Patient-Derived Xenograft and Cell Line Models of Human Primary Cutaneous Diffuse Large B-Cell Lymphoma-Leg Type.

Primary cutaneous diffuse large B-cell lymphoma, leg-type (PCDLBCL-LT) is a rare and aggressive malignancy, mostly occurring on the leg of elderly women (Willemze et al., 2019Willemze R. Cerroni L. Kempf W. Berti E. Facchetti F. Swerdlow S.H. et al.The 2018 update of the WHO-EORTC classification for primary cutaneous lymphomas [published correction appears in Blood 2019;134:1112].Blood. 2019; 133: 1703-1714Crossref PubMed Scopus (663) Google Scholar). PCDLBCL-LT harbors an activated B-cell‒like diffuse large B-cell lymphoma (ABC-DLBCL) signature and is enriched for mutations involving the toll-like receptor, B-cell receptor, and NF-κB pathways (Pham-Ledard et al., 2012Pham-Ledard A. Cappellen D. Martinez F. Vergier B. Beylot-Barry M. Merlio J.P. MYD88 somatic mutation is a genetic feature of primary cutaneous diffuse large B-cell lymphoma, leg type.J Invest Dermatol. 2012; 132: 2118-2120Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar; Zhou et al., 2018Zhou X.A. Louissaint Jr., A. Wenzel A. Yang J. Martinez-Escala M.E. Moy A.P. et al.Genomic analyses identify recurrent alterations in immune evasion genes in diffuse large B-cell lymphoma, leg type.J Invest Dermatol. 2018; 138: 2365-2376Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The high frequency of MYD88L265P mutation in combination with those of CD79B and PIM1 relates these lymphomas to the MCD/C5 subset of diffuse large B-cell lymphomas, which includes ABC-DLBCL occurring in extranodal sites (Danilov et al., 2022Danilov A.V. Magagnoli M. Matasar M.J. Translating the biology of diffuse large B-cell lymphoma into treatment.Oncologist. 2022; 27: 57-66Crossref PubMed Scopus (4) Google Scholar). The combination of rituximab and age-adapted polychemotherapy has improved the prognosis of patients, but refractory disease or relapse occurs in about half of patients. The lack of an experimental model impaired functional studies to decipher PCDLBCL-LT biology and therapeutic resistance mechanisms. In this study, we report the successful expansion of cutaneous B lymphoma cells, thanks to a patient-derived xenograft (PDX), followed by the establishment of a stable PCDLBCL-LT cell line. An explant of a skin biopsy from a leg tumor in an untreated woman aged 85 years with PCDLBCL-LT was subcutaneously transplanted in female immunodeficient NSG (NOD.Cg-Prkdc[scid]Il2rg[tm1Wjll]/SzJ) mouse. Previously, the patient provided written informed consent in accordance with the Declaration of Helsinki and national ethics rules, and the ethical research committee of Bordeaux approved the manipulation of PCDLBCL-LT samples (CER-BDX-2022-18). Engraftment, as a nodule, appeared on day 42, and then it grew exponentially until the mouse killed on day 73 showing a brownish and hemorrhagic subcutaneous tumor without ulceration (Figure 1a). After tumor dissociation, flow cytometry analysis revealed that almost all isolated cells (>98%) expressed CD19, CD20, and CD45, confirming their human B-cell origin (Supplementary Figure S1a). An autopsy revealed splenomegaly, and extracutaneous spreading was confirmed using anti‒HLA-ABC staining in the skin, femur, spleen, kidney, and liver (Figure 1b), as reported at advanced stages of the disease in some patients (Zinzani et al., 2006Zinzani P.L. Quaglino P. Pimpinelli N. Berti E. Baliva G. Rupoli S. et al.Prognostic factors in primary cutaneous B-cell lymphoma: the Italian study group for cutaneous lymphomas.J Clin Oncol. 