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PDF - K, Supplementary Figure 5. Interference of NUP98-fusions with APC/C activity involves primarily CDC20. HEK293 cells were transfected with a control siRNA or with siRNAs against the indicated proteins (CDC20 or CDH1), together with the expression constructs for NUP98 or NUP98-fusion proteins (indicated on the left). Immunoblot analysis shows Securin (SEC) or CyclinB1 (CCNB1) levels after Nocodazole treatment for the indicated time points. Anti-betaactin (betaACT) was used as a loading control.

PDF - 2287K, Supplementary Figure 3. Mitotic index in transfected and in nocodazole arrested HEK293 cells. A, Mitotic index of control untransfected, nocodazole-arrested HEK293 and U937 cells. Cells were labelled with a specific anti-phosphoSer10 antibody and analysed by flow citometry. B, Immunoblot analysis of the expression of the indicated proteins in HEK293 cells transiently transfected with expression constructs for NUP98, NUP98-HOXD13, NUP98-LOC, NUP98-HHEX, treated with nocodazole, and harvested at the indicated time points. An arrowhead indicates the NUP98-HOXD13 band just below that of endogenous NUP98. CyclinB1 (CCNB1), Geminin (GMN) and securin (SEC) amounts were detected C, HEK293 cells, transiently transfected with expression constructs for the indicated proteins, were labelled with a specific anti-phosphoSer10 antibody and analysed by flow citometry.

PDF - 1659K, Supplementary Figure 4. siRNA knock-down experiments. A, Immunoblots showing the effects of siRNAs on CDC20 or CDH1 protein expression. Anti-betaactin was used as a loading control. Graphs representing immunoblot quantitations are shown on the right side. B, Cell cycle analysis using propidium iodide staining and flow citometry on control siRNA-treated HEK293 cells (left side), and on siRNA-treated HEK293 cells expressing the NUP98-HOXD13 oncogene (right side).

PDF - 77K, Legends to Supplementary Figures

PDF - 2289K, Supplemental Figure 1. Expression of NUP98 and of NUP98 fusion oncoproteins in HEK293 cells. A, Schematic representation of the structures of human NUP98 (AAC50366) and of its chimeric oncoproteins. The GLFG repeats and the Rae1-interacting GLEBS domain are shown as black and dark grey boxes, respectively. The N-terminal portion of NUP98 is similar in the studied chimeras, spanning aa 1 to aa 469 in NUP98-HOXD13, and aa 1 to aa 514 in NLOC and NHHEX fusions. The HOXD13 (aa 252 to 335, AAC51635), LOC348801 isoform1 (aa 1 to 178, AAI26363), and HHEX (aa 140 to 270, NP_002720) C-terminal moieties are shown as coloured boxes. The homeodomain regions (HD) of HOXD13 and HHEX are indicated as a grey box within the coloured one. Also shown is the structure of NUP98-HOXD13IQN, which carries mutations of three amino acids within the homeodomain (white), impairing DNA binding. B, Western blot analysis showing the expression levels of the indicated proteins transiently expressed in HEK293 cells (T0), and after Nocodazole treatment at the indicated time points. Immunoblottings were performed with anti-NUP98 and anti-betaactin antibodies as a loading control.

PDF - 1450K, Supplementary Figure 2. Proteasome inhibition rescues NUP98-fusions effects on Securin and Cyclin B degradation. Western blot analysis showing securin (SEC) and cyclin B (CCNB1) levels in HEK293 cells transfected with the indicated constructs after treatment with Nocodazole or Nocodazole and the proteasome inhibitor MG132 for 24 hours. An increase of CCNB1 levels can be appreciated in control cells following MG132 treatment as the result of efficient proteasome inactivation. Anti-betaactin antibody (betaACT) was used as a loading control.

