Acute Lymphoblastic Leukemia ARTICLE Deletions of the long arm of chromosome 5 define subgroups of T-cell acute lymphoblastic Ferrata Storti EUROPEAN HEMATOLOGY Foundation leukemia ASSOCIATION Roberta La Starza,1 Gianluca Barba,1 Sofie Demeyer,2,3 Valentina Pierini,1 Danika Di Giacomo,1 Valentina Gianfelici,4 Claire Schwab,5 Caterina Matteucci,1 Carmen Vicente,2,3 Jan Cools,2,3 Monica Messina,4 Barbara Crescenzi,1 Sabina Chiaretti,4 Robin Foà,4 Giuseppe Basso,6 Christine J. Harrison,5 and Cristina Mecucci1 1Molecular Medicine Laboratory, Center for Hemato-Oncology Research, University of Perugia, Italy; 2Center for Human Genetics, KU Leuven, Belgium; 3Center for the Biology of Disease, VIB, Leuven, Belgium; 4Hematology, Department of Cellular Biotechnologies and Hematology, “Sapienza” University, Rome, Italy; 5Leukaemia Research Cytogenetic Group, Northern Institute for Cancer Research, Newcastle University, Newcastle-upon- Haematologica 2016 6 Tyne, UK; and Pediatric Hemato-Oncology, Department of Pediatrics “Salus Pueri”, Volume 101(8):951-958 University of Padova, Italy ABSTRACT ecurrent deletions of the long arm of chromosome 5 were detected in 23/200 cases of T-cell acute lymphoblastic leukemia. Genomic Rstudies 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 Correspondence: T-cell precursor acute lymphoblastic leukemia, i.e. WT1, ETV6, JAK1, [email protected] 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 Received: February 5, 2016 prevalent in adults (chi-square, P=0.010) and defined a subgroup of Accepted: April 29, 2016 HOXA-positive T-cell acute lymphoblastic leukemia characterized by 130 up- and 197 down-regulated genes. Down-regulated genes included Pre-published: May 5, 2016. TRIM41, ZFP62, MAPK9, MGAT1, and CNOT6, all mapping within the 1.4 Mb common deleted region at 5q35.3. Of interest, besides CNOT6 doi:10.3324/haematol.2016.143875 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 Check the online version for the most updated information on this article, online supplements, acute lymphoblastic leukemia with terminal 5q deletions. In conclusion, and information on authorship & disclosures: interstitial and terminal 5q deletions are recurrent genomic losses identi- www.haematologica.org/content/101/8/951 fying distinct subtypes of T-cell acute lymphoblastic leukemia. Introduction ©2016 Ferrata Storti Foundation Material published in Haematologica is cov- Deletion of the long arm of chromosome 5, del(5q), is the most frequent genom- ered by copyright. All rights reserved to the ic loss in myeloid diseases.1 Two distinct common deleted regions (CDR) were Ferrata Storti Foundation. Copies of articles identified in myelodysplastic syndromes and acute myeloid leukemia. Del(5q), as are allowed for personal or internal use. Permission in writing from the publisher is the sole cytogenetic abnormality, occurs in 10-15% of myelodysplastic syndromes required for any other use. and is known as “the 5q- syndrome”.1 It is characterized by a 1.5 Mb CDR (5q32- q33) where the putative oncosuppressor RPS14 is mapped.2 Del(5q) associated with other cytogenetic changes, often within a complex karyotype, is prevalent in haematologica | 2016; 101(8) 951 R. La Starza et al. acute myeloid leukemia and in high-risk myelodysplastic stitial (I-5q) and terminal (T-5q). I-5q was identified in syndromes.3 Here it is characterized by a ~1 Mb CDR 2.5% of cases with a genomic profile closely resembling (5q31) and haploinsufficiency of the oncosuppressor immature/early T-cell precursor (ETP) ALL. These find- EGR1.4 Conversely, del(5q) has been rarely reported in B- ings indicate that I-5q is a recurrent cytogenetic event in and T- acute lymphoblastic leukemia (ALL).5-8 T-ALL with early differentiation arrest of blasts. T-5q, T-lineage ALL (T-ALL), a heterogeneous group of identified in 9% of cases, clustered within the HOXA cat- leukemias, is characterized by co-occurrence of multiple egory and was characterized by a high incidence of genetic lesions.9 Interestingly, cooperative genetic defects abnormalities involving the CCR4-NOT complex and its have been described in T-ALL, suggesting that perturba- closely related transcription factor BTG1. These observa- tion of specific cell processes are needed for the develop- tions indicate that HOXA over-expression, haplo-insuffi- ment of overt leukemia.10,11 Translocations causing