A structural and 121Sb Mössbauer-spectroscopic study of PrPdSb and NdPdSb

Phase-pure samples of the antimonides PrPdSb and NdPdSb were prepared by arc-melting pieces of the elements and subsequent annealing. The samples were investigated by powder and single crystal X-ray diffraction: NdPtSb type, space group P63mc, a = 458.70(5), c = 780.55(6) pm, wR2 = 0.0272, 244 F2 values, 11 variable parameters for PrPdSb and a = 458.18(4), c = 771.25(6) pm, wR2 = 0.0317, 229 F2 values, 11 variable parameters for NdPdSb. The palladium and antimony atoms form slightly puckered Pd3Sb3 hexagons which are rotated by 60° in every other layer. The rare earth (RE) atoms are coordinated by two Pd3Sb3 hexagons with the RE–Pd shorter than the RE–Sb contacts. The 121Sb Mössbauer spectra at T = 5 K confirm the antimonide character with isomer shifts of −7.55 (PrPdSb) and −7.47 mm · s−1 (NdPdSb). In agreement with the crystal structures, each spectrum could be fitted with one quadrupole split signal.

Congenital Undifferentiated Pure Erythroid Leukemia

INTRODUCTION: Congenital pure erythroid leukemia (M6b) is exceedingly rare with only a few reported cases to date. Because of the extreme rarity, almost nothing is known about the pathogenesis, appropriate therapy and prognosis. Diagnosis of erythroid leukemia is usually based on the positivity for Glycophorin A, Glycophorin C or PAS staining. We report a first case of congenital pure erythroid leukemia expressing E-cadherin in the absence of Glycophorin A, Glycophorin C and PAS staining. We analyzed the cytogenetic abnormalities of this extremely rare disease. RESULTS: The patient was the first daughter of healthy and non-consanguineous Japanese parents, born at 40 weeks of gestation by emergency cesarean section in non-reassuring fetal state after uncomplicated pregnancy. Apgar score was 8/9. Characteristic facial appearance was not recognized. At birth, she presented with marked hepatomegaly, purpura and disseminated intravascular coagulation. White blood cell (WBC) count was 63.5x109/L with blastic cells with vacuoles. Although congenital leukemia was suspected, flow cytometric analyses using CD45 blast gating failed to demonstrate leukemic cells. Karyotype was 46, XX. Fluorescence in situ hybridization (FISH) for trisomy 21 and MLL split signal were negative. GATA1 mutation was not detected. WBC count has gradually decreased within 3-4 weeks with supportive care.However, liver failure, hemophagocytic lymphohistiocytosis and schistocytosis developed. Although treatment with dexamethasone and etoposide has started, multiple nodules appeared in the liver 11 weeks after birth. Liver biopsy demonstrated small round cell tumor with high N/C ratio and vacuoles infiltrating the liver. The tumor cells were immunohistochemically positive for CD43, CD71, E-cadherin, beta-catenin, Ki-67 and c-Myc and negative for CD45, CD20, CD10, PAX5, CD3, CD4, CD8, TdT, CD1a, CD34, CD56, cyMPO, c-kit, CD42b, CD61, Glycophorin A, Glycophorin C, tyrosine hydroxylase, PGP9.5, myogenin, glypican3, NKX2.2, CAM5.2 and Periodic Acid Schiff (PAS) staining etc. Flow cytometric analysis revealed CD43+ CD71+ CD36+ CD58+ cells within large CD45 negative cell population. These cells expressed almost no other hematopoietic cell markers used to screen for leukemia. These cells were indistinguishable from normal erythroblast based on surface markers only. However, flow cytometric cell sorting revealed these cells are blasts with vacuoles. Karyotype of tumor cells has changed to 50, XX, +7, +8, add(15)(q22), +19, add(19)(q13.1-13.3)×2, +21. Based on these results, she was diagnosed with pure erythroid leukemia. Low dose Cytosine Arabinoside improved her clinical symptoms. She is alive at 5 months of age. DISCUSSION: E-cadherin is a selective marker of immature erythroblast. In our case, E-cadherin was key in erythroid lineage assignment. To our knowledge, this is the first reported case of infantile pure erythroid leukemia expressing E-cadherin in the absence of Glycophorin A, Glycophorin C and PAS staining. These results suggest that the tumor cells originated from undifferentiated erythroblast. This disease entity should be recognized. Immunohistochemical staining of c-Myc showed strong positivity. The c-Myc gene is located on chromosome 8. FISH for c-Myc split signal was negative. G-banding and FISH revealed trisomy 8. Overexpression of c-Myc may be involved in the pathogenesis of this undifferentiated pure erythroid leukemia. At birth, karyotype was 46, XX and blasts in peripheral blood decreased with supportive care only. However, we observed changes in karyotype of blasts. We assume that second hit was added during clinical course. Whole exome sequencing analysis is in progress to reveal somatic and germline mutations underlying this unrecognized disease. Disclosures No relevant conflicts of interest to declare.

