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