Publications by authors named "Roberta La Starza"

79 Publications

14q32 rearrangements deregulating BCL11B mark a distinct subgroup of T and myeloid immature acute leukemia.

Blood 2021 04 19. Epub 2021 Apr 19.

University of Perugia, Perugia, Italy.

Acute leukemias (AL) of ambiguous lineage are a heterogeneous group of high-risk leukemias characterized by co-expression 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 Leukemia (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) harbouring four 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, while the other three 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, which was present at diagnosis but not in remission. Deregulation of BCL11B co-occurred with variants at FLT3 and at epigenetic modulators, most frequently DNMT3A, TET2 and/or WT1 gene. Transcriptome analysis identified a specific expression signature, with significant downregulation of BCL11B targets, and clearly separating BCL11B positive AL from AML, T-ALL, and ETP-ALL. Remarkably, ex-vivo drug sensitivity profile identified a panel of compounds with effective antileukemic activity.
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http://dx.doi.org/10.1182/blood.2020010510DOI Listing
April 2021

MYB rearrangements and over-expression in T-cell acute lymphoblastic leukemia.

Genes Chromosomes Cancer 2021 Jul 8;60(7):482-488. Epub 2021 Mar 8.

Hematology and Bone Marrow Transplantation Unit, University of Perugia, Perugia, Italy.

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 ~ 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.
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http://dx.doi.org/10.1002/gcc.22943DOI Listing
July 2021

Comprehensive analysis of mitochondrial and nuclear DNA variations in patients affected by hemoglobinopathies: A pilot study.

PLoS One 2020 22;15(10):e0240632. Epub 2020 Oct 22.

Department of Medicine, Hematology, University of Perugia, Perugia, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240632PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581000PMC
December 2020

New somatic TERT promoter variants enhance the Telomerase activity in Glioblastoma.

Acta Neuropathol Commun 2020 08 25;8(1):145. Epub 2020 Aug 25.

Molecular Medicine Laboratory, Centro di Ricerche Emato-Oncologiche (C.R.E.O.), S. Maria della Misericordia Hospital, University of Perugia, P.le Menghini 9, 06132, Perugia, Italy.

The catalytic activity of human Telomerase Reverse Transcriptase (TERT) compensates for the loss of telomere length, eroded during each cell cycle, to ensure a correct division of stem and germinal cells. In human tumors, ectopic TERT reactivation, most frequently due to hotspot mutations in the promoter region (TERTp), i.e. c.1-124 C > T, c.1-146 C > T, confers a proliferative advantage to neoplastic cells. In gliomas, TERTp mutations (TERTp) mainly occur in oligodendroglioma and glioblastoma. We screened, for TERTp hotspot mutations, 301 adult patients with gliomas and identified heterozygous mutations in 239 cases: 94% of oligodendroglioma, 85% of glioblastoma, and 37.5% of diffuse/anaplastic astrocytoma. Besides the recurrent c.1-124 C > T and c.1-146 C > T, two cases of glioblastoma harbored novel somatic TERTp variants, which consisted of a tandem duplications of 22 nucleotides, i.e. a TERTp c.1-100_1-79dup and TERTp c.1-110_1-89, both located downstream c.1-124 C > T and c.1-146 C > T. In silico analysis predicted the formation of 119 and 108 new transcription factor's recognition sites for TERTp c.1-100_1-79dup and TERTp c.1-110_1-89, respectively. TERTp duplications (TERTp) mainly affected the binding capacity of two transcription factors' families, i.e. the members of the E-twenty-six and the Specificity Protein/Krüppel-Like Factor groups. In fact, these new TERTp significantly enhanced the E-twenty-six transcription factors' binding capacity, which is also typically increased by the two c.1-124 C > T/c.1-146 C > T hotspot TERTp. On the other hand, they were distinguished by enhanced affinity for the Krüppel proteins. The luciferase assay confirmed that TERTp behaved as gain-of-function mutations causing a 2,3-2,5 fold increase of TERT transcription. The present study provides new insights into TERTp mutational spectrum occurring in central nervous system tumors, with the identification of new recurrent somatic gain-of-function mutations, occurring in 0.8% of glioblastoma IDH-wildtype.
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http://dx.doi.org/10.1186/s40478-020-01022-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445914PMC
August 2020

Philadelphia-like acute lymphoblastic leukemia is associated with minimal residual disease persistence and poor outcome. First report of the minimal residual disease-oriented GIMEMA LAL1913.