2006; 24: 1376-1382Crossref PubMed Scopus (186) Google Scholar). To establish PCDLBCL-LT cell line, fresh cells isolated from the xenograft were grown for 45 days in a culture medium containing a cocktail of cytokines (CD40L, IL-2, IL-4, and IL-10), which were thereafter gradually removed (Supplementary Materials and Methods). Then, cells were routinely expanded in RPMI supplemented with fetal bovine serum and human AB serum. These so-called ARSI cells grew in suspension as more or less big clumps with a mean doubling time of about 24 hours (Supplementary Figure S1b and 1c). A continuous expansion for >1 year was achieved with >200 proliferation doublings, suggesting ARSI cell line immortalization (Sugimoto et al., 2004Sugimoto M. Tahara H. Ide T. Furuichi Y. Steps involved in immortalization and tumorigenesis in human B-lymphoblastoid cell lines transformed by Epstein-Barr virus.Cancer Res. 2004; 64: 3361-3364Crossref PubMed Scopus (122) Google Scholar). Clonality of the Ig genes of the PDX and ARSI cell line, assessed by the BIOMED-2 protocol, showed the same heavy and light chain Ig genes monoclonal rearrangements as in the patient’s tumor cells (Supplementary Figure S1d). Immunophenotypes of PDX and ARSI cell line were compared with those of the original tumor (Figure 1c). Patient tumor cells were positive for CD20, BCL2, MUM1, IgM, and BCL6low and negative for CD10, CD30, and IgG, with MYC and Ki-67 expressions in about 60 and 70% of cells, respectively, consistent with an activated B-cell phenotype. PDX cells and ARSI cell line displayed a phenotype similar to that of the initial biopsy, which was stable with time in culture. Next, we analyzed the genetic status of the patient tumor and compared it with that of the ARSI cell line. FISH showed the absence of MYC rearrangement both in patients and in cell line. SNP array and multicolor FISH showed a poorly rearranged genomic profile of the patient tumor cells. This profile remained stable along culture time except for the number of chromosomes X (disomy or monosomy) and 18 (trisomy or tetrasomy) and for copy-neutral loss of heterozygosity at chromosome 7 (Figure 2a‒c and Supplementary Table S1). Next-generation sequencing with a dedicated panel (Mareschal et al., 2017Mareschal S. Pham-Ledard A. Viailly P.J. Dubois S. Bertrand P. Maingonnat C. et al.Identification of somatic mutations in primary cutaneous diffuse large B-cell lymphoma, leg type by massive parallel sequencing.J Invest Dermatol. 2017; 137: 1984-1994Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) revealed mutations of MYD88, CD79B, and PIM1 genes (Supplementary Table S2) in the patient tumor, corresponding to a prototypic PCDLBCL-LT genetic pattern. These mutations were retained in the ARSI cell line, with some changes for IRF4 and PIM1 mutations. The original IRF4H56T mutation (allelic frequency at 20% in the patient’s tumor) was lost in the PDX and in the ARSI cell line, whereas IRF4S104R and IRF4G60K mutations appeared at 7 and 13 months of cell culture, respectively. Of note, the second IRF4 allele was found deleted in the patient’s tumor and ARSI cell line. For PIM1, the original tumor exhibited five mutations recovered in the PDX and ARSI cell line, and an additional mutation (PIM1D226K) appeared in the cell line (Supplementary Table S2). Interestingly, most IRF4 and some PIM1 mutations occurred at DGYW/WRCH sites, which are preferential targets of the activation-induced cytidine deaminase enzyme (Rogozin and Pavlov, 2006Rogozin I.B. Pavlov Y.I. The cytidine deaminase AID exhibits similar functional properties in yeast and mammals.Mol Immunol. 2006; 43: 1481-1484Crossref PubMed Scopus (15) Google Scholar). Our sequential data confirm ongoing aberrant somatic hypermutation of IRF4 and PIM1 genes, which are targets of a sustained activation-induced cytidine deaminase activity previously identified in primary cutaneous large B-cell lymphoma, leg type (Dijkman et al., 2006Dijkman R. Tensen C.P. Buettner M. Niedobitek G. Willemze R. Vermeer M.H. Primary cutaneous follicle center lymphoma and primary cutaneous large B-cell lymphoma, leg type, are both targeted by aberrant somatic hypermutation but demonstrate differential expression of AID.Blood. 