18 th Congress of the European Hematology Association

Molecular Markers in acute lymphoblastic Bolzano, 8Ospedale Oncologico A. Businco, Cagliari, 9Ospedale San Raffaele, 10 Ospedale Maggiore Policlinico, Milano, 11 AOU Careggi, Firenze, 12 ASO S. leukemia Croce e Carle, Cuneo, 13 Ospedale San Giovanni Battista, Torino, Italy

Background: MRD is the most powerful indicator of the risk of relapse, and is S535 increasingly adopted in ALL for the risk-oriented application of stem cell trans - plantation (SCT) in MRD-positive ( pos ) patients. Retrospective analyses indi - NOVEL AND RECURRENT INVOLVED IN STRUCTURAL AND cated a lesser efficacy of SCT in MRD pos cases, however without defining the SEQUENCE VARIATIONS IN RELAPSED CHILDHOOD HIGH HYPER - quantitative MRD ranges associated with SCT failure. Such a definition could DIPLOID ACUTE LYMPHOBLASTIC LEUKEMIA improve the ability to distinguish MRD pos patients for whom SCT is an appro - U Fischer 1,* , C Bartenhagen 2, M Gombert 1, V Okpanyi 1, V Binder 1, S priate choice from those in whom other treatments should be considered Röttgers 3, J Bradtke 3, A Teigler 3, Jochen Harbott 3, Sebastian Ginzel 1,4 , R instead or prior to SCT. 4 2 5 1 1,5 Thiele , M Dugas , J Hu , A Borkhardt , C Chen Aims: To define which post-induction/consolidation MRD levels can negative - 1Department of Pediatric Oncology, Hematology and Clinical Immunology, Uni - ly affect post-transplantation outcome in adult ALL. versity Children’s Hospital, Medical Faculty, Heinrich Heine University, Dues - Methods: The long-term results of a prospective Northern Italy Leukemia seldorf, 2Institute of Medical Informatics, University of Muenster, Muenster, Group trial conducted between 2000 and 2006 and enrolling a total of 304 con - 3Oncogenetic Laboratory, Department of Pediatric Hematology and Oncology, secutive unselected patients with Ph- ALL were reviewed. In this study, SCT Justus Liebig University, Giessen, 4Department of Computer Science, Bonn- was prescribed only to MRD pos patients, regardless of clinical risk class. MRD Rhine-Sieg University of Applied Sciences, Sankt Augustin, Germany, 5Fujian was assessed by RQ-PCR methodology using one-two case-specific sensitive Institute of Hematology, Fujian Medical University Union Hospital, Fuzhou, China molecular probes, examining the bone marrow at weeks 10, 16 and 22, i.e. after chemotherapy cycles no. 3, 5 and 7. MRD results from all three time-points Background: High hyperdiploid acute lymphoblastic leukemia (HH-ALL) is were pooled, and the highest value registered in individual patients was used characterized by 51-67 and nonrandom gains of specific chro - to identify the MRD-negative group (always MRD neg ) and three other subsets mosomes (X, 4, 6, 10, 14, 17, 18, or 21). Occuring in 25-30% of cases, it is the characterized by increasing levels of residual ALL, from low-positive (<10 -4 most frequent numerical cytogenetic alteration in pediatric B-cell precursor [MRD pos1 ]) to positive (10 -4 to <10 -3 [MRD pos2 ]) to strongly positive (≥10 -3 ALL. Pre-leukemic clones are generated already in utero , but cooperating onco - [MRD pos3 ]). MRD neg patients were not offered SCT unless t(4;11)+. For study genic lesions are necessary for overt leukemia. Children suffering from HH-ALL purposes, disease-free survival (DFS) and relapse incidence (RI) were have a good prognosis, but recurrent disease will affect 15-20%. The underly - assessed and compared among different MRD pos groups, specifically in ing genetic mechanisms leading to overt leukemia and relapse remain to be patients selected for SCT at end of consolidation phase after completion of the determined. MRD study as per study design (at least MRD pos2 at week 16 and/or MRD pos1 Aims: The objective of this study was to comprehensively assess and validate at week 22). genetic lesions associated with genesis and relapse of leukemia in a cohort of Results: Of 304 patients treated (median age 35 years, range 16-68; male gen - pediatric patients (n=6) with recurrent HH-ALL. der 57%) 258 (85%) entered complete remission (CR). Sensitive probe(s) were Methods: Whole genome and whole exome