Cytogenetics Aged from 1 Month to 18 Years Diagnosed Between December 1995 and May 2011

Amsterdam, the Netherlands, June 14 – 17, 2012

Cytogenetics aged from 1 month to 18 years diagnosed between December 1995 and May 2011. The mutated genes examined included class I gene involving signaling and RAS pathways (FLT3 -ITD, FLT3 -TKD, C-FMS , C-KIT, NRAS, KRAS, PTPN11, JAK2 V617F), class II affecting transcription and differentiation 0682 (CEBP α, RUNX1 , MLL- PTD, and NPM1 ), class III of tumor suppressor genes (WT-1 and P53) , and class IV of epigenetic regulators ( ASXL1 , DNMT3A , IDH1 THE NUP98-NSD1 FUSION IN ASSOCIATION WITH FLT3-ITD MUTATION and IDH2 ). Mutational analysis was performed with PCR-based assays fol - IDENTIFIES A PROGNOSTICALLY RELEVANT SUBGROUP OF PAEDI - lowed by direct sequencing. Results. One hundred and twenty of 206 patients ATRIC AML, WHICH CAN BE SALVAGED BY ALLOGENEIC TRANSPLANT (58%) were found to have at least one mutation; 51% had class I, 14% had class J Akiki 1, S Dyer 1, D Grimwade 2, A Ivey 2, J Mason 1, K Tawana 3, K Wall 1, M II, 6% had class III and 4% had class IV mutations. The most frequent gene Velangi 3, M Griffiths 1 mutations were FLT 3 (21.6%; FLT3 -ITD 14.9% and FLT3 -TKD 7.4%), RAS 1West Midlands Regional Genetics Laboratory, Birmingham, United Kingdom (15.6%; N-RAS 8.3% and K-RAS 7.4%), and C-KIT 11.5%. Together, 15.5% had 2Cancer Genetics Laboratory, Kings College, London, United Kingdom more than one mutation. Of the 7 patients with mutated genes of epigenetic reg - 3Birmingham Childrens Hospital, Birmingham, United Kingdom ulators, ASXL1 mutations were detected in 2 of 175 patients (E635fsX649 and P835fsX841), DNMT3A mutations in 2 of 168 (W795S and R882H), IDH1 muta - Background. Cytogenetics provides the most powerful independent prognos - tions in 2 of 177 (R132C and R132H), and IDH2 mutation in 1 of 177 (R140Q). tic factor in AML presenting in children and younger adults, providing the frame - The 2 patients with ASXL1 mutations were both of t(8;21), without cooperating work for risk-stratified treatment approaches. However, approximately half the mutation with other 17 genes analyzed. In the present series, 4 patients had cases presenting in this age group fall into the intermediate cytogenetic risk cat - MLL -PTD; two of them, both of FAB M0 subtype, harbored IDH1 gene muta - egory, within which patients have vastly different outcomes. The cytogenetical - tions compared with none of the 173 patients with non- MLL -PTD AML (P < ly cryptic t(5;11)(q35;p15) leading to the NUP98-NSD1 fusion, is a rare but 0.0001). One of the two IDH1 mutated patients had FLT3 -ITD, the other patient recurrent gene rearrangement, recently reported to identify a previously had trisomy 21and RUNX1 mutation. The only one patient harboring IDH2 unrecognised group of young AML patients with a poor prognosis for whom new mutation had normal karyotype and also had FLT3 -ITD and NPM1 mutations. treatment strategies are needed (Hollink et al, Blood 2011). Aims. The aim of Of the 2 patients with DNMT3A mutations, one cooperated with PTPN11 and this study was to determine the frequency of the NUP98-NSD1 fusion in a CEBP α mutations; the other patient had MLL translocation, FLT3 -TKD and series of 54 unselected de novo paediatric AMLs (median age 9yrs, range 0- WT1 mutations. All patients were treated with Taiwan Pediatric Oncology Group- 18yrs) and to develop an RT-qPCR assay to track individual patient response AML 97 protocols. Since the numbers of patients with the 4 gene mutations to treatment. Method. Screening for the NUP98-NSD1 fusions was performed were very small, it precluded the meaningful analysis of the prognostic impact. using reverse-transcription PCR (RT-PCR) together with FISH confirmation of Conclusions. The present study on 18 gene mutations in a relatively large all positive cases. A reverse transcription-quantitative PCR (RT-qPCR) assay cohort of children with de novo AML in Taiwan showed 58% of patients had at was designed to allow sensitive sequential analysis of post treatment materi - least one gene mutation. The frequencies of mutations of epigenetic regulators al, where available. The sensitivity routinely achieved was sufficient to detect were very rare, ASXL1 mutations were associated with t(8;21) and IDH1 muta - normalised NUP98-NSD1 transcripts levels 4 logs below those seen at diag - tions were significantly associated with MLL -PTD. Support by grants NSC-96- nosis. Results. Four positive cases (7%) were identified; three de novo AML 2314-B-195-006-MY3 and MMH-E-99009. and one t-AML following chemotherapy for osteosarcoma two years prior to presentation with AML. All were older children (median age 16yrs, range 13- 18yrs) with very high white blood counts (WBC) at presentation, without 0684 favourable cytogenetic markers. All had a concurrent FLT3- itd and all lacked NPM1 and CEBPa mutations.All four patients received an allograft at 3,4,4 and FROM CYTOGENETICS TO MOLECULAR BIOLOGY: MAPPING OF 13 months post diagnosis respectively. One patient died of transplant related UNIQUE CHROMOSOMAL ABNORMALITIES AT THE NUCLEOTIDE LEV - complications, but was shown to have low level MRD immediately prior to trans - EL plant by retrospective RT-qPCR analysis. Two patients remain alive and in S Pekova 1, T Jancuskova 1, R Plachy 1, D Hardekopf 1, T Liehr 2, A Weise 2, N molecular remission more than two years post-diagnosis, despite demonstrat - Kosyakova 2, J Stika 1, L Zejskova 1, L Sedlackova 1, L Krutilkova 1, R Cmejla 1 ing a high tumour burden prior to transplant, suggesting a potential survival ben - 1Chambon Laboratories, Prague, Czech Republic efit from transplant. The fourth case failed to achieve molecular remission for 2Universitätsklinikum Jena, Jena, Germany the NUP98-NSD1 gene fusion at any of the time points measured pre-or post- transplant and died 11 months post diagnosis of metastases from a pre-exist - Background. Acute myeloid leukemias (AML) in adulthood represent a hetero - ing osteosarcoma. Conclusions. Our data suggests a non-random association geneous entity, characterized by a recurrent chromosomal/genetic abnormality of NUP98-NSD1 with FLT3- itd in a group of older children with high WBC’s with in only 50% of cases. In the remaining individuals, no common molecular abnor - a poor prognosis who may benefit from transplant. The incidence of NUP98- mality can be identified using standard diagnostic screening (AcutePlexX 1, muta - NSD1 rearrangement, potentially as high as 7% of de novo paediatric AML, is tions in NPM1, WT1, CEBPa and others), though unique cytogenetic abnormal - sufficient to recommend routine screening of NUP98-NSD1 in combination with ities can often be detected. As many AML patients are eligible for curative treat - FLT3 -itd for all new diagnostic cases, particularly in the absence of otherwise ment, techniques allowing specific and sensitive minimal residual disease (MRD) favourable cytogenetic markers, to accurately determine risk and allow for con - monitoring are highly needed. Aims. To develop a technique that would allow sideration of early transplant. mapping of cytogenetically identified unique clone-specific abnormalities from the chromosome level to the nucleotide level, enabling us to develop clone-spe - cific quantitative Real-Time PCR assays for sensitive and specific MRD monitor - 0683 ing. Methods. Molecular-cytogenetic techniques (mFISH, mBAND, BAC-FISH), chromosome microdissection, next generation sequencing, long-range PCR and GENE MUTATIONS IN CHILDHOOD ACUTE MYELOID LEUKEMIA WITH direct Sanger sequencing were used to map the chromosomal translocation in SPECIAL REFERENCE ON THE MUTATIONS OF EPIGENETIC REGULA - the der(10)t(3;10)(p21.3;q23) characteristic for the cell line K562. This model cell TORS INCLUDING ASXL1, IDH1/2, AND DNMT3A line was chosen to show the feasibility and reproducibility of the technique as a DC Liang 1, LY Shih 2, HC Liu 1, CP Yang 3, TH Jaing 3, IJ Hung 3, TC Yeh 1, SH proof of principle, with prospective continuation to authentic patient samples. Chen 3, JY Hou 1, YS Shih 4, TH Lin 4, YH Huang 4 After cytogenetic identification of the chromosomal translocation (Figure 1A), the 1Mackay Memorial Hospital, Taipei, Taiwan derivative chromosome was microdissected using a fine needle (Figure 1B). The 2Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan microdissected fragments were directly subjected to whole genome amplification 3Chang Gung Children’s Hospital, Taoyuan, Taiwan (WGA; Figure 1C) and then sequenced on the GS Junior platform for next gen - 4Chang Gung University, Taoyuan, Taiwan eration sequencing (Figure 1D). Obtained reads were aligned to reference sequences of chromosomes 3 and 10, using in-house developed software (Fig - Background and Purpose. Four genes involving the epigenetic regulators, i.e. ure 1E). The last mapped reads from both chromosomes were used as docking ASXL1, DNMT3A, IDH1 and IDH2 , have recently been described in adult acute sites for primers for long-range PCR to amplify the putative breakpoint (Figure 1F). myeloid leukemia (AML) and were associated with poor outcomes. In childhood The long-range PCR products were directly sequenced using Sanger sequenc - AML, the reports on these gene mutations have been very rare, especially ing to reveal the precise nucleotide sequence of the breakpoint (Figure 1G). there has been no report on ASXL1 gene mutations. In addition, comprehen - Results. Using a combination of cytogenetic and molecular approaches, we sive analyses of gene mutations in de novo childhood AML have been limited. mapped the K562-unique translocation in der(10)t(3;10)(p21.3;q23) from the We aimed to determine the genetic alterations in pediatric AML patients with chromosomal level to the nucleotide level (Figure 1). Direct sequencing of this special reference on the mutations of epigenetic regulators. Materials and breakpoint revealed a head-to-head fusion of genes CDC25A and GRID1. The Methods. We analyzed 18 gene mutations in 206 children with de novo AML time demand of the whole procedure, starting from mFISH and ending with the

