Milan, Italy, June 12 – 15, 2014

were associated with overexpression by RQ-PCR. High levels of PRDM16 - Biology 3 expression (greater that three standard deviations above the mean 2- ΔΔ Ct of 10 normal bone marrow controls) were observed in a significant subset of AML with normal karyotype (AML-NK) (12/25;48%) and with adverse cytogenetic P779 prognostic group (3/11;27.3%) but they were also associated with isolated rare translocations (4/10;40%). In 3 cases with a complex karyotype FISH analysis DECREASE EXPRESSION OF MIR-20A PROMOTES CANCER CELL detected an extra copy of PRDM16. Cases with amplification were PROLIFERATION AND PREDICTS POOR SURVIVAL OF ACUTE MYELOID associated with older age, complex karyotype involving 5 and 7 LEUKEMIA 1 1 1 1,* 1 1 1 1 abnormalities, higher WBC and AML compared to cases with PRDM16 P Li , D Ning , X Dahai , Z Nan , L Xiaoliang , W Yang , Z Xiuxian , C Hongli translocations. 1Emergency Department, First Hospital of Jilin University, changchun, China Summary and Conclusion: PRDM16 gene is a frequent target of 1p36 abnormalities in AML. Copy number gain of PRDM16 is a recurrent genetic Background: MicroRNAs (miRNAs) are a family of 19- to 25-nucleotides non- abnormality in AML with 1p36 abnormalities and, although less common, in coding small RNAs that primarily function as gene regulators. Growing AML with complex karyotype but undetectable 1p36 abnormalities. Copy evidences indicate miRNAs play important roles in cancer development, number gain of chromosome 1p36 has not been previously associated with progression, metastasis and may constitute robust biomarkers for cancer PRDM16 overexpression in patients with myeloid malignancies. We also prognosis. To date, although certain miRNAs have been established a clear demonstrated overexpression of PRDM16 in different subgroups of AML without oncogenic role in hematological malignancies, other individual miRNAs PRDM16 rearrangements or amplification, especially in AML-NK subset. Since potentially involved in human leukemogenesis still remain elusive. the poor prognosis associated to PRDM16, the role and prevalence of PRDM16 Aims: This study aimed to determine the clinical characteristics and prognostic expression should be addressed further in a larger cohort of patient. Although significance of microRNA-20a (miR-20a) in adult de novo acute myeloid the limited case series, amplification seems to be associated with distinct leukemia (AML) patients. cytogenetic and clinical features and could reflect a different pathway or role of Methods: The expression levels of miR-20a in bone marrow mononuclear cells PRDM16 in the pathogenesis of these AML patients. Supported by University were measured in 98 newly diagnosed AML patients and 20 cases of normal of Bologna RFO, BolognAil and Coop Reno. healthy donors by real-time quantitative polymerase chain reaction. Kaplan- Meier and Cox proportional regression analyses were utilized to determine the association of miR-20a with survival of patients. The potential functions of miR- P781 20a on proliferation were evaluated by proliferation and flow cytometry analysis. The direct target gene of miR-20a was also identified by luciferase reporter CHARACTERIZATION OF THE RARE TRANSLOCATION T(3;10)(Q26;Q21) assays. IN AN ACUTE MYELOID LEUKEMIA PATIENT Results: miR-20a was expressed at significantly lower levels in the bone T Jancuskova 1,* , R Plachy 1, J Stika 1, D W Hardekopf 1, L Zejskova 1, I Praulich 2, marrow of AML patients compared with normal controls. Patients with lower KA Kreuzer 2, N Kosyakova 3, T Liehr 3, S Pekova 1 miR-20a expression had significantly poorer complete remission (CR) rates 1Laboratory for Molecular Diagnostics, synlab genetics s.r.o., Prague, Czech (Log rank p<0.001), relapse-free survival (RFS, Log rank p<0.001) and overall Republic, 2Department I of Internal Medicine, University at Cologne, Cologne, survival (OS, Log rank p<0.001). Low miR-20a expressers had lower CR rates 3Institute of Human Genetics, Jena University Hospital, Jena, Germany and OS within the Southwest Oncology Group classification. Multivariate analysis revealed that lower miR-20a was an independent predictor of poor Background: Recently, we introduced a flexible strategy for mapping prognosis. MiR-20a restoration could result in low levels of cAMP and weak cytogenetically identified unique abnormalities down to the single nucleotide activity of PKA, thus relieving the inhibitory effect of PKA on mononuclear- level. This strategy has enabled us to design clone-specific assays for sensitive leukemic cell proliferation. Subsequent investigations revealed that miR-20a minimal residual disease (MRD) monitoring in acute leukemia patients directly reduced the endogenous level of myeloid cell leukemia (Jancuskova et al . Leuk Res. 2013) as well as to elucidate regions/ sequence 1 (Mcl-1) in mononuclear-leukemic cell. involved in congenital chromosomal aberrations (unpublished data). Here we Summary and Conclusion: MiR-20a is decreased in AML and correlates with characterize the rare chromosomal translocation t(3;10)(q26;q21), involving AML prognosis. Down-regulation of miR-20a increases the proliferation abilities the MECOM gene (MDS and EVI1 complex located in band 3q26), of AML cells. Our findings suggest miR-20a may represent a novel potential identified in an acute myeloid leukemia (AML) patient. therapeutic target and biomarker for survival of AML patients. Aims: Our aim was to use our strategy to identify the fusion partner on chromosome 10q21 and to characterize the precise nucleotide sequence of the chromosomal breakpoint. P780 Methods: The chromosomal translocation was revealed by standard cytogenetic techniques (G-banding, mFISH), and involvement of the MECOM COPY NUMBER GAINS OF CHROMOSOME 1P36 LEAD TO PRDM16 gene was confirmed by FISH with the use of a commercially available probe OVEREXPRESSION IN AML PATIENTS 1,* 1 1 1 1 1 set. The derivative chromosome 10 was isolated using fine-needle C Baldazzi , E Ottaviani , S Luatti , G Marzocchi , G Ameli , C Papayannidis , microdissection followed by whole genome amplification (WGA). Ten dissected C Gamberini 1, E Franchini 1, M Cavo 1, G Martinelli 1, N Testoni 1 1 fragments were sequenced on the GS-Junior next-generation sequencing Institute of Hematology and Medical Oncology “L. e A. Seràgnoli”, S.Orsola- platform. The reads obtained were aligned to reference sequences of Malpighi Hospital, Bologna, Italy 3 and 10 using in-house developed software. The last mapped reads from both chromosomes were used as docking sites for primers for long- Background: PRDM16 gene (1p36) is rearranged in AML/MDS with range PCR to amplify the putative breakpoint. The long-range PCR products t(1;3)(p36;q21), t(1;21)(p36;q22) and t(1;12)(p36;p13). These translocation were directly sequenced using Sanger sequencing to reveal the precise resulted in PRDM16 overexpression through juxtaposition to the enhancer of nucleotide sequence of the breakpoint. RPN1 at 3q21 or through fusion transcript formation with RUNX1at 21q22 or Results: Using a combination of cytogenetic and molecular approaches, we ETV6 at 12p13, respectively. AML/MDS with t(1;3)(p36;q21) showed similar mapped the t(3;10)(q26;q21) to the single nucleotide level, revealing a fusion clinical and prognostic characteristics with AML/MDS with inv(3)/t(3;3) and EVI1 of the MECOM gene (3q26.2) and C10orf107 (10q21.2). rearrangements. PRDM16 overexpression has been reported in AML in Summary and Conclusion: In AML patients, the MECOM gene can be absence of 1p36 rearrangements, but the mechanisms are still unknown and rearranged with a variety of other partner chromosomes and partner genes. the studies not conclusive. According to the Mitelman database, only one case with a t(3;10)(q26;q21) Aims: To characterize PRDM16 in cases with 1p36 abnormalities and to assess translocation has been reported, but neither the fusion partner of the MECOM PRDM16 expression in a cohort of AML without 1p36 involvement. gene nor DNA sequence were identified. The approach described here opens Methods: The study group was composed of 14 AML and MDS cases with 1p36 up new possibilities in characterizing acquired as well as congenital abnormalities and a cohort of 80 AML without 1p36 involvement by conventional chromosomal aberrations. In addition, DNA sequences of chromosomal cytogenetic (CC) and were analysed by relative RQ-PCR and FISH using 3 BAC breakpoints may be a useful tool for unique molecular MRD target identification probes covering PRDM16 and its flanking regions (BlueGnome Ltd. Cambridge, UK). in acute leukemia patients. Results: We identified 14 cases with 1p36 abnormalities: FISH analysis of 13 available samples identified 4 cases with PRDM16 rearrangement. Two cases showed a t(1;3)(p36;q21), one a t(1;21)(p36;q22) and the last one an add(1)(p36) in CC. In 3 cases the breakpoint was at 5’ of PRDM16, whereas in t(1;21) it was at 3’ of PRDM16, which is a rare event. We were not able to identify the chromosome partner involved in the case with add(1)(p36). Other 3 cases showed copy number gains of PRDM16 identified as the presence from 3 to 5 signals with all the three probes used in interphase FISH. To exclude the amplification of the whole p arm we used a control probe on 1p32 that did not show amplification. Metaphases FISH localized the site of amplification on the der(1)(p36) in two cases, whereas it was localized on unidentified chromosome in the last one. Rearrangements and amplification of PRDM16

