Mutational profiling of acute lymphoblastic leukemia with testicular relapse
- Ling-Wen Ding†1Email author,
- Qiao-Yang Sun†1,
- Anand Mayakonda†1,
- Kar-Tong Tan1,
- Wenwen Chien1, 2,
- De-Chen Lin1, 2,
- Yan-Yi Jiang1,
- Liang Xu1,
- Manoj Garg1, 3,
- Zhen-Tang Lao1, 4,
- Michael Lill2,
- Henry Yang1,
- Allen Eng Juh Yeoh1, 5Email author and
- H. Phillip Koeffler1, 2
© The Author(s). 2017
Received: 1 December 2016
Accepted: 27 February 2017
Published: 2 March 2017
Relapsed acute lymphoblastic leukemia (ALL) is the leading cause of deaths of childhood cancer. Although relapse usually happens in the bone marrow, extramedullary relapse occasionally occurs including either the central nervous system or testis (<1–2%). We selected two pediatric ALL patients who experienced testicular relapse and interrogated their leukemic cells with exome sequencing. The sequencing results and clonality analyses suggest that relapse of patient D483 directly evolved from the leukemic clone at diagnosis which survived chemotherapy. In contrast, relapse leukemia cells (both bone marrow and testis) of patient D727 were likely derived from a common ancestral clone, and testicular relapse likely arose independently from the bone marrow relapsed leukemia. Our findings decipher the mutational spectra and shed light on the clonal evolution of two cases of pediatric ALL with testicular relapse. Presence of CREBBP/NT5C2 mutations suggests that a personalized therapeutic approach should be applied to these two patients.
KeywordsAcute lymphoblastic leukemia ALL Testicular relapse Extramedullary relapse
To the editor
Relapsed acute lymphoblastic leukemia (ALL) is the leading cause of deaths of childhood cancer [1–3]. Although relapse usually occurs in the bone marrow (medullary), extramedullary relapse occasionally occurs, including either in the central nervous system or testis (<1–2%). Involvement of these organs is often associated with an inferior prognosis, perhaps because the blood-brain/blood-testis barrier hinders efficient delivery of chemotherapy, and/or the leukemic cells infiltrated in these immune-privileged sites may escape efficient immune surveillance. Currently, clonal origin and evolution of extramedullary relapse ALL remain poorly understood. To address this, we selected two pediatric ALL patients who experienced testicular relapse and interrogated their leukemic cells with exome sequencing (see Additional file 2: Supplementary Methods).
Patient D483 (5.6 years old at diagnosis) was treated as an intermediate-risk B-ALL [hyperdiploid:56,XY,+X,t(2;14)(p?13;q32),+4,+8,+9,+10,+14,+17,+18,+21,+21, absence of any well-known leukemic fusion oncogene]. He developed bone-marrow (96% blast) and testicular relapse 5 years after induction of remission. Mutations of KRAS (G12D) and CREBBP (S1436C, in histone-acetyltransferase HAT domain) were found in the founding leukemic clone at diagnosis and persisted in bone marrow and testis at relapse (Additional file 1: Table S1). CREBBP encodes a histone/non-histone acetyltransferases which is involved in regulation of glucocorticoid gene expression, its mutation contributes to prednisolone/dexamethasone (glucocorticoid) resistance [4, 5]. Mutation and copy-number deletion of CREBBP are frequent in B-cell lymphoma and ALL [3, 6] and are often associated with disease relapse. A missense mutation (R17Q) of MEF2B (MADS-box transcription enhancer factor-2) was found in both the bone marrow and testicular relapse samples. Missense mutation of MEF2B is frequently detected in diffuse large B-cell lymphoma and contributes to malignant transformation by regulating expression of the proto-oncogene BCL6 . Other mutations included EVX1 (homeobox protein) and OTUD5 (regulates p53 stability by deubiquitinating p53) (Additional file 1: Table S1).
Second patient (case D727, 1.3 years old at diagnosis of B-ALL) harbored a MLL-AF9 fusion gene [t(9;11)] and was treated as a high-risk ALL (82.8% blast in peripheral blood). MLL fusion is often associated with infant-ALL and a poor prognosis. Complete remission was achieved after induction therapy; however, the patient relapsed (91% blast) after a 2.3-year remission. NT5C2 gene (encodes a 5′-nucleotidase involved in purine metabolism) had two mutations in the relapse samples, differencing in their VAF in the bone marrow (34%) and testicle (5%) for R367Q mutation; while D407V mutation was present with a VAF of 7% in bone marrow and 36% in testicular relapse. These two NT5C2 mutations occur as recurrent mutational hotspots in relapse-ALL and they have been functionally validated . These mutations increase the NT5C2 inosine-5-monophosphate-nucleotidase activity; and therefore lead to resistance to one of the chemotherapeutic drugs, 6-mercaptopurine [8, 9] (part of child’s treatment). Additional mutations that occurred in this child’s ALL cells included DUSP13 (phosphatase that regulates JNK/P38 phosphorylation), MAPK8 (JNK1), PPP1R3B (protein phosphatase 1 regulatory subunit 3B), and ALPK3 (alpha-kinase 3) (Additional file 1: Table S1).
Acute lymphoblastic leukemia
Variant allele frequency
This work was funded by the Leukemia Lymphoma Society of America, by the Singapore Ministry of Health’s National Medical Research Council (NMRC) under its Singapore Translational Research (STaR) Investigator Award (NMRC/STaR/0021/2014), Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2013-T2-2-150), the NMRC Centre Grant awarded to National University Cancer Institute of Singapore (NMRC/CG/012/2013) and the National Research Foundation Singapore, and the Singapore Ministry of Education under its Research Centres of Excellence initiatives. This work was also supported by the generous donations from the Melamed Family and Reuben Yeroushalmi.