NUP98 is a recurrent fusion partner in chromosome translocations that cause acute myelogenous leukemia. NUP98, a nucleoporin, and its interaction partner Rae1, have been implicated in the control of chromosome segregation, but their mechanistic contributions to tumorigenesis have been unclear. Here, we show that expression of NUP98 fusion oncoproteins causes mitotic spindle defects and chromosome missegregation, correlating with the capability of NUP98 fusions to cause premature securin degradation and slippage from an unsatisfied spindle assembly checkpoint (SAC). NUP98 fusions, unlike wild-type NUP98, were found to physically interact with the anaphase promoting complex/cyclosome (APC/C)Cdc20 and to displace the BubR1 SAC component, suggesting a possible mechanistic basis for their interference with SAC function. In addition, NUP98 oncoproteins displayed a prolonged half-life in cells. We found that NUP98 stability is controlled by a PEST sequence, absent in NUP98 oncoproteins, whose deletion reproduced the aberrant SAC-interfering activity of NUP98 oncoproteins. Together, our findings suggest that NUP98 oncoproteins predispose myeloid cells to oncogenic transformation or malignant progression by promoting whole chromosome instability. Cancer Res; 74(4); 1079–90. ©2013 AACR.

Sickle cell disease (SCD) is one of the most common severe monogenic disorders in the world caused by a mutation on HBB gene and characterized by hemoglobin polymerization, erythrocyte rigidity, vaso-occlusion, chronic anemia, hemolysis, and vasculopathy. Recently, the scientific community has focused on the multiple genetic and clinical profiles of SCD. However, the lipid composition of sickle cells has received little attention in the literature. According to recent studies, changes in the lipid profile are strongly linked to several disorders. Therefore, the aim of this study is to dig deeper into lipidomic analysis of erythrocytes in order to highlight any variations between healthy and patient subjects. 241 lipid molecular species divided into 17 classes have been annotated and quantified. Lipidomic profiling of SCD patients showed that over 24% of total lipids were altered most of which are phospholipids. In-depth study of significant changes in lipid metabolism can give an indication of the enzymes and genes involved. In a systems biology scenario, these variations can be useful to improve the understanding of the biochemical basis of SCD and to try to make a score system that could be predictive for the severity of clinical manifestations.

Purpose. Implementation of a useful genetic diagnosis for the clinical management of patients with astrocytic tumors. Methods. We investigated 314 prospectively recruited diffuse astrocytic tumours by applying the cIMPACT-NOW Update 3 signature. Results. The cIMPACT-NOW Update 3 (cIMPACT 3+) markers, i.e. alterations of TERT promoter, EGFR, and/or chromosome 7 and 10, characterized 98.5% of IDH-wt cases. Interestingly, it was also found in 57.5% of IDH-mut cases. According to the genomic profile, four genetic subgroups could be distinguished: 1) IDH-wt/cIMPACT 3-negative (=4); 2) IDH-mut/cIMPACT 3-negative (n=14); 3) IDH-mut/cIMPACT 3+ (n=20); and 4) IDH-wt/cIMPACT 3+ (n=276). Multivariate analysis confirmed that IDH1/2 mutations confer a favorable prognosis (IDH-wt, HR 2.91 95%, CI 1.40-6.05), and validated the prognostic value of the cIMPACT 3+ signature. Conclusions. To accurately identify relevant prognostic categories, overcoming the limitations of histopathology and immunohistochemistry, molecular-cytogenetic analyses must be fully integrated into the diagnostic work-up of astrocytic tumors.

Chromothripsis is a mitotic catastrophe that arises from multiple double strand breaks and incorrect re-joining of one or a few chromosomes. We report on incidence, distribution, and features of chromothriptic events in T-cell acute lymphoblastic leukemias (T-ALL). SNP array was performed in 103 T-ALL (39 ETP/near ETP, 59 non-ETP, and 5 with unknown stage of differentiation), including 38 children and 65 adults. Chromothripsis was detected in 11.6% of all T-ALL and occurred only in adult cases with an immature phenotype (12/39 cases; 30%). It affected 1 to 4 chromosomes, and recurrently involved chromosomes 1, 6, 7, and 17. Abnormalities of genes typically associated with T-ALL were found at breakpoints of chromothripsis. In addition, it gave rise to new/rare alterations, such as, the SFPQ::ZFP36L2 fusion, reported in pediatric T-ALL, deletions of putative suppressors, such as IKZF2 and CSMD1, and amplification of the BCL2 gene. Compared to negative cases, chromothripsis positive T-ALL had a significantly higher level of MYCN expression, and a significant downregulation of RGCC, which is typically induced by TP53 in response to DNA damage. Furthermore we identified mutations and/or deletions of DNA repair/genome stability genes in all cases, and an association with NUP214 rearrangements in 33% of cases.