haematologica | 2014; 99(s1) | 283 19 th Congress of the European Hematology Association

although in myeloid malignancies with a t(3;17) translocation over-expressed EVI1 rearranged with MSI2 7. Interestingly, haematological features of all the available cases suggest that GSX2 ectopic expression delineates a genetic subgroup among CD7+ AML. Although the fine mechanisms of GSX2 de- regulation remain to be understood, in our cases we hypothesize that they are related to inactivation of silencers located close to GSX2, at 5’ in patient 1 and at 3’ in patient 2.

1. Kurtzberg J et al. Blood 1989, 73:381-390 2. Harada H et al. Br J Haematol 1995, 90:850-4 3. Cools J et al. Blood 2002, 99: 1776-1784 4. Jeandidier E et al. Cancer Genet 2012 , 205:365-72 5. Lòpez-Juàrez et al. Genes Dev. 2013, 27:1272-1287 6. Matutes E et al. Eur J Haematol 1987, 38:303-9 7. De Weer A et al. Haematologica 2008, 93:1903-7.

Figure 1.

P782 GSX2 DEREGULATION IN CD7+ ACUTE MYELOID LEUKEMIA BEARING 4Q12 TRANSLOCATIONS WITHOUT FUSION GENES D Di Giacomo 1,* , R La Starza 1, V Pierini 1, G Barba 1, F Forghieri 2, E Borlenghi 3, C Mecucci 1 1Haematology and Bone Marrow Transplantation Unit, University of Perugia, Perugia, 2Department of Surgical and Medical Sciences, University of Modena- Reggio Emilia, Modena, 3Department of Haematology, Ospedali Civili, Brescia, Italy

Background: CD7 positivity (CD7+) in approximately 30% of acute myeloid leukemias (AML) is assumed to arise from an early precursor maintaining both Figure 1. myeloid and T-lymphoid potentialities 1. Interestingly, several chromosome translocations in AML were associated with CD7 expression 2,3,4 . One is the rare t(4;12)(q12;p13), with an estimated incidence of under 1% of adult AML, which is characterized by immature phenotype and poor clinical outcome 2. Molecular P783 characterization of t(4;12)(q12;p13) 3 showed that 12p13 breakpoints fell within TELOMERE LENGTH IS SIGNIFICANTLY SHORTENED IN AML PATIENTS ETV6 gene, while 4q12 breakpoints involved either CHIC2 or no genes. IN CYTOGENETIC REMISSION: POSSIBLE IMPLICATIONS ON THE Whatever genomic site recombined with 4q12, the pathogenetic effect 3 ORIGIN OF AML appeared to be linked to ectopic GSX2 . This homeobox gene MS Ventura Ferreira 1,* , M Crysandt 1, P Ziegler 1, J Kaufmann 1, S Hummel 1, is normally expressed only in neural stem cells as a key regulator of 1, 1, 1, 1 5 S Wilop E Jost T H Brümmendorf F Beier neurogenesis . 1Department of Hematology, Oncology and Stem Cell Transplantation, RWTH Aims: To investigate molecular features of translocations with 4q12 breakpoint Aachen University, Aachen, Germany in CD7+ acute myeloid leukemias. FISH and expression studies were performed on bone marrow cells Methods: Background: Telomere length (TL) reflects the replicative history of cells and from two patients with AML-M0 and a 4q12 chromosomal breakpoint. Both had 6 represents an established marker for tissue aging. Critical short telomeres small pseudo-lymphoid blasts with agranular basophilic cytoplasm and CD7 have been found to play an important role in the development of chromosomal expression. Karyotypes were: 37-46, XX, del(3) (p?), t(4;12) (q12;p13), del (5) instability and malignant transformation. Acute myeloid leukemia (AML) is a (q13q31), -15, -16, del (17) (q21q22), del (22) (q?), +mar1, +mar2 [cp9]/46, XX malignant, genetically heterogeneous disease primarily occurring in later in patient 1 and 46, XY, t(4;17) (q12;q22)[12]/46, XY[8] in patient 2. The 4q12 adulthood. Despite of tremendous progress, the origin of AML and especially region was investigated with LSI 4q12 Tricolor Rearrangement Probe (Vysis, the relationship to aging is not well understood. Abbott Molecular) and with homebrew BAC and fosmids (Figure 1). A break Aims: The question whether AML originates from prematurely aged apart FISH assay (RP11-434C1 for the 5’ and RP11-418C2+RP11-297N18 for hematopoietic stem cells (HSC) is still unclear. In the present study, we address the 3’) investigated ETV6 at 12p13 in patient 1; fosmids used for 17q22 in this issue by sequentially analyzing TL in AML patients at diagnosis, in patient 2 are listed in Figure 1. qRT-PCR was performed using Light Cycler 480 remission and during refractory disease or relapse. (Roche) and TaqMan assay probe (Applied Biosystems) Hs00370195_m1for Methods: TL of bone marrow (BM, n=88) and peripheral blood (PB, n=9) of 45 GSX2 gene. ABL1 (Hs00245445_m1) was the endogenous reference control. newly diagnosed AML patients was analyzed using monochrome multiplex RNA from GL15 cell line was our positive control for GSX2 expression quantitative-PCR. PB leukocytes of 87 healthy controls were used for age- (http://www.ncbi.nlm.nih.gov/UniGene/). In silico analysis was performed using adaption of TL. Mean age of the analyzed AML patients was 50.4 years (range ucsc database (http://genome.ucsc.edu/). 21-75). Follow-up included the following timepoints: at diagnosis (n=20), after Results: Breakpoints at 4q12 fell about 15 kb centromeric to GSX2 in patient two cycles of induction chemotherapy (IC, n=42), after three additional cycles 1, and 50kb telomeric to GSX2 in patient 2. Translocation partners were, of consolidation chemotherapy (CC, n=30) and one year after diagnosis (n=5). respectively, ETV6 (12p13) and MSI2 (17q22). GSX2 was significantly over- Results: Newly diagnosed AML patients showed a significantly shortened age- expressed in both cases compared with healthy donors and a group of CD7+ adapted TL (mean±SE: -0.65±0.2 T/S ratio, p=0.001). After induction AML without 4q12 involvement. chemotherapy and compared to TL at diagnosis, TL increased in patients with Summary and Conclusion: GSX2 de-regulation derived from two different complete remission (CR, -0.33±0.1 T/S ratio, n=34), but still remained recombinations involving 4q12 and ETV6 or MSI2 respectively. A moderately shortened compared to age-matched controls (p=0.001). In t(4;17)(q12;q22) with a MSI2 rearrangement has never been reported so far, comparison, patients with persistent AML showed no change in TL (-0.61±0.2