ETV6 Aberrations Are a Recurrent Event in Pediatric Acute Myeloid Leukemia with Poor Clinical Outcome

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease and 30-40% of the patients still die. Prognosis is dependent on relevant genetic aberrations. Although many driving genetic alterations causing AML have been defined, in ~20% of the pediatric AML patients the oncogenic events remain unidentified. The ETS-Variant gene 6 (ETV6) encodes a transcription factor that functions as a tumor suppressor gene and is required for proper hematopoiesis in the bone marrow niche. Point mutations, deletions and translocations can lead to silencing of the gene, resulting in loss of transcriptional repression activity. ETV6 aberrations strongly associate with leukemia. In pediatric B-cell precursor acute lymphoblastic leukemia, translocation ETV6/RUNX1 occurs in ~25% of cases. Mutations in ETV6 are identified in ~25% of early immature T-cell ALL and also reported as event in adult AML (Van Vlierberghe et al, J Exp Med 2011; Barjesteh van Waalwijk van Doorn-Khosrovani et al, Oncogene 2005). We previously reported that pediatric AML patients can be divided in three clusters based on HOX-expression; (1) low HOXA/B expression, (2) high HOXA and low HOXB expression, and (3) high HOXA/B expression, and identified new repetitive genetic abnormalities in the third cluster, especially in NUP98. Cluster 1 is mainly represented by core-binding factor (CBF) AML, but in ~20% of these cases we did not find specific genetic abnormalities. Helton et al presented ETV6 aberrations in pediatric CBF-AML at ASH 2011, identified with whole genome sequencing, and with poor clinical outcome. We hypothesized that ETV6 aberrations might reduce the number of patients without known driving abnormality, especially in the low HOXA/B cluster. We screened a large representative de novo pediatric AML cohort for ETV6 mutations in exons 2-8 with direct sequencing, for ETV6 deletions by multiplex ligation-dependent probe amplification and for ETV6 translocations using split signal FISH, and analyzed outcome. In a well-characterized de novo pediatric AML cases with available gene-expression data, 6/275 (2.2%) patients had mutations affecting the predicted amino acid sequence of ETV6 and one had a silent mutation, 4/259 (1.5%) had an ETV6 deletion and 6/65 (9.2%) patients an MNX1/ETV6 translocation. Additionally, we identified 3 cases with a positive split signal FISH suggestive of a break in which ETV6 is involved, and a similar gene expression profile was found in these three cases. The aberrations of ETV6 were seen in patients of all three HOX-groups; n=9, n=6 and n=4 for cluster 1, 2 and 3 respectively. In patients with an ETV6 mutation (n=6) or deletion (n=4) 13 and 38 genes, respectively, were significantly up-regulated, including CLDN5,DPEP1 and BIRC7. This is consistent with the up-regulated genes in functional studies silencing ETV6 in LOUCY cells (Van Vlierberghe et al, J Exp Med 2011). High expression of BIRC7 has been associated with poor prognosis in adult acute leukemia (El-Mesallamy et al, Leuk Res 2011). The median age of patients with an ETV6-mutation or deletion (n=10) was 11.3 years (range 4.0-15.3) and 40% were female. Median WBC was significantly lower (15.1x109/L vs 47.0x109/L, p<0.01) in comparison to other pediatric AML cases. Other cytogenetic aberrations found in the ETV6-mutated or deleted cases were RUNX1/RUNX1T1 (n=3), PML/RARA (n=1), MLL/AF6 (n=1) and one case with an NPM1-mutation. Six out of ten patients encountered a relapse and one patient died of treatment-related mortality. The median age for patients with an ETV6-split signal FISH (n=9) was significantly lower compared to other pediatric AML cases (median 1.1 years vs 9.8, p<0.01), median WBC and sex did not differ. Five out of 9 encountered relapsed/refractory disease whereas 2 cases died of treatment-related mortality. The 3-yr pOS for all ETV6-aberrated patients taken together (n=19) was 37±11% vs 65±3% for the other pediatric AML patients (n=242, p<0.01); the 3-yr pEFS was 26±10% vs 46±3% (p=0.07), and 3-yr pCIR 47±13% vs 37±3% (p=0.24). We conclude that ETV6 aberrations are rare but recurrent in pediatric AML. ETV6 aberrations predict a poor survival, although there was no evidence for an increased relapse incidence in this small cohort. Disclosures No relevant conflicts of interest to declare.