haematologica | 2012; 97(s1) | 281 17 th Congress of the European Hematology Association actual sequence of the breakpoint was approximately 6 weeks. This is optimal ed chromosome 21 in this family. By using this map, a diagnostic test for car - timing for a standard clinical setting, when the laboratory receives the first follow- riership analysis will be made available for screening family members at risk up samples one month after diagnosis. Summary and Conclusions. Current of developing MDS/AML themselves or as potential transplantation donors. technologies of molecular cytogenetics and molecular biology open new vistas Affected family members, especially the patient diagnosed with ALL, will also in the detection and identification of unique, clone-specific genetic abnormalities be examined. It is important to realize that adequate screening of families for in patients with AML. As only half of the individuals with AML can be molecular - predisposition to MDS/AML not only involves mutation analysis, but that other ly MRD followed-up using recurrent genetic abnormalities, there is still a sizeable methods (e.g. aCGH, FISH) and genetic counseling should also be considered. proportion of patients who might benefit from the identification of the “finger prints”of their malignant cells for the design of clone-specific MRD Real-Time PCR assays. Our work clearly shows that “walking”from the chromosomal level to the 0686 nucleotide level is feasible and readily applicable for eligible AML patients. Acknowledgements. The work was supported by the Grant Agency of Ministry of HIGH RESOLUTION MICROARRAY-BASED GENOMIC PROFILING FOR Industry and Trade of the Czech Republic and in part by the Monika-Kutzner THE IDENTIFICATION OF SMALL GENETIC LESIONS AND SUBSEQUENT Foundation. DESIGN OF MINIMAL RESIDUAL DISEASE TARGETS IN ACUTE LYM - Reference PHOBLASTIC LEUKEMIA Plachy R. et al., 2011; Blood, 118:1083, Abstract 2526. A Simons , M Stevens-Kroef, S van Reijmersdal, R Pfundt, E Waanders, R Kui - per, A Geurts van Kessel Radboud University Medical Center Nijmegen, Nijmegen, Netherlands

Background. In acute lymphoblastic leukemia (ALL) specific genomic abnor - malities provide important clinical information. We previously demonstrated that microarray-based genomic profiling allows the detection of focal genomic loss - es, frequently harboring clinically relevant ALL-related genes, such as EBF1 , CDKN2A/B , PAX5 , ETV6 , BTG1 and IKZF1. The latter gene was recently shown to be associated with a high relapse rate in patients with ALL (Kuiper et al., 2010, Leukemia). The ability to detect focal lesions, however, largely depends on the resolution of the platform used.The analysis of minimal residual disease (MRD) at early time points during therapy has been widely implemented as an accurate prognostic parameter to determine treatment strategy. MRD targets usually include immunoglobulin and/or T-cell receptor rearrangements detect - ed in the major clone at diagnosis. Accurate array-based mapping of recurrent abnormalities on ALL may facilitate the development of ALL-specific MRD tar - gets, as we recently described for IKZF1 (Venn et al., 2012 Leukemia). Aims. We evaluated the performance of a novel high resolution genome-wide microar - ray platform (Cytoscan HD, Affymetrix) for the detection of ALL-specific genom - ic abnormalities. Due to the high coverage of probes on this platform, we also evaluated its suitability for accurate identification of breakpoints covering dele - Figure 1. The outline of the technique used to walk from the chromosome tions of leukemia-specific genes, which could subsequently serve as markers level to the nucleotide level. in sensitive PCR-based MRD tests. Methods. The Cytocan HD platform (2.7 mil - lion probes) was used for genomic profiling of 8 patients with ALL. Genomic lesions affecting recurrent gene loci (see above) were evaluated and the posi - tions of the breakpoints were accurately mapped. To confirm the focal genom - 0685 ic losses identified by the CytoScan HD, the samples were also evaluated with a commercially available multiplex ligation-dependent probe amplification RUNX1 DISRUPTED BY A COMPLEX REARRANGEMENT OF CHROMO - (MLPA) test (MRC Holland) and breakpoint-spanning PCR. Results. In 8 ALL- SOME 21 CAUSES FAMILIAL PREDISPOSITION TO HEMATOLOGICAL patients tested we identified 21 deletions (sizes down to 10 kb) encompassing MALIGNANCIES genes commonly affected in ALL, which were all validated by MLPA. Eleven of S Snijder 1, T Os 1, A Polstra 1, K Huijsdens-van Amsterdam 1, C Mellink 1, O these deletions were smaller than 100 kb, which is below the detection level of Mook 1, W Kloosterman 2, C Huisman 1 most commonly used micro-array platforms. Next, we analyzed the resolution 1Academic Medical Center, Amsterdam, Netherlands with which the breakpoints in the 21 deletions could be mapped by using the 2University Medical Center, Utrecht, Netherlands Cytoscan HD microarray data. In 8 of the 21 deletions covering leukemia genes both breakpoints were located in a region less than 1 kb. These data can be very Background. Clinicians are increasingly aware of familial predisposition to helpful in designing leukemia-specific PCR primer sets in individual patients. By myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). In a applying this approach we were able to identify such primer sets in 7 of the 8 substantial part of these families there is a mutation in the RUNX1 gene on patients and for which 5 deletion-specific PCR products were validated, so far. chromosome 21q22. Chromosomal rearrangements, like deletions, involving This demonstrates the additive value of the Cytoscan HD platform in designing the RUNX1 locus are less frequently described. Aims and Methods. In our hos - PCR-based targets which can be used for MRD analysis. Summary and Con - pital, a male patient (age 28) presented with mild thrombocytopenia and abnor - clusions. We conclude that the Cytoscan HD microarray platform has improved mal platelet aggregation. Family history revealed five relatives with leukemia detection rate of small focal genomic losses, including IKZF1 , as compared to (3 AML, 1 ALL and 1 of unknown origin). Our patient and his mother were not other platforms. Additionally, we demonstrated the power of this platform for affected with leukemia. To elucidate the underlying genetic aberration in this accurately identifying breakpoints of genomic losses for development of patient- family, blood and bone marrow samples of the index patient and his mother specific targets for MRD analysis. were examined by conventional karyotyping, fluorescence in situ hybridization (FISH), Agilent 180k microarray (AMADID 023363), Nimblegen capture array (custom made) and mutation analysis. Results. Although karyotyping and 0687 mutation analysis of both the index patient and his mother showed normal results, interphase FISH revealed 3 signals for the RUNX1 locus. Array CGH NUCLEOPORIN GENES INVOLVEMENT IN HAEMATOPOIETIC DISOR - demonstrated 3 minor aberrations (2 gains and 1 loss) of distinct regions of DERS chromosome 21q22, but could not explain the extra FISH signal. Therefore, it V Nofrini 1, R La Starza 2, V Pierini 2, B Crescenzi 2, D Beacci 2, G Barba 2, C Har - was speculated that an intra-chromosomal rearrangement with a breakpoint rison 3, C Mecucci 2 through RUNX1 had resulted in a split signal. This was confirmed by capture 1University of Perugia, Perugia, Italy array data, which demonstrated a fusion of the 5’ RUNX1 breakpoint to a chro - 2Hematology and Bone Marrow Transplantation Unit, University of Perugia, mosome 21 sequence 11 Mb upstream (same orientation). Summary and Perugia, Italy Conclusions. Based on the results so far, it was concluded that a complex 3Northern Institute for Cancer Research, Newcastle University, Newcastle, rearrangement with several breakpoints on chromosome 21 resulted in the United Kingdom disruption of the RUNX1 gene. Currently, further investigations by using a nov - el next-generation sequencing technique are ongoing. We will perform genome- Background. Nucleoporins (NUPs), a group of ~30 proteins, constitute the wide long mate-pair sequencing in order to delineate an exact map of the affect - Nuclear Pore Complex, a large multi-protein channel which perforates the