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T/S ratio, n=8). TL of patients in CR remained stable following three additional be performed in myeloid malignancies with chromosome 3q abnormalities. cycles of consolidation chemotherapy (-0.23±0.1 T/S ratio, n=26, p=0.03) as Furthermore, FISH should be performed in cases with 7q abnormalities when well as after one year (-0.25±0.1 T/S ratio, n=5, p=0.28). Patients with relapse the quality of metaphases is suboptimal. Supported by University of Bologna after consolidation chemotherapy remained substantially shortened (-1.0±0.2 RFO, BolognAIL and Coop Reno. T/S ratio, n=4, p=0.04). To exclude treatment related effects as a predominant reason for TL shortening, we longitudinally followed selected individual patients. We found no evidence for accelerated TL shortening in CR after induction (- P785 0.28±0.1 T/S ratio, n=24), after consolidation chemotherapy (-0.18±0.1 T/S ratio, n=24) and at one year follow-up (-0.25±0.1 T/S ratio, n=5) supporting the DEVELOPMENT OF MOLECULAR BEACON BASED PCR ASSAY FOR notion that chemotherapy does not influence TL during CR. EASY DETECTION AND QUANTIFICATION OF CHIMERIC TRANSCRIPTS Summary and Conclusion: In this study we show that TL of AML at diagnosis OF AML-ETO [T(8;21)] IN ACUTE MYELOID LEUKEMIA PATIENTS R Arora 1,* , S Jetly 1, S Bakhshi 2, S saluja 3, D Saluja 1 is substantially short and increases - reaching cytogenetic remission - mostly 1 due to a shift from leukemic cells (with shortened TL) to non-clonal cells. Biomedical Sciences, Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, 2Medical Oncology, All India Institiute of Medical sciences, However, age-adapted TL of patients in cytogenetic remission is still significantly 3 short in first CR and remains stably shortened after further treatment up until Hematology, Vardhaman Mahavir Medical college and Safdarjung hospital, one year of follow-up. In summary, our data provides first evidence that AML New Delhi, India possibly originates from HSC with prematurely shortened TL. The degree of TL shortening found in non-clonal cells during first CR can be translated into Background: Acute myeloid leukemia (AML) is a malignant disease of blood approx. 22.6 years of additional aging. arising as a result of cancer transformation and disorders in differentiation of hematopoietic cells on the level of myeloid cell precursor cells. Tumor cells of such patients often contain mutant forms of certain oncogenes, and therefore P784 mutations in these genes are believed to be responsible for malignization of the hematopoietic system. In 20% of patients with AML, leukemic cells carry a COMPLEX CHROMOSOMAL REARRANGEMENTS LEADING TO EVI1 translocation between chromosomes 21 and 8 resulting in formation of the OVEREXPRESSION ARE RECURRENT MECHANISMS IN CASES WITH chimeric oncogene AML1-ETO, producing a fused protein AML1-ETO, which VARIOUS 3Q ABNORMALITIES has the activity of a transcription factor. Molecular rearrangements are constant C Baldazzi 1,* , E Ottaviani 1, S Luatti 1, G Marzocchi 1, G Ameli 1, C Papayannidis 1, and occur in exon 5 of the AML1 gene and in exon 2 of the ETO gene. Protein C Gamberini 1, E Zuffa 1, M Cavo 1, G Martinelli 1, N Testoni 1 AML1-ETO contains the N-terminal region of AML1 protein, which includes the 1Institute of Hematology and Medical Oncology “L. e A. Seràgnoli”, S.Orsola- DNA and Core Binding Factor β ( CBF β)- binding Runt Homology Domain (RHD), Malpighi Hospital, Bologna, Italy whereas the C-terminal part belongs to ETO protein including its four Nervy Homology Region (NHR) domains. AML1 activates transcription and thus Background: Chromosomal rearrangements involving 3q26 region are a promots differentiation of granulocytes due to transactivation of series-specific recurrent finding in myeloid malignancies. These abnormalities lead to up- target genes while formation of the fused protein, AML1-ETO, results in the regulation of EVI1 gene that has been associated with a very poor prognosis. replacement of the AML1 activation domain by the ETO repressor domain. The EVI1 is also overexpressed in different subgroups of AML without cytogenetic fused protein AML1-ETO binds with DNA via the RHD domain of the AML1 abnormalities of 3q26, such as AML with -7/7q- abnormalities, which are also moiety and effectively recruits corepressor complex via the NHR domain of the most frequent additional abnormalities to EVI1 rearrangements. In some ETO moiety, and thus inhibits the expression of AML1 target genes instead of cases, FISH analysis revealed cryptic EVI1 rearrangements. activating them. Patients having AML-ETO translocation have a relatively Aims: To identify EVI1 rearrangements in cases with myeloid malignancies favourable prognosis and thus its detection helps the clinician to take better and 3q or -7/7q- abnormalities. therapeutic decisions. Cytogenetic analysis by G-banding (karyotyping) is Methods: The cases were selected according to the presence of 3q required at the time of diagnosis of all AML patients; however this method is abnormalities or -7/7q- by conventional cytogenetics (CC) and were analysed often time-consuming