Danika Di Giacomo

New MLLT10 gene recombinations in pediatric T-acute lymphoblastic leukemia

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.

SQSTM1-NUP214: a new gene fusion in adult T-cell acute lymphoblastic leukemia

In patients with T-cell lymphoblastic leukemia (T-ALL) chromosome rearrangements and gene mutations are used as diagnostic markers for genetic classification and prognostic stratification.[1][1] Gene deregulation in T-ALL is determined by ectopic or over-expressed oncogenes, gain or loss of function

Nucleoporin genes in human diseases

Nuclear pore complexes (NPCs) are large channels spanning the nuclear envelope that mediate nucleocytoplasmic transport. They are composed of multiple copies of ~30 proteins termed nucleoporins (NUPs). Alterations in NUP genes are linked to several human neoplastic and non-neoplastic diseases. This review focuses on NUPs, their genes, localization, function in the NPC and involvement in human diseases.

MN1-ETV6 fusion gene arising from MDS with 5q-.

An isolated 5q− Myelodysplastic Syndrome (MDS), “5q− synrome”, is characterized by a favorable prognosis whereas a eletion of chromosome 5q associated with one additional abnorality seems to confer a shorter median survival even though the rognostic impact of diverse aberrations in addition to 5q− has not een established [1]. Translocation t(12;22)(p13;q11)/MN1-ETV6 has been found in nly 2 cases of MDS [2,3]. The putative MN1-ETV6 transcription facor has transforming activity in vitro and may induce Acute Myeloid eukemia (AML) in mice [4,5]. Here we report the first case of MDS with 5q− and trisomy 21 t diagnosis which developed into secondary MN1-ETV6 positive ML.

Expression of leukemia-associated Nup98 fusion proteins generates an aberrant nuclear envelope phenotype

Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98s N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

DDX3X-MLLT10 fusion in adults with NOTCH1 positive T-cell acute lymphoblastic leukemia.

MLLT10 (also known as AF10 ), at chromosome 10 band p12, is emerging as a promiscuous gene. Six partners have been reported to date: PICALM(CALM )/11q14, MLL /11q23, NAP1L1 /12q21, HNRNPH1 /5q35, DDX3X /Xp11.3 and NUP98 /11p15.[1][1],[2][2] All fusions retain the MLLT10 octapeptide motif-leucine-

NUP98/11p15 translocations affect CD34+ cells in myeloid and T lymphoid leukemias.

We assessed lineage involvement by NUP98 translocations in myelodysplastic syndromes (MDS), acute myeloid leukaemia (AML), and T-cell acute lymphoblastic leukaemia (T-ALL). Single cell analysis by FICTION (Fluorescence Immunophenotype and Interphase Cytogenetics as a Tool for Investigation of Neoplasms) showed that, despite diverse partners, i.e. NSD1, DDX10, RAP1GDS1, and LNP1, NUP98 translocations always affected a CD34+/CD133+ hematopoietic precursor. Interestingly the abnormal clone included myelomonocytes, erythroid cells, B- and T- lymphocytes in MDS/AML and only CD7+/CD3+ cells in T-ALL. The NUP98-RAP1GDS1 affected different hematopoietic lineages in AML and T-ALL. Additional specific genomic events, were identified, namely FLT3 and CEBPA mutations in MDS/AML, and NOTCH1 mutations and MYB duplication in T-ALL.

Deletions of the long arm of chromosome 5 define subgroups of T-cell acute lymphoblastic leukemia

Recurrent deletions of the long arm of chromosome 5 were detected in 23/200 cases of T-cell acute lymphoblastic leukemia. Genomic studies identified two types of deletions: interstitial and terminal. Interstitial 5q deletions, found in five cases, were present in both adults and children with a female predominance (chi-square, P=0.012). Interestingly, these cases resembled immature/early T-cell precursor acute lymphoblastic leukemia showing significant down-regulation of five out of the ten top differentially expressed genes in this leukemia group, including TCF7 which maps within the 5q31 common deleted region. Mutations of genes known to be associated with immature/early T-cell precursor acute lymphoblastic leukemia, i.e. WT1, ETV6, JAK1, JAK3, and RUNX1, were present, while CDKN2A/B deletions/mutations were never detected. All patients had relapsed/resistant disease and blasts showed an early differentiation arrest with expression of myeloid markers. Terminal 5q deletions, found in 18 of patients, were more prevalent in adults (chi-square, P=0.010) and defined a subgroup of HOXA-positive T-cell acute lymphoblastic leukemia characterized by 130 up- and 197 down-regulated genes. Down-regulated genes included TRIM41, ZFP62, MAPK9, MGAT1, and CNOT6, all mapping within the 1.4 Mb common deleted region at 5q35.3. Of interest, besides CNOT6 down-regulation, these cases also showed low BTG1 expression and a high incidence of CNOT3 mutations, suggesting that the CCR4-NOT complex plays a crucial role in the pathogenesis of HOXA-positive T-cell acute lymphoblastic leukemia with terminal 5q deletions. In conclusion, interstitial and terminal 5q deletions are recurrent genomic losses identifying distinct subtypes of T-cell acute lymphoblastic leukemia.