Availability of data and materials
The datasets supporting the conclusions of this manuscript are included within the article and its online supplementary table. Please contact author for raw sequence data requests.
LWD, QYS, AEJY, and HPK conceived and designed the research study and wrote the manuscript. LWD and QYS performed the experiments. AM, KTT, LWD, and HY performed the bioinformatics analysis. WC, DCL, YYJ, XL, MG, ZTL, and ML analyzed and interpreted data. All authors read, revised, and approved the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The study was approved by Institutional Review Board and was conducted in accordance with provision of the Declaration of Helsinki. Patients’ samples were collected with informed consent.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Mar BG, Bullinger LB, McLean KM, Grauman PV, Harris MH, Stevenson K, Neuberg DS, Sinha AU, Sallan SE, Silverman LB, Kung AL, Lo Nigro L, Ebert BL, Armstrong SA. Mutations in epigenetic regulators including SETD2 are gained during relapse in paediatric acute lymphoblastic leukaemia. Nat Commun. 2014;5:3469.View ArticlePubMedPubMed CentralGoogle Scholar
- Ma X, Edmonson M, Yergeau D, Muzny DM, Hampton OA, Rusch M, Song G, Easton J, Harvey RC, Wheeler DA, Ma J, Doddapaneni H, Vadodaria B, Wu G, Nagahawatte P, Carroll WL, Chen IM, Gastier-Foster JM, Relling MV, Smith MA, Devidas M, Guidry Auvil JM, Downing JR, Loh ML, Willman CL, Gerhard DS, Mullighan CG, Hunger SP, Zhang J. Rise and fall of subclones from diagnosis to relapse in pediatric B-acute lymphoblastic leukaemia. Nat Commun. 2015;6:6604.View ArticlePubMedPubMed CentralGoogle Scholar
- Ding LW, Sun QY, Tan KT, Chien W, Thippeswamy AM, Eng Juh Yeoh A, Kawamata N, Nagata Y, Xiao JF, Loh XY, Lin DC, Garg M, Jiang YY, Xu L, Lim SL, Liu LZ, Madan V, Sanada M, Fernandez LT, Preethi H, Lill M, Kantarjian HM, Kornblau SM, Miyano S, Liang DC, Ogawa S, Shih LY, Yang H, Koeffler HP. Mutational landscape of pediatric acute lymphoblastic leukemia. Cancer Res. 2017;77(2):390–400.View ArticlePubMedGoogle Scholar
- Mullighan CG, Zhang J, Kasper LH, Lerach S, Payne-Turner D, Phillips LA, Heatley SL, Holmfeldt L, Collins-Underwood JR, Ma J, Buetow KH, Pui CH, Baker SD, Brindle PK, Downing JR. CREBBP mutations in relapsed acute lymphoblastic leukaemia. Nature. 2011;471(7337):235–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Pasqualucci L, Dominguez-Sola D, Chiarenza A, Fabbri G, Grunn A, Trifonov V, Kasper LH, Lerach S, Tang H, Ma J, Rossi D, Chadburn A, Murty VV, Mullighan CG, Gaidano G, Rabadan R, Brindle PK, Dalla-Favera R. Inactivating mutations of acetyltransferase genes in B-cell lymphoma. Nature. 2011;471(7337):189–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Inthal A, Zeitlhofer P, Zeginigg M, Morak M, Grausenburger R, Fronkova E, Fahrner B, Mann G, Haas OA, Panzer-Grumayer R. CREBBP HAT domain mutations prevail in relapse cases of high hyperdiploid childhood acute lymphoblastic leukemia. Leukemia. 2012;26(8):1797–803.View ArticlePubMedPubMed CentralGoogle Scholar
- Ying CY, Dominguez-Sola D, Fabi M, Lorenz IC, Hussein S, Bansal M, Califano A, Pasqualucci L, Basso K, Dalla-Favera R. MEF2B mutations lead to deregulated expression of the oncogene BCL6 in diffuse large B cell lymphoma. Nat Immunol. 2013;14(10):1084–92.View ArticlePubMedPubMed CentralGoogle Scholar
- Tzoneva G, Perez-Garcia A, Carpenter Z, Khiabanian H, Tosello V, Allegretta M, Paietta E, Racevskis J, Rowe JM, Tallman MS, Paganin M, Basso G, Hof J, Kirschner-Schwabe R, Palomero T, Rabadan R, Ferrando A. Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL. Nat Med. 2013;19(3):368–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Meyer JA, Wang J, Hogan LE, Yang JJ, Dandekar S, Patel JP, Tang Z, Zumbo P, Li S, Zavadil J, Levine RL, Cardozo T, Hunger SP, Raetz EA, Evans WE, Morrison DJ, Mason CE, Carroll WL. Relapse-specific mutations in NT5C2 in childhood acute lymphoblastic leukemia. Nat Genet. 2013;45(3):290–4.View ArticlePubMedPubMed CentralGoogle Scholar
- Popic V, Salari R, Hajirasouliha I, Kashef-Haghighi D, West RB, Batzoglou S. Fast and scalable inference of multi-sample cancer lineages. Genome Biol. 2015;16:91.View ArticlePubMedPubMed CentralGoogle Scholar