as it requires culturing of leukemic cells and their capture by FISH using EVI1 breakapart probes (Cytocell, UK), and by relative RQ-PCR. at metaphase. FISH analysis for AML1-ETO fusion can also be performed using Results: We analysed 97 AML, 28 SMD and 9 MPD cases with 3q (n=75) or - locus-specific probes. Dual color probes against AML1 and ETO resulting in 7/7q- (n=60) abnormalities. Among cases with 3q abnormalities, 24 showed fusion-signals are commercially available and commonly used but are not cost inv(3)/t(3;3), 13 balanced 3q26 translocations, 11 balanced 3q21 translocations effective. qPCR is a quick and sensitive method that is recommended for and 27 various 3q abnormalities involving different loci from 3q26. EVI1 detection of the fusion transcript but is highly expensive and requires rearrangements were detected in 32/37 (86.5%) cases with 3q26 abnormalities, infrastructure as well as expertise. Most of these methods are therefore not whereas 5/37 (13.5%) had 3q26 abnormalities without EVI1 involvement performed routinely in developing countries and therefore, it becomes suggesting the presence of other genes implicated in leukemogenesis. imperative to develop a cost effective and highly sensitive method for the Unexpectedly, 4/11 (36.4%) cases with balanced t(3q21) displayed EVI1 detection of AML-ETO translocation [t(8;21)] in Acute Myeloid Leukemia patients rearrangements. To better characterize these latter cases, metaphase FISH in developing countries. analyses was performed and revealed that EVI1 rearrangements were the Aims: To develop a PCR based easy to visualize quantitative assay for consequence of complex mechanisms involving multiple breakpoints on 3q detection of AML-ETO translocation [t(8;21)] in Acute Myeloid Leukemia arm that masked 3q26 region involvement. Multiple breakpoints were also patients. identified in a t(3;8)(q26;q22) by interphase FISH suggesting that this Methods: Peripheral blood samples were collected from untreated AML mechanism can occur in apparent classical 3q26 translocation, too. EVI1 patients from Safdarjung and AIIMS hospital. Cost effective RNA isolation was rearrangement was also detected in 2/27 (7.4%) cases with various 3q standardized from these blood samples by comparing expression of four abnormalities: metaphase FISH revealed a t(3;6)(q26;q25) and a housekeeping genes in leukocytes. In-house primers and molecular beacons t(1;3;13)(p34;q26;q14) that were cryptic because of suboptimal quality of were designed for the detection of AML-ETO transcript and PCR assay was metaphases. All cases with EVI1 rearrangements detected by FISH showed standardized for quantification of transcript using beacon strategy. The assay overexpression by RQ-PCR compared to 10 normal bone marrow samples. was evaluated against qPCR using published primers. Among cases with 7/7q- abnormalities, only one case demonstrated EVI1 Results: Using the technique of molecular beacons, we successfully developed involvement by FISH. A further examination of the karyotype allowed the a cost effective and highly specific and sensitive reverse transcriptase-PCR identification of a sub-clone with 3q abnormality harbouring EVI1 gene assay for AML-ETO detection. Our assay could detect as low as 100fg of AML- amplification (>50 copies) not previously identified. Gene amplification is a ETO chimeric DNA and shows a linear increase in fluorescence with increase mechanism leading to overexpression that has been rarely described for EVI1. in template concentration (Figure 1). The specificity of our assay was confirmed Other 8 cases showed elevated EVI1 expression without EVI1 rearrangements. using competition experiments. The clinical evaluation of the assay has been Thirty-nine cases showed EVI1 rearrangement. In 12 (30.8%) cases it was the carried out using 100 leukemic samples. sole cytogenetic abnormality; -7/7q- was found in 25 cases (64.1%); del(5q) was Summary and Conclusion: We envisage that the current method is as seen in 7 cases (17.9%) and complex karyotype in 10 cases (25.6%). The sensitive and specific as commercially available method. Since the method is majority of patients were diagnosed as de novo AML or MDS (61.5%), median highly cost effective, and does not require expensive infrastructure and age was 51 years; 24 were male and 15 female. Only 5 of 24 treated patients expertise, it can be used in resource poor settings. (20.8%) achieved a complete cytogenetic remission after more than one cycle of chemotherapy. The median survival of EVI1 rearranged cases was 10.2 months, with an overall survival of 33% at 1 year, and 5% at 3 and 5 years. Summary and Conclusion: Although 3q26 abnormalities are strictly associated with EVI1 rearrangements, FISH and RQ-PCR identified cases with 3q26 abnormalities involving EVI1 and cases with EVI1 rearrangements without involvement of 3q26 by CC. These events could be the consequence of a low quality of metaphases, but they were often the result of complex chromosomal rearrangements that occurred frequently in t(3q21) cases. Because of poor prognosis of EVI1 overexpression, a screening for EVI1 rearrangements should