2006; 107: 4926-4929Crossref PubMed Scopus (43) Google Scholar). Because IRF4 and PIM1 are respectively involved in the activation of the B-cell receptor and NF-κB signaling pathways (Maffei et al., 2021Maffei R. Fiorcari S. Benatti S. Atene C.G. Martinelli S. Zucchini P. et al.IRF4 modulates the response to BCR activation in chronic lymphocytic leukemia regulating IKAROS and SYK.Leukemia. 2021; 35: 1330-1343Crossref PubMed Scopus (11) Google Scholar; Nihira et al., 2010Nihira K. Ando Y. Yamaguchi T. Kagami Y. Miki Y. Yoshida K. Pim-1 controls NF-κappaB signalling by stabilizing RelA/p65.Cell Death Differ. 2010; 17: 689-698Crossref PubMed Scopus (94) Google Scholar), such mutational changes may contribute to the selection of a subclone with an advantage for cell immortalization or proliferation. Our lymphopanel analysis cannot exclude that aberrant somatic hypermutation also targeted other relevant oncogenes. The gene expression profile of the ARSI cell line was evaluated by RNA sequencing along with those of the SU-DHL-4 and OCI-Ly3 cell lines used as prototypic germinal center B-cell‒like diffuse large B-cell lymphomas and ABC-DLBCL, respectively. As expected, unsupervised clustering analysis revealed that the ARSI cell line was classified into ABC-DLBCL together with the OCI-Ly3 cell line profile (Figure 2d). To identify the molecular pathways activated in the ARSI cell line, we compared its gene expression profile signature with that of healthy PBMC controls. Gene set enrichment analysis showed an enrichment of genes related to cell proliferation and NF-κB in the ARSI cell line (Figure 2e), in agreement with their high proliferation rate and the PCDLBCL-LT signature. These data provide evidence that the PDX cells and ARSI cell line largely capture and retain the phenotypic and genomic features of the most common PCDLBCL-LT subtype. The rate of progression or relapse of patients after rituximab and age-adapted polychemotherapy underscore the need of selecting and evaluating new therapeutic strategies. In patients with PCDLBCL-LT, this has been hampered by the advanced age of patients and the rarity of this aggressive disease, making clinical trials difficult. In such instances, the development of preclinical models may offer an opportunity for drug screening. Ibrutinib, a Bruton tyrosine kinase inhibitor, was found effective in ABC-DLBCL cases with combined MYD88 and CD79A/B mutations (Wilson et al., 2015Wilson W.H. Young R.M. Schmitz R. Yang Y. Pittaluga S. Wright G. et al.Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma.Nat Med. 2015; 21: 922-926Crossref PubMed Scopus (823) Google Scholar). We therefore tested the cytotoxic effect of ibrutinib (range = 0.15‒50.103 nM) on the ARSI cell line after 72 hours and compared it with the effect on the ABC-DLBCL nodal OCI-Ly3 cell line carrying the MYD88L265P mutation. We observed that ARSI cells were more sensitive to ibrutinib (half-maximal inhibitory concentration = 2.6 nM) than OCI-Ly3 cells, for which the half-maximal inhibitory concentration could not be determined under our conditions (Figure 2f). Although PIM1 mutations have been proposed to contribute to ibrutinib resistance (Kuo et al., 2016Kuo H.P. Ezell S.A. Hsieh S. Schweighofer K.J. Cheung L.W. Wu S. et al.The role of PIM1 in the ibrutinib-resistant ABC subtype of diffuse large B-cell lymphoma.Am J Cancer Res. 2016; 6: 2489-2501PubMed Google Scholar), both cell lines expressed PIM1 mutations. More interestingly, CARD11, a scaffold protein that interacts with BCL10 and MALT1 downstream to Bruton