Independent Development of Lymphoid and Histiocytic Malignancies from a Shared Early Precursor

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Letters to the Editor 955 Independent development of lymphoid and histiocytic malignancies from a shared early precursor

Leukemia (2016) 30, 955–958; doi:10.1038/leu.2015.193 later owing to progressive disease. An elaborate case report is available in the Supplementary Material. We isolated genomic DNA from skin fibroblasts, from Histiocytic neoplasms are tumors that develop from macrophages a bone marrow sample taken at the time of T-ALL diagnosis and dendritic cells and are also called histiocytes. Histiocytic and from biopsy specimens from the non-Langerhans cell sarcoma is a malignant tumor derived from mature histiocytes histiocytosis and the histiocytic sarcoma, after informed that are characterized by the expression of one or more histiocytic consent and approval by the medical ethics committee/ markers (CD163, CD68 or lysozyme) and the absence of institutional board (CMO, study 2012/271) of the Radboudumc. Langerhans cell, follicular dendritic cell and myeloid cell markers To investigate the clonal relationship between the malignancies, (CD1a, langerin, CD21, CD35, CD33, CD13 and myeloperoxidase).1,2 we determined the TR gene rearrangements (Supplementary Non-Langerhans cell histiocytosis describes a very diverse group Methods). All samples showed a VDJ-rearranged TRB gene, of histiocytic proliferations for which the cells do not meet the an incomplete DJ TRB gene rearrangement and two (biallelic) criteria for Langerhans cells (CD1a+, Langerin+ and S100+).3 VJ-rearranged TRG genes (Figure 1d). The four clonal rearrange- The genetic etiology of these tumors has not been described as ments were of identical size in the three specimens, which yet, but considering the cell types involved, the presumption is suggests that the three tumors originate from the same that these tumors derive from a myeloid stem cell. Both histiocytic precursor cell. sarcoma and non-Langerhans cell histiocytosis are very rare and To get insight into the clonal development, we performed high- mostly affect adults. A few pediatric and young adult cases have resolution copy number analysis (SNP6.0) and whole-exome been described, and in these cases, the histiocytic tumor often co- sequencing on all three tumor samples and skin fibroblasts occurred or developed subsequently to lymphoid neoplasms such (Supplementary Methods). The data are deposited at the as lymphoma and B-lineage or T-lineage acute lymphoblastic European Genome-Phenome Archive under accession number leukemia (T-ALL) (Pagni et al.,4 Feldman et al.,5 Kumar et al.6 and EGAS00001001353. All tumors carried a similar somatic loss of McClure et al.,7 and references therein). Hematopoiesis is approximately 1.7 Mb affecting the CDKN2A/B locus. However, as postulated as the unidirectional maturation of stem cells into copy number aberrations in the ileum sample were barely detected, lineage-committed cells. However, immunoglobulin and T-cell presumably owing to low percentage of malignant cells fi receptor gene rearrangements (TR rearrangements) typical for in the sample, we isolated DNA from a formalin- xed lymphoid cells were also identified in histiocytic tumors sample of the non-Langerhans cell histiocytosis in which the 2 4 5 6 tumor cell percentage was 480% and repeated the copy (Vos et al., Pagni et al., Feldman et al., Kumar et al., fi fi McClure et al.7 and Brunner et al.,8 and references therein). number analysis on a lower-resolution formalin- xed, paraf n- This apparent lineage switch of lymphoid cells might have embedded (FFPE)-compatible Affymetrix Oncoscan array. occurred through (1) transdifferentiation, defined as the trans- With this approach, we established that indeed all samples carried deletions affecting the CDKN2A/B locus (Figure 2a, formation of one cell type into another without passing an Supplementary Figure 1 and Supplementary Table 1). The intermediate pluripotent state or progenitor cell type, or (2) de- T-ALL showed a distinct biallelic loss at this locus. In addition, differentiation, defined as the transformation via an intermediate clear differences in copy number profiles between the three pluripotent state or progenitor cell type. Alternatively, these tumors were observed. These included a deletion of part of tumors may have developed from a multipotent stem cell that is chromosome 8q in the non-Langerhans cell histiocytosis, capable of differentiating into both myeloid and lymphoid 9–11 and acquired segmental duplications of a large part of precursors. In this letter, we describe a child with three chromosome 5, chromosome 11p and chromosome 22q in the tumors from two different lineages in which genetic analysis histiocytic sarcoma. shows that the tumors were derived from a common precursor Whole-exome sequencing on all samples identified an average cell showing lineage plasticity for both lymphoid and myeloid of 47 424 variants per sample with an average coverage of development. 94 × and 88% of the exome targets covered more than We encountered a young boy who presented with de novo 20 × (Supplementary Table 2). After exclusion of all variant calls T-ALL at 4 years of age. During T-ALL treatment, he presented with detected in the non-malignant sample (skin fibroblasts), we were a non-Langerhans cell histiocytosis in the ileum and a histiocytic able to confirm 17 high-confident variant calls using Sanger sarcoma in the liver, which disseminated to the spleen, intestine sequencing in at least one of the four samples. FFPE-derived DNA and lungs (Figure 1a). Immunophenotyping of the T-ALL showed a was used for validations in the non-Langerhans cell histiocytosis. premature T-lineage population (CD1a+, CD3+, CD2+, CD5+, CD7+, Six variants were found to be present in the germline and the HLA-DR+, cyCD79a+, TdT+, partial CD34+ (33%), weak partial CD4+ remaining 11 variants were somatic mutations, present in at least (10%) and CD10 − ,CD8− ,CD117− )andfluorescence in situ one of the tumor samples (Figure 2a and Supplementary Table 3). hybridization was negative for BCR-ABL1, ETV6-RUNX1 or MLL- A total of six somatic mutations were present in all three translocations. The non-Langerhans cell histiocytosis was devoid of malignancies and were thus relatively early events. Three CD1a, S100, CD34 or CD117 expression, while CD68 was positive. mutations were observed in T-ALL and histiocytic sarcoma, but Figures 1b and c show the histology of both non-Langerhans cell were absent in the non-Langerhans cell histiocytosis, including a histiocytosis and histiocytic sarcoma at two time points. Hematoxylin truncating mutation in the cyclin gene CCND3 and a missense and eosin staining demonstrated a progression in mitotic activity mutation in the insulin-like growth factor receptor gene IGF1R. and atypical multinuclear cells in the histiocytic sarcoma. The patient Two missense mutations at highly conserved positions in showed no adequate response to treatment and died 6 months KIAA1644 and C6orf211, two genes of unknown function, were