haematologica | 2014; 99(s1) | 285 19 th Congress of the European Hematology Association

Figure 1. (A) OS in five groups of CN-AML patients: R2high/noFLT3-ITD, R2low/noFLT3-ITD, R2low/FLT3-ITD, R2high/FLT3-ITD and CEBPAmut. (B) OS in five groups of CN-AML patients: NPM1mut/noFLT3-ITD, NPM1wt/noFLT3-ITD, NPM1wt/FLT3-ITD, NPM1mut/FLT3-ITD and CEBPA - mut. Summary and Conclusion: In our study we found that the expression level Figure 1. Graphical representation of fluorescent intensity of product of R2 was elevated compared to HVs suggesting that not only NPM1 mutation obtained by carring in-house standardized AML-ETO specific PCR with but also its splice variant expression might play some role in the process of the different concentrations of positive close (TA vector containing AML-ETO tumorigenesis. As the R2 represents a truncated form of NPM1 gene, this insert), with 50nM beacon added to each, measured on Elisa reader (at isoform mostly localizes in the nucleoplasm, and thus might also have a 550,590nm). biological impact in the malignant cells. In our cohort of cases survival differences seen between the established ELN groups according to a NPM1 /FLT3 -ITD stratification were less impressive than between groups stratified according to R2 expression/ FLT3 -ITD mutational status. In summary, P786 the expression of R2 might be of biological importance for CN-AML patients. ANALYSIS OF NPM1 SPLICE VARIANTS REVEALS DIFFERENTIAL Moreover, R2 splice variant provides prognostic value for CN-AML patients EXPRESSION PATTERNS OF PROGNOSTIC VALUE IN ACUTE MYELOID and it might give information in addition to the NPM1 mutational status. LEUKEMIA M Zajac 1,* , A Dolnik 2, M Schunn 2, S Correa 2, 3, 4, E Glodkowska-Mrowka 5, R Chakkarappan Sundaram 2, O Jankowska 6, R Schlenk 2, K Dohner 2, P787 L Bullinger 2, K Giannopoulos 1,6 HIGH LEVELS OF HOTAIRM1, A LONG INTERGENIC NON-CODING RNA 1Experimental Hematooncology Department, Medical University of Lublin, RELATED TO HOX GENES, IS ASSOCIATED WITH POOR PROGNOSIS Lublin, Poland, 2Department of Internal Medicine III, University of Ulm, Ulm, AND A DISTINCTIVE MICRORNA SIGNATURE IN INTERMEDIATE-RISK Germany, 3Institute of Biophysics Carlos Chagas Filho, Federal University of 4 ACUTE MYELOID LEUKEMIA Rio de Janeiro, Stem-cell Laboratory, Bone Marrow Transplantation Unit, 1,* , 2, 1, 1, 3, 5 M Diaz Beya A Navarro M Pratcorona M Nomdedeu R M Risueño National Cancer Institute, Rio de Janeiro, Brazil, Department of Immunology, 2, 1 6 M Monzo J Esteve Medical University of Warsaw, Warsaw, Department of Hematooncology and 1Hematology Department,, Hospital Clínic, IDIBAPS, Barcelona, Spain, Bone Marrow Transplantation Unit, Medical University of Lublin, Lublin, Poland 2Molecular Oncology and Embryology Laboratory, Human Anatomy Unit,, School of Medicine, University of Barcelona, 31Josep Carreras Leukaemia Background: The process of mRNA splicing has been reported to play an Research Institute, Barcelona, Spain important role in human disease development and many cancer-related genes are regulated by alternative splicing. In addition, first analyses of alternatively Background: During the last years non-coding RNAs (ncRNAs) have emerged splicing in bone marrow of AML samples identified novel splice variants specific as key regulators of diverse cellular processes. They are classified according for AML patients in comparison to normal cells. to their size in short ( i.e. microRNAs) and long ( i.e. lincRNAs) ncRNAs. Aims: Since splicing variants play an important role in cellular functioning the LincRNAs are long ncRNAs located in intergenic regions with multiple current study focuses on the characterization of NPM1 splicing variants regulatory functions including gene expression regulation. Interestingly, an expression as well as its impact on the biology and prognosis of AML patients. active crosstalk between microRNAs and lincRNAs has been shown. LincRNAs Methods: For the first cohort of patients (104 samples) qRT-PCR was performed are known to be deregulated in some cancers but their potential importance in and total expression (Rt) as well as levels of the three splice variants of NPM1 acute myeloid leukemia (AML) is so far unknown. HOX genes play an important were evaluated: R1 (exons 1- 9 and 11-12), R2 (exons 1- 10), R3 (exons 1-7, 9 role in hematopoiesis and are deregulated in AML. Some lincRNAs, including and 11-12). We found prognostic significance of the expression level of R2, HOTAIRM1, HOTTIP and HOTAIR, are located in the HOX genomic regions, therefore we decided to validate it in independent cohort of AML patients. We but their expression level and prognosis role have not been studied in patients consolidated 104 patients previously analyzed with 97 patients from the new with AML. Since the intermediate risk cytogenetic (IR)-AML is a group with cohort of total 201 cases and preformed the final analysis for R2. The existence highly diverse prognosis; we analyzed the prognosis value of expression of of R2 at the protein level was evaluated with the use of Western Blot technique. these lincRNAs in this subgroup of patients. To investigate whether R2 might disrupt localization of the NPM1 wild type protein, Aims: To investigate whether the expression of HOX-related lincRNAs is immunohistochemistry analysis for NPM1 in 23 AML cases was performed. associated with molecular characteristics, miRNA expression, and clinical Results: Total expression as well as expression of R1 and R3 were significantly outcome in IR-AML. higher in 104 AML patients compared to healthy volunteers (HVs) with a median Methods: We have analyzed bone marrow samples from 77 IR-AML patients expression of 8.59 vs 0.93 (p=0.001), 1.73 vs 0.55 (p=0.014), and 2.54 vs 0.11 (median age, 52; 51% males) who received intensive chemotherapy following (p<0.001), respectively. We evaluated the existence of R2 at the protein level CETLAM trials in a single institution. Forty-two patients harbored NPM1 in AML patients samples and AML cell lines. We found that the expression of mutation ( NPM1 mut, 54%), 35 FLT3 -ITD (45%) and 7 patients, a biallelic R2 was significantly higher in all AML patients compared to HVs with a median CEBPA mutation (9%). The expression of HOTAIRM1 (Hs03296533_g1), expression of 1.64 vs 0.33 (p=0.009). High R2 expression was associated with HOTAIR (Hs03296631_m1) and HOTTIP (Hs00955374_s1) was analyzed longer OS when CN-AML patients were analyzed (880 vs 438 days, p=0.028). using TaqMan ® Gene Expression Assay (Applied Biosystems). Statistical Longer OS was observed in CN-AML patients with high R2 expression without analysis was performed with BRB Array Tools, SPSS v15.0.1. MaxStat program concomitant FLT3 -ITD mutations compared to the rest of groups. Most from R software was used to determine the optimal cutoff point. importantly, in CN-AML cases survival differences seen between the Results: HOTTIP and HOTAIRM1 , but not HOTAIR , were expressed in most established ELN groups according to a NPM1 /FLT3 -ITD stratification were less of our AML samples. HOTAIRM1 expression was associated with NPM1 mut impressive (p=0.03) than between groups stratified according to R2 (p=0.005). In the entire cohort, high HOTAIRM1 expression was associated with expression/ FLT3 -ITD mutational status (p=0.003). Multivariate analysis shorter 5-year survival (OS) (p=0.003, 14±18% vs .44±14%), shorter 5-year revealed R2 expression (HR, 0.470; 95%>CI, 0.098 to 0.842; p=0.042), FLT3 - disease-free survival (p=0.002, 52±12% vs .91±26%), and a higher risk of ITD alteration (HR, 3.325; 95%>CI, 3.000 to 3.650; p<0.001), and age (HR, relapse at 5 year (p=0.012, 85±25% vs .35±14%). This effect was maintained 1.035; 95%>CI, 1.023 to 1.047, p=0.004), but not WBC or NPM1 mut as both within the subgroup of NPM1 mutated patients (OS, p=0.013, 20% ±24% significant variables for OS. Finally, in cases with high R2 expression we were vs . 52±18% ) and within subgroup lacking favorable molecular features ( i.e. , able to determine a cytoplasmic localization of NPM1 even in the absence of absence of NPM1 mutation and CEBPA biallelic mutation and/or FLT3 -ITD) NPM1 mutation. Therefore, we provide further evidence that the cytoplasmic (OS, p=0.017, 10±18% vs .40±15%). In the multivariate analysis including age, localization of NPM1 might depend not only on its mutational status, but might WBC, NPM1 mut, FLT3 -ITD as covariates, HOTAIRM1 expression level showed also be influenced by the distribution of its splice variants. independent prognostic significance (p=0.003; HR=3.052, 95% CI: 1.5-6.3)

286 | haematologica | 2014; 99(s1) Milan, Italy, June 12 – 15, 2014 both in the entire cohort and the unfavorable molecular subgroup (p=0.017; 49 vs 25.1, p<0.01, Mann-Whitney). As shown in Fig.1 the highest expression HR=2.8, 95% CI: 1.2-6.5). Supervised analysis by means of t-test based on was reported in NPM mut/ FLT3 -ITD genotype AML in both bulk and multiplex permutations revealed a distinctive 33-miRNA signature which CD34+CD38- leukemic cell populations (mean value of MedFI 55.1 and 93.6, correlated with HOTAIRM1 expression. Importantly, a positive correlation was respectively). observed between HOTAIRM1 and miR-196b (p<0.001), a miRNA located in the same HOX genomic region.

