Acute Myeloid Leukemia M2 Research Articles

Reduced serum retinol-binding protein levels in patients with de novo acute myeloid leukaemia

Retinol-binding protein (RBP) has been used as a nutritional index for children with acute myeloid leukaemia (AML) in previous studies. However, no studies have yet examined RBP levels in AML patients from all age groups. In this study, AML patients presented with lower RBP concentrations than healthy control subjects and patients with benign haematopathies. A negative association was observed between serum RBP level and peripheral white blood cell count in M4 and M5 AML patients. Moreover, patients carrying the FLT3-ITD mutation and young patients had lower RBP levels than those lacking this mutation and elderly patients. In conclusion, these observations suggest that aberrant retinol levels may be associated with AML.

Acute promyelocytic leukemia relapsing into acute myeloid leukemia-M2 with normal cytogenetics

The use of all trans-retinoic acid (ATRA) and combination chemotherapy has made acute promyelocytic leukemia (APL) a potentially curable leukemia. Late sequelae of the treatment of APL have therefore become an important consideration in the overall treatment strategy. We report a patient with APL who achieved complete clinical and molecular remission after treatment with daunorubicin and ATRA. Three years later, she developed acute myeloid leukemia (AML), M2 subtype without any evidence of relapse of the APL clone. Karyotypic analysis showed a normal female karyotype.

DNMT3A mutation is a poor prognosis biomarker in AML: Results of a meta-analysis of 4500 AML patients

Somatic DNA methyl transferase 3A (DNMT3A) mutations have been recognized recently as recurrent molecular aberrations in acute myeloid leukemia (AML). The precise role of these mutations in leukemogenesis remains elusive but a number of studies have already been conducted to study their potential prognostic value in AML patients with variable results. We performed a meta-analysis on published data from over 4500 AML patients to provide robust evidence supporting DNMT3A mutation testing in clinical setting for AML patients. Our meta-analysis showed that DNMT3A mutations were associated with M4 and M5 AML subtypes. Those mutations conferred significantly worse prognosis with both shorter OS (p=0.0004) and shorter RFS (p=0.002). Notably, DNMT3A mutations appeared to be an independent adverse prognostic factor also in younger patients with normal cytogenetics AML (OS (p=0.01) and RFS (p=0.0005)) and also in the subgroup of patients with high risk genotypes defined according to the criteria of the European Leukemia Net (ELN) (OS (p=0.002)). Therefore, DNMT3A mutational status can improve the risk stratification of AML patients in the setting of integrated mutational profiling.

Clinical Significance of Serum p53 and Epidermal Growth Factor Receptor in Patients with Acute Leukemia

Pretreatment serum p53 and epidermal growth factor receptor (EGFR) were assessed using enzyme-linked immunosorbent assay (ELISA) in patients with acute leukemia to analysis their roles in characterization of different subtypes of the disease. Serum samples from thirty two patients with acute myeloid leukemia (AML) and fourteen patients with acute lymphoid leukemia (ALL) were analysed, along with 24 from healthy individuals used as a control group. The results demonstrated a significant increase of serum p53 and EGFR in patients with AML (p<0.0001) compared to the control group. Also, the results showed a significant increase of both markers in patients with ALL (p<0.05, p<0.0001 respectively). Sensitivities and specificities for these variables were 52% and 100% for p53, and 73.9%, 95.8% for EGFR. Serum p53 and EGFR could successfully differentiate between M4 and other AML subtypes, while these variables failed to discriminate among ALL subtypes. A positive significant correlation was noted between p53 and EGFR. Negative significant correlations were observed between these variables and both of hemoglobin (Hg) content and RBC count. Mutant p53 and EGFR are helpful serological markers for diagnosis of patients with AML or ALL and can aid in characterization of disease. Moreover, these markers may reflect carcinogenesis mechanisms.