282 | haematologica | 2012; 97(s1) Amsterdam, the Netherlands, June 14 – 17, 2012 nuclear envelope and mediates macromolecular import and export between interpretation of copy number gains and losses. Results. CNAs were detect - nucleus and cytoplasm. NUP98 /11p15 and NUP214 /9q34 are involved in the ed in a low proportion (2/30) of patients with CML-CP thus subclassification of pathogenesis of haematopoietic disorders. In chromosomal translocations or cases being resistant or responding to imatinib therapy proved to be impossi - inversions with different partner genes both produce abnormal fusion proteins ble. Investigating patients with CML having lyBC at diagnosis or at any follow- with oncogenic properties. Although rearrangements are frequently cryptic, con - up time points, we did not find CNAs in their samples acquired during CP; how - ventional cytogenetics may be predictive of NUP98 involvement in around 20- ever, in all but one samples withdrawn during lyBC, we observed several CNAs; 30% of cases of Acute Myeloid Leukemias/Myelodysplastyc Syndromes with an an average of 22 probes per sample showed abnormal patterns. IKZF1 was the 11p15 chromosome change. NUP98 maintains its 5’-end in all fusions while most frequently affected gene followed by PAX5 , CDKN2 , MIR31 , MIR24-2 , partners always contribute with the 3’-end. Aims. To investigate the involvement ETV6 and EBF1 . CNAs were detected in 73% of patients with pALL; CDKN was of 30 NUP genes in patients with diverse haematological disorders and abnor - the most frequently affected gene followed by ETV6 , PAX5 , MIR31 , IKZF1 , mal karyotypes involving chromosomal regions/arms containing one or more RB1 , BTG1 , EBF1 , IKZF3 and MIR23A . Comparing the CNA profile of diagn os - NUP genes. Methods. The study included 194 cases (145 myeloid and 49 lym - tic and relapsed samples of patients with pALL, in one-third of cases we found phoid neoplasms) collected from files of the Laboratory of Cytogenetics and patterns referring to clonal evolution process (i.e. cells harbouring both original Molecular Genetics of the University of Perugia (Prof. Cristina Mecucci). An and newly acquired abnormalities recurred). In two-thirds of cases, cell popu - additional series of 25 cases with myeloid neoplasms with 11p15 changes was lations dominating at different time points were not clonally related but inde - provided by Prof. Christine Harrison (Leukemia Research Cytogenetics Group, pendently arisen suggesting the presence of a preleukemic cell pool. Conclu - Northern Institute for Cancer Research, Newcastle University). Genomic clones sions. MLPA has been used for investigating patients with CML for the first time were selected to test each NUP with a double colour break apart test. The fol - and it proved to be an efficient tool for detecting transition not only from chron - lowing NUPs were investigated: TMEM48 /1p32, 25 cases; TPR /1q31, 21 cas - ic phase to lymphoblastic crisis but vice versa as well. Evaluating samples from es; NUP133 /1q42, 20 cases; NUP358 /2q13, 16 cases ; NUP35 /2q32, 17 cas - children with ALL, combined application of various MLPA probe mixes allowed es; NUP210 /3p25, 14 cases; SEC13 /3p25 14 cases ; NUP54/4q21, 3 cases ; us to detect a panel of CNAs broader than ever before. Identification of the ori - NUP155 /5p13 3 cases; NUP153 /6p22, 13 cases; NUP43/ 6q24, 23 cases; gin of the malignant clone at relapse will have great impact on future treatment NUPL2 /7p22, 18 cases; POM121 /7q11.2 18 cases; NUP205 /7q33 22 cases; strategies in pALL. In this study, if the original leukemic clone recurred at relapse GLE1 /9q34, 11 cases; NUP188 /9q34, RP11-98H23 (5’), RP11-167N5 (3’) 11 the patient died within a short period of time while the occurrence of a clonally cases; NUP214 /9q34, 11 cases; NUP160 /11p11.5, RP11-346F1 (5’), RP11- independent cell population (i.e. a ‘new disease’) was associated with optimal 112L21 (3’) 10 cases; NUP98 /11p15.5 35 cases; AAAS /12q13, 7 cases; response to the administered ALL-REZ BFM 2002 protocol. NUP107 /12q15 8 cases; NUP37 /12q23 8 cases; NUPL1 /13q12, 13 cases; NUP93 /16q13, 4 cases; NUP88 /17p13, 27 cases; NUP85 /17q25, 9 cases; SEH1L /18p11.2 9 cases; NUP62 /19q13.33, 1 case; RAE1 /20q13 11 cases; 0689 NUP50 /22q13, 10 cases. Results. NUP98 was the only NUP gene shown to be directly involved in structural chromosomal rearrangements. Other NUPs A CONTRIBUTION TO THE CYTOGENETIC INVESTIGATION OF MONO - underwent deletions or gains, most of them as expected were according to CLONAL B-CELL LYMPHOCYTOSIS chromosome losses or gains as described in the karyotypes. Our NUP98 break I Kostopoulos 1, G Paterakis 2, G Androutsos 3, D Pavlidis 3, S Papadhimitriou 3, apart FISH test showed 5/35 (14%) positive cases among myeloid malignan - O Tsitsilonis 4 cies with a breakpoint at 11p (between p11 and pter). Additional FISH studies 1G.Gennimatas Athens Regional General Hospital, Athens, Greece confirmed a NUP98 fusion and characterized the partner gene as: HOXA /7p15 2G.Gennimatas Athens Regional General Hospital ( Flow Cytometry Laborato - in 2 cases, NSD1 /5q35.1 and HOXC /12q13, one case each. In the fifth case ry), Athens, Greece the NUP98 3’ probe was deleted, suggesting rearrangement/fusion of the 3G.Gennimatas Athens Regional General Hospital ( Haematology Laboratory), retained 5’-end. Unfortunately the study could not be finalized due to lack of bio - Athens, Greece logic material. Summary and Conclusions. According to our results NUP98 4Department of Animal and Human Physiology, Faculty of Biology, University in karyotypes from myeloid malignancies with visible 11p15 abnormalities was of Ath, Athens, Greece the only NUP gene found to be directly involved in chromosomal rearrange - ments. Therefore, due to multiple known fusion partners of NUP98 , FISH Background. Monoclonal B-cell lymphocytosis (MBL) has recently attracted a screening is recommended for precise diagnosis of these patients and for fusion significant research interest as the prelude of chronic lymphoproliferations. partners characterization. However, despite the advances and wide application of flow cytometry which allow for the identification of the disorder in an increasing number of patients, the cytogenetic investigation of MBL remains a technical challenge and the rel - 0688 evant data is scarce. Aims. In this study, we have applied interphase FISH (i- FISH) for the investigation of chromosomal aberration frequently found in chron - DETECTION OF LYMPHOBLASTIC TRANSFORMATION IN CHRONIC ic lymphoproliferations in patients with documented MBL. Methods. 39 men and MYELOID LEUKEMIA AND REVEALING THE CLONAL ORIGIN OF 21 women with MBL, according to the currenty established diagnostic criteria, RELAPSE IN PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA USING were included in this study. Flow cytometry and i-FISH were performed on a MLPA peripheral blood sample. In cases with a B-cell count <15% of WBC, the i-FISH D Alpar 1, D de Jonge 2, S Savola 3, H Yigittop 3, B Kajtar 4, L Kereskai 4, L Pajor 4, study was performed on purified B-cells after immunomagnetic separation tar - K Szuhai 2 geting CD19. The presence of 13q-,+12, -11/11q-, 17/17p-,-6/6q- , t(11;14) and 1University of Pecs, Pecs, Hungary rearrangement of the BCL2 and IGH genes, was investigated in all cases, 2Department of Molecular Cell Biology, Leiden University Medical Center, Lei - regardless of the morphological features or the phenotype. Results. High-sen - den, Netherlands sitivity flow cytometry showed a chronic lymphocytic leukemia (CLL)-like 3MRC-Holland, Amsterdam, Netherlands (CD5+CD23+), a mantle cell lymphoma (MCL)-like (CD5+CD23-) and an “atyp - 4Department of Pathology, Faculty of Medicine, University of Pecs, Pecs, Hun - ical”phenotype in 44, 5, and 11 cases, respectively. i-FISH was positive for gary t(11;14) in 3 cases (all with a MCL phenotype), +12 in 3 cases (all with CLL phe - notype) and 13q-, involving 13q14, in 23 cases (19 with CLL and 3 with “atyp - Background. Multiplex ligation-dependent probe amplification (MLPA) is a ical”phenotype. In one case, +12 was concurrent with hemizygous 13q-. Of the robust technique to simultaneously detect copy number abnormalities (CNAs) rest 22 cases with 13q-, the deletion was hemizygous in 16 (15 of which with at several loci. CNA profile provides valuable information for risk assessment CD5+CD23+, 1 with CD5-CD23- B-cell population), homozygous in 4 (all of various hematological malignancies. Aims. We used MLPA to stratify sam - CD5+CD23+) and hemizygous coexisting with homozygous in 2 cases ples from CML patients in chronic phase (CP) or in lymphoblastic transforma - (CD5+CD23+). During the follow-up period (at least 24 months), evolution into tion (lyBC) and to discriminate CML-CP cases responding or resistant to ima - “clinical”CLL (RAI stage 0) was observed in only one case (CD5+CD23+, with tinib therapy. Furthermore, a large number of prognostically relevant CNAs 13q deletion and no other aberrations upon diagnosis of the CLL). Conclu - were screened in pediatric acute lymphoblastic leukemia (pALL) using matched sions. i. The most frequent finding in this series of MBL cases was 13q14 dele - samples of diagnosis and relapse. Methods. We investigated bone marrow tion. It was observed mainly - but not exclusively - in CD5+CD23+ phenotype, samples from 30 patients with CML-CP without evidence of blastic crisis (15 at a rate comparable to that in clinical CLL. On the other hand, the absence of responder and 15 resistant to imatinib therapy), from 9 patients with CML under - aberrations denoting an adverse prognosis in overt lymphoproliferations - such gone lymphoblastic transformation and from 49 patients diagnosed with pALL. as deletion of the ATM or p53 genes - implies that CD5+CD23+ MBL correspond Ethical Committee approval and written informed consent have been obtained to the low risk CLL. ii. Evidence for clonal evolution (namely homozygous and for the study. Two different MLPA kits were applied allowing to detect gene concurrent hemizygous/homozygous 13q14 deletion or the coexistence of locus centered CNAs at 58 different loci. Tumor cell ratio measured by flow 13q14 deletion with +12) indicates genetic instability in MBL already at the pre - cytometry and cytogenetic data were also considered at the calculation and clinical stage, but the biological and clinical significance of this phenomenon