Figure 1. Summary and Conclusion: Our results indicate that NPM1 and FLT3 -ITD Figure 1. Prognostic value of HOTAIRM1 levels. mutations correlate with higher CD123 expression levels in AML. Although CD123 was globally positive in most cases of AML in our series, it was markedly Summary and Conclusion: In this series of IR-AML patients, HOTTIP and expressed in AML carrying mutations of both NPM1 and FLT3 either on HOTAIRM1 were expressed in most patients. The expression level of the HOX- leukemic bulk cells as well as on CD34+CD38- putative LSCs, making this AML related lincRNA HOTAIRM1 showed independent prognostic value. subtype a suitable candidate for CD123-targeted immunotherapy. Interestingly, HOTAIRM1 expression levels showed a strong positive correlation with its neighboring gene miR-196b. Nonetheless, confirmation of the prognostic impact of this lincRNA and search of potential underlying mechanisms P789 accounting for this prognostic effect is warranted. Acknowledgments: ISCIII RH13/00205, SEHH, FIS13/00999 THE PROGNOSTIC VALUE OF LEUKEMIC STEM CELLS IN PEDIATRIC AML B De Caluwé 1,* , R Wouters 2, V De Haas 3, J G te Marvelde 4, B De Moerloose 5, 6, 1, 3, 7, 1, 2, P788 E S de Bont B Denys A De Jong G Kaspers J Philippé J Cloos V H van der Velden 4 CD123 (IL-3R α) IS CONSISTENTLY EXPRESSED ON ACUTE MYELOID 1Department of Clinical Biology, Microbiology and Immunology, University of LEUKEMIA (AML) CARRYING NUCLEOPHOSMIN (NPM1) GENE Ghent, Ghent, Belgium, 2Pediatrics, VU University Medical Center, Amsterdam, MUTATION 3DCOG, The Hague, 4Immunology, Erasmus Medical Center, Rotterdam, L Brunetti 1,* , I Gionfriddo 1, R Roberta 1, F Mezzasoma 1, F Milano 1, C Vetro 2, Netherlands, 5Pediatrics, University of Ghent, Ghent, Belgium, 6Pediatrics, F Di Raimondo 2, B Falini 1, MP Martelli 1 UMCG, Groningen, 7Pediatric Oncology/Hematology and Hematology, VU 1Hematology, University of Perugia, Perugia, 2Hematology, University of University Medical Center, Amsterdam, Netherlands Catania, Catania, Italy Background: Pediatric acute myeloid leukemia (AML) patients exhibit a high Background: Antibody-based immunotherapy is a promising strategy to target risk of relapse (30-40%) and therapy related mortality (5-15%), resulting in a 5 and eliminate chemoresistant leukemic cells. Bispecific T cell engaging year overall survival rate of only 65-70%. Because of the rarity of AML in antibodies (BiTEs) are a novel class of antibodies which recruit in vivo the children, little is known about the pathogenesis and few adequate prognostic patient’s own cytotoxic T cells and retarget them directly at specific surface parameters have been identified. The cancer stem cell model proposes that antigens on leukemic cells. The interleukin-3 receptor a chain (CD123) has AML cells are hierarchically organized into compartments that contain leukemic been identified as a potential immunotherapeutic target because it is stem cells (LSC) which have unlimited self-renewal capacity and are capable overexpressed in AML compared to normal hematopoietic stem cells. Notably, of propagating leukemia and relapse. Although in adult AML it has convincingly CD123 is selectively overexpressed not only on AML blasts, but also on been demonstrated that the number of LSC (CD34+CD38-) at diagnosis is an CD34+CD38- AML leukemic stem cells (LSCs) that are considered to be major important prognostic factor 1, this has not thoroughly been investigated in players in chemotherapy drug resistance and leukemia relapse. CD123 as pediatric AML. immunotherapeutic target in AML has been validated in preclinical studies with Aims: Despite complete remission rates of about 70%, the majority of pediatric either CD123xCD3 bispecific antibodies or CD123 CAR-engineered T cells and patients with AML relapses within 3-5 years from diagnosis. Therefore, there is clinical trials with these agents are expected in the next future. Nucleophosmin great need of identifying more sensitive prognostic factors that can predict (NPM1) mutation is the most common genetic lesion in AML accounting for relapse (originating from LSC outgrowth). We explored whether the number of about 30% of cases. Immunohistochemical studies, have previously shown LSC at diagnosis, detected with flow cytometry, is correlated with the risk of that in NPM1 -mutated as well as in FLT3-ITD AMLs, CD123 is more frequently relapse and time to relapse. expressed. However, a comprehensive and quantitative evaluation of CD123 Methods: Within the framework of the DB-AML01 treatment protocol (a expression in AML has not been reported. collaboration between BSPHO and DCOG), serial samples from diagnosis till Aims: Here, we quantified CD123 expression by flow cytometry correlating end of treatment were collected. Sixty-seven Dutch and 42 Belgium diagnostic expression intensity with cytogenetic and molecular disease characteristics in samples were available for flow cytometric analysis. Our initial analyses order to identify patient cohorts suitable for CD123-targeted therapy. included the Dutch samples. Stem cells were analyzed using the labeling CD45- Methods: CD123 expression levels were evaluated by flow cytometry in fresh CD34-CD117-CD38-CD2+56-CD123-CD7-HLA DR. LSC were defined as samples from 130 consecutive adult AML patients at diagnosis, using an antibody CD34+/CD38- cells (fixed cut-off values 10³ and 3x10³, respectively) and combination including CD34-FITC/CD123-PE/CD45-PerCP-Cy5.5/CD38-APC, expressed as a percentage of the total amount of CD34+ cells. For all analyses acquiring at least 50000 events and reporting both percentage of CD123 positive the Infinicyt software was used. Statistics were performed using SPSS (version cells (PPC) and CD123 median fluorescence intensity (MedFI) in both bulk and 22.0). CD34+CD38- cell population. Blasts were gated using both FSC/SSC and Results: Mann Whitney U-Test showed that the percentage of LSC was CD45/SSC dot-plots. For all patients NPM1 gene mutation status was assessed significantly associated with the risk of relapse (p=0,034) and the risk of adverse by immunohistochemistry, Western Blot and/or molecular analysis. FLT3 event (relapse or death) (p=0,023). ROC curve analysis showed that at a cut- mutational status was assessed by RT-PCR. 60/130 (46%) carried NPM1 gene off of 23,5%, relapse can be predicted with a sensitivity of 69% and a specificity mutation; 27/93 (25%) were FLT3 -ITD mutated. of 75%. Kaplan-Meier survival analysis showed that this percentage was Results: According to karyotype (n=94), CD123 was more expressed in AML significantly associated with relapse free survival (p=0,006). Neither the with normal karyotype than in AML with one or more karyotype abnormalities percentage of blasts, the percentage of CD34+ cells, the number of white blood (mean values of MedFI 36.2 vs 19.5, p<0.01, Mann-Whitney). According to cells (WBC) or the presence of genetic abnormalities (inv(16), FLT3, NPM1 NPM1 gene status (n=130), NPM1 -mutated (NPMmut) AML showed brighter and t(8;21) at diagnosis proved to be significantly associated with the risk of CD123 expression as compared to NPM1 wild type (NPMwt) (mean values of relapse or relapse free survival. ROC curve analysis showed that at a cut-off MedFI 43.3 vs 22.1, p<0.01, Mann-Whitney). According to FLT3 gene status of 17,1% LSC, the occurrence of an adverse event can be predicted with a (n=93), FLT3 -ITD AML were associated with higher CD123 expression when sensitivity of 76% and a specificity of 72%. Kaplan-Meier survival analysis compared to the FLT3 wild-type (FLT3wt) counterparts (mean values of MedFI showed that this percentage was significantly associated with event free survival

