Marco Togni

CBFA2T3-GLIS2 fusion transcript is a novel common feature in pediatric, cytogenetically normal AML, not restricted to FAB M7 subtype

Pediatric cytogenetically normal acute myeloid leukemia (CN-AML) is a heterogeneous subgroup of myeloid clonal disorders that do not harbor known mutations. To investigate the mutation spectrum of pediatric CN-AML, we performed whole-transcriptome massively parallel sequencing on blasts from 7 CN-AML pediatric patients. In 3 patients we identified a recurrent cryptic inversion of chromosome 16, encoding a CBFA2T3-GLIS2 fusion transcript. In a validation cohort of 230 pediatric CN-AML samples we identified 17 new cases. Among a total of 20 patients with CBFA2T3-GLIS2 fusion transcript out of 237 investigated (8.4%), 10 patients (50%) did not belong to the French-American-British (FAB) M7 subgroup. The 5-year event-free survival for these 20 children was worse than that for the other CN-AML patients (27.4% vs 59.6%; P = .01). These data suggest that the presence of CBFA2T3-GLIS2 fusion transcript is a novel common feature of pediatric CN-AML, not restricted to the FAB M7 subtype, predicting poorer outcome.

Infants with acute myeloid leukemia treated according to the Associazione Italiana di Ematologia e Oncologia Pediatrica 2002/01 protocol have an outcome comparable to that of older children

Children under the age of one year (i.e. infants) with acute myeloid leukemia (AML) represent a distinct subgroup of patients with peculiar clinical and biological characteristics.[1][1] As compared to older children, a higher prevalence of unfavorable clinical and cytogenetic/molecular features has

Whole transcriptome sequencing of a paediatric case of de novo acute myeloid leukaemia with del(5q) reveals RUNX1-USP42 and PRDM16-SKI fusion transcripts.