Platelet-derived growth factor receptor B (PDGFRB) gene rearrangements define a unique subgroup of myeloid and lymphoid neoplasms frequently associated with eosinophilia and characterized by high sensitivity to tyrosine kinase inhibition. To date, various PDGFRB/5q32 rearrangements, involving at least 40 fusion partners, have been reported. However, information on genomic and clinical features accompanying rearrangements of PDGFRB is still scarce. Here, we characterized a series of 14 cases with a myeloid neoplasm using cytogenetic, single nucleotide polymorphism array, and next-generation sequencing. We identified nine PDGFRB translocation partners, including the KAZN gene at 1p36.21 as a novel partner in a previously undescribed t(1;5)(p36;q33) chromosome change. In all cases, the PDGFRB recombination was the sole cytogenetic abnormality underlying the phenotype. Acquired somatic variants were mainly found in clinically aggressive diseases and involved epigenetic genes (TET2, DNMT3A, ASXL1), transcription factors (RUNX1 and CEBPA), and signaling modulators (HRAS). By using both cytogenetic and nested PCR monitoring to evaluate response to imatinib, we found that, in non-AML cases, a low dosage (100–200 mg) is sufficient to induce and maintain longstanding hematological, cytogenetic, and molecular remissions.

Introduction:The spread of Covid-19 has worsened the prognosis of oncology patients, interrupting or delaying life-saving therapies and contextually increasing the risk of severe SARS-CoV-2 infections. Acute lymphoblastic leukemia (ALL) is the most frequent cancer in pediatric age and the management of this disease with concomitant SARS-COV-2 infection represents a challenging situation.Case presentation:We present the case of a 6-year-old female newly diagnosed with ALL during a documented SARS-CoV-2 infection. Our patient was admitted 20 days after SARS-CoV-2 detection for evening-rise fever. Laboratory testing showed severe neutropenia while chest x-ray detected moderate pulmonary involvement. Acute lymphoblastic leukemia diagnosis was made through morphological and molecular analysis on bone marrow aspirate. Given the stability of the blood count and clinical conditions, antiviral therapy with Remdesivir and Convalescent Plasma was started before antileukemic treatment, obtaining a rapid resolution of the infection.Conclusion:In our experience, the treatment with Remdesivir and Convalescent Plasma led to a rapid resolution of Sars-Cov-2 infection. Our case did not present any adverse event to the therapy. Thus, this treatment could be considered in patients with malignancies, in order to accelerate the resolution of the infection and begin immunosuppressive treatment safely. Further studies are required to confirm this hypothesis.

T-cell acute lymphoblastic leukemias (T-ALL) are immature lymphoid tumors localizing in the bone marrow, mediastinum, central nervous system, and lymphoid organs. They account for 10–15% of pediatric and about 25% of adult acute lymphoblastic leukemia (ALL) cases. It is a widely heterogeneous disease that is caused by the co-occurrence of multiple genetic abnormalities, which are acquired over time, and once accumulated, lead to full-blown leukemia. Recurrently affected genes deregulate pivotal cell processes, such as cycling (CDKN1B, RB1, TP53), signaling transduction (RAS pathway, IL7R/JAK/STAT, PI3K/AKT), epigenetics (PRC2 members, PHF6), and protein translation (RPL10, CNOT3). A remarkable role is played by NOTCH1 and CDKN2A, as they are altered in more than half of the cases. The activation of the NOTCH1 signaling affects thymocyte specification and development, while CDKN2A haploinsufficiency/inactivation, promotes cell cycle progression. Among recurrently involved oncogenes, a major role is exerted by T-cell-specific transcription factors, whose deregulated expression interferes with normal thymocyte development and causes a stage-specific differentiation arrest. Hence, TAL and/or LMO deregulation is typical of T-ALL with a mature phenotype (sCD3 positive) that of TLX1, NKX2-1, or TLX3, of cortical T-ALL (CD1a positive); HOXA and MEF2C are instead over-expressed in subsets of Early T-cell Precursor (ETP; immature phenotype) and early T-ALL. Among immature T-ALL, genomic alterations, that cause BCL11B transcriptional deregulation, identify a specific genetic subgroup. Although comprehensive cytogenetic and molecular studies have shed light on the genetic background of T-ALL, biomarkers are not currently adopted in the diagnostic workup of T-ALL, and only a limited number of studies have assessed their clinical implications. In this review, we will focus on recurrent T-ALL abnormalities that define specific leukemogenic pathways and on oncogenes/oncosuppressors that can serve as diagnostic biomarkers. Moreover, we will discuss how the complex genomic profile of T-ALL can be used to address and test innovative/targeted therapeutic options.