Haematologica 2021 Jun 1;106(6):1559-1568. Epub 2021 Jun 1.

GIMEMA Data Center, Fondazione GIMEMA Franco Mandelli Onlus, Rome, Italy.

Early recognition of Ph-like acute lymphoblastic leukemia cases could impact on the management and outcome of this subset of B-lineage ALL. To assess the prognostic value of the Ph-like status in a pediatric-inspired, minimal residual disease (MRD)-driven trial, we screened 88 B-lineage ALL cases negative for the major fusion genes (BCR-ABL1, ETV6-RUNX1, TCF3-PBX1 and KTM2Ar) enrolled in the GIMEMA LAL1913 front-line protocol for adult BCR/ABL1-negative ALL. The screening - performed using the BCR/ABL1-like predictor - identified 28 Ph-like cases (31.8%), characterized by CRLF2 overexpression (35.7%), JAK/STAT pathway mutations (33.3%), IKZF1 (63.6%), BTG1 (50%) and EBF1 (27.3%) deletions, and rearrangements targeting tyrosine kinases or CRLF2 (40%). The correlation with outcome highlighted that: i) the complete remission (CR) rate was significantly lower in Ph-like compared to non-Ph-like cases (74.1% vs 91.5%, p=0.044); ii) at time point 2 (TP2), decisional for transplant allocation, 52.9% of Ph-like cases vs 20% of non-Ph-like were MRD-positive (p=0.025); iii) the Ph-like profile was the only parameter associated with a higher risk of being MRD-positive at TP2 (p=0.014); iv) at 24 months, Ph-like patients had a significantly inferior event-free and disease-free survival compared to non-Ph-like patients (33.5% vs 66.2%, p=0.005 and 45.5% vs 72.3%, p=0.062, respectively). This study documents that Ph-like patients have a lower CR rate, EFS and DFS, as well as a greater MRD persistence also in a pediatric-oriented and MRD-driven adult ALL protocol, thus reinforcing that the early recognition of Ph-like ALL patients at diagnosis is crucial to refine risk-stratification and to optimize therapeutic strategies.
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http://dx.doi.org/10.3324/haematol.2020.247973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168510PMC
June 2021

Blockade of Oncogenic NOTCH1 with the SERCA Inhibitor CAD204520 in T Cell Acute Lymphoblastic Leukemia.

Cell Chem Biol 2020 06 7;27(6):678-697.e13. Epub 2020 May 7.

University of Parma, Department of Medicine and Surgery, Parma 43126, Italy. Electronic address:

The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL).
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http://dx.doi.org/10.1016/j.chembiol.2020.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305996PMC
June 2020

Insights into Genetic Susceptibility to Melanoma by Gene Panel Testing: Potential Pathogenic Variants in ACD, and .

Cancers (Basel) 2020 04 19;12(4). Epub 2020 Apr 19.

Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, Maryland, MD 20892, USA.

The contribution of recently established or candidate susceptibility genes to melanoma missing heritability has yet to be determined. Multigene panel testing could increase diagnostic yield and better define the role of candidate genes. We characterized 273 and negative probands through a custom-designed targeted gene panel that included and Co-segregation, loss of heterozygosity (LOH)/protein expression analysis, and splicing characterization were performed to improve variant classification. We identified 16 (5.9%) pathogenic and likely pathogenic variants in established high/medium penetrance cutaneous melanoma susceptibility genes ( and ), including two novel variants in and 4 in . We also found four deleterious and five likely deleterious variants in (3.3%). Thus, including potentially deleterious variants in increased the diagnostic yield to about 9%. Inclusion of rare variants of uncertain significance would increase the overall detection yield to 14%. At least 10% of melanoma missing heritability may be explained through panel testing in our population. To our knowledge, this is the highest frequency of putative deleterious variants reported in melanoma families, suggesting a possible role in melanoma susceptibility, which needs further investigation.
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http://dx.doi.org/10.3390/cancers12041007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226507PMC
April 2020

Targeting cytokine- and therapy-induced PIM1 activation in preclinical models of T-cell acute lymphoblastic leukemia and lymphoma.