next-generation-sequencing was available for 200 (77.5%) CR patients, of whom 141 completed consolidation applied to analyze matched sample sets of six children with recurrent HH-ALL and 59 did not (early SCT 13, relapse 41, toxicity 5). Of 141 evaluable patients, taken at initial diagnosis and/or relapse and remission. Paired-end genomic 136 completed the MRD study, 64 being MRD neg (47%), 21 MRD pos1 (15.5%), libraries were sequenced on a Genome Analyzer IIx or a HiSeq 2000 (Illumi - 17 MRD pos2 (12.5%) and 34 MRD pos3 (25%). With a minimum observation na). Reads were aligned against the reference genome (GRCh37) using time of 4 years and a maximum close to 12 years, estimated 6-year DFS and BWA. Unique reads were analyzed with GASV to detect translocations and RI were 57% and 32% in MRD pos1 , 46% and 50% in MRD pos2 and 15% and inversions. Somatically acquired variations not present in the Database of 76% in MRD pos3 cohorts, respectively (all P’s <0.0001). Of all 72 MRD pos Genomic Variants were reported. FREEC was employed to detect copy num - patients, 44 (61%) underwent SCT as per protocol design (allogeneic SCT 26, ber alterations. Targeted enrichment of whole exomic regions was carried out “hypercycles” with autologous blood stem cell rescue 18). Although 6-year DFS employing SeqCap EZ libraries (Roche) and 100 bp single reads were rate was improved after allogeneic SCT (42% vs 20% with autologous SCT, sequenced on a HiSeq 2000. Mutations were called and LOH detected using P=0.09), MRD level was highly influential for post-transplantation outcome an in-house bioinformatic pipeline. Putative somatically acquired mutations (DFS 48% in MRD pos1-2 group [n=25] vs 16% in MRD pos3 group [n19], P=0.025; were validated by PCR, Sanger sequencing and FISH analysis. RI 42% vs 69%, P=0.13), and the best overall result was obtained with allo - Results: We detected and validated 11 interchromosomal translocations affect - geneic SCT in MRD pos1-2 group (DFS 60% [n=15] vs 18% in MRD pos3 group ing genes coding for (ART4, C12orf60, MACROD2, TBL1XR1, LRRN4, [n=11], P=0.08; RI 23% vs 64%, P=0.09). KIAA1467, ELMO1) and several miRNAs (e.g. miR1200), lincRNAs and nuclear Summary / Conclusion: In this prospective study, about one half of MRD pos1- RNAs involved in splicing (U6, U13). A MACROD2/KIAA1467-fusion potential - 2 patients were salvaged by SCT, mainly by allogeneic rather than autologous ly encoded a novel chimeric . All other rearrangements presented loss- SCT (DFS 60%). Because of the poorer SCT results in MRD pos3 group, patients of-function or -expression alterations. Most translocations were associated with with post-induction/consolidation MRD ≥10 -3 should receive further/experimen - copy number alterations. One case showed oscillating copy numbers at clus - tal therapy and not proceed to SCT until the MRD signal is brought below the tered breakpoints indicative of shattering/rejoining of chromosomal fragments, 10 -3 cutoff. termed chromothripsis. Furthermore, deletions, inversions and loss-of-het - erozygosity were detected. Exome sequencing revealed recurring mutations of CREBBP and members of the Ras family of small GTPases. One patient S537 expressing wildtype NRAS and KRAS harbored mutated PAR4, a tumorsup - pressor that is downregulated by oncogenic RAS for efficient transformation. COMPARISON OF NEXT-GENERATION SEQUENCING AND ASO-PCR Further mutations (non synonymous coding, splice site mutations or gained METHODS FOR MRD DETECTION IN ACUTE LYMPHOBLASTIC stop codons) were detected and validated in various transcription factors and LEUKEMIA 1 2 2 2 1,* signaling molecules involved in differentiation, cell cycle regulation and apop - G Malnassy , V Carlton , M Moorhead , M Faham , W Stock 1 2 tosis. Relapse was associated with partial chromosomal gain (1q), loss (4q), a University of Chicago, Chicago, Sequenta, Inc., South San Francisco, Unit - novel translocation (t(4;7)), deletions (IKZF1), increasing dominance of a chro - ed States mothriptic clone and selection for RAS and CREBBP alterations. Summary / Conclusion: Our data indicate a central role for RAS and CREBBP Background: The clinical