Identification of GSX2 and AF10 as NUP98 partner genes in myeloid malignancies

Identification of GSX2 and AF10 as NUP98 partner genes in myeloid malignancies

Pseudo Chediak-Higashi Anomaly

A 9-year-old girl was admitted to hospital with a 1-month history of fever, weight loss, epistaxis, and abdominal pain. The girl’s parents were non-consanguineous. Her medical history was unremarkable. Upon admission she weighed 27 kg (25th-50th percentile), was 131 cm tall (50th percentile), and was pale. Physical examination showed multiple cervical and inguinal microlymphadenopathies, without hepatosplenomegaly. Complete blood count findings were as follows: hemoglobin 7.9 g/dL; white blood cell count: 12.3x109/L; platelet count: 9.2×109/L. Her peripheral blood smear showed 43% blast cells, 42% lymphocytes, 8% monocytes, 6% neutrophils, and 1% eosinophils. Bone marrow aspiration showed hypercellularity, with 45% myeloblasts, 15% promyelocytes, 7% myelocytes, 4% metamyelocytes, 1% eosinophils, 2% neutrophils, 15% lymphocytes, and 11% normoblasts. Wright staining of a bone marrow smear showed large (2-4 μm) round-to-oval, hyaline-structured eosinophilic granules (pseudo Chediak-Higashi [PCH] granules) within 15% of the blast cells, promyelocytes, and myelocytes (Figure 1). Some of the granules appeared to be in vacuoles, giving them a haloed appearance. The blasts were myeloperoxidase (MPO)-positive, and periodic acid-Schiff (PAS)-negative. Immunophenotypic analysis of the leukemic cells showed proliferation of CD13 (75%), CD33 (59%), CD34 (95%), CD117 (68%), and HLA DR (44%). Figure 1 Wright staining of a bone marrow smear shows large eosinophilic granules (PCH granules) within the blast and myeloid cells. The patient was diagnosed with as type M2 acute myeloid leukemia (AML). Cytogenetic analysis of a bone marrow sample showed 45,XX,t(8;21)(q22;q22). Cerebrospinal fluid biochemistry was normal, without detectable cells, based on cytocentrifuge analysis. The patient was treated according to the Berlin-Frankfurt-Munster (BFM) 2004 treatment protocol for AML. She is being followed without chemotherapy for two years and she does not have any problems. PCH anomaly was first described in 1964 by Didisheim et al. [1]. Later, VanSlyck and Rebuck [2] reported similar granules in the leukemic cells of 2 patients with AML-M4, and used the term, pseudo Chediak-Higashi anomaly of acute leukemia, because of the resemblance of the granules to those seen in patients with inherited Chediak-Higashi syndrome. PCH anomaly is characterized by the presence of large cytoplasmic eosinophilic granules in leukemic blast cells, promyelocytes, and myelocytes. It is most often observed in patients with AML subtypes M2, M3, M4, and M5, but it is also associated with chronic myeloid leukemia, myelodysplastic syndrome, and mixed-lineage leukemias [1,2,3,4,5,6,7]. Most reported cases of PCH anomaly in leukemia patients are in adults; there are only a few reported childhood cases. Ultrastructural studies of PCH anomaly have shown that the granules are strongly MPO positive, with variable positivity for PAS, Sudan black, and high-iron diamine, and without an obviously crystalline structure [5,8]. Electron microscopic studies suggest that these granules are formed by the fusion of azurophilic granules [9]. Some studies propose that these granules should be considered a morphological variant of Auer bodies [10]; however, the pathophysiology of PCH anomaly remains unknown. The clinical significance of this abnormality has yet to be been determined, because PCH anomaly is not always associated with disseminated intravascular coagulation or any other characteristic clinical picture. Additional research is required to establish the therapeutic and prognostic relevance of PCH anomaly.

Abstract 5588: Functional drug sensitivity and resistance profiling of AML patient cells defines a disease-specific combination of druggable signal addictions.

Abstract Numerous underlying molecular events have been described in acute myeloid leukemia (AML), but still, the fundamental disease mechanisms are poorly understood. Several targeted therapies have been investigated for improved AML therapy but have not succeeded to date, likely due to the inability to identify the patient subgroups that are most likely to benefit. Here, we describe functional profiling of AML patient cells ex vivo with a drug sensitivity and resistance (DSRT) platform in order to distinguish disease- and patient-specific molecular vulnerabilities and individualized therapeutic strategies. The oncology drug collection covers 306 anti-cancer agents including 131 approved, 107 investigational and 68 experimental compounds. Each compound is tested in a dose response series allowing for calculation of drug sensitivity scores. The functional exploration of AML patient samples was accompanied with comprehensive molecular profiling and clinical background data to link drug sensitivities to molecular aberrations and predictive biomarkers. Comparison of the drug sensitivity profiles of 24 AML patient and 5 control samples revealed that targeted inhibitors often exhibit no to little effect in the controls and the majority of AML patient samples with only a subset of patients showing very selective responses, indicating that cancer specific vulnerabilities can be detected with the DSRT platform. Clustering of the drug responses among patient and control samples identified distinct subgroups of patients and drugs. Each of these groups could be defined by a separate drug class, implying that the linked samples were addicted to the corresponding signaling networks. Specifically, we saw select addictions to drug classes such as dasatinib/VEGFR TKIs, MEK inhibitors, rapalogs, JAK inhibitors, mTOR inhibitors in 6 (25 %), 5 (21%), 5 (21%), 5 (21%), and 6 (25%) of the patients, respectively. Together, these sensitivities highlight a set of disease-specific and individualized signaling nodes that can serve as starting points for personalized combination therapy strategies in AML. Further, integration of DSRT results with molecular and clinical profiling link some of the selective drug responses to clinically actionable markers. For example, dasatinib sensitivity was found to be predominant in AML M5 patients, suggesting that this drug is a promising therapeutic candidate for relapsed and refractory AML M5 patients. In conclusion, the DSRT platform can be used as a functional interrogation of patient cells leading to the identification of druggable vulnerabilities that can be used for personalized medicine strategies. Furthermore, insight into disease-specific signal addictions and establishment of how these are linked can aid in deconvoluting the complex molecular disease mechanisms of cancers and generate strategies for novel drug development. Citation Format: Tea Pemovska, Bhagwan Yadav, Riikka Karjalainen, Evgeny Kulesskiy, Mika Kontro, Muntasir Mamun Majumder, Laura Turunen, Ida Lindenschmidt, Anna Lehto, Jonathan Knowles, Caroline Heckman, Kimmo Porkka, Tero Aittokallio, Olli Kallioniemi, Krister Wennerberg. Functional drug sensitivity and resistance profiling of AML patient cells defines a disease-specific combination of druggable signal addictions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5588. doi:10.1158/1538-7445.AM2013-5588