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(p=0,002). Kaplan-Meier survival analysis also showed that the amount of WBC breakpoints involved the Zinc finger E-box Binding homeobox 2 ( ZEB2 ) gene. was significantly associated with event free survival (p=0,002). ZEB2 exon 2 was fused in-frame to BCL11B exon 2. Western blotting detected 2 ZEB2-BCL11B splicing isoforms with or without BCL11B exon 3. Breakpoints at 6q25 fell in a region where no genes have been mapped. ZEB2-BCL11B and BCL11B expression was high in t(2;14) and t(6;14) AML, respectively. SNP analysis confirmed all karyotypic changes and detected non-recurrent copy number variations. High Throughput Sequencing did not find any other common fusion or mutation. Genes like DNMT3A, TET2, WT1, EP300 were involved in 6/8 patients. Gene Expression Profiling identified a specific BCL11B translocation signature. Differential expression of 14/39 BCL11B targets indicated common downstream targets in t(2;14) and t(6;14). No cryptic BCL11B translocations were found. Summary and Conclusion: Two recurrent translocations, (2;14)(q22.3;q32) and t(6;14)(q25;q32), deregulated BCL11B in a CD2+, FLT3+, AML and have a specific gene expression signature. In the t(2;14) the BCL11B involved in the ZEB2/BCL11B fusion was overexpressed. Notably the ZEB2-BCL11B protein and BCL11B share a RRKQXXP NuRD interaction motif at the N- terminal 1,8 and the same C-terminal containing BCL11B C2H2-zinc finger DNA binding domain. The mechanism of BCL11B up-regulation in the t(6;14) remains to be determined. A super enhancer mapped at 6q25, however, is an interesting candidate. In our study we showed that as a consequence of these translocations BCL11B and ZEB2-BCL11B may act as similar transcription factors. References 1. Cismasiu VB et al. Oncogene 2005;24:6753-64 2. Kadoch C et al. Nat Genet. 2013;45:592-601 3. De Keersmaecker K et al. Nat Med. 2010;16:1321-7 4. Gutierrez A et al. Blood 2011;118:4169-73 5. Przybylski GK et al. Leukemia 2005;19:201-8 6. Gesk S et al. Leukemia 2003;17:738-45 7. Abbas S et al. Haematologica 2014;doi:10.3324/haematol.2013.095604 8. Verstappen G. et al. Hum Mol Genet. 2008;17:1175-83 Figure 1. Summary and Conclusion: The percentage of stem cells, detected at diagnosis with flow cytometry, is a prognostic factor for relapse in pediatric P791 AML patients. Our current studies focus on refinement of the analysis strategy which will be applied to the 42 Belgian patients at diagnosis. In addition, the PSEUDO-EXHAUSTION OF T CELLS IN RELAPSED AML F S Lichtenegger 1,2,* , F M Schnorfeil 1,2, K Emmerig 1,2, M Schlueter 1,2, value of measuring LSC in the MRD setting will be evaluated. Furthermore, J S Neitz 1,2, R Draenert 3, W Hiddemann 1, M Subklewe 1,2 these studies will be extended in the recently started NOPHO-DBH AML 2012 1Department of Internal Medicine III, Klinikum der Universität München, trial. 1van Rhenen A et al (2005). Clin Cancer Res 11: 6520-6527. This study 2Clinical Cooperation Group Immunotherapy, Helmholtz Zentrum München, was supported by KIKA, the Netherlands. 3Division of Clinical Infectiology, Department of Internal Medicine IV, Klinikum der Universität München, Munich, Germany P790 Background: The prognosis of acute myeloid leukemia (AML), particularly BCL11B UP-REGULATION IN ACUTE MYELOID LEUKEMIA WITH CD2 T- when associated with adverse chromosomal or molecular aberrations, is poor ANTIGEN EXPRESSION AND FLT3 MUTATION due to a high relapse rate after induction chemotherapy. Postremission L Brandimarte 1,* , R La Starza 1, P Gorello 1, K De Keersmaecker 2, Z Kalender 3, therapy for elimination of minimal residual disease remains a major challenge. C Matteucci 1, I Lahortiga 4, D Di Giacomo 1, V Pierini 1, E Gottardi 5, F Daraio 5, Immunotherapeutic strategies aim at the stimulation of AML-specific + M Negrini 6, B Crescenzi 1, G Barba 1, L Elia 7, L Luciano 8, A Ambrosetti 9, immunity, especially of CD8 T cells. However, the functionality of these cells N Testoni 10 , D Raspadori 11 , I Wlodarska 12 , P Vandenberghe 12 , S Aerts 12 , in AML patients is not well described. T cell exhaustion has been suggested J Cools 2, C Mecucci 1 to contribute to immune evasion in various solid and hematological 1Hematology and Bone Marrow Transplantation Unit, University of Perugia, malignancies. Primarily demonstrated in chronic viral infections, exhausted Perugia, Italy, 2Center for Human Genetics and Center for the Biology of T cells are characterized by an increased expression of several inhibitory Disease, 3Department of Human Genetics and Flanders Interuniversity Institute molecules, reduced proliferation and an impaired capability of cytokine for Biotechnology, 4Center for Human Genetics and Center for the Biology of secretion and cytotoxicity. Disease, Leuven, Belgium, 5Department of Clinical and Biological Sciences, Aims: To characterize T cell phenotype and function in AML patients at different University of Turin, Turin, 6University of Ferrara, Ferrara, 7Department of stages of disease. Cellular Biotechnologies and Hematology, Sapienza University, Rome, Methods: CD8 + and CD4 + T cells from AML patients at primary diagnosis, 8University of Federico II, Naples, 9Department of Hematology, Policlinico GB with refractory disease, at relapse and at relapse after allogeneic stem cell Rossi, Verona, 10 ’’Seràgnoli’’ Institute of Hematology, Bologna University transplantation (alloSCT) (23, 4, 9 and 7 individuals, respectively) were School of Medicine, Bologna, 11 Department of Hematology, University of Siena, analyzed by flow cytometry-based assays. Surface expression of CD244, Siena, Italy, 12 Center for Human Genetics, Leuven, Belgium CD160, PD-1, TIM-3 and LAG-3 was determined. T cell proliferation and production of the cytokines IFN- γ, TNF- α and IL-2 were measured in response Background: BCL11B/ 14q32 belongs to kruppel zinc finger family of to different stimuli. Results were compared to healthy controls (HC) (30 transcription factors directly binding a GC-rich consensus sequence in target individuals), while untreated HIV-infected patients (10 individuals) served as genes. It interacts with the Nucleosome Remodelling Deacetylase (NuRD) positive controls for an exhausted T cell state. complex and is a subunit of mammalian SWI/SNF, the most frequently mutated Results: In HIV-infected patients, we observed a pronounced upregulation of chromatin-regulatory complex in human cancer. 1,2 In hematological diseases, the inhibitory molecules CD244, CD160 and PD-1 on CD4 + and CD8 + T cells BCL11B might behave as tumor suppressor gene or oncogene. It is as well as globally impaired cytokine production, clearly indicating T cell downregulated by mono-allelic deletions or mutations in 10-15% of T-ALL 3,4 exhaustion. In contrast, T cells from AML patients at primary diagnosis showed and in cases with inv(14)(q11q32)/t(14;14)(q11;q32). 5,6 Over-expression an expression pattern of inhibitory surface molecules that was similar to T occurred in 4 AML cases. 7 cells from age-matched HCs. Interestingly, AML patients with a relapse after Aims: To investigate AML with 14q32 reciprocal translocations alloSCT showed a 3- and 6-fold increased overall expression of PD-1 on CD8 + Methods: Karyotyping identified t(2;14)(q22;q32) and t(6;14)(q25;q32) (p=0.0084) and CD4 + (p<0.0001) T cells, respectively. This PD-1 expression respectively in 6 and 2 FLT3 mutated AML, with CD2 positivity. Other expressed pattern correlated to an increased proportion of memory T cells, which have T-antigens were CD7 and/or cyCD3. Fluorescence in situ hybridization (FISH), an inherently higher expression of PD-1. Both, relapsed patients after RT-PCR, cloning and sequencing investigated partner gene breakpoints. qRT- conventional chemotherapy and relapsed patients after alloSCT, displayed a PCR (Light Cycler 480, Roche) evaluated BCL11B expression. Affymetrix shift from the naïve towards the memory T cell compartment, indicating SNPa, (Cytogenetics Whole-Genome 2.7M Array Platform), Exome and RNA reinforced T cell differentiation. Functionally, no defect in T cell proliferation in sequencing (Hiseq 2000, Illumina) were performed. FISH studied cryptic any of the AML patient cohorts was detected. Of note, however, we observed BCL11B rearrangements in 689 cases. a 2-fold decrease (p=0.0068) in IFN- γ production by CD4 + T cells exclusively Results: 14q32 breakpoints fell at the BCL11B gene 5’ end in all cases. 2q22.3 in patients at primary diagnosis.