management of patients with acute lymphoblastic regulated pathways as assisting oncogenic lesions in pathogenesis and relapse leukemia (ALL) relies on accurate prediction of relapse risk to inform treatment of HH-ALL. Additional mutations indicate disturbance of B-cell differentiation, decisions (Pui et al , JCO 2011). The measurement of minimal residual disease enhanced proliferation, suppression of cell death and a possible role of regu - (MRD) has emerged as the most important predictor of outcome in ALL (Cam - latory RNAs in HH-ALL. pana, Hematology Am Soc Hematol Educ Program 2010). Allele-specific oligonucleotide (ASO)-PCR can be used to assess MRD; however, this tech - nique requires preparation of clonotype-specific primers for each individual S536 which is laborious and time-consuming. We developed the LymphoSIGHT™ platform, a high-throughput sequencing method, which universally amplifies DIFFERENT MINIMAL RESIDUAL DISEASE (MRD) LEVELS PREDICT antigen-receptor segments and can identify all leukemia-specific POST-TRANSPLANTATION OUTCOME IN MRD+ ACUTE LYMPHOBLAS - sequences at diagnosis, allowing monitoring of disease progression and clon - TIC LEUKEMIA (ALL) al evolution during therapy (Faham et al , Blood 2012). 1,* 2 2 3 2 2 R Bassan , O Spinelli , E Oldani , T Intermesoli , M Tosi , B Peruta , Aims: In this study, we compared the sequencing and ASO-PCR methods for E Borlenghi 4, E Pogliani 5, E Bona 6, V Cassibba 7, A Scattolin 1, C Romano 8, F measuring MRD in follow-up samples and analyzed the extent of clonal evo - Ciceri 9, A Cortelezzi 10 , G Gianfaldoni 11 , D Mattei 12 , E Audisio 13 , A Rambaldi 2 lution present in diagnostic and follow-up samples from 37 ALL patients. 1 2 Ospedale dell’Angelo, Mestre-Venezia, Ospedale Papa Giovanni XXIII, Methods: Using the sequencing assay, we analyzed diagnostic blood and 3Ospedale Papa Giovanni XXXIII, Bergamo, 4Spedali Civili, Brescia, 5Nuovo bone marrow samples from 37 ALL patients for clonal rearrangements of Ospedale San Gerardo, Monza, 6Ospedale Civile, Vicenza, 7Ospedale Civile, immunoglobulin (IGH-VDJ, IGH-DJ, IGK) and T cell receptor (TRB, TRD, TRG)

224 | haematologica | 2013; 98(s1) Stockholm, Sweden, June 13 – 16, 2013 genes. Clonal rearrangements had been previously detected in all 37 patients S538 using ASO-PCR methods. We assessed MRD at the IGH and/or TRG in 99 follow-up samples, and analysis of the concordance between MRD results ERG DELETION EXERTS A CD2-DEPENDENT POSITIVE PROGNOSTIC IMPACT ON IKZF1-DELETED CHILDHOOD ALL obtained by the sequencing method and ASO-PCR is ongoing. Finally, we 1,2 2 1 3 1 looked for cases with clone evolution and assessed the frequency of evolved M Zaliova , O Zimmermanova , P Dörge , C Eckert , A Möricke , M Zimmermann 4, J Stuchly 2, A Teigler-Schlegel 5, R Koehler 6, C Bartram 6, clones over time in serial samples. 7 7 1 2 1 1,* Results: Sequencing detected the presence of a high-frequency clonal L Karawajew , P Rhein , G Cario , J Zuna , M Schrappe , M Stanulla 1Department of Pediatrics, University Hospital Schleswig-Holstein, Kiel, Ger - rearrangement of at least one receptor (“calibrating receptor”) in 100% of the 2 37 ALL patients; 36 patients had at least 2 calibrating receptors at diagnosis, many, CLIP-Molecular genetics, Department of pediatric hematology and oncology, 2nd Faculty of Medicine, Charles University in Prague, Prague, Czech 29 patients had at least 3 calibrating receptors, and 12 patients had more than 3 3. The TRG assay was the most frequent gene rearrangement: at least one Republic, Pediatric Hematology and Oncology, Charite University Hospital Berlin, Berlin, 4Pediatric Hematology and Oncology, Hannover Medical School, TRG clonal rearrangement was detected in 29 of the diagnostic ALL samples. 