Blood 2020 05;135(19):1685-1695

Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell acute lymphoblastic lymphoma (T-LBL) are aggressive hematological malignancies that are currently treated with high-dose chemotherapy. Over the last several years, the search toward novel and less-toxic therapeutic strategies for T-ALL/T-LBL patients has largely focused on the identification of cell-intrinsic properties of the tumor cell. However, non-cell-autonomous activation of specific oncogenic pathways might also offer opportunities that could be exploited at the therapeutic level. In line with this, we here show that endogenous interleukin 7 (IL7) can increase the expression of the oncogenic kinase proviral integration site for Moloney-murine leukemia 1 (PIM1) in CD127+ T-ALL/T-LBL, thereby rendering these tumor cells sensitive to in vivo PIM inhibition. In addition, using different CD127+ T-ALL/T-LBL xenograft models, we also reveal that residual tumor cells, which remain present after short-term in vivo chemotherapy, display consistent upregulation of PIM1 as compared with bulk nontreated tumor cells. Notably, this effect was transient as increased PIM1 levels were not observed in reestablished disease after abrogation of the initial chemotherapy. Furthermore, we uncover that this phenomenon is, at least in part, mediated by the ability of glucocorticoids to cause transcriptional upregulation of IL7RA in T-ALL/T-LBL patient-derived xenograft (PDX) cells, ultimately resulting in non-cell-autonomous PIM1 upregulation by endogenous IL7. Finally, we confirm in vivo that chemotherapy in combination with a pan-PIM inhibitor can improve leukemia survival in a PDX model of CD127+ T-ALL. Altogether, our work reveals that IL7 and glucocorticoids coordinately drive aberrant activation of PIM1 and suggests that IL7-responsive CD127+ T-ALL and T-LBL patients could benefit from PIM inhibition during induction chemotherapy.
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http://dx.doi.org/10.1182/blood.2019003880DOI Listing
May 2020

Design of a Comprehensive Fluorescence in Situ Hybridization Assay for Genetic Classification of T-Cell Acute Lymphoblastic Leukemia.

J Mol Diagn 2020 05 4;22(5):629-639. Epub 2020 Mar 4.

Hematology and Bone Marrow Transplantation Unit, Department of Medicine, Molecular Medicine Laboratory, University of Perugia, Perugia, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jmoldx.2020.02.004DOI Listing
May 2020

Cytogenetic/mutation profile of chronic lymphocytic leukemia/malignant melanoma collision tumors of the skin.

Mol Cytogenet 2018 16;11. Epub 2018 Jan 16.

1Molecular Medicine Laboratory, Hematology and Bone Marrow Transplantation Unit, University of Perugia, Hospital S. Maria della Misericordia, Piazzale Menghini n.9, 06132 Perugia, Italy.

Background: Collision tumors are rare entities that consist of two histologically distinct tumor types arising in the same anatomic site. An association between chronic lymphocytic leukemia (CLL) and malignant melanoma (MM) has been already described. Up to now, they have been documented only at positive regional lymph nodes while we focused on collision tumor in a skin lesion.

Case Presentation: We characterized the genomic profile of a skin CLL/MM collision tumor in a patient with a 9-years story of CLL. Typical high-grade genomic biomarkers featured the CLL: the immunoglobulin heavy variable genes were unmutated; a clonal del(11q), involving and , was present in the peripheral blood (PB) and skin lesion, while a subclonal large del(13q)/D13S319 was detected only in the PB. Interestingly, the del(13q) clone, increased from 10% to 46% from diagnosis to relapse. , , and were wild type. The MM lesion carried a and a promoter mutation.As the family story was consistent with a genetic predisposition to cancer, we performed mutational analysis of genes involved in familial melanoma and CLL, and of and . No germinal mutation known to predispose to CLL, MM, or breast cancer was found. Interestingly, conventional cytogenetic detected a constitutional t(12;17)(p13;p13).