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

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

Clinical characteristics in adult acute myeloid leukemia with isocitrate dehydrogenase gene mutation

To explore the prevalence and clinical characteristics of isocitrate dehydrogenase (IDH)1 R132 and IDH2 R140/R172 gene mutations in acute myeloid leukemia (AML) patients. Polymerase chain reaction (PCR) and direct sequencing were used to sequence exon 4 of IDH gene in 570 AML patients from 2005 to 2011. In a cohort of 570 patients, AML IDH gene mutation was found in 90 (15.79%) patients. IDH1 and IDH2 mutations were detected in 27 (4.74%) patients and 63 (11.05%) patients respectively. None of them had the combined mutations of IDH1 and IDH2. The highest frequency of IDH mutations was found in AML M1 (according to the FAB scheme) compared with all other subtypes (P < 0.01). The median age was 53 years in mutated group versus 40 years in wild-type group (P = 0.010). Mutated and wild-type groups had no significant difference in gender, white blood cell count at diagnosis, hemoglobin count and bone marrow blast percentage, excepting for blood platelets level (median 52×10(9)/L vs 31×10(9)/L, P < 0.01). IDH gene mutations were associated with cytogenetically normal (CN)-AML, NPM1 mutations and particularly with the genotype of mutated NPM1 without FLT3-ITD. IDH gene mutations had no significant correlation with WT1, FLT3-TKD and MLL-PTD mutations. IDH mutated patients had a lower complete remission rate than unmutated in non-M3 patients (58.1% vs 77.9%, P < 0.05). And the patients with mutant IDH gene were associated with a shorter overall survival (28.4% vs 51.3%, P < 0.01). IDH gene mutations are more prevalent in elder AML patients and it may constitute a molecular marker for a poor prognosis in AML.

The eukaryotic translation initiation factor eIF4E is a direct transcriptional target of NF-κB and is aberrantly regulated in acute myeloid leukemia

The eukaryotic translation initiation factor eIF4E is a potent oncogene elevated in many cancers, including the M4 and M5 subtypes of acute myeloid leukemia (AML). Although eIF4E RNA levels are elevated 3- to 10-fold in M4/M5 AML, the molecular underpinnings of this dysregulation were unknown. Here, we demonstrate that EIF4E is a direct transcriptional target of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) that is dysregulated preferentially in M4 and M5 AML. In primary hematopoietic cells and in cell lines, eIF4E levels are induced by NF-κB activating stimuli. Pharmacological or genetic inhibition of NF-κB represses this activation. The endogenous human EIF4E promoter recruits p65 and cRel to evolutionarily conserved κB sites in vitro and in vivo following NF-κB activation. Transcriptional activation is demonstrated by recruitment of p300 to the κB sites and phosphorylated Pol II to the coding region. In primary AML specimens, generally we observe that substantially more NF-κB complexes associate with eIF4E promoter elements in M4 and M5 AML specimens examined than in other subtypes or unstimulated normal primary hematopoietic cells. Consistently, genetic inhibition of NF-κB abrogates eIF4E RNA levels in this same population. These findings provide novel insights into the transcriptional control of eIF4E and a novel molecular basis for its dysregulation in at least a subset of M4/M5 AML specimens.

Prognostic significance of Bcl-2 and p53 protein expressions and Ki67 proliferative index in diffuse large B-cell lymphoma