Fusion genes are frequently detected in childhood acute myeloid leukaemia (AML) and other haematopoietic cancers. Recent decades have witnessed the identification, through chromosomal analysis techniques or reverse transcription polymerase chain reaction (RT-PCR), of several fusion genes influencing either proliferation/apoptosis or differentiation ability of AML cells (Gianfelici et al, 2012). The advent of next generation sequencing (NGS) has dramatically improved our ability to investigate the mutational profile of cancer cells, and several studies used NGS to identify novel mutations and chromosomal alterations in haematological neoplasms (Masetti et al, 2013). Along this line of research, we performed whole-transcriptome sequencing (WTS) of blast cells from a paediatric AML patient with del(5q) to elucidate the mutational profile of this rare type of childhood leukaemia associated with resistance to chemotherapy and grim survival (Van den Berghe et al, 1985). An 8-year-old child was diagnosed with AML FrenchAmerican-British (FAB) type M5b. Clinical presentation was characterized by hyperleucocytosis (96 18 x 10/l), anaemia (7 6 g/l) and thrombocytopenia (105 x 10/l). Bone marrow (BM) smears showed 60% blasts; conventional cytogenetics on BM cells revealed an abnormal karyotype, 46, XY, del(5) (q14q34), in 20 metaphases. No recurrent genetic abnormality involving KMT2A (also termed MLL), CBFB, NPM1 and FLT3 genes was found. The patient was enrolled in the Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP) AML 2002/01 Protocol. After first induction course, BM evaluation on day +21 documented induction failure (88% blasts). Two cycles of salvage treatment allowed the patient to achieve complete remission. Thereafter, he received allogeneic haematopoietic transplantation from a human leucocyte antigen (HLA)-identical unrelated donor. 5 years after the allograft, the patient is alive and disease-free. The massively parallel sequencing of the transcriptome allowed us to map 50 3 million reads at a mean depth of 20X. Giving value to only concordant results emerging from the use of three distinct algorithms, namely Chimerascan, deFuse and FusionMap, two fusion transcripts, RUNX1USP42 and PRDM16-SKI, were identified in the patient. Perusal of the reads mapping these chimeric transcripts showed that the first was an in-frame fusion, while PRDM16SKI was a novel out-of-frame fusion (Fig 1A). RT-PCR analysis and Sanger sequencing confirmed the presence of both chimeric transcripts (Fig 1B). To date, the cryptic t (7;21) leading to the fusion between RUNX1 and USP42 has been reported only once in children (Paulsson et al, 2006), while seven adult cases of myeloid neoplasms harbouring the RUNX1-USP42 transcript have been published (Table I), suggesting that this genetic abnormality is a rare, but non-random, feature of myeloid malignancies in adults. To assess the recurrence of RUNX1-USP42 fusion transcript in childhood AML, we examined 132 children with cytogenetically normal (CN) de novo AML enrolled in the AIEOP AML 2002/01 Protocol. No additional patient was found to carry this chimeric transcript. To the best of our knowledge, this is the second paediatric case in which the RUNX1-USP42 fusion transcript has been detected and, unprecedentedly, it was observed at diagnosis together with del(5q), whereas in the case described by Paulsson et al (2006) the 5qoccurred later in the disease course. In our case, the fusion occurred between exon 6 of RUNX1 and exon 3 of USP42 (Fig 1A-B). However, as previously reported, owing to different breakpoints and alternative splicing in RUNX1, several splice-variants of RUNX1-USP42 chimeric transcript may be generated by the cryptic t(7;21) (Foster et al, 2010) (Table I). This notwithstanding, all RUNX1-USP42 isoforms harbour both the catalytic domain (UCH) of USP42 and the highly conserved Runt homology domain (RHD) of RUNX1, which mediates DNA binding and heterodimerization of RUNX1 with CBFb. Several mechanisms through which this chimeric transcript could contribute to the leukaemogenesis have been proposed: (i) dominant negative inhibition of wild-type RUNX1 transcription activation activity (Paulsson et al, 2006; Panagopoulos et al, 2013), (ii) USP42-mediated stabilization of RUNX1 from ubiquitin-proteasome degradation (Paulsson et al, 2006), and iii) ectopic over-expression of USP42 and deregulation of TP53-dependent cell-cycle arrest (Gigu ere & H ebert, 2011). In accordance with the last hypothesis, through RTqPCR analysis, we demonstrated an over-expression of USP42 in t(7;21)-positive cells when compared with other CN-AML cells lacking this translocation, and CD34+ haematopoietic progenitors (P = 0 02) (Fig 1C). This finding was further validated through gene expression analysis performed on the WTS data (Fig 1D). Taken together, these correspondence

Genomic complexity and dynamics of clonal evolution in childhood acute myeloid leukemia studied with whole-exome sequencing

Despite significant improvement in treatment of childhood acute myeloid leukemia (AML), 30% of patients experience disease recurrence, which is still the major cause of treatment failure and death in these patients. To investigate molecular mechanisms underlying relapse, we performed whole-exome sequencing of diagnosis-relapse pairs and matched remission samples from 4 pediatric AML patients without recurrent cytogenetic alterations. Candidate driver mutations were selected for targeted deep sequencing at high coverage, suitable to detect small subclones (0.12%). BiCEBPα mutation was found to be stable and highly penetrant, representing a separate biological and clinical entity, unlike WT1 mutations, which were extremely unstable. Among the mutational patterns underlying relapse, we detected the acquisition of proliferative advantage by signaling activation (PTPN11 and FLT3-TKD mutations) and the increased resistance to apoptosis (hyperactivation of TYK2). We also found a previously undescribed feature of AML, consisting of a hypermutator phenotype caused by SETD2 inactivation. The consequent accumulation of new mutations promotes the adaptability of the leukemia, contributing to clonal selection. We report a novel ASXL3 mutation characterizing a very small subclone (<1%) present at diagnosis and undergoing expansion (60%) at relapse. Taken together, these findings provide molecular clues for designing optimal therapeutic strategies, in terms of target selection, adequate schedule design and reliable response-monitoring techniques.

NUP98-fusion transcripts characterize different biological entities within acute myeloid leukemia: a report from the AIEOP-AML group

NUP98-fusion transcripts characterize different biological entities within acute myeloid leukemia: a report from the AIEOP-AML group