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ic, and gene expression profiles resembling those of Burkitt lymphoma (BL), but lacks MYC rearrangements according to standard methods of detection, such as fluorescence in situ hybridization (FISH). Alternatively, these tumors carry an 11q-arm aberration characterized by proximal gains and telomeric losses.4 In comparison with BL, BLL-11q seems to have more complex karyotypes, a certain degree of cytological pleomorphism, sporadically a follicular pattern and a high incidence of nodal presentation.4,5 Very similar cases have also been reported in the post-transplant set- ting,6 although its incidence in other immunocompromised conditions, such as human immunodeficiency infection, is still unclear.7,8
BLL-11q has been incorporated in the revised World Health Organization (WHO) classification as a provisional category1 because its precise taxonomy as a particular vari- ant of BL, diffuse large B-cell lymphoma (DLBCL) or a dis- tinct form of high-grade B-cell lymphoma (HGBCL) is still controversial.1,4-6,9-11 The clarification of the biological nature of this uncommon subtype of lymphoma is clini- cally relevant because of increasing interest in defining the most appropriate management strategies for specific sub- types of lymphomas in pediatric and young adult patients.12 Recent DNA copy number alteration and next- generation sequencing studies have provided a compre- hensive catalog of genomic aberrations in BL and DLBCL which clearly distinguish these entities.13-17 In this study we performed an integrated analysis of genomic and mutational alterations with a complete annotation of clin- ical and pathological features of BLL-11q with the goal of obtaining insights to refine the understanding of the pathogenesis of these tumors and improve their diagnosis.
Methods
Sample selection and DNA/RNA extraction
To identify BLL-11q cases we initially reevaluated the presence of MYC translocations in 95 cases diagnosed as BL, atypical BL or HGBCL, not otherwise specified (NOS), in our Hematopathology Unit between 2000-2016. Three consultation cases from centers belonging to the Sociedad Española de Hematología y Oncología Pediátricas (SEHOP) were also analyzed. Cases were reviewed by three pathologists (BG-F, EC, ESJ). DNA and RNA were extracted using standard protocols (Qiagen, Hilden, Germany). This study was approved by the Institutional Review Board of the Hospital Clinic of Barcelona. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki.
Immunohistochemistry and fluorescence in situ hybridization
Immunohistochemical analysis using a panel of antibod- ies detecting common B- and T-cell markers as well as LMO2 and MYC was performed and interpreted as previ- ously reported (Online Supplementary Table S1).18,19
MYC breaks and MYC/IGH fusions were analyzed by FISH using the XL MYC BA Probe (Metasystems, Altlussheim, Germany) and LSI IGH/MYC/CEP 8 Tri- Color Dual Fusion Probe Kit (Vysis-Abbott, Abbott Park, IL, USA) respectively. The 11q alteration was studied with a custom FISH probe using BAC clones (Invitrogen Inc.) for proximal gains (RP11-414G21-spectrum green) and ter- minal losses (RP11-629A20-spectrum red) combined with CEP11-spectrum aqua (Vysis-Abbott Inc.). The FISH con- stellation in a normal case is characterized by two signals
per probe, while the pattern corresponding to the 11q gain/loss or gain/amplification/loss aberration would be two blue, three up to five green signals and one red signal. The probe was tested in an independent series of eight non-Hodgkin B-cell lymphomas and four MYC-negative HGBCL with lack of the 11q alteration by array analysis and all showed the normal pattern described above.
Copy number analysis
DNA was hybridized on Oncoscan FFPE or SNP array platform (ThermoFisher Scientific, Waltham, MA, USA) and analyzed as described previously (Online Supplementary Methods).2 Published copy number data on MYC-positive BL20 and DLBCL13 were rean- alyzed for comparison.
Sequencing approaches
The mutational status of 96 B-cell lymphoma-related genes (Online Supplementary Table S2) was examined by target next-gen- eration sequencing in ten BLL-11q cases and four MYC-negative 11q-negative cases using a NGS SureSelect XT Target Enrichment System Capture strategy (Agilent Technologies, Santa Clara, CA, USA) before sequencing in a MiSeq instrument (Illumina, San Diego, CA, USA) (Online Supplementary Methods). Additionally, analysis of hotspots of mutation in ID3, TCF3 and CCND3 genes, ETS1 exon 1 (transcript NM_005238) and verification of variants in specific cases was performed by Sanger sequencing using primers described in Online Supplementary Table S3.
Gene expression analysis
Cell of origin was determined by Lymph2Cx assay (Nanostring, Seattle, WA, USA) as previously published.21 Gene expression lev- els of MYC and ETS1 were investigated by real-time quantitative polymerase chain reaction (Online Supplementary Methods) using Taqman assays described in Online Supplementary Table S4.
Results
Identification of cases with Burkitt-like lymphoma with 11q aberration
To identify BLL-11q cases we reevaluated the presence of MYC translocations in 95 cases diagnosed as having BL, atypical BL or HGBCL, NOS. We confirmed the presence of MYC rearrangements in 78 cases (82.1%), of which 67 (70.5%) were classified as BL. Since the 11q aberration has been found mainly in children and young adults (<40 years old),4 we analyzed the 60 patients under 40 years and the 35 older patients separately (Online Supplementary Figure S1).
In the younger cohort (n=60), the 46 (76.7%) cases with MYC translocations were classified as having BL. To find BLL-11q cases, we initially used the Oncoscan platform in the remaining 14 MYC-negative patients and detected the presence of the 11q gain/loss alteration in eight of them. Additionally, we found a copy number pattern consistent with the presence of 11q alteration in three recent consul- tation cases from SEHOP (Online Supplementary Figures S1 and S2). Then, among those BLL-11q cases we were able
haematologica | 2019; 104(9)
Statistical methods
Burkitt-like lymphoma with 11q aberration
The χ2 method was used for categorical variables and Student t- tests for continuous variables. Non-parametric tests were applied when necessary. The P-values for multiple comparisons were adjusted using the Benjamini–Hochberg correction. Survival curves were estimated with the Kaplan-Meier method. Statistical analyses were carried out with SPSS v22 and R software v3.1.3.
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