haematologica | 2012; 97(s1) | 283 17 th Congress of the European Hematology Association requires further investigation. iii. The presence of t(11;14) among cases with suspected but not confirmed. Anyway all patients with fever, splenomegaly, consistent phenotype is perhaps equivalent to the diagnosis of the recently thrombocytopenia and high levels of ferritin, in whom bone marrow aspirate described “indolent”MCL. The follow-up of these cases at the clinical and genet - does not document leukemia, should be rapidly screened on peripheral blood ic level may provide important clues on the mechanisms of oncogenesis in lymphocytes by flow-cytometry for the expression of perforin and the ability to human lymphomas. degranulate. The combination of these two assays, widely accessible to many haematology or immunology laboratories, can predict the presence of mutations in the PRF1 or in the UNC13D, STX11, STXBP2 genes providing a robust sup - 0690 port to the diagnosis and justification for high-risk chemo-immunotherapy including indication to allogeneic SCT. The question about indication to SCT for FAMILIAL HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS IN ADULTS patients with milder clinical picture or even in a pre-symptomatic phase remains E Sieni 1, V Cetica 1, A Piccin 2, F Gherlinzoni 3, FC Sasso 4, A Bosi 5, D Pende 6, open. M Aricò 1 1Meyer Children’s Hospital, Florence, Italy 2Haematology Department and BMT Unit, San Maurizio Regional Hospital, 0691 Bolzano, south Tyrol, Italy 3Haematology Division, S. Maria di Ca’ Foncello Hospital, Treviso, Italy HIGHLY SENSITIVE MONITORING OF MINIMAL RESIDUAL DISEASE IN 4Dept. of Internal and Experimental Medicine, Second University of Naples, FLT3-ITD-POSITIVE ACUTE MYELOID LEUKEMIA PATIENTS WITH A Naples, Italy MUTATION SPECIFIC QUANTITATIVE PCR METHOD 5Haematology Department, Azienda Ospedaliero Universitaria Careggi, Flo - J Schiller , I Praulich, M Hallek, KA Kreuzer rence, Italy University at Cologne, Cologne, Germany 6IRCCS A.O.U. San Martino-IST, Genoa, Italy Background. Minimal residual disease (MRD) monitoring in patients with acute Background. Familial Hemophagocytic lymphohistiocytosis (FHL) is a rare myeloid leukemia (AML) can predict relapse clearly in advance and therefore immune deficiency with defective cytotoxic function. The age at onset is usu - allows early therapeutic intervention. Moreover, recent studies have highlight - ally young and the natural course is rapidly fatal if untreated. A later onset of ed the significance of personalized treatment on the basis of MRD status for the disease has been sporadically reported even in adolescents and adults. improving outcome in AML. The FLT3 internal tandem duplication ( FLT3 -ITD) Aims. To report all patients diagnosed with FHL at an age of 18 year or older occurs in 13-35% of all AML. Clinically, FLT3 -ITDs have been strongly associ - and enrolled in the Italian Registry of HLH. Methods. A retrospective data col - ated with poor outcome. However, due to the high sequence variability of indi - lection was performed. FHL was defined according to the diagnostic criteria vidual FLT3 -ITD commonly a universal PCR approach is applied which has a established by the Histiocyte Society. Patient information were collected on low sensitivity (approx. 1: 5x10 2). Aims The aim of this study was to investi - specific forms. Biological samples were collected for immunological studies gate individual FLT3 -ITD in detail with respect to prognosis. As well as, to devel - (flowcytometry analysis of perforin expression and degranulation assay, cellu - op a novel cDNA-based, highly sensitive quantitative real-time reverse tran - lar cytotoxicity) and direct sequencing of currently known FHL-related genes. scription polymerase chain reaction (qRT-PCR) assays for the detection of the Data were pooled in a common database and analysed. Results. A total of nine FLT3 -ITD mutation level. Methods. We investigated FLT3 -ITD length and posi - patients were diagnosed with FHL based on the finding of biallelic mutations tion in 39 AML cases. On the basis of individual FLT3 -ITD, mutation-specific for - in one FHL-related gene. They included 6 males and 3 females, from eight unre - ward or reverse primers were designed. The expression of FLT3 -ITD was deter - lated families; their age ranged between 18 years and 43 years (median, 25.1 mined using complementary DNA samples at different points in time diagnosis years). Seven families were of Italian origin and one was from Colombia. Fam - and subsequent treatment. Results. From a total of 409 newly diagnosed AML ily history was unremarkable in six families at the time of the diagnosis. Their patients 54 (13%) were FLT3 -ITD positive. Retrospectively we analyzed ITD genetic diagnoses were: FHL2 (n=6), FHL3 (n=1), FHL5 (n=1), XLP1 (n=1). Iso - mutation of FLT3 in 39 available cases. Eleven patients had extra insertions of lated asymptomatic hypertransaminasemia followed by neurologic symptoms, 2-38 base pairs between two repeats. The length of ITD ranged from 3 to 144 lymphoproliferative-like manifestations, fulminant mononucleosis, mono-like base pairs (median 45). Within our cohort patients with FLT3 -ITD ≥45 base episodes were the first manifestations of the disease in four patients; a clinical pairs had significantly higher relapse rates ( P=0.03) and a worse overall sur - onset with full-blown rapidly progressive FHL was detected in only three vival ( P=0.03). For the FLT3 -ITD quantification we developed patient-specific patients. Outcome, molecular and functional data are reported in the Table 1. qRT-PCR for 31 individuals with mutation-specific forward or reverse primers. Our method could be applied to 97% of FLT3 -ITD positive patients and yield - Table 1. ed similar results when compared to other high sensitive assays for molecular markers like MLL -PTD, NPM1 mutations or PML -RARA (correlation: r=0.99; 0.99 and 0.63, respectively). In one cases a co-amplification of the wild-type could not be avoided resulting in lower sensitivity (1:10 3). MRD negativity pre - dicted lasting remission independent of allo-SCT ( N=7) or non-allo-SCT ( N=9). All paired diagnostic/relapsed samples showed FLT3 -ITD positivity. Compared with bone marrow samples FLT3 -ITD analyses appeared to be equivalently sensitive in peripheral blood. Conclusions. We conclude that highly sensitive detection of individual FLT3 -ITD posses equal prognostic power in AML like established molecular MRD markers. Using this approach MRD guided treat - ment decisions appear to be justified and should be incorporated in future stud - ies.

