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The MLLT10 gene, located at 10p13, is a known partner of MLL and PICALM in specific leukemic fusions generated from recurrent 11q23 and 11q14 chromosome translocations. Deep sequencing recently identified NAP1L1/12q21 as another MLLT10 partner in T-cell acute lymphoblastic leukemia (T-ALL). In pediatric T-ALL, we have identified 2 RNA processing genes, that is, HNRNPH1/5q35 and DDX3X/Xp11.3 as new MLLT10 fusion partners. Gene expression profile signatures of the HNRNPH1- and DDX3X-MLLT10 fusions placed them in the HOXA subgroup. Remarkably, they were highly similar only to PICALM-MLLT10-positive cases. The present study showed MLLT10 promiscuity in pediatric T-ALL and identified a specific MLLT10 signature within the HOXA subgroup.

Mutations at the protein-coding region of TERT gene (chromosome 5p15.33) have been well characterized in patients affected by a constitutional telomere syndrome, including diskeratosis, aplastic anemia and pulmonary fibrosis (Calado RT, Hematology, ASH 2009:338). In rare cases somatic mutations may occur in de novo MDS/AML (Calado RT, Young NS. Blood 2008;111:4446). Mutations involving the regulatory region of TERT gene have been recently identified in a consistent proportion of familial and sporadic melanoma and in a subset of tumors originating from tissues with low rate of self-renewal (glioblastoma, liposarcoma, oligodendroglioma bladder cancer, upper urinary tract cancer). These mutations create new binding motifs for Ets/TCF transcription factors, thus increasing TERT gene transcription (Killela PJ et al, PNAS 2013;110:6021; Horn S et al, Science 2013;339:959). As far as we know TERT promoter mutations were never described in MDS. Material and Methods Index Case. A 52 years old woman was selected because of a complex phenotype including abnormal skin pigmentation, familial pulmonary fibrosis, osteoarthritis, and a refractory cytopenia with multilineage dysplasia (RCMD, WHO 2008) with a 47,XX,+8 karyotype. Screening Cases. Mutational analysis was extended to a cohort of 115 patients (72 males; 44 females, median age 69) referred to the Laboratory of Cytogenetics, Hematology Department,University of Perugia, Italy. Cytogenetics was normal in 49 cases (43%). Abnormalities included: isolated del(5q) (seven cases, 6%); del(5q) plus one additional change (three cases, 3%); isolated del(20q) (fourteen cases, 12%); trisomy 8 (five cases, 4%); monosomy 7 (two cases, 2%); -Y (three cases, 3%); del(11q) (two cases, 2%); complex karyotype (twenty-four cases, 21%); other aberrations (six cases, 5%). Genomic DNA was extracted from bone marrow (BM) of all cases and from peripheral blood T lymphocytes of index case using Salting out method. In all cases TERT promoter was screened using PCR based DHPLC assay (Wave system; MD Transgenomic Inc. Omaha, NE). The 274bp amplicon was amplified with forward primer 5'GTCCTGCCCCTTCACCTTC3' and reverse primer 5'AGCACCTCGCGGTAGTGG3' using Robust Start Taq KAPA2G (Biosystems, Woburn, MA). DNA from abnormal elution profiles were re-amplified and sequenced by Sanger method (ABI 3500 Genetic Analyzer, Applied Biosystems). Variations were detected using Finch TV v. 1.4.0. In the index case 23 amplicons encompassing all 16 exons of TERT gene were also screened (NC_000005.9). Mutations cloning was carried out after RNA extraction (Trizol, Invitrogen, Life Technologies, Paisley, UK), reverse transcription (ThermoscriptTM RT-PCR System, Invitrogen) and amplification (TERT_2CF (5'-CAGCGCTACTGGCAAATGCG-3' Ta-61,4°C; and TERT_2543R (5'-GGCACTGGACGTAGGACTTG-3' Ta-61,4°C). PCR products were sub-cloned into pGEM-T easy vector (Promega, Madison, WI, USA) and sequenced. Results Index Case. Patient was a compound heterozygous for two germline variations: a nonsense mutation c.1209C>A p.C403* (exon 2) and a missense mutation c.2455C>T p.R819C (exon 8). A putative somatic A>C transition 57 bp before the ATG start codon was detected only in BM cells (Table). Screening Cases. DHPLC analysis showed three patients (2.6%) with a two-peak elution profile. Sequencing revealed a 10 bp duplication (c.1-110_1-101) in case 2; a c.1-124 C>T point mutation in case 3; a point mutation c.1-78 C>T in case 4 (Table). Comment We showed that TERT gene promoter variations are recurrent events in 2.6% of MDS patients. Only low/int1 risk MDS (IPSS) were affected in this series. The c.1-57A>C, previously reported as a germline activating variation in familial melanoma (Horn S et al, Science 2013;339:959), was likely a somatic mutation in our index case, thus supporting its role in clonal MDS proliferation. We first found the melanoma activating c.1-124 C>T point mutation (Huang FW et al, Science 2013;339:957; Killela PJ et al, PNAS 2013;110:6021) in a MDS with isolated del(5q). The new variation of our case n.4 does not appear to introduce a new transcription factor binding site (http://www.cbrc.jp/research/db/TFSEARCH.html), whereas the 10bp duplication of case 2 indicates an hypothetical binding site for Ikaros. Further results from ongoing functional studies in these cases will be presented. Disclosures: No relevant conflicts of interest to declare.