To the Editor Here we present a case of multiple granulocytic sarcomas with monocytic differentiation involving the skin and retroperitoneal area. A 19-year-old man presented with the complaints of generalized arthralgia and fixed non-tender nodular lesions (Figure 1). Physical examination revealed generalized immobile, painless violaceous erythematous nodules on his back, bilateral arms and legs, and joints. The largest nodule was 3x2 cm, located on his right posterolateral upper leg. Abnormal laboratory values included hemoglobin of 84 g/L, hematocrit of 27.2%, erythroid sedimentation rate of 58 mm/h, and lactate dehydrogenase of 613 IU/L. Blood smear revealed hypochromia and microcytosis, and bone marrow was normocellular without blasts. However, monocyte and macrophage marker CD163 was positive in 10%-15% of the bone marrow cells. The pathological examination of the nodular lesions revealed medium-sized, irregular-shaped blastic cells with frequent mitotic activity, which infiltrated the dermis. The tumor tissue contained tingible body macrophages with nuclear debris. In immunohistochemical study, these cells were positive for CD163, lysozyme, CD4, and CD43, while CD117, MPO, CD68, langerin, CD1a, S–100, CD35, CD21, clusterin, fascin, factor 13a, CD123, TCL–1, TdT, CD3, CD20, and CD30 were all negative. The Ki–67 proliferation index of the neoplastic cells was 80%. In situ hybridization study with the EBER probe for Epstein-Barr virus was negative. Figure 1 Multiple violaceous skin nodules. Abdominal and thorax computerized tomography (CT) was performed, which revealed a right retroperitoneal mass with blurred margins. A CT-guided biopsy was performed. The biopsy showed pleomorphic cell infiltration with large mononuclear and binuclear cells with clear nucleoli, which were CD163-positive, CD117-negative, and focal MPO-positive. These atypical pleomorphic, monocytic-histiocytic cells did not have blastic morphology. A high dose of cytosine-arabinoside and idarubicin was started. The nodular lesions disappeared 20 days after starting chemotherapy. Arthralgia, dyspnea, and respiratory distress developed consequently. Right pleural effusion was demonstrated in the chest X-ray. The cytological examination of the pleural fluid revealed blastic infiltration. Since the retroperitoneal mass persisted in follow-up abdominal CT, a new chemotherapy regimen containing fludarabine, cytosine arabinoside, and idarubicin was started. However, pleural effusion persisted and required Pleurocan evacuation. Methotrexate (Mtx) was given intrapleurally accompanied with systemic high-dose Mtx. Tumor lysis syndrome developed 28 days after this regimen and required intubation; the patient died due to cardiac arrest 3 days later. Granulocytic sarcoma (GS) is the tumoral infiltration of extramedullary sites with immature myeloid precursor cells; it is associated with 3%-8% of acute myeloid leukemia (AML) cases and rarely with chronic myeloproliferative disorders [1]. Monocytic differentiation of GS is seen mostly in AML M4 and M5 subtypes according to the French-American-British (FAB) classification [2]. Any extramedullary anatomic site may be involved with GS and it might rarely precede but usually occurs simultaneously with acute leukemia. CD163 positivity is controversial in GS. While Lau et al. claimed that CD163 is a highly specific marker for cells of monocyte/macrophage lineage, they did not demonstrate CD163 positivity in GS in their series [2]. Backe et al., however, demonstrated CD163 positivity in myeloid leukemias with monocytic differentiation [3], while Benet et al. demonstrated CD163 positivity in 52% of their cases of leukemia cutis [4]. The prevalence of skin involvement in myeloid malignancies is approximately 3%, and it is usually associated with cases with monocytic differentiation. It is difficult to identify the origin of the tumor when it is not associated with bone marrow infiltration, namely “aleukemic leukemia cutis” [4]. In a series of 173 patients with leukemia cutis, 7.5% of the patients with leukemic skin infiltration had no underlying myeloid neoplasm [4). Skin is one of the most common sites of involvement in GS, with approximately one-quarter of patients with GS having skin involvement [4]. The presented patient had arthralgia and multiple granulocytic nodules on various parts of the body and joints without bone morrow involvement. The morphologic appearance of the skin lesions was typical for leukemia cutis in its color and nodular form, as is immunohistochemistry with monocytic differentiation. Nevertheless, most of the aleukemic GS of skin is known to be associated with infiltration with cells of monocytic differentiation [5]. In conclusion, myeloid leukemias might present with granulocytic sarcomas, particularly in skin without bone marrow involvement. Multiple skin lesions of nodular pattern with violaceous color might suggest an underlying myeloid neoplasm. The positivity of monocytic markers such as CD68 and CD163 is typical for leukemia with a predilection to the skin. Patients with aleukemic granulocytic sarcoma might have a very aggressive course, with evolution to tumor lysis syndrome. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

Blast cell deficiency of CD11a as a marker of acute megakaryoblastic leukemia and transient myeloproliferative disease in children with and without Down syndrome

The classification of acute myeloid leukemia (AML) FAB subtype M7 relies on immunophenotypic assessment. CD41 is expressed throughout all stages of maturation of megakaryocytes and has therefore been described as a specific blast cell marker in AML M7 as well as in transient myeloproliferative disease (TMD) of patients with Down syndrome (DS). However, technical difficulties underlie the need for new markers for these entities. We evaluated the expression of human lymphocyte function-associated antigen 1 (CD11a) in a large cohort of pediatric AML and TMD patients (n = 91) of the Austrian AML-BFM 98 and 2004 studies. We found a consistent deficiency of CD11a as assessed by mean fluorescence intensity in all patients with non-DS AML M7 (n = 8) and M6 (n = 1), all cases of classical DS-AML (n = 12) as well as TMD (n = 15) that was statistically significant in comparison to non-DS AML M0-M5 patients (n = 55; P < 0.001, sensitivity 100%). Only three of 55 Non-DS M0-5 patients were CD11a deficient (specificity 95%). Monocytic leukemias (M4/5) and normal monocytes typically showed a high CD11a expression, FAB types M1/2 and normal neutrophils an intermediate expression level, while all M3 leukemias were rather low in CD11a expression. We conclude, that deficiency of CD11a expression should be added to the diagnostic criteria of AML-M7, classical DS-AML and TMD.