0692

CLONAL EVOLUTION OF PROGNOSTICALLY ADVERSE MOLECULAR AND CYTOGENETIC FEATURES DURING THE DISEASE COURSE IN CHRONIC LYMPHOCYTIC LEUKEMIA I Praulich , C Krings Rocha, J Schiller, M Hallek, KA Kreuzer University at Cologne, Cologne, Germany

Background. It has been shown that chronic lymphocytic leukemia (CLL) Conclusions. the concept that later onset of FHL is possible has been repeat - patients displaying a mutation within the tumor suppressor gene TP53 or a edly brought to the attention of adult haematologists.Nevertheless adult patients chromosomal deletion 17p (del(17p)) have an adverse prognosis when com - are at most considered as potentially affected by the so-called “secondary”form pared to CLL patients who do not exhibit these anomalies. Furthermore, there of HLH rather than by genetic FHL with several implications.These data con - is growing evidence that complex chromosomal aberrations, and especially firm that FHL may present beyond the paediatric age. The clinical criteria may transclocations detected in the neoplastic clone are associated with a similar be not completely fulfilled and unusual or not specific manifestations are com - inferior outcome even if the patients otherwise exhibit prognostically favourable mon. Based on our experience we suggest adult specialists to consider FHL factors. Aims. The aim of this study was to investigate clonal evolution in CLL in the differential diagnosis of patients with cytopenia and liver or central nerv - patients with prognostically adverse molecular and cytogenetic features dur - ous system disorders, especially when a clonal lymphoproliferative disease is ing the course of the disease. Methods. Cytogenetic analysis and molecular

284 | haematologica | 2012; 97(s1) Amsterdam, the Netherlands, June 14 – 17, 2012 genetic analysis of TP53 and immunoglobulin heavy chain variable (IgHV) negative. This novel JAK2V617F/ABL AS-LAMP assay was validated on DNA mutational status were applied to a cohort of 100 CLL patients. Results. In our extracted from granulocytes of patients affected by PV (n=6), ET (n=7), other cohort, we identified a total of 23 patients (23%) with progressive disease. Of hematological malignancies (n=7) and demonstrated 100% concordance with these, 8 patients showing either a TP53 mutation and a del(17p) (35%) or a sole conventional Allele Specific PCR (ASO-PCR). Furthermore, the high sensitivi - TP53 mutation (n=7, 30%) or a sole del(17p) (n=1, 4%) or complex chromoso - ty level of the assay allowed to correctly detect the JAK2V617F mutation on 17 mal abnormalities (≥ three structural or numerical anomalies) without del(17p) DNA samples extracted directly from peripheral blood, avoiding the step of (n=7, 30%). Eighteen patients of the population with progressive CLL (78%) granulocytes isolation. Interestingly, a direct relationship between the amount exhibited an unmutated IgHV. In 6 cases, the observed lesion could not be of mutant target tested and the height of peaks obtained after melting analysis detected in a previous investigation. Out of these cases in 3 patients adel(17p) has been consistently observed. Moreover, all the negative controls, (ET=5), evolved together with a TP53 mutation, in one case a TP53 mutation evolved (MF-Pos-ET=3), (healthy donors=10), proved negative for JAK2V617F muta - without a del(17p) and in two cases complex chromosomal aberrations devel - tion but showed a correct amplification of the ABL gene. This feature allows to oped. The mean duration for the development of adverse parameters was 21 ensure that the absence of JAK2V617F amplification does not depend on a low months. In most cases (n=4) those occurred after treatments with either fludara - quality DNA or the presence of wrong reaction conditions or inhibitors. Conclu - bine and/or cyclophosphamide and/or rituximab or bendamustine and/or ritux - sions This Real-Time AS-LAMP assay is a new robust and rapid test to detect imab. However, in two patients clonal evolution was detectable without any the JAK2V617F mutation that can lead to a further development of a fully quan - causative therapy for CLL. Summary and Conclusions. We conclude that titative assay. clonal evolution of prognostically adverse molecular or cytogenetic lesions can occur in patients with initially favourable risk profile. It appears that this helds true not only for established parameters such as del(17p) and TP53 mutations 0694 but also for newer prognostic factors such as a complex karyotype. Biological - ly, an overall genetic instability may account for this phenomenon as well as a DOUBLE HIT B-CELL LYMPHOMAS mitogenic or mutagenic effect caused by cytostatic drugs. It can therefore be T Obukhova 1, E Baryakh 1, Y Lorie 1, G Alimova 1, I Kleina 1, S Chernysh 2, G discussed, whether assessment of adverse risk factors including conventional Petrova 3, T Kolosheinova 1, A Magomedova 1, E Parovichnikova 1, A Kovrigina 1, karyotyping should be generally performed at an earlier point of the disease E Domracheva 1 course and might be repeated if clinical signs of progression are evident. 1National Center for Hematology, Moscow, Russian Federation 2Botkin Hospital, Moscow, Russian Federation 3Russian Cancer Research Center, Moscow, Russian Federation 0693 Background . Double-hit lymphomas are defined by a chromosomal break - REAL-TIME MONITORING OF THE JAK2V617F MUTATION BY ULTRA- point affecting C-MYC/8q24 locus (cytogenetic marker of Burkitt lymphoma) in RAPID, HIGH SENSITIVE FLUORESCENT AS-LAMP combination with another recurrent breakpoint such as BCL2/18q21, G Minnucci 1, G Amicarelli 1, E D’Agostini 1, R Mesturini 1, S Salmoiraghi 2, O BCL6/3q27 and CCND1/11q13. These tumors are known to behave aggressive - Spinelli 2, F Bonelli 1, F Colotta 1, A Rambaldi 3 ly with poor prognosis.AIM. In order to evaluate the cytogenetic, morphologic 1DiaSorin SpA, Gerenzano (VA), Italy and immunologic peculiarities of these cases, clinical course of the disease and 2Ospedali Riuniti di Bergamo, Bergamo, Italy response to treatment we analyzed retrospective data in our cytogenetic data - 3USC Ematologia, Ospedali Riuniti, Bergamo, Italy base. Patients and Methods. For 12-years period in the National Center for Hematology double hits were revealed in 13 cases of B-cell lymphomas, 8 Background. The molecular detection of the JAK2V617F mutation is part of woman and 5 men, mean age 59 years (37-79). In 10 patient conventional the diagnostic work-up of Chronic Myeloproliferative Neoplasms (MPNs), as cytogenetic analysis and FISH were performed, in 3 cases only paraffin-embed - recommended by the 2008 WHO classification criteria. We recently described ded tissues were available for FISH-assay. FISH studies were performed using an innovative, non PCR method based on an Allele Specific-Loop mediated a commercially available probes: LSI C-MYC/IGH, cep 8 DF TP, LSI C-MYC isothermal AMPlification (AS-LAMP) that specifically amplifies JAK2V617F BAP, LSI BCL-2/IGH DF TP, LSI BCL-2 BAP, LSI BCL-6 BAP, LSI CCND1/IGH mutated DNA with a high sensitivity under isothermal conditions (Minnucci G DF TP and LSI CCND1 BAP (Abbott), XL IGL BAProbe and XL IGK BAP (Meta - et al Haematologica 2012, Epub ahead of print ). Aims. To improve the reliabil - Systems). ity of the JAK2V617F detection by implementing an internal control reaction to the AS-LAMP and to allow Real-Time monitoring. Table 1. Characteristics of 13 patients with Double-hit lymphomas.

Results. 7 primary cases were diagnosed as DLBCL in 6 pts, FL in 1 and ALL in 1. Secondary C-MYC rearrangements were observed in 2 relapsed patients, Figure 1. Melting analysis of duplex JAK2V617F/ABL LAMP. 1 with FL and 1 MCL, after 9 years and 8 months of response, respectively. Two patients with MGUS and MM progressed to C-MYC+ plasmablastic lymphoma after 2 and 5 years after initial diagnosis. In one patient with DLBCL C-MYC Methods. The isothermal AS-LAMP reaction has been further optimized by the rearrangement was not revealed by FISH at the diagnosis and was detected addition of an intercalating dye (Yo-Pro-1, Invitrogen) for fluorescent Real-Time after 8 months in the disease progression. In 7 cases C-MYC translocations monitoring. Moreover, an internal control reaction has been implemented for were found in coexistence with BCL-2 rearrangements (ALL - 2, FL - 1, DLB - simultaneous amplification of the endogenous Abelson gene (ABL) DNA in the CL - 4), in 3 DLBCL patients - with BCL-6, in 3 patients (MCL -1, MM -2) with same tube. Target and internal control amplified products are characterized by CCND1 translocations. The most common types of C-MYC alterations were different melting temperatures and can be therefore distinguished by a melting translocations with non-Ig-partners (7 patients), translocations with IGH in 3 cas - analysis. Results. The duplex JAK2V617F/ABL AS-LAMP assay detects in es including one complex translocation t(8;11;14)(q24;q13;q32) and transloca - only 30 minutes the JAK2V617F mutation down to 0.05% (mutant DNA from tions with IGL in 3 patients. Double hits involving two IGH loci were observed UKE-1 into wild type DNA from B-JAB, 25 ng total). The analytical specificity is in one untreated DLBCL patient within tetraploid clone. High proliferation rate 100%, since 315 DNA replicates from wild type cell lines resulted JAK2V617F (Ki-67~100%) was observed in all cases except the patient with MCL (~40%).

haematologica | 2012; 97(s1) | 285 17 th Congress of the European Hematology Association