Accepted article preview online 23 July 2015; advance online publication, 11 August 2015

Figure 1. Clinical ﬁndings in a patient who presented with a T-ALL, non-Langerhans cell histiocytosis and histiocytic sarcoma. (a) Fluorodeoxyglucose positron emission tomography computed tomography (FDG-PET CT) scan showed several masses in the liver spleen and intestine. Frontal view on the left and side view on the right. (b and c) Hematoxylin and eosin staining of resected tissue at diagnosis (b)andat autopsy (c) demonstrated a clear progression in mitotic activity and atypia with increase of multinucleated tumor cells (arrowheads). (d)T-cell receptor gene rearrangement patterns show clonal relationship between the tumors. The PCRs of the different TR targets were performed in duplicate; a monoclonal and a polyclonal control were included in each experiment. Note that some peaks display a pink signal, which indicates product overloading. The T-ALL showed clonal VDJ-rearranged TRB genes (C 264 bp), two incomplete (DJ) clonal TRB gene rearrangements (C/C 179/304 bp) and two VJ-rearranged TRG genes (C/C 160/171 bp). This rearrangement pattern ﬁts to a monoclonal T-cell population, but does not fully exclude the presence of an additional small T-ALL clone. In the non-Langerhans cell histiocytosis and histiocytic sarcoma samples, four identical-sized clonal TR rearrangements were detected. This rearrangement pattern is consistent with the presence of a monoclonal population in these tumors, which is clonally related to the T-ALL. HS, histiocytic sarcoma; NLCH, non-Langerhans cell histiocytosis.