Abstract 1431 Background T-cell Acute Lymphoblastic Leukemia (T-ALL) affects about 15% of children with ALL. MLLT10 at 10p12 encodes for a transcription factor and is involved in leukemogenic fusions with PICALM or MLL in about 10% of childhood T-ALL with overexpression of HOXA cluster genes. In case of gene fusion MLLT10 retains the “octapeptide motif-leucine zipper” (OM-LZ) domain that appeared to be essential for leukemic transformation in mouse models (Deshpande AJ et al. Leukemia 2011) as it interacted with critical components of the chromatin modifying machinery, such as H3K79 methyltransferase hDOT1L (Okada Y et al. Cell 2005). Recently, in a case of early T-cell precursor ALL, NAP1L1 at 12q21 was identified by whole-genome sequencing as a new MLLT10 fusion partner gene (Zhang J et al. Nature 2012). We identified two new fusion transcripts involving MLLT10 in 2 cases of pediatric T-ALL suggesting MLLT10 is a promiscuous fusion partner gene in T-ALL. Aim Characterization of new MLLT10 fusion transcripts in 2 pediatric cases of T-ALL. Methods Total RNAs were extracted from cryopreserved bone marrow cells and retrotranscribed with MLLT10 specific reverse primer using 5'-RACE kit (Invitrogen). cDNAs were amplified in nested PCR using AAP/AUAP (Abridged Anchor Primer, Abridged Universal Amplification Primer, Invitrogen) as forward and MLLT10 specific primers as reverse. To confirm fusion transcripts we performed RT-PCR experiments using Thermoscript RT-PCR System (Invitrogen). cDNAs were amplified in nested PCR using HNRNPH1 and DDX3X specific primers as forward in patient 1 and 2 respectively and MLLT10 specific primers as reverse in both samples. PCR products were subcloned into pGEM-T easy vector (Promega) and sequenced. Whole genome analysis was performed applying Combined-Interphase Fluorescence In Situ Hybridization (CI-FISH) for 32 candidate genes as previously described (Gorello P et al. Haematologica 2010); Single Nucleotide Polymorphisms (SNPs) were performed following manifacturer's instructions (Affymetrix); Gene Expression Profiling (GEP) was applied to investigate the hypothesis that both new MLLT10 fusion genes shared leukemogenic properties with other MLLT10 fusions, particularly PICALM-MLLT10. Results In patient 1 the karyotype was 46,XX,inv(10)(p12q?)/46,XX. CI-FISH on bone marrow nuclei showed MLLT10 rearrangements (55%) and IKZF1 deletions (10%). The 10q disruption was located at 10q25.3 in a region of about 12 kb flanked by fosmid G248P87999G12, retained on inv(10), and RP11–411P18 translocated to chromosome 5. SNPs analysis was normal for copy number and LOH profile. In patient 2 cytogenetics failed. CI-FISH revealed a 9p deletion, with loss of PAX5 (9p13), CDKN2A/B (9p21) and JAK2 (9p24) in about 75% of interphase nuclei. MLLT10 break apart was abnormal in about 60% of nuclei, with break within RP11–418C1 spanning MLLT10 exons 1–3. SNPs analysis showed a 47,5Mb loss at 9p24.3-p11.2 and a 33,51Mb gain at 17q21.32-q25.3. 5'-RACE-PCR showed HNRNPH1 in patient 1 and DDX3X in patient 2 as two new MLLT10 partner genes in T-ALL. RT-PCR, cloning and sequencing confirmed these results. We found 2 in frame splicing variants in both cases: HNRNPH1 exon 11 (nt.1324)-MLLT10 exon 15 (nt. 2097) and HNRNPH1 intron 10 (nt. 6701)-MLLT10 exon 15 (nt. 2097) in patient 1; DDX3X exon 2 (nt. 958)-MLLT10 exon 3 (nt. 510) and DDX3X exon 1 (nt. 900)-MLLT10 exon 4 (nt. 590) in patient 2 (nucleotide numbers refer to GenBank accessions NM_005520.2 and NC_000005.9 for HNRNPH1, NM_001356.3 for DDX3X and NM_004641.3 for MLLT10). GEP showed that the new MLLT10 fusions were similar to 4 cases with PICALM-MLLT10 fusion but different from 5 other T-ALL, i.e. 2 with MLL translocations, 2 with inv(7)(p15;q34)/TCRB-HOXA and 1 with SET-NUP214 fusion, with a HOXA signature. Conclusions These two cases add new insights in multiple genomic recombinations affecting MLLT10 in pediatric T-ALL. Both new partner genes, i.e. HNRNPH1 and DDX3X, are involved in RNA processing and have not been reported to be involved in any genomic specific translocations, so far. The presence of the MLLT10 OM-LZ domain in both new fusions as well as the GEP of leukemic cells suggest that these MLLT10 recombinations activate the same leukemogenic pathways as identified for PICALM-MLLT10 and suggest promiscuity of MLLT10 in recombinations with leukemogenic genes. Disclosures: No relevant conflicts of interest to declare.