The GNAS1 gene in myelodysplastic syndromes (MDS)

GNAS1 gene is located at the long arm of chromosome 20 (q13.32). GNAS1 gene deletion has never been investigated in MDS. A GNAS1 activating mutation (R201) was recently found in MDS. We applied FISH and DHPLC plus sequencing to investigate GNAS1 gene in MDS cases with and without del(20q) at karyotype.

Involvement of a member of the histone cluster 1 at 6p21 in NUP98-positive MDS/AML

HIST1H1T, one of the eleven members of the histone H1 gene family (H1A, H1B, H1C, H1D, H1E, H1T, H1F0, H1FNT, H1FOO, HILS1 and H1FX), is considered a germinal variant as it is specifically expressed in early meiotic spermatocytes until late spermatids in mammals [1]. Information on the involvement of this family in malignancy are still scarce. Members of the histone cluster 1 have never been reported in myeloid malignancies, so far, though HIST1H1B, C, D and E mutations were observed in chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) [2]. A deregulation of HIST1H1T was found in carcinomas of prostate, head and neck, bladder and endometrium [2]. In addition, a pathogenetic role has been attributed to the rs2051542 HIST1H1T polymorphism because of its association with lung carcinoma [3]. We here provide the first evidence of HIST1H1T gene involvement in human myeloid leukemia as a new partner of the promiscuous NUP98 gene. In 2012, a 38-year-old Jamaican woman with adult T-cell leukaemia lymphoma (ATLL) HTLVþ was treated with combination chemotherapy (VCAP-AMO-VECP, six cycles), obtaining complete remission. Maintenance therapy was a-interferon (3MU) and zidovudine (600mg/day) for 10 months. Three years later, in April 2015, the patient developed secondary myelodysplastic syndrome (MDS), with trilinear cytopenia and macrocytosis (WBC 2.67 10/l, PMN 18%, Hb 93 g/l, PLT 80 10/l, MCV 121 fl). Bone marrow aspirate showed RAEB1 with trilinear dysplasia and 7% blasts. Karyotype was 46,XX,t(6;11)(p21;p15)[10]/46,XX[12] (Figure 1(A)). No therapy was administered. In August 2015, patient developed frank AML with 48% bone marrow blasts CD34 , CD117, CD33þ, CD13þ, CD14 , CD16 and HLA-DR. Karyotypic evolution was observed: 47,XX,t(6;11)(p21;p15),þ8[13]/ 46,XX,t(6;11)(p21;p15) [7]. Mutational analysis (primers in Supplementary Table 2) showed FLT3 mutation (D835H) at the AML diagnosis, but not at the MDS phase. No other mutations were found in ASXL1, RUNX1, SETBP1, SRSF2, TET2, EZH2, DNMT3A, IDH1-2, SF3B1, JAK2, HRAS, WT1, KIT, NRAS and KRAS genes. Induction therapy used idarubicin 12mg/m/day, 3 days, cytosine arabinoside 100mg/m/dayX2, 7 days, VP16 100mg/m/day, 5 days. Consolidation therapy was idarubicin (10mg/m/day, 2 days), cytosine arabinoside (100mg/m/day 2, 7 days) followed by cytarabine (1 g/m/day 2, 4 days). Complete remission was obtained and in February 2016 the patient underwent successful syngeneic peripheral blood stemcell transplantation from her HLA-identical twin after conditioning with busulphan and fludarabine. To characterize the t(6;11)(p21;p15) chromosomal translocation, FISH was performed on bone marrow cells. At least 200 interphase nuclei and/or seven abnormal metaphases were analyzed in each experiment. A breakapart FISH assay with RP11-348A20 (NUP98 exons 1–27) and CTD-3234F16 (NUP98 exons 13–32) indicated the 11p15 breakpoint fell within NUP98 exons 1–13 in 75% of cells at AML diagnosis, with RP11-348A20 showing three hybridization signals: on normal 11, on derivative 11 and on derivative 6. Homebrew BAC and PAC clones (Supplementary Table 1) investigated the 6p breakpoint, which was identified within clone RP1-221C16 at 6p22.2. Figure 1(B) shows the reciprocal translocation in a double fusion experiment. RP1-221C16 clone encompasses the 30-end of the hemochromatosis gene (HFE) and H4C, H1T, H2BC, H2AC, H1E, H2BD, H2BE and H4D genes, members of histone gene cluster 1, on chromosome 6p21. The hybridization pattern indicated that the breakpoint localized to the most telomeric RP1-221C16 region, where two putative partner genes, HIST1H1T and HIST1H2BC, were mapped with appropriate centromere–telomere orientation. Total RNA was extracted by Trizol reagent