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Summary and Conclusion: We show that T cells of newly diagnosed and Results: Overall, in 7/12 relapsed patients (Pt) (58.3%), mutations within the relapsed AML patients are fully functional. Moreover, we demonstrate enhanced PML-RARA fusion transcript were detected. In these seven patients we T cell differentiation in relapsed AML patients. We therefore hypothesize that performed mutation analysis also in samples taken at primary diagnosis. In bulk T cells in AML are in a status of activation, not exhaustion. Thus, none of the cases these mutations were detectable at initial diagnosis and thus immunotherapies that aim at eliciting tumor-specific immune responses, e.g. all were acquired mutations. All mutations were missense mutations with dendritic cell based vaccines, may be particularly suited for postremission mutation loads of 10-100%. Two of seven patients were treated with ATRA and therapy. had a mutation in the RARA region of PML-RARA . One of these mutations was previously described (p.Ser287Leu), whereas the other was novel (p.Val135Leu). Five of seven patients were treated with ATRA and ATO and P792 harbored mutations in the PML region of PML-RARA. All five patients had at least two and up to four mutations in the PML region of PML-RARA (Pt1: PROSPECTIVE LONG-TERM MINIMAL RESIDUAL DISEASE MONITORING p.Gly269Ser + p.Val301Met + p.Ala303Val; Pt2: p.Leu81Pro + p.Glu277Lys + ON PERIPHERAL BLOOD IN RUNX1-RUNX1T1 ACUTE MYELOID p.Ala303Thr + p.Gln354Arg; Pt3: p.Ala263Ser + p.Glu296*; Pt4: p.Ala125Thr LEUKEMIA: RESULTS OF THE FRENCH CBF-2006 TRIAL + p.Arg282His + p.Val287Ile; Pt5: p.Glu281X + p.Gly317Asp) . All PML C Willekens 1,* , O Blanchet 2, A Renneville 1, P Cornillet-Lefebvre 3, E Jourdan 4, 5 6 7 8 2 1 mutations were located in functional domains and were not described before. C Pautas , R Guièze , N Vey , H Dombret , N Ifrah , C Preudhomme , In three patients material from different relapse time points was available. In the N Boissel 8 1 2 first patient three relapses occurred and all three mutations in the PML region Centre Hospitalier Regional Universitaire, Lille, Centre Hospitalier of PML-RARA were for the first time detectable at the time point of third relapse Universitaire, Angers, 3Centre Hospitalier Universitaire, Reims, 4Centre 5 after treatment with ATRA and ATO. The second patient had two relapses. Four Hospitalier Universitaire, Nimes, Centre Hospitalier Universitaire, Créteil, mutations in the PML region of PML-RARA were detected at first relapse after 6Centre Hospitalier Universitaire, Clermont-Ferrand, 7Institut Paoli Calmette, 8 treatment with ATRA and were not detectable at the time point of second relapse Marseille, Hopital Saint Louis, Paris, France after treatment with ATRA and ATO. The third patient also had two relapses (no treatment information available). Mutations in the PML region of PML-RARA Background: Core-binding factor acute myeloid leukemias (AML) are were detected at first relapse and were not detectable at second relapse. associated with a favorable prognosis. However, around one third of patients Summary and Conclusion: We demonstrate a high incidence of acquired will present hematological relapse. In AML with translocation t(8;21)(q22;q22), mutations within the PML-RARA fusion transcript in relapsed APL patients. quantification of RUNX1-RUNX1T1 fusion transcript with real-time quantitative Although functional data is still missing, these mutations may confer resistance polymerase chain reaction (RQ-PCR) allows to monitor minimal residual to ATRA and ATO. These data may have implications for further treatment disease (MRD) during and after therapy. In the CBF2006 trial, we showed that strategies of such patients. early MRD response was a major prognostic factor (Jourdan E. et al, Blood 2013). Until now, the prognosis of MRD long-term follow-up has been rarely explored in prospective trials. Aims: The main objective of the study was to assess the usefulness of prospective MRD monitoring on peripheral blood (PB) by RQ-PCR to predict relapse. Methods: In the multicenter CBF-2006 study, 96 patients with t(8;21) AML in complete remission after intensive chemotherapy entered a prospective peripheral blood (every 3 months) and bone marrow (every year) MRD monitoring for 2 years. Transcript ratios were normalized to ABL as ( RUNX1- RUNX1T1/ABL ) x 100 and complete molecular response (CMR) was defined by a transcript ratio ≤0.001% on peripheral blood. Prognostic impact of CMR and loss of CMR on cumulative incidence of relapse (CIR) and overall survival (OS) were evaluated in a time-dependent manner (Mantel-Byar analyzes). Results: During the 2-year follow up, 77 patients reached CMR. Median time between complete remission and CMR was 2.5 months [IQ: 1.4 – 4.6] up to 16 months. As expected, CMR achievement was associated with significantly reduced incidence of relapse (3-year CIR: 23% vs 51%, P=.001) and better overall survival (3-year OS: 90% vs 56%, P=.008). However, time to CMR was not predictive of CIR or OS. Among the 77 patients who achieved CMR, 23 patients presented a positive MRD (>0.001%) with a median time of 6.9 months [IQ, 3.9 – 13.3] up to 23 months after CMR achievement. Loss of CMR was confirmed on a second sample in 13/23 patients (57%). Cumulative incidence of relapse was significantly higher in patients with a confirmed loss of CMR when compared to patients with a non confirmed loss of CMR (3-years CIR, 83% vs 14%, p=.03) with no difference in overall survival. In patients with confirmed loss of CMR, time between first positive MRD and hematological relapse was 3.9 months [IQ: 3.3 – 6.9]. Summary and Conclusion: Contrarily to early MRD reduction on therapy, time to CMR achievement is not predictive of relapse in patients with RUNX1- RUNX1T1 AML. Monitoring peripheral blood MRD every 3 months up to 2 years after CR allowed to predict and anticipate hematological relapse.

P793 FREQUENT MUTATIONS IN PML-RARA IN RELAPSED ACUTE PROMYELOCYTIC LEUKEMIA A Fasan 1,* , C Haferlach 1, E Sirch 1, W Kern 1, T Haferlach 1, S Schnittger 1 1MLL Munich Leukemia Laboratory, Munich, Germany

Background: Two therapeutic agents, ATRA and arsenic trioxide (ATO), induce differentiation of promyelocytes in vivo and clinical remission of acute promyelocytic leukemia (APL) patients. However, mutations in the RARA and PML parts of the PML-RARA fusion gene have been reported to confer resistance to ATRA (Imaizumi et al. , Blood 1998) and ATO (Goto et al. , Blood 2011), respectively. Aims: The evaluation of mutations within the PML-RARA fusion gene in relapsed APL. Methods: We screened 12 cases with APL who showed hematologic or molecular relapse for mutations within the PML-RARA fusion transcript. Amplified PML-RARA fusion transcript was analyzed by direct Sanger sequencing. The detection limit of this analysis was 1:10 allowing the detection of 1 mutated allele in a background of 10 wildtype alleles. Eight patients were male and four patients were female with a median age of 47.15 years (range: 16.7-73.2 years).

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