Conclusions: Our data are consistent with distinct genetic landscape of the two tumors which were characterized by specific disease-related abnormalities. CLL cells carried poor prognostic imbalances, i.e. large deletions of the long arm of chromosomes 11 and 13, while in MM cells two functionally linked mutations, i.e. and a promoter occurred. Although, known germline variations predisposing to MM and/or CLL were ruled out, genetic counseling suggested the proband family was at high risk for MM.
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http://dx.doi.org/10.1186/s13039-017-0353-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771154PMC
January 2018

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

Leuk Lymphoma 2017 11 9;58(11):2765-2767. Epub 2017 May 9.

a Molecular Medicine Laboratory, Department of Medicine , CREO, University of Perugia , Perugia , Italy.

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http://dx.doi.org/10.1080/10428194.2017.1312375DOI Listing
November 2017

The EMT transcription factor Zeb2 controls adult murine hematopoietic differentiation by regulating cytokine signaling.

Blood 2017 01 28;129(4):460-472. Epub 2016 Sep 28.

Department of Internal Medicine III, Hematology/Oncology/Rheumatology, University of Bonn, Bonn, Germany.

Epithelial-to-mesenchymal-transition (EMT) is critical for normal embryogenesis and effective postnatal wound healing, but is also associated with cancer metastasis. SNAIL, ZEB, and TWIST families of transcription factors are key modulators of the EMT process, but their precise roles in adult hematopoietic development and homeostasis remain unclear. Here we report that genetic inactivation of Zeb2 results in increased frequency of stem and progenitor subpopulations within the bone marrow (BM) and spleen and that these changes accompany differentiation defects in multiple hematopoietic cell lineages. We found no evidence that Zeb2 is critical for hematopoietic stem cell self-renewal capacity. However, knocking out Zeb2 in the BM promoted a phenotype with several features that resemble human myeloproliferative disorders, such as BM fibrosis, splenomegaly, and extramedullary hematopoiesis. Global gene expression and intracellular signal transduction analysis revealed perturbations in specific cytokine and cytokine receptor-related signaling pathways following Zeb2 loss, especially the JAK-STAT and extracellular signal-regulated kinase pathways. Moreover, we detected some previously unknown mutations within the human Zeb2 gene (ZFX1B locus) from patients with myeloid disease. Collectively, our results demonstrate that Zeb2 controls adult hematopoietic differentiation and lineage fidelity through widespread modulation of dominant signaling pathways that may contribute to blood disorders.
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http://dx.doi.org/10.1182/blood-2016-05-714659DOI Listing
January 2017

MYB deregulation from a EWSR1-MYB fusion at leukemic evolution of a JAK2 (V617F) positive primary myelofibrosis.

Mol Cytogenet 2016 1;9(1):68. Epub 2016 Sep 1.

Hematology and Bone Marrow Transplantation Unit, University of Perugia, C.R.E.O., Piazzale Menghini n.9, 06132 Perugia, Italy.

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 JAK2 (V617F) 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 JAK2 (V617F), TET2, and SRSF2 mutations, as well as PMF morphological features persisted.

Conclusions: Rapid leukemic transformation of JAK2 (V617F) 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.
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http://dx.doi.org/10.1186/s13039-016-0277-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009546PMC
September 2016

RNA sequencing unravels the genetics of refractory/relapsed T-cell acute lymphoblastic leukemia. Prognostic and therapeutic implications.

Haematologica 2016 08 5;101(8):941-50. Epub 2016 May 5.

Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University, Rome, Italy

Despite therapeutic improvements, a sizable number of patients with T-cell acute lymphoblastic leukemia still have a poor outcome. To unravel the genomic background associated with refractoriness, we evaluated the transcriptome of 19 cases of refractory/early relapsed T-cell acute lymphoblastic leukemia (discovery cohort) by performing RNA-sequencing on diagnostic material. The incidence and prognostic impact of the most frequently mutated pathways were validated by Sanger sequencing on genomic DNA from diagnostic samples of an independent cohort of 49 cases (validation cohort), including refractory, relapsed and responsive cases. Combined gene expression and fusion transcript analyses in the discovery cohort revealed the presence of known oncogenes and identified novel rearrangements inducing overexpression, as well as inactivation of tumor suppressor genes. Mutation analysis identified JAK/STAT and RAS/PTEN as the most commonly disrupted pathways in patients with chemorefractory disease or early relapse, frequently in association with NOTCH1/FBXW7 mutations. The analysis on the validation cohort documented a significantly higher risk of relapse, inferior overall survival, disease-free survival and event-free survival in patients with JAK/STAT or RAS/PTEN alterations. Conversely, a significantly better survival was observed in patients harboring only NOTCH1/FBXW7 mutations: this favorable prognostic effect was abrogated by the presence of concomitant mutations. Preliminary in vitro assays on primary cells demonstrated sensitivity to specific inhibitors. These data document the negative prognostic impact of JAK/STAT and RAS/PTEN mutations in T-cell acute lymphoblastic leukemia and suggest the potential clinical application of JAK and PI3K/mTOR inhibitors in patients harboring mutations in these pathways.
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http://dx.doi.org/10.3324/haematol.2015.139410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967573PMC
August 2016

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

Haematologica 2016 08 5;101(8):951-8. Epub 2016 May 5.

Molecular Medicine Laboratory, Center for Hemato-Oncology Research, University of Perugia, Italy

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.
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http://dx.doi.org/10.3324/haematol.2016.143875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967574PMC
August 2016

Cryptic 13q34 and 4q35.2 Deletions in an Italian Family.

Cytogenet Genome Res 2015 9;147(1):24-30. Epub 2015 Dec 9.

Regional Reference Centre for Inherited Bleeding Disorders, University Hospital of Parma, Parma, Italy.

Variations of DNA sequences in the human genome range from large, microscopically visible chromosome anomalies to single nucleotide changes. Submicroscopic genomic copy number variations, i.e. chromosomal imbalances which are undetectable by conventional cytogenetic analysis, play an intriguing clinical role. In this study, we describe the clinical consequences of the concurrent presence of an interstitial deletion in 13q34 and a terminal deletion in 4q35.2 in an Italian family. The index patient, a 19-year-old male, as well as his 12-year-old sister are carriers of both deletions, one of maternal and the other of paternal origin. The phenotype includes language delay, multiorgan involvement and bleeding diathesis with mild deficiency of factors X and VII. In the sister, the concomitant presence of Noonan syndrome may partly explain the clinical symptoms. The deleted region on chromosome 13 involves several genes (ATP11A, MCF2L, F7, F10, PROZ, PCID2, CUL4A, and LAMP1); some of these seem to play a role in the proband's phenotype. The terminal deletion in 4q35.2 contains other OMIM genes (FRG1, FRG2 and DBET); moreover, the 4q region is reported as a susceptibility locus for Crohn's disease, diagnosed in the proband's father. To our knowledge, this is the first report of a family with these 2 submicroscopic copy number changes. We tried to relate the clinical phenotype of the proband and his family to the molecular function of the involved genes.
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http://dx.doi.org/10.1159/000442068DOI Listing
June 2016

Targeted sequencing identifies associations between IL7R-JAK mutations and epigenetic modulators in T-cell acute lymphoblastic leukemia.

Haematologica 2015 Oct 23;100(10):1301-10. Epub 2015 Jul 23.

Center for Human Genetics, KU Leuven, Belgium Center for the Biology of Disease, VIB, Leuven, Belgium

T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia.
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http://dx.doi.org/10.3324/haematol.2015.130179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591762PMC
October 2015

Molecular Cytogenetics Detect an Unbalanced t(2;13)(q36;q14) and PAX3-FOXO1 Fusion in Rhabdomyosarcoma With Mixed Embryonal/Alveolar Features.