Acute leukemias (ALs) of ambiguous lineage are a heterogeneous group of high-risk leukemias characterized by coexpression of myeloid and lymphoid markers. In this study, we identified a distinct subgroup of immature acute leukemias characterized by a broadly variable phenotype, covering acute myeloid leukemia (AML, M0 or M1), T/myeloid mixed-phenotype acute leukemia (T/M MPAL), and early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Rearrangements at 14q32/BCL11B are the cytogenetic hallmark of this entity. In our screening of 915 hematological malignancies, there were 202 AML and 333 T-cell acute lymphoblastic leukemias (T-ALL: 58, ETP; 178, non-ETP; 8, T/M MPAL; 89, not otherwise specified). We identified 20 cases of immature leukemias (4% of AML and 3.6% of T-ALL), harboring 4 types of 14q32/BCL11B translocations: t(2,14)(q22.3;q32) (n = 7), t(6;14)(q25.3;q32) (n = 9), t(7;14)(q21.2;q32) (n = 2), and t(8;14)(q24.2;q32) (n = 2). The t(2;14) produced a ZEB2-BCL11B fusion transcript, whereas the other 3 rearrangements displaced transcriptionally active enhancer sequences close to BCL11B without producing fusion genes. All translocations resulted in the activation of BCL11B, a regulator of T-cell differentiation associated with transcriptional corepressor complexes in mammalian cells. The expression of BCL11B behaved as a disease biomarker that was present at diagnosis, but not in remission. Deregulation of BCL11B co-occurred with variants at FLT3 and at epigenetic modulators, most frequently the DNMT3A, TET2, and/or WT1 genes. Transcriptome analysis identified a specific expression signature, with significant downregulation of BCL11B targets, and clearly separating BCL11B AL from AML, T-ALL, and ETP-ALL. Remarkably, an ex vivo drug-sensitivity profile identified a panel of compounds with effective antileukemic activity.

We investigated MYB rearrangements (MYB-R) and the levels of MYB expression, in 331 pediatric and adult patients with T-cell acute lymphoblastic leukemia (T-ALL). MYB-R were detected in 17 cases and consisted of MYB tandem duplication (tdup) (= 14) or T cell receptor beta locus (TRB)-MYB (= 3). As previously reported, TRB-MYB was found only in children (1.6%) while MYB tdup occurred in both age groups, although it was slightly more frequent in children (5.2% vs 2.8%). Shared features of MYB-R T-ALL were a non-early T-cell precursor (ETP) phenotype, a high incidence of NOTCH1/FBXW7 mutations (81%) and CDKN2AB deletions (70.5%). Moreover, they mainly belonged to HOXA (=8), NKX2-1/2-2/TLX1 (=4), and TLX3 (=3) homeobox-related subgroups. Overall, MYB-R cases had significantly higher levels of MYB expression than MYB wild type (MYB-wt) cases, although high levels of MYB were detected in similar to 30% of MYB-wt T-ALL. Consistent with the transcriptional regulatory networks, cases with high MYB expression were significantly enriched within the TAL/LMO subgroup (P = .017). Interestingly, analysis of paired diagnosis/remission samples demonstrated that a high MYB expression was restricted to the leukemic clone. Our study has indicated that different mechanisms underlie MYB deregulation in 30%-40% of T-ALL and highlighted that, MYB has potential as predictive/prognostic marker and/or target for tailored therapy.