tyrosine kinase and spleen tyrosine kinase to activate NF-κB signaling, is mutated in the OCI-Ly3 (CARD11L251P) but not in the ARSI cell line. This mutation occurring in the coiled‒coil domain of CARD11 has been proposed as a mechanism of resistance to B-cell receptor inhibition in diffuse large B-cell lymphomas (Fox et al., 2018Fox L.C. Yannakou C.K. Ryland G. Lade S. Dickinson M. Campbell B.A. et al.Molecular mechanisms of disease progression in primary cutaneous diffuse large B-cell lymphoma, leg type during ibrutinib therapy.Int J Mol Sci. 2018; 19: 1758Crossref PubMed Scopus (17) Google Scholar) and could explain the less sensitivity of OCI-Ly3 cells to ibrutinib than ARSI cells. Altogether, these data indicate that the genetic characteristics of different preclinical models may be account for drug resistance and may contribute to screening for drug efficacy. By overcoming the difficulties of obtaining live lymphoma B cells, in this study, we succeeded to establish the PDX and cell line models recapitulating the most common PCDLBCL-LT subtype. This original cell line may contribute to deciphering the physiopathology of PCDLBCL-LT, facilitating target identification and preclinical drug screening. No dataset was generated. Martina Prochazkova-Carlotti: http://orcid.org/0000-0002-7719-7679 Audrey Gros: http://orcid.org/0000-0002-8958-3745 Elodie Richard: http://orcid.org/0000-0003-3778-1210 Floriane Cherrier: http://orcid.org/0000-0002-4181-0283 Elodie Laharanne: http://orcid.org/0000-0002-0337-3537 Yamina Idrissi: http://orcid.org/0000-0002-8654-5380 Camille Baron: http://orcid.org/0000-0002-0875-7126 Sandrine Poglio: http://orcid.org/0000-0001-8783-0709 Océane Ducharme: http://orcid.org/0000-0001-8801-2900 Sarah Menguy: http://orcid.org/0000-0002-9214-8592 Anne Pham-Ledard: http://orcid.org/0000-0003-2960-6084 Marie Beylot-Barry: http://orcid.org/0000-0001-6150-1229 Jean-Philippe Merlio: http://orcid.org/0000-0002-0305-0850 Laurence Bresson-Bepoldin: http://orcid.org/0000-0002-8829-9945 The authors state no conflict of interest This work was supported by INSERM, University of Bordeaux, Ligue Régionale contre le cancer (comité de Gironde), Groupe Interrégional de Recherche Clinique et Innovation- Cancéropôle Grand Sud-Ouest- Centre Hospitalier Universitaire de Bordeaux 2018/78 to AG (grant PROTEOM). The authors thank the donors for agreeing to have their cells used for research. We acknowledge Benoît Rousseau and Julien Izotte from the A2 Animal facility at Bordeaux university (Bordeaux, France) and Atika Zouine and Vincent Pitard for technical assistance at the flow cytometry facility, CNRS UMS 3427 INSEM US 005, UB’Facsility at Bordeaux University. Conceptualization: MPC, AG, JPM, LBB; Funding Acquisition: AG, LBB; Investigation: MPC, EL, AG, CB, YI, SM, LBB; Methodology: ER, FC, SP, LBB; Resources: OD, APL, MBB; Supervision: LBB; Writing - Original draft preparation: MPC, AG, JPM, LBB; Writing – Review and Editing: MPC, AG, JPM, LBB Antibodies used for immunolabeling are described in Supplementary Table S3. The human CD40L multimer kit was from Miltenyi Biotec (Paris, France). IL-2, IL-4, and IL-10 were supplied by PeproTech (Neuilly-sur-Seine, France). DAPI was from Fischer Scientific (Bordeaux, France). Ibrutinib was provided by Bio-Techne (Rennes, France). Biopsy from the patient with primary cutaneous diffuse large B-cell lymphoma-leg type was collected in RPMI (Fischer Scientific) supplemented with 10% fetal bovine serum (Dominique Dutscher, Bernolsheim, France) and 1% penicillin‒streptomycin (Fischer Scientific) at the Oncodermatology Department of Université de Bordeaux (Bordeaux, France). The patient provided written informed consent in accordance with the Declaration of Helsinki and national ethics rules. Our institutional