5 IGH-VDJ was the second most informative receptor, with clonal rearrange - Hannover, Oncogenetic laboratory, Pediatric Hematology and Oncology, Uni - versity of Giessen, Giessen, 6Department of Human Genetics, University of Hei - ments being detected in 23 patients. We analyzed follow-up samples for the 7 presence of MRD. Sequencing detected MRD levels of >10% in 4 samples, 1- delberg, Heidelberg, Department of Hematology, Oncology and Tumor 10% in 4 samples, 0.1-1% in 8 samples, 0.01-0.1% in 12 samples, 0.001-0.01% Immunology, HELIOS-Clinic Berlin-Buch, Berlin, Germany in 8 samples, and <0.001% in 11 samples. MRD was undetectable by sequenc - ing in 45 samples. Being able to track more than one clonal sequence allows Background: B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) is a the follow up of the dynamics of these clones. In at least one patient, the rel - clinically and biologically heterogeneous disease. Based on the presence of rou - ative frequencies of the 3 clonal sequences changed dramatically over the dis - tinely screened recurrent genetic aberrations, most cases can be classified into ease course (Figure 1). genetically defined subgroups. Despite this success, a still significant propor - Summary / Conclusion: This high-throughput sequencing method enables tion of patients falls into the so-called “B-other” category with largely unknown MRD detection without the need for development of patient-specific reagents. genetic background and heterogeneous outcome. Importantly, the majority of The sequencing method allows monitoring of clonal dynamics over time, which relapses occur within patients who lack any known strong prognostic feature. may provide valuable insight into tumor evolution and resistance to therapy at To improve the prognosis of this group, identification of new and more accurate the individual clone level. Concordance data between sequencing and ASO- risk predictors is of ongoing interest. PCR will be presented. Aims: Using SNP microarray analysis, intragenic deletions of the ERG gene on 21q22.3 ( ERG del) were recently described as a recurrent abnormality in childhood ALL. However, detailed characterization of this aber - ration in the context of large modern clinical trials has not been performed so far. In our study we aimed to assess the incidence of ERG del in childhood ALL and to evaluate its clinical value as a prognostic marker. Methods: We developed a multiplex PCR assay to screen ERG del on the genomic level and screened 1323 patients treated on the multicentre ALL-BFM 2000 protocol. A commercially available multiplex ligation-dependent probe amplification (MLPA) kit was used to analyze IKZF1 deletions. Other clinical and laboratory parameters were acquired by routine diagnostic procedures. Results: We identified 60 cases with ERG del, all exclusively within BCP-ALL. The majority of positive patients were observed in the “B-other” subgroup (44/403). Interestingly, in at least 1/3 of cases the deletion was bi-/oligoclonal and it was significantly associated with higher age, CD2-positivity and IKZF1 deletion. We found no or only weak associations of ERG del alone with prog - nosis. However, when combined with CD2 status, the CD2-positive/ ERG del Figure 1. Dynamics of clone frequency over time in one ALL patient across patients demonstrated superior outcome. This effect was significant even with - 5 samples. Colors indicate three different clonal sequences. The 5 sam - in cases harbouring IKZF1 deletions which have previously been shown to ples are shown on the X axis. The clone frequency is shown on the Y have a negative prognostic impact in the ALL-BFM 2000 trial population. In axis. addition, we analyzed stability of ERG del between diagnosis and relapse in all six CD2-negative/ ERG del relapses. In 3/6 cases the ERG deletions were lost and in the remaining 3 cases the relapse deletions differed from those found at diagnosis. Summary and Conclusions: To conclude, we describe a high incidence of ERG del in B-other ALL. Our data suggest that the ERG locus is specifically prone to deletion in this subgroup. Nevertheless, the deletion does not seem to play a driving role in the pathogenesis of the disease. We show that com - bined with CD2-positivity the ERG del confers a superior prognosis which even overcomes the negative impact of concurrent IKZF1 deletions. Support: P302/12/G101; UNCE204012

haematologica | 2013; 98(s1) | 225 18 th Congress of the European Hematology Association