The hemoglobin disorders are the most common single gene disorders in the world. Previous studies have suggested that they are deeply geographically structured and a variety of genetic determinants influences different clinical phenotypes between patients inheriting identical β-globin gene mutations. In order to get new insights into the heterogeneity of hemoglobin disorders, we investigated the molecular variations on nuclear genes (i.e. HBB, HBG2, BCL11A, HBS1L and MYB) and mitochondrial DNA control region. This pilot study was carried out on 53 patients belonging to different continents and molecularly classified in 4 subgroup: β-thalassemia (β+/β+, β0/β0 and β+/β0)(15), sickle cell disease (HbS/HbS)(20), sickle cell/β-thalassemia (HbS/β+ or HBS/β0)(10), and non-thalassemic compound heterozygous (HbS/HbC, HbO-Arab/HbC)(8). This comprehensive phylogenetic analysis provided a clear separation between African and European patients either in nuclear or mitochondrial variations. Notably, informing on the phylogeographic structure of affected individuals, this accurate genetic stratification, could help to optimize the diagnostic algorithm for patients with uncertain or unknown origin., Author summary Worldwide most frequent genetic diseases are represented by a very heterogeneous group of congenital hemolytic anaemias which include β-thalassemia and sickle cell anaemia. They are deeply geographically structured and characterized by high complexity of the genetic background associated with variable clinical severities. Due to this heterogeneity we investigated the nuclear and mitochondrial DNA marker variations in patients with hemoglobinopathies in order to provide the first comprehensive genetic stratification of these hereditary disease. This study contributes to detail the multifaceted picture of hemoglobin disorders.

T-cell acute lymphoblastic leukemia (T-ALL) results from deregulation of a number of genes via multiple genomic mechanisms. We designed a comprehensive fluorescence in situ hybridization (CI-FISH) assay that consists of genomic probes to simultaneously investigate oncogenes and oncosuppressors recurrently involved in chromosome rearrangements in T-ALL, which was applied to 338 T-ALL cases. CI-FISH provided genetic classification into one of the well-defined genetic subgroups (ie, TAL/LMO, HOXA, TLX3, TLX1, NKX2-1/2-2, or MEF2C) in 80% of cases. Two patients with translocations of the LMO3 transcription factor were identified, suggesting that LMO3 activation may serve as an alternative to LMO1/LMO2 activation in the pathogenesis of this disease. Moreover, intrachromosomal rearrangements that involved the 10q24 locus were found as a new mechanism of TLX1 activation. An unequal distribution of cooperating genetic defects was found among the six genetic subgroups. Interestingly, deletions that targeted TCF7 or TP53 were exclusively found in HOXA T-ALL, LEF1 defects were prevalent in NKX2-1 rearranged patients, CASP8AP2 and PTEN alterations were significantly enriched in TAL/LMO leukemias, and PTPN2 and NUP214-ABL1 abnormalities occurred in TLX1/TLX3. This work convincingly shows that CI-FISH is a powerful tool to define genetic heterogeneity of T-ALL, which may be applied as a rapid and accurate diagnostic test.

The aim of this study was to describe the mutational spectrum of hemoglobinopathies during the period 1988-2015 in Umbria, Central Italy, which has never been considered endemic for these conditions. Twenty-four different β-globin gene mutations were identified in 188 patients and eight different α-globin gene mutations in 74 patients. Sixty percent β-thalassemia (β-thal), 85.0% sickle cell disease, 44.0% Hb S (HBB: c.20A>T)/β-thal and 85.0% compound heterozygotes for hemoglobin (Hb) variant-carrying patients were diagnosed or molecularly characterized in the last 3 years. Moreover, most homozygous or compound heterozygous patients (84.5%) came from foreign countries, while only 15.5% were of Italian origin. These data are in accordance with the increasing foreign resident population in Umbria, which has nearly doubled in 10 years (2004-2014). Different from β-globin gene variations, no increasing trend in α defects was observed in our study cohort. Consistently, 58.0% of patients have an Italian origin, suggesting no broad influence of foreign migration in the α-globin genes genetic background. As few defects are prevalent in each country of origin or ethnic group, their knowledge may provide a proper strategy for the identification of mutations in immigrant individuals in a non-endemic region and be important for carrier identification and prenatal screening.