Improved Outcome in Pediatric Relapsed Acute Myeloid Leukemia: Results of a Randomized Trial on Liposomal Daunorubicin by the International BFM Study Group

In pediatric relapsed acute myeloid leukemia (AML), optimal reinduction therapy is unknown. Studies suggest that liposomal daunorubicin (DNX; DaunoXome; Galen, Craigavon, United Kingdom) is effective and less cardiotoxic, which is important in this setting. These considerations led to a randomized phase III study by the International Berlin-Frankfurt-Münster Study Group. Patients with relapsed or primary refractory non-French-American-British type M3 AML who were younger than 21 years of age were eligible. Patients were randomly assigned to fludarabine, cytarabine, and granulocyte colony-stimulating factor (FLAG) or to FLAG plus DNX in the first reinduction course. The primary end point was status of the bone marrow (BM) sampled shortly before the second course of chemotherapy (the day 28 BM). Data are presented according to intention-to-treat for all 394 randomly assigned patients (median follow-up, 4.0 years). The complete remission (CR) rate was 64%, and the 4-year probability of survival (pOS) was 38% (SE, 3%). The day 28 BM status (available in 359 patients) was good (≤ 20% leukemic blasts) in 80% of patients randomly assigned to FLAG/DNX and 70% for patients randomly assigned to FLAG (P = .04). Concerning secondary end points, the CR rate was 69% with FLAG/DNX and 59% with FLAG (P = .07), but overall survival was similar. However, core-binding factor (CBF) AML treated with FLAG/DNX resulted in pOS of 82% versus 58% with FLAG (P = .04). Grade 3 to 4 toxicity was essentially similar in both groups. DNX added to FLAG improves early treatment response in pediatric relapsed AML. Overall long-term survival was similar, but CBF-AML showed an improved survival with FLAG/DNX. International collaboration proved feasible and resulted in the best outcome for pediatric relapsed AML reported thus far.

Recurrence of a t(8;21)-Positive Acute Myeloid Leukemia in the Form of a Granulocytic Sarcoma Involving Cranial Bones: A Diagnostic and Therapeutic Challenge

Granulocytic sarcoma (GS) is a rare extramedullary solid tumor defined as an accumulation of myeloblasts or immature myeloid cells. It can cooccur with or precede the acute myeloid leukemia (AML) as well as following treated AML. The incidence of GS in AML patients is 3–8% but it significantly rises in M2 FAB subtype AML. This variety of AML harbors t(8;21) in up to 20–25% of cases (especially in children and black ones of African origin) and, at a molecular level, it is characterized by the generation of a fusion gene known as RUNX1-RUNX1T1. Approximately 10% of M2 AML patients will develop GS, as a consequence, the t(8;21) and the relative transcript represent the most common cytogenetic and molecular abnormalities in GS. FLT3-ITD mutation was rarely described in AML patients presenting with GS. FLT3 ITD is generally strongly associated with poor prognosis in AML, and is rarely reported in patients with t(8;21). GS presentation is extremely variable depending on organs involved; in general, cranial bones and sinus are very rarely affected sites. We report a rare case of GS occurring as a recurrence of a previously treated t(8;21), FLT3-ITD positive AML, involving mastoid bones and paravertebral tissues.

Association of cup-like nuclei in blasts with FLT3 and NPM1 mutations in acute myeloid leukemia

The purpose of this study was to investigate the correlation of mutations of the fms-like tyrosine kinase (FLT3) and nucleophosmin (NPM1) genes with the cup-like nuclear morphology of blasts in patients with acute myeloid leukemia (AML). We retrospectively reviewed peripheral blood (PB) and bone marrow (BM) slides of 208 patients prepared at the time of diagnosis of AML based on the results of testing for mutations of both NPM1 exon 12 and FLT3. We investigated the association between this phenotype and hematologic findings, disease markers, and mutations in NPM1 exon 12, FLT3-internal tandem duplication (ITD), and tyrosine kinase domain (TKD) genes. Cup-like nuclei were found in 44 patients (21.2 %) diagnosed with AML. This morphology was associated with high blast counts in the PB and BM; AML type, especially AML M1 (FAB classification); low CD34 expression; and mutation of FLT3-ITD, -TKD, NPM1 regardless of other mutations (p < 0.05 for all). However, FLT3-ITD or TKD mutation alone (nine cases, p = 0.228) was not associated, and NPM1 mutation alone (14 cases, p = 0.036) was weakly associated with cup-like nuclei. Mutation of both NPM1 and FLT3-ITD or TKD (17 cases, p < 0.001) was strongly correlated with the cup-like nuclear morphology. AML with cup-like nuclei is strongly associated with co-occurring mutations of both NPM1 and FLT3-ITD or TKD. Therefore, testing for both mutations is recommended for patients with the cup-like nuclear morphology.