Morphological and immunological features of 9 cases were those of Burkitt Methods. For microarray-based genomic profiling we used the 250k SNP array lymphoma. All relapsed and progressive patients have died of disease with a platform of Affymetrix. In order to rule out false positive results we used the fol - median survival after C-MYC revelation of 4,5 months. Among primary patients lowing interpretation criteria: (i) the threshold for copy number aberrations was 5 died of disease in 1,4, 7, 8 and 12 months after diagnosis, one died in induc - set at >5 Mb, (ii) smaller gains or losses were interpreted as aberrant in case tion and two are alive on treatment (1 pt) or disease free (1 pt) with median OS they coincided with known (recurrent) aberrations as reported in the literature of 7 months. Conclusions. Double-hit lymphomas are highly aggressive or in “Atlas of Genetics and Cytogenetics in Oncology and tumors with poor prognosis affecting, in our selection, predominantly woman Haematology”(http://atlasgeneticsoncology.org/) and (iii) the threshold for aUPD and could be diagnosed in both primary and relapsed/progressive patients with was set at >10 Mb and to telomeric. Karyotyping and interphase FISH were per - acute lymphoblastic leukemia, follicular, mantle cell, diffuse large B-cell lym - formed using standard cytogenetic methods. Results. 47 of the 87 MDS phomas and multiply myeloma. Diagnosis of these tumors is only possible on patients had a normal karyotype. Of interest, in 7 of these 47 (15%) patients the basis of cytogenetic analysis. C-MYC translocations with IGH or IGL that with a normal karyotype, acquired UPD and/or focal abnormalities (< 5 Mb) are typical for Burkitt lymphoma are rare, and rearrangements with non-Ig chro - encompassing genes known to be involved in MDS tumorgenesis were mosomal partners are predominated. observed. Through microarray-based genomic profiling, almost all numerical abnormalities as observed by karyotyping were detected. In addition, acquired UPD and focal (<5 Mb) CNAs were observed in the karyotypically abnormal 0695 group. Microarray-based genomic profiling also allows the identification of unbalanced translocations and a recently described phenomenon of genomic ATYPICAL BCR-ABL1 REARRANGEMENTS DETECTED BY DNA instability, fulfilling the definition of chromothripsis. As expected, balanced chro - SEQUENCING mosomal abnormalities such as t(3;3)(q21;q26) and t(6;9)(p24;q34), which are L Chauffaille , K Miyashiro, R Sacramento, W Keller, D Cantagalli, G Santos, H present in a minority of MDS patients, were not identified. Summary and Con - Silva clusions. We demonstrate that microarray-based genomic profiling allows the Fleury, São Paulo, Brazil identification of almost all copy number abnormalities also observed by kary - otyping. In addition, we show that microarray-based genomic profiling allows Background. BCR-ABL1 quimeric gene is detected in 90-95% of chronic the detection of novel focal CNAs and acquired UPDs in patients with both nor - myeloid leukemia cases (CML), in 20% of adult acute lymphoblastic leukemia mal and abnormal karyotypes. The prognostic value of novel CNAs and (ALL), 5% of child ALL and 2% of acute myeloid leukemia (AML). During treat - acquired UPDs has to be evaluated in prospective clinical trials. ment, the quantification of BCR-ABL1 transcripts is important for disease mon - itoring. However, some cases show atypical rearrangements that are not detect - ed by the primers usually used for the most frequent subtypes as: e13a2 0697 (b2a2)/e14a2 (b3a2) that code 210kDa protein and e1a2, for the 190 kDa pro - tein. Aims. The objective of this study is to relate the result of DNA sequenc - GENOMIC BREAKPOINTS, LABORATORY AND CLINICAL FEATURES OF ing to detect the variant cases. Methods. Cases in which Ph chromosome was MLL-TET1 REARRANGEMENT IN ACUTE LEUKEMIAS: THREE NEW CAS - detected by karyotype but no BCR-ABL1 rearrangement was found by real ES AND LITERATURE REVIEW time PCR (RqPCR) were selected for the present study. Direct DNA sequenc - TS Park 1, SY Cho 1, MJ Kim 1, HJ Yoon 1, SH Oh 2, EH Cho 3, S Lee 4, E Baek 5, ing was performed with primers for exons 1 (5’- CGG TTG TCG TGT CCG AGG JH Choi 5, S Bohlander 6, L Lode 7, S Richebourg 7, R Marschalek 8, C Meyer 8 -3’) and 13 (5’- TTC AGA AGC TTC TCC CTG ACA T -3’) in BCR gene and in 1Kyung Hee University School of Medicine, Seoul, South-Korea exons 10 and 11 (5’- TCA TGG TCC AGA GGA TCG CTC -3’) in ABL1 gene. 2Inje University College of Medicine, Busan, South-Korea After the RNA extraction from peripheral blood or marrow samples, reverse 3Greencross Reference Laboratory, Yongin-city, South-Korea transcription and amplification by nested PCR of BCR-ABL1 transcript was 4Kyungpook National University, Daegu, South-Korea performed, followed by direct sequencing using ABI PRISM 3130 ( Applied 5Hanyang University Kuri Hospital, Kuri, South-Korea Biosystems ). Results. Five rare rearrangements were detected: e1a3 (58yr, 6University of Munich Hospital, Munich, Germany male, AML), e13a3 (b2a3) (52yr, female, CML), e19a2 (46yr, female, ALL); 7Service de Biologie/Hématologie, CHU, Nantes, France while in a patient with CML (female, 29yr) another rare subtype, e13a4 (b2a4), 8Goethe-University of Frankfurt, Frankfurt/Main, Germany was detected simultaneously to a frequent one, e13a2; and a deletion of exon 7of ABL (41yr, male, CML). Summary and Conclusions. BCR-ABL1 Background. Recently, rapid developments in new technology such as the rearrangements in which BCR gene is juxtaposed to ABL1 exon 3 and not to whole genome/exome sequencing have revealed that some novel gene muta - exon 2 are rare. Apparently, only five cases of CML with subtype e1a3 and ten tions such as DNMT3A , IDH1 , IDH2 and TET2 contribute to the main process with e13a3 have been described so far. We add more five cases to these of leukemogenesis. Among these, TET2 , one of the TET family gene located described. In summary, the association of different methods (RqPCR, kary - on 4q24, is becoming more important through its highly frequent mutation in otype and sequencing) was of fundamental importance to detect the rearrange - various myeloid neoplasms such as myeloproliferative neoplasm (MPN), ment and plan the disease monitoring. myelodysplastic syndromes (MDS), MDS/MPN and acute myeloid leukemia (AML). On the other hand, the TET1 gene, located on 10q22, has been rarely reported as a partner gene for MLL rearrangement in AML and acute lym - 0696 phoblastic leukemia (ALL) patients since its first report in 2002. Aims. There has been a total of nine documented instances of t(10;11)(q22;q23), where all IDENTIFICATION OF CHROMOSOMAL ABNORMALITIES BY MICROAR - except in two cases were detected in AML patients. Nonetheless, so far there RAY-BASED GENOMIC PROFILING AS COMPARED TO KARYOTYPING IN has been little research with respect to the common features of the MLL-TET1 PATIENTS WITH MYELODYSPLASTIC SYNDROME (MDS) rearrangement itself. Therefore the authors sought to provide a molecular, lab - M Stevens-Kroef 1, A Simons 2, N ElIdrissi-Zaynoun 2, M Linssen-Wiersma 2, A oratory, and clinical characterization of the MLL-TET1 rearrangement through Siepman 2, P Muus 2, M MacKenzie 2, J Jansen 2, R Kuiper 2, A Geurts van Kes - literature review and three new AML patients. Methods and Results. Includ - sel 2 ing three new cases, this study analyzes a total of 12 acute leukemia cases of 1Radboud University Medical Centre, Nijmegen, Netherlands t(10;11)(q22;q23) that are known so far, detected in 10 AML and 2 ALL patients. 2Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands Six out of 10 AML patients (60%) have shown association with FAB-M4/M5 sub - types. There were seven male and five female patients and the median age was Background. Cytogenetic anomalies are routinely used as diagnostic, prog - 38.0 (age range: 1 month ~ 67 years). Among nine patients with available chro - nostic and therapeutic markers in the clinical management of myelodysplastic mosome study results, three patients have shown sole chromosomal abnormal - syndrome (MDS) (Greenberg et al . Blood 89:2079,1997). Although karyotyp - ity of t(10;11), and the remaining six patients had accompanied various addi - ing is generally considered as the gold standard in the genetic diagnosis of tional chromosomal abnormalities. MLL-TET1 rearrangement was confirmed in MDS, 40-60% of the patients exhibit a normal karyotype. Intrinsically, the res - eight out of 12 cases through molecular methods including Southern blot (1 olution of karyotyping is limited by its capacity to detect only those copy num - case), long distance inverse-polymerase chain reaction (LDI-PCR) (6 cases), ber changes that are microscopically visible (5-10 Mb in size). In contrast, and reverse transcriptase-PCR (6 cases). The various genomic breakpoints of microarray-based genomic profiling analyses allow a genome-wide detection TET1 gene in MLL-TET1 rearrangement would be also described. Conclu - of copy number alterations (CNAs), down to 100 kb in size, and regions of sions. As there were only six out of 12 patients with available clinical out - acquired uniparental disomy (UPD). Such analyses also overcome some of the comes, this study may certainly lack sufficient amount of data. Nevertheless, other major limitations of karyotyping such as low success rate due to inade - the results show that four of them reached complete remission (CR). Mean - quate metaphase yield and/or poor banding quality. Aims. We have compared while, three patients including one who did not reach CR died at an average of karyotyping and SNP-based microarray analyses with respect to the detection 18 months after initial diagnosis, showing little difference with the generally yield for genetic abnormalities in bone marrow samples of 87 MDS patients. known poor prognosis of MLL rearrangements. Further clinical and molecular