Pediatr Blood Cancer 2015 Dec 14;62(12):2238-41. Epub 2015 Jul 14.

Hematology and Bone Marrow Transplantation Unit, University of Perugia, Perugia, Italy.

Distinguishing between alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) is crucial because treatment and prognosis are different. We describe a case of paratesticular rhabdomyosarcoma (RMS), which was classified as mixed ERMS/ARMS. Fluorescence in situ hybridization (FISH) detected losses of 3'PAX3 and 5'FOXO1, suggesting they had undergone an unbalanced rearrangement that probably produced the PAX3-FOXO1 fusion. Double-color FISH and reverse transcription-polymerase chain reaction (RT-PCR) revealed PAX3-FOXO1, which is characteristic of high-risk RMS. This finding highlights the importance of supplementing histology with genetics so that atypical RMS is appropriately classified and patients are correctly stratified and treated.
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http://dx.doi.org/10.1002/pbc.25664DOI Listing
December 2015

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

Leuk Res 2015 Jul 7;39(7):769-72. Epub 2015 May 7.

Laboratory of Molecular Medicine, CREO, University of Perugia and A.O. Perugia, 06132 Perugia, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.leukres.2015.04.014DOI Listing
July 2015

Metachronous cardiac and cerebral sarcomas: case report with focus on molecular findings and review of the literature.

Hum Pathol 2015 Mar 9;46(3):482-7. Epub 2014 Dec 9.

Department of Experimental Medicine, Pathological Anatomy and Histology Unit, Medical School, University of Perugia, I-06129 Perugia, Italy.

Although multiple primary malignancies are relatively rare, they have increased in frequency over the last decades, partly because of advances in diagnosis and therapy. This report describes for the first time the case of a patient with past occupational exposure to asbestos and no family history of cancer who developed 2 rare primary malignancies: a cardiac sarcoma and a gliosarcoma 11 months later. Molecular-cytogenetic studies did not identify common lesions to these 2 rare metachronous sarcomas. The gliosarcoma was associated with monosomy 10 and underlying PTEN monoallelic loss, which has been recurrently observed. In the cardiac sarcoma, MDM2 amplification and CDKN2AB/9p21 biallelic deletion suggested intimal sarcoma. No causal relationship was found between cardiac sarcoma and asbestos exposure, although MDM2 abnormalities were linked to malignant mesothelioma.
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http://dx.doi.org/10.1016/j.humpath.2014.10.028DOI Listing
March 2015

Genetic profile of T-cell acute lymphoblastic leukemias with MYC translocations.

Blood 2014 Dec 30;124(24):3577-82. Epub 2014 Sep 30.

Hematology Unit, University of Perugia, Polo Unico S.M. Misericordia, Perugia, Italy;

MYC translocations represent a genetic subtype of T-lineage acute lymphoblastic leukemia (T-ALL), which occurs at an incidence of ∼6%, assessed within a cohort of 196 T-ALL patients (64 adults and 132 children). The translocations were of 2 types; those rearranged with the T-cell receptor loci and those with other partners. MYC translocations were significantly associated with the TAL/LMO subtype of T-ALL (P = .018) and trisomies 6 (P < .001) and 7 (P < .001). Within the TAL/LMO subtype, gene expression profiling identified 148 differentially expressed genes between patients with and without MYC translocations; specifically, 77 were upregulated and 71 downregulated in those with MYC translocations. The poor prognostic marker, CD44, was among the upregulated genes. MYC translocations occurred as secondary abnormalities, present in subclones in one-half of the cases. Longitudinal studies indicated an association with induction failure and relapse.
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http://dx.doi.org/10.1182/blood-2014-06-578856DOI Listing
December 2014

Multiple EWSR1-WT1 and WT1-EWSR1 copies in two cases of desmoplastic round cell tumor.

Cancer Genet 2013 Nov 4;206(11):387-92. Epub 2013 Nov 4.