NUP98 is a recurrent fusion partner in chromosome translocations that cause acute myelogenous leukemia. NUP98, a nucleoporin, and its interaction partner Rae1, have been implicated in the control of chromosome segregation, but their mechanistic contributions to tumorigenesis have been unclear. Here, we show that expression of NUP98 fusion oncoproteins causes mitotic spindle defects and chromosome missegregation, correlating with the capability of NUP98 fusions to cause premature securin degradation and slippage from an unsatisfied spindle assembly checkpoint (SAC). NUP98 fusions, unlike wild-type NUP98, were found to physically interact with the anaphase promoting complex/cyclosome (APC/C)Cdc20 and to displace the BubR1 SAC component, suggesting a possible mechanistic basis for their interference with SAC function. In addition, NUP98 oncoproteins displayed a prolonged half-life in cells. We found that NUP98 stability is controlled by a PEST sequence, absent in NUP98 oncoproteins, whose deletion reproduced the aberrant SAC-interfering activity of NUP98 oncoproteins. Together, our findings suggest that NUP98 oncoproteins predispose myeloid cells to oncogenic transformation or malignant progression by promoting whole chromosome instability. Cancer Res; 74(4); 1079–90. ©2013 AACR.

We set up a diagnostic double-color double-fusion fluorescence in situ hybridization (DCDF-FISH) assay to investigate a case of a de novo acute myeloid leukemia (AML)-M4 bearing an inv(11)(p15q22). DCDF-FISH detected the NUP98-DDX10 rearrangement as two fusion signals, at the short and the long arms of the inv(11). Reverse transcription-polymerase chain reaction (RT-PCR) and cloning experiments confirmed the NUP98-DDX10 fusion and identified two splicing fusion isoforms: the known "type II fusion," originating from the fusion of NUP98 exon 14 to DDX10 exon 7 and a new in-frame fusion transcript between NUP98 exon 15 and DDX10 exon 7, which we termed "type III fusion."

Background Although Philadelphia-negative myeloproliferative neoplasms (Ph-MPN) are usually not aggressive, the type and the number of molecular lesions impact greatly on leukemic transformation. Indeed, the molecular background underlying progression is still largely unexplored even though ASXL1, IDH1/2, SRSF2, and TP53 mutations, together with adverse karyotypic changes, place the patient at high risk of leukemic transformation. Case presentation Our patient, a 64-year old man with a diagnosis of JAK2V617F primary myelofibrosis (PMF) had an unusually rapid leukemic transformation. Genomic profiling showed that TET2 and SRSF2 mutations were also present. At leukemic transformation, the patient developed a complex chromosome rearrangement producing a EWSR1-MYB fusion. Remarkably, the expression of MYB and of its target BCL2 was, respectively, ≥4.7 and ≥2.8 fold higher at leukemic transformation than after chemotherapy, when the patient obtained the hematological remission. At this time point, the EWSR1-MYB fusion disappeared while JAK2V617F, TET2, and SRSF2 mutations, as well as PMF morphological features persisted. Conclusions Rapid leukemic transformation of JAK2V617F PMF was closely linked to a previously undescribed putative EWSR1-MYB transcription factor which was detected only at disease evolution. We hypothesize that the EWSR1-MYB contributed to leukemia transformation through at least two mechanisms: 1) it sustained MYB expression, and consequently deregulated its target BCL2, a putative onco-suppressor gene; and 2) ectopic EWSR1-MYB expression probably fulfilled its own oncogenic potential as demonstrated for other MYB-fusions. As our study confirmed that MYB is recurrently involved in chronic as well as leukemic transformation of PMF, it appears to be a valid molecular marker for tailored treatments. Electronic supplementary material The online version of this article (doi:10.1186/s13039-016-0277-1) contains supplementary material, which is available to authorized users.

In an adult case of B-cell acute lymphoblastic leukemia (B-ALL) with a complex karyotype, both chromosomes 14 were involved in unbalanced rearrangements, specifically, der(14)t(13;14)(q21;q21) and dup(14)(q11q32). Fluorescence in situ hybridization (FISH) detected two CEBPE-IGH rearrangements at the dup(14). One was found at the duplication breakpoint and the other derived from insertion of CEBPE into an apparently normal IGH locus. Hypotheses to account for these unusual chromosomal rearrangements are discussed. This case provides the first evidence that chromosome duplication and cryptic insertion produce the CEBPE-IGH fusion and that more than one CEBPE-IGH recombination can occur in a leukemic cell. Our findings confirm that deregulated CEBPE plays a crucial role in the pathogenesis of CEBPE-IGH positive B-ALL.