286 | haematologica | 2012; 97(s1) Amsterdam, the Netherlands, June 14 – 17, 2012 researches on acute leukemias with MLL-TET1 rearrangement would be nec - results in this clinically defined syndrome. The most prevalent ones are FAN - essary. In the near future, the authors plan to conduct a functional study of the CA, FANCC, FANCG, and FANCD2. At the molecular level, a fundamental MLL-TET1 fusion gene to know the role of the rare MLL- related fusion genes defect in DNA repair underlies this complex phenotype. FA-A is the most com - in leukemogenesis. mon group, accounting for approximately 65% of all affected individuals. The mutation spectrum of the FANCA gene, located on chromosome 16q24.3, is highly heterogeneous, including point mutations, small insertions/deletions, 0698 splicing mutations, and large intragenic deletions. FA-A is usually associated with private FANCA mutations in individual families. Thus, the number of differ - THE WHOLE BLOOD MULTIPLEX DETECTION OF THE HERITABLE ent pathogenic variants described for the FANCA gene is very high consider - THROMBOPHILIA MUTATIONS F5-LEIDEN (F5G1691A) AND PROTHROM - ing the relatively low number of patients. Here we describe three patients (2 BIN (F2G20210A) USING A SIMULTANEOUS APPLICATION OF FLUORO - females, 11 and 17 years old and 1 male, 23 years old) with Fanconi anemia METRIC PRIMERS AND REAL-TIME PCR with similar clinical presentation: BMF, ren arquatus and renal ectopy and abnor - C Blessing , P Bignell, P Wright mal skin pigmentation (café-au-lait) without skeletal abnormality. In one of the John Radcliffe, Oxford, United Kingdom female patients the persistent Ductus arterious, vesico-ureteral reflux and pto - sis were also present. The molecular analysis of FANCA gene using the MLPA A full thrombophilia screen includes direct testing of F5G1691A and F2G20210A kit Fanconi anemia P-031, showed homozygous deletion of exon 3 in all three mutations, which increase a patient’s risk to developing deep-vein thrombosis. patients. Molecular analysis of the flanking regions of exon 3 precisely defines The Molecular Hematology Laboratory, at the John Radcliffe Hospital, ana - unique deletion of 2040 bp and insertion of C (1788_3824insC). The break - lyzes 2,000 thrombophilia samples per-year. These mutations are routinely points are in intron 3 and 4 in the Alu repetitive sequence. We conducted a famil - detected via a multi-stage process of: DNA extraction, PCR amplification, over- ial analysis and proved that the mutation was inherited from the parents. These night restriction and interpretation by gel-electrophoresis. This method is robust, are the first three patients homozygous for deletion of ex3 described to date. reliable and cost-effective for weekly, batched assays. However, the nature of The patients are not related but they came from the same region from Mace - batched assays causes a two-day method to generate turnaround times of up donia. Determination of family pathogenic variants is the ultimate confirmation to two weeks. DNA extraction and long-term storage is costly.The 3M TM Inte - of diagnosis and is necessary for molecular prenatal or preimplantation tests grated Cycler was evaluated to determine if this new technology could provide and mutation carrier detection. The homozygous deletion of exon 3 of FANCA service improvement. The simultaneous application of real-time PCR and flu - gene is a founder mutation of Fanconi anemia patients inMacedonia. Our find - orescently labelled primers, produces a method which offers the possibility of ing has very strong implication in diagnostic and carrier screening strategy for detecting these mutations from whole-blood, within two hours. This rapid pro - BMF and Fanconi anemia in Macedonian patients and in comprehensive genet - cessing time is due, in-part, to the removal of a lengthy DNA extraction ic counseling. process.The 3M TM Integrated Cycler utilizes the Focus Simplexa TM direct chemistry to monitor PCR amplified fluorescence levels of four independent primer pairs. These primers are specific to F5 and F2 wild-type and mutant 0700 genotypes. The intensity of the fluorescence is quantified by four dedicated lazer-channels, over the course of 45 PCR cycles and is then compared to IDENTIFICATION OF THE FIRST MUTATION IN BRE MOTIF OF b-GLOBIN b established thresholds; C T values are based on a panel of control samples.This GENE AND ITS POLYGENIC INHERITANCE WITH TWO OTHER -GLOBIN Simplexa TM kit contains four sets of primer pairs and a master-mix, which must MUTATIONS IN PATIENTS OF THE SAME FAMILY be combined specifically for each run. This system also requires a one-part A Inati 1, H Abbas 1, M Souaid 2, S Koussa 2, A Taher 2, T Abi Nasr 2, D Chui 3 patient sample and three-parts PBS buffer dilution to be made before use. The 1Lebanese American University, Byblos, Lebanon primer-mix and samples are pipetted into the universal-disk, as indicated by the 2Chronic Care Center, Beirut, Lebanon program software. Once the disk is loaded, it is sealed and placed onto the 3Boston University School of Medicine, Boston, United States of America 3M TM Cycler for analysis using pre-programmed cycling conditions.Thirty-three samples of known genotypes were used to establish the initial whole blood C T Background. Thalassemia is one of the most common inherited genetic dis - values. To audit the strength of these reference thresholds against the gel-elec - eases in the Mediterranean region. Patients with heterozygous mutations in trophoresis method, a whole-blood, blind panel of 46 patient samples, 6 known beta thalassemia, and to a lesser extent alpha-thalassemia, suffer from mild positive controls and 2 negative controls were used; totalling 78 individual microcytic anemia. While the anemia is mild and confers no clinical threat in car - results for each SNP, over four separate runs.Two separate samples were run riers, homozygosity of the mutation often leads to major thalassemic disease. five times in the same assay and a further six samples were run four times in Mutations and variants of α-globin and β-globin chains usually segregate inde - different assays, to determine the intra and inter-assay variation of C T. The C T pendently, and there are rare reports of co-inheritance. Clinical outcomes of β values showed maximum results of 4.0% and 4.4% respectively; both in the F2 and α-globin homozygous mutations vary significantly. Aims. A 7 year-old boy wild-type detection channel. There is a positive correlation of 97% between the presented to our clinic with persistent mild microcytic anemia and recurrent 3M TM Cycler and the gel-electrophoresis method.Once the threshold limits infections, but with repeatedly normal iron profile and with normal HPLC . This were established, the 3M® Cycler was capable of identifying the F5G1691A and prompted further genetic investigation of underlying β-and α-globin gene muta - F2G20210A in wild-type, heterozygous and homozygous states. In this setting, tions. Methods. Upon receiving IRB approval and parents’ consent, DNA sam - where DNA samples are not needed for further investigation, family studies or ples were extracted from blood withdrawn from proband, parents and sibling. archive, the assay may be useful for the rapid, simultaneous detection of the Genetic analysis with multiplex gap-PCR tests were conducted in the Chronic two SNP’s. Further examination is required to establish sample acceptance Care Center, Lebanon and reconfirmed in Boston University, USA for single a- criteria, including: white-cell-count, sample-condition and anticoagulant-status, globin gene deletions of rightward ( -a 3.7 ) and leftward (-a 4.2 ) types, deletion MED 20.5 and their corresponding effect on C T levels. of two a-globin genes in cis of the( — ) and -( α) , as well as for β-globin gene single point mutations and single nucleotide polymorphisms . Results. The father’s a-globin genotype was (aa Hph / aa), while the mother’s a-globin 0699 genotype showed another mutation which was (- a 3.7 /aa). The a Hph mutation is a deletion in the IVSI donor splice site which is a 5-base pair deletion of (- FANCONI ANEMIA FOUNDER MUTATION IN MACEDONIAN PATIENTS GTGAG) in a2-globin genes. The a 3.7 deletion is a single a-globin deletion and S Madzunkova 1, S Kocheva 2, D Plasevska-Karanfilska 1 is the most common form of a-thalassemia mutation in the US and the Asia. The 1Research Center for Genetic Engineering and Biotechnolgy MASA, Skopje, proband and his sibling inherited both a-globin mutations from each parent, con - Macedonia comitantly, and their a-globin genotypes were (- a 3.7 / a Hph a). While the father’s 2University Children’s Hospital, Skopje, Macedonia β-globin genotype was normal, the mother had a novel mutation (G>C) at nucleotide (-39) in BRE motif. Notably, genetic analysis showed that the Fanconi anemia (FA) is a rare autosomal recessive disorder (1-5/1 000 000 live proband and his sibling also inherited the β-globinmutation (G>C) from the births) genetically and phenotypically heterogeneous, defined by cellular hyper - mother. A list of mutations in all four members of the family is found in Table 1. sensitivity to DNA cross-linking agents. Clinically FA is characterized by vari - Conclusions. This is the first report to show a mutation in BRE motif, where able developmental abnormalities that may affect skeletal morphogenesis as TFIIB binds to the β-globin gene promoter. Moreover, the (- a 3.7 / aa) genotype well as any of the major organ systems, stem cell loss, causing progressive segregated with the BRE-motif mutation of β-globin in the mother and both sib - bone marrow failure (BMF) and sterility, and profound predisposition to neopla - lings. The polygenic segregation, although rare in beta and alpha thalassemias, sia mostly leukemia and solid tumors. FA is the most frequent inherited cause led to a mild microcytic anemia without iron deficiency and normal HPLC. More - of BMF.The FA complementation system consists of 15 FANC genes. There are over, carrying two different a-globin mutations in proband and sibling conferred evidences that all 15 gene products operate in a common molecular pathway no fulminant a-thalassmia disease, despite a concomitant β-globin mutation. to preserve genomic integrity. Biallelic disruption of any one of these genes Further studies to decipher the clinical significance of this novel mutation in BRE