A Novel t(2;10)(q31;p12) Balanced Translocation in Acute Myeloid Leukemia

We describe a case of acute myeloid leukemia M5 showing a balanced t(2;10) (q31;p12) translocation. This has never been described before as the sole cytogenetic abnormality in a bone marrow cell clone at onset. Using fluorescence in situ hybridization with properly designed bacterial artificial chromosome probes, we mapped the breakpoint regions on both derivative chromosomes 2 and 10: der(2) and der(10), respectively. The MPP7 gene, disrupted by the breakpoint on chromosome 10, was juxtaposed upstream of both HNRNA3 and NFE2L2 genes on chromosome 2, without the formation of any fusion gene. Using real-time quantitative polymerase chain reaction, we tested the possible disregulation of any of the breakpoint-associated genes as a consequence of the translocation, but we found no statistically significant alteration. Considering the potential role of this clonal cytogenetic abnormality in leukemogenesis, we speculate that this translocation could have an impact on additional genes mapping outside the breakpoint regions. However, the limited amount of RNA material available prevented us from testing this hypothesis in this present case.

Dysregulation of the Imprinted DLK1-DIO3 Locus in Promyelocytic Leukemia

Abstract 3500Small non-coding RNAs (sncRNAs) have emerged as important regulators of multiple cellular processes, with a growing body of evidence suggesting their dysregulation and possible pathogenic role in many malignancies, including acute myeloid leukemia (AML). Previous high throughput sncRNA transcriptome sequencing studies have focused on the 15–30 nucleotide fraction that primarily consists of microRNAs (miRNAs), thus excluding many larger, potentially important sncRNAs, such as small-nucleolar RNAs (snoRNAs) and piwiRNAs. To address this “sequencing gap”, we extended our transcriptome sequencing to include RNA species of 15–75 nucleotides in length. Here, we report the small RNA transcriptome sequencing of 28 cases of AML. As a control, we also sequenced the small RNA transcriptome in CD34+ progenitors (n = 4) and promyelocytes (n=2) from healthy donors. The most common class of sncRNA detected in all samples was small-nuclear/snoRNAs (∼40% of reads), followed by miRNAs (∼17% of reads), ribosomal/transfer RNAs (15% of reads), unannotated/miscellaneous sncRNAs (∼15% of reads), and piwiRNAs (∼10% of reads). Partek Genomics Suite was used to identify significantly dysregulated sncRNAs using a p-value<0.05, false discovery rate of 5%, and a minimum 2 fold-change from normal CD34+ cells. Compared to normal CD34+ cells, expression of 158 sncRNAs was significantly increased and 29 were significantly decreased in de novo AML samples.The most striking example of dysregulation was observed in acute promyelocytic leukemia (M3 AML). The DLK1-DIO3 locus at 14q32.2 contains 41 snoRNA genes belonging to the SNORD112–114 family, in addition to a large number of miRNAs. Compared with normal CD34+ cells or non-M3 AML, 31 of the 41 snoRNAs in this locus were massively upregulated (6–2,000 fold) in M3 AML samples. Concordantly large increases (10–1,000 fold) of 20 different miRNAs in this locus were also observed in M3 AML. We extended these findings to an independent cohort of M3 and non-M3 AML cases (n = 187) for which RNA transcriptome sequencing was available (RJW and TJL, personal communication, on behalf of the TCGA AML analysis group). Analysis of this data confirmed massive up-regulation of sncRNAs in the DLK1-DIO3 locus that was restricted to M3 AML. We considered the possibility that the increase in sncRNAs in this locus was secondary to a promyelocyte-specific expression pattern in M3 AML cells, but expression of sncRNAs in normal promyelocytes was lower than that observed in normal CD34+ cells, making this hypothesis unlikely.The DLK1-DIO3 locus is one of the best characterized imprinted regions in the human genome. The paternally-derived protein coding genes in this locus (DLK1, RTL1 and DIO3) showed no dysregulation in M3-AML in contrast to the aberrantly expressed, maternally-derived sncRNAs. While the function of the sncRNAs in this region is largely unknown, there is evidence they may contribute to stem cell pluripotency (Stadtfeld et al. Nature 2010, Liu et al. JBC 2010). Since imprinting at this locus is controlled by methylation of several differentially methylated regions (DMRs), we analyzed array-based methylation data from the TCGA cohort to determine if imprinting was disrupted. No difference in methylation status of the DMRs was observed between M3 and non-M3 AML. Moreover, based on expressed germline single nucleotide polymorphisms, mono-allelic expression of sncRNAs in this locus was preserved. Together, these data show that imprinting of the DKL1-DIO3 locus is not disrupted in M3-AML samples, and that dysregulation of the sncRNAs in this region occur through an imprinting-independent mechanism. In summary, extended small RNA transcriptome sequencing is a valuable new tool to analyze malignant cells; it successfully identified massive dysregulation of sncRNAs in the DKL1-DIO3 locus in M3 AML. The contribution of the aberrantly expressed sncRNAs within this locus to the pathogenesis of M3 AML will require additional study. Disclosures:No relevant conflicts of interest to declare.