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. publisher: Elsevier articletitle: NUP98/11p15 translocations affect CD34+ cells in myeloid and T lymphoid leukemias journaltitle: Leukemia Research articlelink: http://dx.doi.org/10.1016/j.leukres.2015.04.014 content_type: article copyright: Copyright © 2015 Elsevier Ltd. All rights reserved. ispartof: Leukemia Research vol:39 issue:7 pages:769-72 ispartof: location:England status: published

NPM1 gene mutations are the most frequent genetic lesion in the 60% of adult acute myeloid leukemias (AMLs) with normal karyotype and no evidence of typical fusion genes (BCR/ABL1, PML/RARA, AML1/ETO, CBFB/MYH11, DEK/CAN). Using direct sequencing we previously identified six different heterozygous mutants within exon 12 encoding the nucleophosmin C-terminus. Because of these mutations the shuttling protein nucleophosmin is aberrantly delocalized in the cytoplasm of leukemic cells (NPMc+). Here, we designed and tested a denaturing high-performance liquid chromatography (DHPLC) assay to detect NPM1 mutated variants. To assess specificity, sensitivity, reliability, and reproducibility, we analyzed DNA from 120 primary adult AMLs and compared DHPLC results with immunohistochemistry and sequencing. All electropherogram profiles in the 26 NPMc+ leukemias were different from the wild type, indicating 100% sensitivity. Sequencing categorized mutations A, B, and D, and all mutation A cases gave identical elution profiles. The other mutations showed typical chro-matograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1 mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML.

We performed cytogenetic and molecular studies on an adult patient with refractory anemia with an excess of blasts with an add(11)(p15). Multicolor fluorescence in situ hybridization (FISH) identified the extra material on 11p as belonging to chromosome 15. Metaphase FISH with probes for chromosomes 5, 11, and 15 revealed a complex four-break rearrangement. Clone RP5-1173K1, containing exons 10–20 of the NUP98 gene, gave three fluorescence signals on the normal 11, the der(5), and the der(15). 3′-RACE-PCR identified an in-frame fusion between NUP98 and NSD1, which was confirmed by RT-PCR. Two different spliced forms, that is, NUP98 exon 11/NSD1 exon 6 and NUP98 exon 12/NSD1 exon 6, were detected. The reciprocal NSD1/NUP98 was not found. A dual-color experiment with RP5-1173K1 and CTC-549A4, spanning the entire NSD1 gene, indicated an insertion of NUP98 into the NSD1 locus. This is the first report of an adult with myelodysplastic syndrome (MDS) harboring an NUP98/NSD1 fusion resulting from insertion of 5′-NUP98 into the NSD1/5q35 locus. © 2004 Wiley-Liss, Inc.

A PDGFRB -positive acute myeloid malignancy with a new t(5;12)(q33;p13.3) involving the ERC1 gene

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.

Multiple lesions in genes that are involved in cell cycle control, proliferation, survival and differentiation underlie T-cell acute lymphoblastic leukaemia (T-ALL). We translated these biological insights into clinical practice to improve diagnostic work-ups and patient management. Combined interphase fluorescence in situ hybridization (CI-FISH), single nucleotide polymorphism (SNP), and gene expression profiles (GEP) were applied in 51 children with T-ALL who were stratified according to minimal residual disease (MRD) risk categories (AIEOP-BFM ALL2000). CI-FISH identified type A abnormalities in 90% of patients. Distribution of each was in line with the estimated incidence in childhood T-ALL: 37.5% TAL/LMO, 22.5% HOXA, 20% TLX3, 7.5% TLX1, and 2.5% NKX2-1. GEP predictions concurred. SNP detected type B abnormalities in all cases, thus linking type A and B lesions. This approach provided an accurate, comprehensive genomic diagnosis and a complementary GEP-based classification of T-ALL in children. Dissecting primary and secondary lesions within MRD categories could improve prognostic criteria for the majority of patients and be a step towards personalized diagnosis.

We investigated TET2 deletion in 418 patients with hematological malignancies. Overall interphase FISH detected complete or partial TET2 monoallelic deletion (TET2del) in 20/418 cases (4.7%). TET2del was very rare in lymphoid malignancies (1/242 cases; 0.4%). Among 19 positive myeloid malignancies TET2del was associated with a 4q24 karyotypic abnormality in 18 cases. In AML, TET2del occurred in CD34-positive hematopoietic precursors and preceded established genomic abnormalities, such as 5q− and −7/7q−, which were the most frequent associated changes (Fisher's exact test P = 0.000).