haematologica | 2012; 97(s1) | 287 17 th Congress of the European Hematology Association motif of β-globin gene are warranted. 0702

Table 1. Distribution of mutations in a- and -globin genes among family COMPARISON OF THE BCR-ABL GENE EXPRESSION BY THE GENEX - members. PERT SYSTEM AND STANDARDIZED RQ-PCR METHOD IN THREE POL - ISH MOLECULAR LABORATORIES IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA TREATED WITH TKIS T Sacha 1, M Zawada 1, A Leszczynska 2, I Solarska 3, I Florek 1, S Czekalska 1, D Cwynar 1, W Prejzner 2, M Szalajko 2, K Borg 3, K Warzocha 3, A Skotnicki 1 1Jagiellonian University Hospital, Kraków, Poland 2Medical University of Gdansk, Department of Hematology, Gdansk, Poland 3Institute of Haematology and Transfusion Medicine, Warszawa, Poland

Background. The GeneXpert System was introduced to facilitate the molec - ular monitoring of therapy with tyrosine kinase inhibitors (TKI) of patients suf - fering from chronic myeloid leukemia. Aims. The aim of the study was to eval - uate the novel rapid PCR GeneXpert System with internationally standardized 0701 BCR-ABL1/ABL1 RQ-PCR methodology Methods. The comparison of the BCR-ABL1 gene expression consisted of paired BCR-ABL1/ABL1 expression A CERTIFIED PLASMID REFERENCE MATERIAL FOR THE STANDARDI - analysis performed in three Polish Molecular Laboratories by RQ-PCR TaqMan SATION OF BCR-ABL1 MRNA QUANTIFICATION BY REAL TIME QUANTI - methodologies aligned to International Standard (IS) and by the GeneXpert TATIVE PCR BCR-ABL System. 90 patients with chronic phase chronic myeloid leukemia H White 1, L Deprez 2, P Corbisier 2, S Mazoua 2, S Trapmann 2, L Fletcher 3, H expressing e13a2 or e14a2 BCR-ABL1 transcripts and treated with tyrosine El Housini 4, DW Kim 5, E Oppliger Leibundgut 6, H Pfiefer 7, M Müller 8, G kinase inhibitors were evaluated. Patients were subdivided into three groups Romeo 9, K Zoi 10 , H Schimmel 2, N Cross 1, H Emons 2 according to BCR-ABL1 transcript level estimated by standardized RQ-PCR 1National Genetics Reference Lab (Wessex), Salisbury, United Kingdom method. Group 1 consisted of patients with BCR-ABL1/ABL1 ratio below 0,1% 2Institute for Reference Materials and Measurements (IRMM), Geel, Belgium [IS], group 2 with BCR-ABL1/ABL1 ratio between 0,1% and 1,0% [IS], and 3Dept. of Genetics and Molecular Pathology, Adelaide, Australia group 3 with BCR-ABL1/ABL1 ratio between 1,0% and 10,0% [IS]. Results. 4Medical Genetics Department, Erasme Hospital, Brussels, Belgium The comparable results of two evaluable techniques were observed in 32% of 5Molecular Genetics Research Institute, The Catholic University of Korea, all samples. The Wilcoxon test revealed statistically significant differences Seoul, South-Korea between the results of GeneXpert System and standardized RQ-PCR BCR- 6Dept. of Haematology, University Hospital Bern, Bern, Switzerland ABL1/ABL1 method (p< 0,0001) (Figure 1). The mean BCR-ABL1/ABL1 gene 7Goethe University, Frankfurt, Germany expression evaluated by the GeneXpert System was 1 log lower than in the RQ- 8III. Medizinische Klinik, Mannheim, Germany PCR method. The comparable results of both techniques in group 1, 2 and 3 9Royal Perth Hospital, Perth, Australia were noted in 35%, 15% and 17% respectively. The differences were statisti - 10 Haematology Research Laboratory, Foundation of Biomedical Research, cally significant in each evaluated group. Conclusions. The GeneXpert sys - Academy of A, Athens, Greece tem provides very easy to use and rapid diagnostic platform, which could be very useful in evaluating CML patients’ response to TKI therapy. Improvement Background. Serial quantification of BCR-ABL1 mRNA is an important therapeu - of system sensitivity and the development of proposed already a reagent lot- tic indicator for patients with CML and Philadelphia-positive ALL, but there is sub - specific conversion factor to the international standard could further enhance stantial variation in the qRT-PCR protocols employed by testing laboratories. The the reliability of results obtained by the use of GeneXpert in patients with CML principal difference is the use of different internal control genes (CG) to normalise monitored for the response to TKI therapy. results . For positive specimens, raw results are expressed as a ratio of BCR- ABL1/CG transcripts whereas for negative specimens the number of CG tran - scripts indicates the sensitivity with which BCR-ABL1 can be excluded for that sample. Determination of the number of BCR-ABL1 and CG transcripts is typical - ly performed by reference to a plasmid calibrator, however different calibrators are in use worldwide and currently no standard exists to which they can be aligned. Comparative studies have demonstrated that the use of different plasmid calibra - tors and different batches of the same calibrator give rise to significant variation in assay results. Aims. To help standardise the reporting of copy numbers of BCR- ABL1 (e14a2) and CG ( ABL1 , BCR and GUSB ) transcripts we sought to develop an internationally accepted plasmid certified reference material (CRM). Methods. Fragments specific for the fusion transcript BCR-ABL1 e14a2 and control genes BCR and GUSB were amplified and cloned into pUC18 in a 1:1:1 ratio. For the CRM production according to ISO Guide 34:2009 by the IRMM, the plasmid was lin - earised, diluted to six different concentrations and copy numbers were assigned following 18 independent digital (Fluidigm) PCR measurements results for each dilu - tion. For confirmation of the identity the entire plasmid was sequenced. The mate - rial was characterised for inter unit-heterogeneity, stability during dispatch and stor - age in accordance with ISO Guide 35:2006. A commutability study to assess the utility of the six CRMs was performed by nine BCR-ABL1 testing laboratories using the six plasmid dilutions, two common cDNAs and their own local methods. Results. The copy numbers of the 6 calibrants were assigned as 1.08 ± 0.12×10 6, 1.08 ± 0.11×10 5, 1.03 ± 0.10×10 4, 1.02 ± 0.09×10 3, 1.04 ± 0.10×10 2 and 10.0 ± 1.5 copies/µl (with a coverage factor k = 2 used for the expanded uncertainty . Data from the commutability study assessing the utility of the CRM were collated and the calibration standard curves were evaluated for their gradient and coefficient of determination ( r2 ). In total, 171/174 (98.3%) calibration curves showed acceptable gradients (range -3.0 to -3.6); BCR-ABL1 (n= 84), ABL1 (n =36 ), BCR (n =23 ) and GUSB (n =28 ). 3/174 (1.7%) calibration curves were rejected because their gradi - ent was less than -3.60 (-3.61 (n=1), -3.62 (n=1). All calibration curves had an r2 above 0.99. The CRMs proved to be commutable for all methods employed. Sum - Figure 1. mary. We conclude that this set of 6 plasmid CRMs with defined DNA copy num - bers can be used to standardise the reporting of transcript numbers of BCR-ABL1 and three control genes; ABL1, BCR and GUSB in e14a2 RQ-PCR monitoring tests. The plasmid dilutions with the CRM code ERM-AD623a-f, can be obtained from the IRMM or its authorised distributors (http://irmm.jrc.ec.europa.eu).

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