MYC Translocation Partner Gene Determines Survival in Large B-Cell Lymphoma with MYC- or Double Hit MYC/BCL2 Translocation

Abstract 542 MØP and AOG shared the first authorship. Background: In large B-cell lymphoma (LBCL) chromosomal translocations involving the MYC protooncogene (8q24) with or without concurrent BCL2 translocation (double hit) have been associated with inferior survival. We recently found in a prospective cohort of LBCL patients that double hit MYC/BCL2 translocations had no impact on overall survival (Pedersen et al., Eur.J.Haematol. 2012). However, further stratification of patients with double hit MYC/BCL2 translocation indicated an inferior survival related to immunoglobulin MYC translocation partner gene (MYC-IG). We sought to confirm this in a larger prospective cohort of LBCL patients. Materials and methods: All patients diagnosed with LBCL (diffuse large B-cell lymphoma, DLBCL, or B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, BCLU), at Dept. of Pathology and subsequently treated at Dept. of Hematology, Copenhagen University Hospital in Herlev, were prospectively collected from 2009–2011. Tumors were classified according to morphology and immunophenotype (2008 WHO classification). Chromosomal translocations were examined with FISH, including BCL2, MYC, MYC/IGH, kappa and lambda probes. Cases which were MYC/IGH fusion signal positive or MYC split signal positive + kappa or lambda split signal positive were classified as MYC-IG. Clinical data were collected from patient files. A total of 237 patients (163 primary LBCL, 49 transformed LBCL, 25 relapsed LBCL) were included. Results: MYC translocation was found in 28/225 patients, with translocation partner gene MYC-IG in 12/24 patients and MYC-nonIG in 12/24 patients. Double hit MYC/BCL2 was found in 23/228 patients, with translocation parter gene MYC-IG in 9/19 patients and MYC-nonIG in 10/19 patients. Cox regression models were performed for calculating p-values and survival curves (Fig. 1+2). The presence of MYC translocation or MYC/BCL2 double hit translocation showed no correlation with survival. However, stratification according to MYC translocation partner gene showed an inferior overall survival related to MYC-IG compared to MYC-nonIG (p=0.03), and to MYC translocation negative (Fig. 1). Among patients with double hit MYC/BCL2 translocation, a similar picture evolved where MYC-IG/BCL2 had an inferior overall survival compared to MYC-nonIG/BCL2 (p=0.006) and MYC/BCL2 translocation negative cases (Fig. 2). Most patients were treated with standard Rituximab containing chemotherapy and treatment was comparable between the groups. Conclusion: MYC translocation, with or without concurrent BCL2 translocation, was associated with inferior survival only if MYC had immunoglobulin translocation partner gene, in this prospective cohort of LBCL patients. This suggests that prognostic stratification by MYC and MYC/BCL2 translocations should include examination of MYC translocation partner genes. An overrepresentation of transformed cases which was observed in the MYC-nonIG group could lead to an underestimation of the prognostic effect of MYC-IG. Disclosures: No relevant conflicts of interest to declare.