Figure 2. Shared and unique mutations reveal clonal relationships. (a) Data matrix showing copy number aberrations and single- nucleotide variants in the T-ALL, non-Langerhans cell histiocytosis (NLCH) and histiocytic sarcoma (HS) samples. (b)Schematic of the evolution of T-ALL, NLCH and HS in this patient. A precursor cell with rearranged T-cell receptor genes and carrying a CDKN2A/B deletion and six additional missense mutations evolved in the NLCH, which carried a unique additional deletion of the q-arm of chromosome 8, to a precursor cell that acquired three additional mutations and further developed into the T-ALL and the HS. The T-ALL acquired an additional TRB DJ rearrangement and a second deletion in CDKN2A/B. The timeline below indicates the dates of diagnosis of the three tumors. The red bar speciﬁes ALL10 high-risk treatment; dark red bar signiﬁes the adjusted treatment for histiocytic sarcoma.

exclusively present in the histiocytic sarcoma. None of the switching, such as PAX5, C/EBPα, C/EBPβ and PTEN.4,5–7,12 In animal mutations were present in the catalog of somatic mutations in models, knockout of PAX5 can dedifferentiate B-cells13 while cancer database (COSMIC). Owing to the low tumor load C/EBPα- and C/EBPβ-enforced inhibition of PAX5 leads to rapid of our original non-Langerhans cell histiocytosis sample, its transdifferentiation into macrophages.14 In addition, silencing and exome sequencing data were not informative, and mutations reactivation of the EBF1/PAX5 pathway in Myc-induced hemato- unique for this sample could not be identified. Based on poietic tumors gives an oscillation between B-lymphoid and the identified copy number changes and mutations, we could myeloid lineages.15 The PTEN+/ − Ink4a/Arf (CDKN2A)− / − mouse confirm that all three malignancies were derived from a shared model shows an outgrowth of biphenotypic myelolymphoid precursor cell. The additional abnormalities that were detected in cells and, subsequently, lymphoblastic B-cell lymphomas only one or two tumors allowed us to derive the clonal relation and histiocytic sarcomas with biallelic loss of PTEN.12 In our (Figure 2b). patient, the initial diagnosis was a T-lineage rather than B-lineage Previous studies on histiocytic sarcomas have focused on the leukemia, and no PTEN or PAX5 mutations were identified in any of trans- or de-differentiation models and described, yet not in all the samples. These data suggest that oncogenesis in these tumors cases, aberrations in genes involved in lineage commitment and may be heterogeneous and that detailed protein and RNA-based

© 2016 Macmillan Publishers Limited Leukemia (2016) 947 – 1002 Letters to the Editor 958 studies will be needed to reveal the details of this apparent 2Department of Pathology, Radboud University Medical Center, lineage switch. Nijmegen, The Netherlands; Even though several studies put forward the possibility of 3Princess Máxima Center for Pediatric Oncology, De Bilt, transdifferentiation and plasticity in the hematopoietic system,10 The Netherlands and based on the small number of aberrations analyzed in each study, 4Department of Pediatric Oncology, Radboud University Medical a common precursor as the source of these histiocytic neoplasms Center and Radboud Institute for Molecular Life Sciences, Nijmegen, cannot be ruled out. Our comprehensive genetic analysis of all The Netherlands three tumors in one patient enabled us to retrospectively map the E-mail: [email protected] order of events leading to the presentation of these tumors. We 5These authors contributed equally to this work. have shown that the tumors share TR gene rearrangements, as well as several genomic abnormalities, signifying clonal relationship between the tumors. The absence of T-ALL specific REFERENCES lesions in the non-Langerhans cell histiocytosis and the histiocytic 1 Pileri SA, Grogan TM, Harris NL, Banks P, Campo E, Chan JK et al. Tumours of sarcoma indicates that these tumors did not develop from the histiocytes and accessory dendritic cells: an immunohistochemical approach to predominant T-ALL clone through transdifferentiation or de- classification from the International Lymphoma Study Group based on 61 cases. differentiation. 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A map for lineage restriction of progenitors during hematopoiesis: the essence of the myeloid-based model. We acknowledge Prof Dr Jacques van Dongen and Prof Dr Robert Arceci for Immunol Rev 2010; 238:23–36. their expert advice on the treatment of this patient. We thank Dr Charles Mullighan 11 Slamova L, Starkova J, Fronkova E, Zaliova M, Reznickova L, van Delft FW et al. for helpful discussions and Ingrid Vogelaar, Christian Gilissen and Eugène Verwiel CD2-positive B-cell precursor acute lymphoblastic leukemia with an early switch for technical assistance. We thank the members of the Genomic Disorders Group to the monocytic lineage. Leukemia 2014; 28:609–620. Nijmegen and the Radboud Genomics Technology Center for their technical 12 Cobaleda C, Jochum W, Busslinger M. Conversion of mature B cells into T cells by support. EW is a KWF fellow from the Dutch Cancer Society (KUN2012-5366), dedifferentiation to uncommitted progenitors. Nature 2007; 449: 473–477. 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Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)