aided in establishing a diagnosis of Philadelphia positive de novo acute myeloid leukemia.

Overexpression of CD158 and NKG2A Inhibitory Receptors and Underexpression of NKG2D and NKp46 Activating Receptors on NK Cells in Acute Myeloid Leukemia

Natural killer (NK) cells are innate immune system cells that are actively involved in immune-surveillance of tumor cells. Recognition of tumors by NK cells occurred via natural cytotoxicity receptors and killer cell immunoglobulin-like receptors. Some ligands of the activating receptors seem to be present on malignant cells from patients with acute myeloid leukemia. The aim of the study was to evaluate the expression of activating receptors such as NKG2D, DNAM-1, NKp30, and NKp46, and inhibitory receptors such as NKG2A, CD158b, CD158a, and CD158e1 on NK cells from patients with newly diagnosed acute myeloid leukemia before and after stimulation with IL-2 and IL-12. Patients were divided into two groups: group 1 AML M3, and group 2 non-M3 AML. Flow cytometry was performed on whole PBMC to evaluate NK cell receptors. Twenty one AML patients, aged 26-78years, and 11 matched healthy individuals were studied. NKG2D, and NKp46 expression was decreased in group 1 (p <0.019). Patients in Group 2 showed underexpression of the activating receptors NKp46. Differences after stimulation of NK cells with IL-2 and IL-12 were observed only in Group 2, in which a significant decrease in the expression of NKp46 receptor was found (p <0.0016). Patients in groups 1 and 2 showed overexpression of the inhibitory receptors CD158b (p <0.007) and NKG2A (p <0.01). NKG2D receptor expression is decreased in patients with AML M3. In addition, patients with all FAB types of AML have overexpression of inhibitory receptors such as CD158b and NKG2A and decreased expression of the activating receptor NKp46.

Analysis of chromosome karyotypes in 80 patients with M2 subtype acute myeloid leukemia in Xinjiang Uygur Autonomous Region

Objective To explore the chromosome abnormality characteristics and ethnic differences in patients with M2 subtype acute myeloid leukemia (AML-M2) in Xinjiang Uygur Autonomous Region. Methods The chromosome karyotypes of 80 cases of AML-M2 were detected by using 24 hours short-term culture method and R banding technique. The chromosome abnormality characteristics and ethnic differences were analyzed. Results In 80 patients, 48.75 % (39/80) patients had abnormal chromosome karyotype, with t(8;21) abnormaled in 48.71 % (19/39), including pure t(8;21) [15.38 % (6/39)] and t(8;21) with other chromosomal abnormalities [33.3 % (13/39)], witch majority with t(8;21) and lack of sex chromosome (-X or -Y) [23.08 % (9/39)]. 51.28 % (20/39) patients with the exception of t(8;21) chromosomal abnormalities. The differences of abnormal chromosome karyotype in Han and Uygur patients were statistically significant [62.50 % (25/40) vs 35.00 % (14/40), χ2 = 16.66, P< 0.01]. Conclusion The chromosome karyotype characteristics of Han and Uygur M2 patients exist differences. Key words: Leukemia; Karyotyping