Blast Asparagine Synthetase Deficiency in Acute Myeloid Leukemia

AbstractAbstract 4333 IntroductionL-asparaginase (L-Aspa) is one of the standard components of acute lymphoid leukemia (ALL) therapy. Its efficacy as an antineoplasic agent is due to its capacity to deplete plasmatic asparagine, starving the leukemic cells and subsequently inhibiting protein synthesis.In vitro studies have already shown the sensitivity to L-Aspa of leukemic cells from patients with acute myeloid leukemia (AML), depending on the French-American-British (FAB) subtype (Zwaan et al., 2000; Okada et al., 2003). Despite promising results in clinical trials (Capizzi et al., 1988; Wells et al., 1993), L-Aspa has only been used scarcely in the treatment of AML, mainly because of the adverse effects observed commonly that impair its use.The existence of a new formulation of L-Aspa (L-Aspa encapsulated in heterologous red blood cells) with a better safety profile allows considering its use for the treatment of AML (Godfrin et al., 2012).The sensitivity to L-Aspa may be inversely correlated to their expression of asparagine synthetase (ASNS, an enzyme catalyzing the intracellular synthesis of asparagine). The aim of this study was to investigate the potential of L-Aspa for AML treatment by determining the leukemic cell expression of ASNS and their sensitivity to L-Aspa. Materials and methodsStudies were performed on an AML cell line (HL-60) and on leukemic cells isolated from the bone marrow of AML patients by Ficoll density gradient.The IC50 (concentration inhibiting 50% of cell viability) was determined in vitro by incubating various concentrations of L-Aspa with the cells and measuring the cell viability with a cell counting kit (CCK-8 viability assay).ASNS expression was determined by western-blot. ResultsAs a preliminary, determination of IC50 for the HL-60 cell line demonstrated that these cells were equally sensitive to L-Aspa than the ALL cell line MOLT-4 in vitro(0.23 IU/mL versus 0.19 IU/mL, respectively). The expression of ASNS in the HL-60 cell line was low in comparison with other cancer cell lines.Concerning blasts isolated from AML patients, 2/3 patients displayed an IC50 < 0.01 IU/mL whereas 1/3 displayed an IC50 as low as 0.13 IU/mL, which means a high sensitivity to L-Aspa. All these patients were negative for ASNS expression.On 6 patients tested for ASNS expression, 5 had their blasts negative whereas one was positive. Patients with blasts negative for ASNS expression were all affected by FAB M5 or M1 AML whereas patient with blasts positive was diagnosed with a M2 AML. ConclusionBoth AML cell line and cells isolated from AML patients were sensitive to L-Aspa. In all clinical cases, high sensitivity and/or lack of ASNS expression were linked to a FAB M5 or M1 AML, consistent with data from literature suggesting a higher sensitivity of M1, M4 and M5 AML subtypes.The only clinical case positive for ASNS expression was a FAB M2 AML, consistent with literature that indicates an in vitro resistance of the M2 AML subtype to L-Aspa.Globally, these results suggest that L-Aspa is effective for killing AML cells with low/no ASNS expression. Thus, thanks to its low toxicity profile, L-Aspa may be considered as a potential therapy for FAB subtypes or individuals characterized by low/no ASNS expression. Based on the epidemiology of AML subtypes (Selter et al., 2011), L-Aspa therapy may be beneficial for 50% of patients with AML.These results finally highlight the necessity of the determination of the grade and/or the expression of ASNS in leukemic cells to select AML patients at potential for L-Aspa therapies. Disclosures:Berlier:ERYTECH Pharma: Employment. Aguera:ERYTECH Pharma: Employment. Campion:ERYTECH Pharma: Employment. Gay:ERYTECH Pharma: Employment. Godfrin:ERYTECH Pharma: COO Other.

R115777(tipifarnib) Improves Early Survival when Used As Maintenance Therapy for Elderly or Relapsed/Refractory Patients with Acute Myelogenous Leukemia in Remission