Hematology and Bone Marrow Transplantation Unit, University of Perugia, Perugia, Italy. Electronic address:

To provide new insights into the genomic profile of desmoplastic round cell tumors (DSRCT), we applied fluorescence in situ hybridization (FISH) and metaphase comparative genomic hybridization (M-CGH) to two newly diagnosed cases. FISH detected multiple subclones bearing one to three copies of der(11)t(11;22)(p13;q12) and/or der(22)t(11;22)(p13;q12) in both patients. This peculiar genomic imbalance might result from derivative chromosome duplication due to non-disjunction and/or mitotic recombination between normal and derivative chromosomes 11 and 22. Concomitant loss of normal chromosomes (i.e., 11 in patient 1 and 22 in patient 2) caused loss of the WT1 or EWSR1 wild-type allele. M-CGH identified other genomic imbalances: gain at chromosome 3 in both cases and chromosome 5 polysomy in patient 1. Common genomic events (i.e., trisomy 3 and extra EWSR1-WT1 and WT1-EWSR1 copies) probably contributed to disease pathogenesis and/or evolution of DSRCT. Our study demonstrated that an integrated molecular cytogenetic approach identified EWSR1-WT1 cooperating molecular events and genetic markers for prognosis. Thus, FISH and M-CGH might well be applied in a large series of patients to elucidate the genomic background of DSRCT.
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http://dx.doi.org/10.1016/j.cancergen.2013.10.005DOI Listing
November 2013

The hereditary hyperferritinemia-cataract syndrome in 2 italian families.

Case Rep Pediatr 2013 4;2013:806034. Epub 2013 Dec 4.

Pediatric Oncology and Hematology Section, Santa Maria della Misericordia Hospital, Località Sant'Andrea delle Fratte, 06156 Perugia, Italy.

Two 8- and 9-year-old brothers were referred to the Pediatric Oncology Unit, Perugia General Hospital, because of hyperferritinemia. Both had a history of bilateral cataract and epilepsy. Genetic investigation revealed two distinct mutations in iron haemostasis genes; homozygosity for the HFE gene H63D mutation in the younger and heterozygosity in the elder. Both displayed heterozygosity for C33T mutation in the ferritin light chain iron response element. A 7-year-old boy from another family was referred to our unit because of hyperferritinemia. Genetic analyses did not reveal HFE gene mutations. Family history showed that his mother was also affected by hyperferritinemia without HFE gene mutations. Magnetic resonance imaging in the mother was positive for iron overload in the spleen. Cataract was diagnosed in mother and child. Further genetic investigation revealed the C29G mutation of the ferritin light chain iron response element. C33T and C29G mutations in the ferritin light chain iron response element underlie the Hereditary Hyperferritinemia-Cataract Syndrome (HHCS). The HFE gene H63D mutation underlies Hereditary Haemochromatosis (HH), which needs treatment to prevent organ damages by iron overload. HHCS was definitively diagnosed in all three children. HHCS is an autosomal dominant disease characterized by increased L-ferritin production. L-Ferritin aggregates accumulate preferentially in the lens, provoking bilateral cataract since childhood, as unique known organ damage. Epilepsy in one case and the spleen iron overload in another could suggest the misleading diagnosis of HH. Consequently, the differential diagnosis between alterations of iron storage system was essential, particularly in children, and required further genetic investigation.
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http://dx.doi.org/10.1155/2013/806034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3867874PMC
December 2013

Linking genomic lesions with minimal residual disease improves prognostic stratification in children with T-cell acute lymphoblastic leukaemia.

Leuk Res 2013 Aug 2;37(8):928-35. Epub 2013 Jun 2.

Hematology Unit, University of Perugia, Polo Unico S.M. Misericordia, Perugia, Italy.

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.
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http://dx.doi.org/10.1016/j.leukres.2013.04.005DOI Listing
August 2013

New MLLT10 gene recombinations in pediatric T-acute lymphoblastic leukemia.

Blood 2013 Jun 14;121(25):5064-7. Epub 2013 May 14.

Hematology and Bone Marrow Transplantation Unit, University of Perugia, Perugia, Italy.

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.
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http://dx.doi.org/10.1182/blood-2013-02-487256DOI Listing
June 2013