review board approved the manipulation of primary cutaneous diffuse large B-cell lymphoma-leg type samples (CER-BDX-2022-18). In vivo experiments were performed according to ethical criteria approved by the French Ministry of Agriculture (agreement APAFIS#29572-2021020517035043, version 4). A 2-mm3 piece of the fresh biopsy was subcutaneously grafted in the inguinal fat pad of a female immunodeficient NSG (NOD.Cg-Prkdc[scid]Il2rg[tm1Wjll]/SzJ) mouse from the animal facility aged 8 weeks. Tumor volume was determined weekly by measuring the length and width of the tumor with a caliper and calculating the volume using the following formula: V = lw2/2, where l is the length, and w is the width. When the tumor volume reached 2,000 mm3, the mouse was killed. After dissection, one half of the tumors were formalin fixed, and the other half was dissociated for cell culture. Different organs were removed and formalin fixed for immunohistochemistry experiments. After dissection, the tumor was mechanically dissociated. The percentage of CD19pos and CD20pos cells was determined by flow cytometry. In the beginning, cells were cultured at a high density (1‒2 million/ml) in RPMI 1640 containing 10% human serum AB (Institut de Biotechnologies Jacques Boy, Reims, France), 10% fetal bovine serum (Dominique Dutscher), 1 μg/ml CD40L, 20 ng/ml IL-2, 10 ng/ml IL-4, 50 ng/ml IL-10, and 1% penicillin‒streptomycin for 45 days. Cytokines were then gradually removed from the culture medium, and the cells were routinely diluted at 0.5 million/ml with fresh culture medium three times a week for 2 years. The percentage of B cells in the tumor or in the culture was estimated on the basis of CD19posCD20pos CD45pos cell surface expression by flow cytometry using Canto II cytometer (BD Biosciences, Le Pont-de-Claix, France) and DIVA (Data-Interpolating Variational Analysis) software. Cell doubling time was determined using the CellTrace violet Cell proliferation Kit (Fisher Scientific) according to the manufacturer’s instructions. Briefly, 106 cells were stained with carboxyfluorescein succinimidyl ester-violet (5 μM) for 20 minutes at 37 °C, and after rinsing in the culture medium, cells were seeded in six-well plates. Cell proliferation was followed for 6 days by measuring cell fluorescence (λex = 405 nm, λem = 450 nm) by flow cytometry. Analyses were performed using FlowJo (version 10.8) software. Immunostainings were performed on 3-μm thick formalin-fixed paraffin-embedded mice organ sections. Cells in culture were fixed with 4% formalin, pre-embedded in 2% agarose, and further embedded in a paraffin block. Protein expression was revealed using primary antibodies described in Supplementary Table S3 and Impress IgG polymer kit peroxidase (Vector Laboratories, Eurobio Scientific, Les Ulis, France) with 3’3-diaminobenzidine as a chromogen. Sections were then counterstained with hematoxylin (Vector Laboratories). Hematoxylin-Eosin-Saffron‒stained sections were analyzed in parallel. Images were obtained using a NIKON Eclipse C1 microscope coupled with a camera NIKON DS-F12 and NIS BR imaging software, version 4.0 (Nikon, Champigny Sur Marne, France). To determine the half-maximal inhibitory concentration of ibrutinib, 20,000 ARSI or OCI-Ly3 (DMSZ, Braunschweig, Germany) cells per well were seeded in 96-well plates, with various concentrations of ibrutinib for 72 hours. Ibrutinib cytotoxicity was determined using the luminescent cell viability assay CellTiter-Glo Cell viability assay (Promega, Charbonnières-les-Bains, France). Luminescence levels were quantified using the FlexStation 3 (Molecular Devices, San Jose, CA). Results were analyzed using GraphPad Prism software, version 9 (Graphpad Software, San Diego, CA). Metaphase chromosomes preparations were done