AbstractAbstract 676Although maintenance therapy has been shown to be of value in Acute Lymphoid Leukemia, its role in non M3 Acute Myeloid Leukemia (AML) has never been proven. Studies have been limited by issues related to efficacy as well as toxicity. The Eastern Cooperative Oncology Group (ECOG) trial, E2902, was a randomized phase III intergroup trial of the Farnesyl Transferase Inhibitor, R115777(tipifarnib), as maintenance therapy for AML. Patients(pts) with AML in remission after requiring salvage therapy or over age 60 in first remission were eligible. A confirmatory bone marrow exam prior to randomization was required to demonstrate a complete remission (CR) or morphologic remission(MR). Absolute neutrophil count >1000/mm3, platelet count >=50,000/mm3, normal renal, and hepatic function were required. Consolidation or post remission therapy prior to enrollment was allowed but not required. Pts who had received an allogeneic transplant in this remission were ineligible.Pts were randomized to either receive tipifarnib twice daily (BID) or to observation. The main objective of the study was to compare disease free survival (DFS) in the two groups. The study was designed with 84% power to detect a 76% relative increase in the median DFS from 3.4 months to 6 months (mo). The final analysis was to take place when 111 events had taken place. Between August 2004 and December 2009, 144 pts were accrued to the study. The median age of the pts is 70 (range 28–86). The majority of pts enrolled on the study (67%) were in first CR. Seventy three pts were enrolled onto the treatment arm and 71 pts were enrolled onto the observation arm. When the study initially opened, tipifarnib was given at a dose of 400 mg BID with dose reductions and interruptions as per protocol for toxicity. Based on the first interim analysis, toxicity data, the starting dose was decreased to 300 mg BID. Significant hematologic toxicity was seen in patients at both 300 and 400 mg. The majority of patients experienced >=grade 3 neutropenia or thrombocytopenia. There was 1 episode of febrile neutropenia. Non-hematologic toxicities were minimal.At the time of 110 events (90 relapses and 100 deaths), the final analysis of the study was conducted. The relapse rate was 60.3% on tipifarnib vs 64.8% for observation. Median DFS and 10 and 12 mo DFS and overall survival (OS) are documented in Table 1. Kaplan Meier curves for DFS and OS are presented. As the difference in median DFS is not considered significant (9.3 vs 5.8 mo, log rank p value 0.21), in July 2011, pts and physicians were informed of the lack of evidence of benefit so they could decide if they wished to continue drug. However an unplanned analysis of the data demonstrates a statistically significant improvement in OS at 10 months in pts who received tipifarnib. (70% vs 48%, p=0.05). While the benefit is subsequently lost, given a median survival measured in months, an increase in the number of pts alive and/or without disease at 10–12 mo is of value, particularly in the absence of noteworthy toxicity. Future studies are needed to better determine the role of maintenance therapy in pts with AML.TipifarnibObservationMedian DFS (months)9.3 (95% CI:5.7, 13.3)5.8 (95% CI: 3.7, 8.8)p=0.21OS rate at 10 mo70% (95% CI: 58%, 79%)48% (95% CI: 36%, 60%)p=0.05OS rate at 12 mo59% (95% CI: 47%, 70%)44% (95% CI: 32%, 55%)p=0.07DFS rate at 10 mo47% (95% CI: 36%, 59%)32% (95% CI: 21%, 43%)p=0.07DFS rate at 12 mo39% (95% CI: 28%, 50%)30% (95% CI: 20%, 41%)p=0.26 [Display omitted] [Display omitted] Disclosures:Larson:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy; Ariad: Consultancy, Research Funding.

Microrna-143 Blocks ERK5 Signaling During Granulocytic Differentiation of Hematopoietic Stem Cells and Is Downregulated in AML

AbstractAbstract 3516Mitogen-activated protein kinase (MAPK) pathways are a family of related and sometimes interconnected pathways and one of the most studied. Over the last years, extensive work has established that these proteins play a critical role in G-CSF mediated maturation of neutrophil granulocytes. Understanding the mechanisms by which the MAPK pathways are regulated represents an important area of investigation. MicroRNAs, a class of small non-coding RNAs, have been found to play an important role in the regulation of diverse cellular processes by binding to target mRNAs leading to their translational repression. Deregulation of certain microRNAs, thereby, may lead to disrupted signal pathways, such as MAPK-signaling, and to tumorigenesis. However, the role of microRNAs in hematopoietic differentiation and development of leukemia remains largely unknown. In this study we performed a global screen to identify microRNAs involved in G-CSF-regulated MAPK-pathways in primary human CD34+ hematopoietic progenitor cells. Here we found microRNA-143 (miR-143) to be frequently upregulated in G-CSF stimulated CD34+ cells with a strong correlation to CD15 expression. We could also show the granulopoietic association of miR-143 in several hematopoietic cell line models and acute myeloid leukemia (AML) patient samples. Especially, AML patient samples FAB M4 and M5, which show monocytic phenotypes, had a significant lower expression level of miR-143 compared to the AML FAB types M0, M1, M2, and M3. In general, miR-143 expression was shown to be downregulated in AML patient samples in comparison to normal CD34+ hematopoietic progenitor cells. Most interestingly, we show that miR-143 expression is upregulated in acute promyelocytic leukemia (APL) patients after ATRA treatment. By in silico prediction we found MAPK protein family members (eg. MAPK1, MAPK3 and MAPK7) as predicted targets of miR-143. Western blot analysis of AML patient samples and G-CSF stimulated CD34+ cells clearly show an inverse correlation of miR-143 and MAPK7 (ERK5) protein expression. Finally, by transient overexpression of miR-143 we could show a strong downregulation of ERK protein expression in NB4 cells. Our study suggest that miR-143 upregulation by G-CSF may be an important regulatory step for permitting neutrophil differentiation. MicroRNA-143 blocks ERK5 signaling in G-CSF-induced granulopoiesis of CD34+ hematopoietic stem cells, is downregulated in myelo-monocytic acute myeloid leukemia subtypes, and upregulated after ATRA treatment in APL patients. This information may prove useful for the understanding of conditions in which neutrophil proliferation/differentiation balancing is dysregulated, such as in myeloid leukemia and myelodysplastic disorders. Disclosures:No relevant conflicts of interest to declare.