NUP98/JARID1A Is a New Recurrent Genetic Abnormality in Pediatric Acute Megakaryoblastic Leukemia with a Distinct HOX-Gene Expression Pattern

Abstract 537 Introduction: Cure rates in pediatric AML are currently in the 60–70% range despite treatment with intensive chemotherapy. To improve prognosis new treatment targets need to be identified, hence there is a need to better understand the underlying biology. It is hypothesized that AML results from at least two types of mutations which non-randomly collaborate in leukemogenesis. The type-I aberrations confer a proliferative advantage, type-II mutations lead to impairment of hematopoietic differentiation (Kelly et al, 2002). We recently described NUP98/NSD1 as recurrent event in cytogenetically normal AML (Hollink et al, 2011). Patients with NUP98/NSD1 had dismal outcome, and a stem-cell phenotype characterized by overexpression of homeobox (HOX) A and –B genes. Using split-signal FISH on 122 pediatric AML cases without driving oncogenic mutation, 26 NUP98- rearranged cases were identified, including 1 patient with acute megakaryoblastic leukemia (AMKL). We previously reported a patient with fusion of JARID1A, located on chromosome 12p13, to NUP98, located on chromosome 11p15, in a non-Down Syndrome (DS) AMKL case (Van Zutven et al, 2006). Therefore, a large series of non-DS AMKL patients was screened for NUP98/JARID1A and for other abnormalities, including the novel CBFA2T3/GLIS2 translocation (Gruber et al, ASH2011; #757). Methods: Samples from 105 pediatric non-DS AMKL cases, diagnosed between 1998 and 2011, were obtained from the DCOG, the AML-BFM SG, the Saint-Louis Hospital in Paris, and the COG. AMKL is more common in DS patients, therefore we also screened a series of DS AMKL (n=16). Centrally reviewed clinical and cell-biological data were provided by these study groups. Translocation of NUP98/JARID1A, MLL-rearrangements, RBM15/MKL1, and CBFA2T3/GLIS2 were identified using RT-PCR, as well as molecular characterization including hospots for the following mutations: FLT3, KIT, RAS, PTPN11, NPM1, WT1, and CEBPA. HOXA and –B expression levels were analyzed using gene expression profiling (Affymetrix) in 274 pediatric AML patients (Balgobind et al, 2011) including 9 AMKL patients, and validated with quantitative real-time PCR (n=37). Results: NUP98/JARID1A translocations were identified in 11 patients (11%). Four other patients had a NUP98- aberration with unknown translocation partner based on split signal FISH. We identified 16/105 patients with RBM15/MKL1, 13/105 with CBFA2T3/GLIS2 translocation, and 13/96 harbouring an MLL-rearrangement. Hence, specific non-random abnormalities could be defined in 61% of pediatric AMKL cases. Only 3/45 cases harboured a type-I mutation, all localized in the RAS gene. Comparing NUP98/JARID1A positive cases with negative cases in pediatric AMKL, no significant differences in patient characteristics including sex, age, and white blood cell count (WBC) were found. Considering prognosis, 5-year pEFS (22±14% vs. 36±6%, p=0.50) did not differ significantly from all other AMKL patients, nor did the cumulative incidence of relapse (56±19% vs. 54±7% p=0.9). CBFA2T3/GLIS2 translocated patients also did not differ from other AMKL patients (pEFS 19±16% vs. 36±6%, p=0.63). However, 5-year pEFS for RBM15/MKL1 translocated patients was significantly better (73±13% vs. 28±6%, p=0.043), but not in multivariate analysis adjusted for age and WBC. Gene expression analysis showed significantly higher HOXA5/A9/A10 and HOXB2/B3/B4/B5/B6 expression in NUP98/JARID1A compared to other pediatric AML cases. We did not identify any NUP98/JARID1A cases in the 16 DS AMKL patients. Discussion and conclusion: NUP98/JARID1A is a recurrent cryptic translocation in approximately 11% of pediatric AMKL cases. In 61% of all AMKL cases a type-II mutation could now be identified. Similar to NUP98-NSD1 a stem-cell phenotype was detected with persistent HOXAB-gene expression. Although NUP98/JARID1A did not influence prognosis, outcome in pediatric AMKL is unsatisfactory. NUP98 is known to recruit CREBBP/p300 resulting in histone acetylation, and transcriptional activation of HOX genes (Wang et al, 2007), suggesting that histone acetyltransferase inhibitors may be active. Moreover, JARID1A is unable to demethylate H3K4me2/3, which also results in sustained up regulation of HOX genes. This may provide options for targeted therapy. Disclosures: No relevant conflicts of interest to declare.