S539 HIGH HYPERDIPLOIDY (HEH) AMONG ADOLESCENTS AND ADULTS WITH ACUTE LYMPHOBLASTIC LEUKAEMIA (ALL): CYTOGENETIC FEATURES, CLINICAL CHARACTERISTICS AND OUTCOME L Chilton 1,* , C Harrison 1, R Ketterling 2, J Rowe 3, M Tallman 4, A Goldstone 5, A Fielding 6, A Moorman 1 1Leukaemia Research Cytogenetics Group, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom, 2Depart - ment of Cytogenetics, Mayo Clinic, Rochester, MN., United States, 3Department of Hematology, Shaare Zedek Medical Center, Jerusalem, Israel, 4Memorial Sloan Kettering Cancer Center, New York, United States, 5Department of Haematology, UCLH, 6Department of Haematology, Royal Free and UCMS, London, United Kingdom

Background: HeH is the massive clonal gain of chromosomes resulting in a modal number of 51-65 chromosomes. The pattern of chromosome gain is non- random and distinctive with 8 chromosomes accounting for >75% gains. HeH is the most prevalent genetic subtype in paediatric ALL occurring in 33% patients and is associated with a good prognosis. We have previously reported the incidence and outcome of HeH among adolescents and adults treated on UKALLXII/ECOG2993 both with and without Philadelphia positive (Ph+) disease (Blood, 2007;109:3189 & 2009;113:4489). Among paediatric patients, several cytogenetic features, including the presence of specific trisomies, have been asso - ciated with outcome. In particular, +4, +10, +17, +18 and high modal number have been associated with improved survival. Aims: There are no previous studies of HeH in adults describing the pattern of chromosome gain or the relationship with outcome. As adolescent and adult ALL patients generally have an inferior outcome compared to children the prognostic relevance of specific trisomies may be more relevant in this group. Results: Among 1232 BCP-ALL patients treated on UKALLXII/ECOG2993 (1993- 2006) tested for HeH, 159 (13%) were positive. The incidence of HeH was simi - lar among Ph+ (48/340, 14%) and Ph- (111/892, 12%) patients. Ph+ HeH patients were older than Ph- HeH patients (38 v 26 years, P=0.001). Although ~60% of Ph- patients were <25 years, ~25% were 40-59 years. Ph+ HeH had sig - nificantly lower modal numbers whereas the distribution for Ph- cases was simi - lar to paediatric ALL with a peak at 55 chromosomes. There were significant dif - ferences in the pattern of chromosome gain: Ph+ karyotypes were more likely to have +2, +15 and +der(22)t(9;22)(q34;q11), and less likely to have +10, +17 and +18. Gains of chromosomes 4, 6, 14, 21 and X were equally prevalent in both groups. The median follow-up time for the 111 Ph- HeH patients was 8 years. The majority (106, 96%) achieved a complete remission (CR) with 31 (29%) cases sub - sequently relapsing and 17 (16%) dying in first remission. The 5 year survival was 58% (95% CI 48-66%). Patients who had gained a chromosome 4, 10, 14 or X had a significantly improved outcome: HR (95% CI) 0.41 (0.22-0.72), 0.55 (0.31- 0.98), 0.56 (0.32-0.99) and 0.49 (0.27-0.90) respectively. However, only +4 and +10 remained significant (P=0.003 & 0.027 respectively) in a multivariate model with age and white cell count. There was no evidence to suggest a relationship between modal chromosome number and outcome. Survival data was only avail - able for 26 Ph+ patients treated prior to the introduction of imatinib (median fol - low-up 11 years). All patients, bar one, achieved CR (96%) but 9 (35%) subse - quently relapsed and 10 (40%) died in first remission. Thus the 5 year survival was 38% (20-56). There was little evidence to suggest outcome heterogeneity accord - ing to specific trisomies or modal chromosome number; except for a borderline result for chromosome 4 [0.45 (0.18-1.16)]. Summary / Conclusion: In conclusion, the cytogenetic landscape of Ph- HeH ALL is almost identical to that observed in paediatric ALL whereas it is distinctive from Ph+ HeH ALL. There is significant evidence to suggest outcome heterogeneity according to the presence of specific trisomies in Ph- HeH ALL. Further studies based on more contemporary cohorts will be needed to evaluate these results among young adults treated on paediatric or paediatric-like protocols.

226 | haematologica | 2013; 98(s1)