ciency of genes at T-5q, and deregulation of the CCR4- TAL/LMO, TLX1, TLX3, MYB, MEF2C, NKX2-1/2, or NOT complex are characteristic of this subgroup of HOXA over-expression define distinct gene expression leukemia. signatures and are known as “type A” abnormalities.6,12-14 These rearrangements co-occur with multiple mutations and imbalances, named “type B” abnormalities, which Methods activate oncogenic signaling cascades, including JAK/STAT, PI3K/AKT, and RAS/MEK/ERK. The most We investigated the incidence of del(5q) in a cohort of 66 adult prevalent type B abnormalities are loss-of-function muta- (≥18 years) and 134 pediatric T-ALL patients from the Italian tions and genomic losses, indicating that tumor suppres- (GIMEMA LAL-0496 and LAL-0904 and AIEOP LLA-2000) and sor genes, such as CDKN2A/B, PHF6, LEF1, PTEN, WT1, UK clinical trials (MRC, ALL2003 and ALL97) (Online ETV6, and PTPN2, play pivotal roles in the initiation of T- Supplementary Table S1, Online Supplementary Information).10,16,17 All ALL.9,15 patients or their parents/guardians gave informed consent to To provide insights into the prevalence and specific fea- sample collection and molecular analyses, in agreement with the tures of del(5q), in T-ALL, we screened a series of 200 T- Declaration of Helsinki. The study was approved by the local ALL cases by fluorescence in situ hybridization (FISH). We bio-ethical committee (research project 2014-025). found two distinct types of recurrent 5q deletions: inter- Combined interphase FISH identified T-ALL-associated Table 1. Clinical, hematologic and molecular-cytogenetic features of 5 cases of T-ALL with interstitial 5q deletion. N. Sex/Age Immunophenotype Karyotype ETP-ALL Genetic CI-FISH CNV Follow-up related mutations category (months) 1 F/9 undefined: CD34+, n.a. DNM2, WT1, HOXA SET-NUP214[80%] LOSS: CREBBP/16p13.3, 64† CD33+ LPHN2 del(5)(q31q33) [87%] JAKMIP2/5q32, SYNCRIP/6q14.3 del(6)(q15q21) [82%] del WT1 [85%] 2 F/16 ETP 46,XX[15] RUNX1, JAK3, HOXA HOXA-translocation [60%] LOSS: 5q14.3-q32,12p13.2-p12.3, 63† ETV6, PHF6 del(5)(q14q31) [55%] 14q32.11-q32.12,16q22.2-q24.3 del ETV6/CDKN1B [59%] GAIN: 1q32.1-q44,19q13.2-q13.4 CN-LOH: 6q12-q27 3 F/27 pro-T n.a. WT1, PHF6 HOXA SET-NUP214[93%] LOSS: 2p14-p13.3,3q26.31-q26.33,5q11.1- 12† del(5)(q31) [99%] q12.2,5q21.1-q22.1,5q23.2,5q31.1- del(6)(q21) [98%] q31.3,5q31.3-q32,6q21,6q22.31,6q22.33,7p12.2- del WT1 [85%] p11.2,9q34.11-q34.13,11p14.1-p13,11p11.2- del RB1 [96%] p11.12,11q14.1-q23.3,12q14.1-q23.3,13q13.3- del ATM [99%] q14.3,13q21.33-q31.1,14q12,16q21-q22.1,17p13.2- del TP53 [99%] p13.1,19q13.32 CN-LOH: 1p36.33-p36.11 4 F/49 pro-T 46,XX[15] JAK3,JAK1, MEF2C RUNX1-translocation LOSS: 5q31.1 7† PHF6 [82%] GAIN: Xp22.33-p11.23 del(5)(q31) [84%] CN-LOH: 9q33.2-q34.3 gain DDX3X [46%] 5 M/37 ETP 48,XY,del(5) n.a. unclassified del(5)(q31)[93%] LOSS: 1q25.1, 2p13.2, 3q13.1-q13.3, 1† (q31q33),+8, del(6)(q16q21)[93%] 5q21.3-33.2, 6q15-q22.3, 9q22.3-q33.2, del(9q),der(12),del(13q), del(9)(q32/TAL2[97%] 11q14.2-q23.2, 12q13.1, 13q13.3-q21.3, +21[10] del RB1[99%] 16p13.3, 20p11.2 del(20)(p11)[92%] GAIN: 21q11.2-q22.3 trisomy 21[95%] N.: patient number; F: female; M: male; CI-FISH: combined interphase fluorescence in situ hybridization (the percentage of cells with abnormal CI-FISH is indicated between brackets); CNV: copy number variations were determined by haloplex analysis (case n.1) or single nucleotide polymorphism array (cases n. 2-5); n.a.: not available; ETP: early T-cell precursor; †dead. Only mutations typically asso- ciated with immature/ETP-ALL were reported. In patient n. 1 the panel of antibodies tested did not allow to stage of differentiation of leukemic blasts to be determined. Patients n. 2 and 3 were studied by transcriptome sequencing and gene expression profiling. 952 haematologica | 2016; 101(8) Deletion of chromosome 5 long arm in T-ALL genomic rearrangements which classified cases into defined sub- Results and Discussion groups (Online Supplementary Table S2).10,16,17 Deletions of 5q were detected by FISH with LSI EGR1/D5S23, D5S721 Dual Colour Incidence and distribution of del(5q) in T-cell acute probe, RP11-182E4/RP11-453D13 and CTB-31E20/RP11- lymphoblastic leukemia 266N12 for rearrangements of TLX3, RP11-117L6 for NPM1 abnormalities, and RP1-240G13 for deletions of the subtelomer- FISH and single nucleotide polymorphism array ic 5q region.