using the standard protocol: incubation with 0.1 μg/ml Colcemid (Gibco, Waltham, MA) for 4 hours, hypotonic shock with 0.075M potassium chloride for 20 minutes at 37 °C, and fixation with cold Carnoy’s fixative (3:1 (v:v) methanol to glacial acetic acid). At least three fixative changes were applied, and these cytogenetic pellets were stored in fixative at ‒20 °C until use. A multicolor FISH experiment was conducted according to the manufacturer’s instructions (MetaSystems, Compiègne, France) using 24XCyte kit on metaphase spreads. Briefly, slides with metaphase spreads were prepared 1 week before hybridization, and every slide was checked for metaphase concentration. Slides were rehydrated by decreasing ethanol series (100, 70, 50, 30%) and 0.1× saline-sodium citrate (SSC), then treated with 2× SSC at 70 °C for 30 minutes, and let cool down for the next 20 minutes. Finally, slides were transferred to 0.1× SSC, denatured in 0.07 M sodium hydroxide, passed in 0.1× SSC and 2× SSC at 4 °C, and dehydrated by increasing ethanol series. Each step was performed at room temperature for 1 minute, if not specified. Slides were let air dried before applying probe. Probe mixture was denatured by incubating at 75 °C for 5 minutes, then put on ice briefly, and incubated at 37 °C for 30 minutes. Prehybridized probe was applied onto the denatured chromosome preparation and overlaid with a coverslip, sealed with rubber cement, and let hybridize for 3 days at 37 °C in a humidified chamber (ThermoBrite System, Abbott Molecular, Des Plaines, IL). Posthybridization washes were realized in 0.4× SSC at 72 °C for 2 minutes, then in 2× SSC/0.05% Tween-20 for 3 minutes, and in PBS for the next 2 minutes at room temperature. Finally, slides were left air dried and mounted with Vectashield (Vector Laboratories) containing DAPI to counterstain metaphase spreads. Metaphase finder and image acquisition were performed using the fluorescence-based microscopic scanning system Metafer (MetaSystems, Amplitech SAS, Compiegne, France). This scanning system is based on a fully motorized Axio Imager Z2 microscope (Zeiss, Rueil Malmaison, France) equipped with a motorized eight-slide scanning stage (Märzhäuser Wetzlar, Wetzlar, Germany) and high-resolution monochrome camera CoolCube 1m (MetaSystems). The slide scanning and automated multicolor FISH acquisition are performed using 40× objective and appropriate individual excitation and emission filter sets. Metaphase images were then treated and analyzed manually with Isis software (MetaSystems) to establish karyotypes. We have acquired and collected a maximum of abnormal karyotypes (between 10 and 30 karyotypes) per sample, especially if two or more cytogenetic subclones were present. We define a subclone as an abnormal karyotype present at least in three metaphases. DNA and RNA were extracted using the QiAmp DNA mini kit (Qiagen, Hilden, Germany) and the Direct-Zol RNA miniprep kit (Zymo Research, Ozyme, Saint-Cyr l’Ecole, France), respectively, according to manufacturers’ instructions. For evaluation of B-cell clonality, the human Ig rearrangements were studied using the BIOMED-2 protocol as described previously (van Dongen et al., 2003van Dongen J.J.M. Langerak A.W. Brüggemann M. Evans P.A.S. Hummel M. Lavender F.L. et al.Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2567) Google Scholar). DNAs extracted from the initial tumor of the patient and ARSI cell line were analyzed using BeadChip Infinium CytoSNP-8x850K, version 1.2 (Illumina, Évry, France), which contains nearly 850,000 SNPs with a 15× redundancy, giving an overall effective resolution of approximately 18 kb. Briefly, DNAs were amplified, fragmented, precipitated, and hybridized on the microarray before sample extension and staining, according to the manufacturer’s instructions. Then, the microarray was scanned on the Next Seq 550Dx (Illumina), and the analysis was performed using BlueFuse Multi Software. Analyses were retained if the call rate was >0.955. For aberration analysis, we used the log R ratio parameter (on the basis of intensity information) in combination with the B-allele frequency metric (on the basis of the genotype information) providing powerful data and allowing us to detect copy-neutral loss of heterozygosity. The validated aberrations are presented according to International System for Human Cytogenomic Nomenclature 2020 standard. A lymphopanel was designed to identify alterations within 36 genes important for lymphomagenesis on the basis of literature data and on whole-exome sequencing of relapsed/refractory diffuse large B-cell lymphoma sequencing and previously used to characterize a primary cutaneous large B-cell lymphoma, leg type cohort (Ducharme et al., 2019Ducharme O. Beylot-Barry M. Pham-Ledard A. Bohers E. Viailly P.J. Bandres T. et al.Mutations of the B-cell receptor pathway confer chemoresistance in primary cutaneous diffuse large B-cell lymphoma leg type.J Invest Dermatol. 2019; 139: 2334-2342.e8Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar; Mareschal et al., 2017Mareschal S. Pham-Ledard A. Viailly P.J. Dubois S. Bertrand P. Maingonnat C. et al.Identification of somatic mutations in primary cutaneous diffuse large B-cell lymphoma, leg type by massive parallel sequencing.J Invest Dermatol. 2017; 137: 1984-1994Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The lymphopanel was designed with Ion Ampliseq technology (Fisher Scientific) and covered 75.08 kb. These genes include ARID1A, B2M, BCL2, BRAF, BTK, CARD11, CCND3, CD58, CD79A, CD79B, CDKN2A, CDKN2B, CIITA, CREBBP, CXCR4, EP300, EZH2, FOXO1, GNA13, ID3, IRF4, MEF2B, MYC, MYD88, NOTCH1, NOTCH2, PIM1, PLCG2, PRDM1, SOCS1, STAT6, TCF3, TNFAIP3, TNFRSF14, TP53, and XPO1. Amplified libraries were sequenced with the IonS5 on 530 Chips, and data analysis was performed with Torrent Suite software, version 5.10 (Fisher Scientific). Reads were mapped to the human hg19 reference genome. The Variant Caller detected point mutations with a variant allele frequency ≥2% for single nucleotide variation and ≥5% for short insertion/deletion. Variant Call Format files were annotated by ANNOVAR (Wang et al., 2010Wang K. Li M. Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (8550) Google Scholar). BAM sequences were also checked if necessary using Alamut Visual Software (SOPHiA Genetics, Saint-Sulpice, Switzerland). Library preparation, capture, sequencing, and alignment have been done by IntegraGen SA (Evry, France). Libraries were prepared with NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Evry, France) for Illumina protocol according to supplier instructions. mRNA molecules using poly-T oligo attached magnetic beads from 100 ng total RNA (with the Magnetic mRNA Isolation Kit from New England Biolabs) were purified and fragmented using divalent cations under elevated temperature. Cases were sequenced in pooled libraries on the NovaSeq6000 platform (paired-end 100 bp reads, Illumina). The quality of reads was assessed for each sample using FastQC (version 0.11.4). RNA-sequencing reads were aligned to GRCh38 using the STAR aligner (version 2.7.10a) (Dobin et al., 2013Dobin A. Davis C.A. Schlesinger F. Drenkow J. Zaleski C. Jha S. et al.STAR: ultrafast universal RNA-seq aligner.Bioinformatics. 2013; 29: 15-21Crossref PubMed Scopus (21979) Google Scholar). The number of reads associated with each gene in the Gencode annotation, version 31, was obtained. Data normalization was performed with the Bioconductor DESeq2 package (Love et al., 2014Love M.I. Huber W. Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 2014; 15: 550Crossref PubMed Scopus (38077) Google Scholar) to import raw counts for each sample into R statistical software, version 4.1.2, and extracted the count matrix. We used the Bioconductor edgeR package (version 3.36.0) for differential analysis of sequence read count data. The normalized expression matrix from the 1,000 most variant genes (based on SD) was used to classify the samples according to their gene expression patterns. We used FactoMineR to perform Hierarchical Clustering on Principle Components (with euclidean distance and ward’s method). Preranked analysis was performed using the gene set enrichment analysis software, version 4.1.0, to calculate the normalized enrichment score and false discovery rate (Subramanian et al., 2005Subramanian A. Tamayo P. Mootha V.K. Mukherjee S. Ebert B.L. Gillette M.A. et al.Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci USA. 2005; 102: 15545-15550Crossref PubMed Scopus (29202) Google Scholar). A normalized enrichment score corresponds to the enrichment score, which reflects the degree to which a gene set is overrepresented at the top or bottom of a ranked list of genes. The normalization is based on the gene-set enrichment scores for all dataset permutations. Genes were preranked according to t-test statistic value obtained, thanks to limma R package. Gene sets that obtained the highest normalized enrichment score with a normalized P < 0.005, a false discovery rate < 0.005, and a Family-wise error rate P < 0.045 were considered significantly enriched. We used the Kyoto Encyclopedia of Genes and Genomes and custom NFKB gene list (Compagno et al., 2009Compagno M. Lim W.K. Grunn A. Nandula S.V. Brahmachary M. Shen Q. et al.Mutations of multiple genes cause deregulation of NF-kappaB in diffuse large B-cell lymphoma.Nature. 2009; 459: 717-721Crossref PubMed Scopus (865) Google Scholar) and a B-cell receptor gene list.Supplementary Table S1mFISH and SNParray Analyses of Patient Tumor and Patient-Derived Cell Line According to Time in the CultureSampleSNP ArraymFISH KaryotypePatient tumorarr[GRCh37]2q24.32q34(164485597_210073419)x1,Not available3p21.31-p14.3(49441091_58232485)x1,6pter-p25.2(108666_2832331)x1,7q31.31-q36.3(118850537_159124481)x1, 9p21.3(21728683_21979204x1,21984661_22056499x0),9p21.3-p21.2(22077543_25717386)x1, 14q24.1(68370656_68692290)x0, 14q32.33(106082680_107078986)x1,18x3,19q12q13.43(30090883_59095126)x3,Xq28(148561332_155236747)x3ARSI 7 moarr[GRCh37]2q24.3q34(164436645_210077614)x1,47, XX, der(2)del(2q)(WCP2+), der(3)t(3;12)(WCP12+,WCP3+), der(6)add(6)(q2?6)(WCP6+,WCP19+),der(7)t(7;12)(WCP7+,WCP12+), der(12)t(3;12)(WCP3+,WCP12+),+18 [21]3p21.31p14.3(49389842_58228082)x1,6p25.3-p25.2(304135_2842830)x1, 7p21.3p14.3(11394751_35247446)x2 mos hmz,7q31.31q36.3(118701195_159126310)x1, 9p21.3(21728683_21944317x1,21944818_22062134x0),9p21.3-p21.2(21062134_25717537)x1,14q24.1(68370656_68711163)x0, 14q32.33(106082680_107083638)x1,18x3,19q12q13.43(29808196_59036479)x3,Xq28(148562599_155141985)x3ARSI 13 moarr[GRCh37]2q24.3-2q34(164965571_210118415)x1,46∼48,X[6], +X[13], der(2)del(2q) (WCP2+)[19],der(3)t(3;12) (WCP12+,WCP3+)[19], der(6)add(6)(q2?6)(WCP6+,WCP19+)[19],der(7)t(7;12)(WCP7+,WCP12+)[19], der(12)t(3;12)(WCP3+,WCP12+)[19],+18[19],+18[8][cp19]3p21.31-p14.3(49317338_58278244)x1,6pter-p25.2(108666_2876581)x1,7p21.3p14.3(63494_34283049)x2 mos hmz,7q31.31-q36.3(119270386_15926310)x1,9p21.3(21728683_21944317x1,21944818_22062134x0),9p21.3-p21.2(21062134_25745702)x1,14q24.1(68413835_68793554)x0, 14q32.33(106079698_107242489)x1,18x3,19q12qter(29262931_59097160)x3,Xq28(148525485_155236747)x3Abbreviation: mFISH, multicolor FISH. Open table in a new tab Supplementary Table S2List of the Somatic Mutations Detected by the LymphopanelGeneTranscriptMutationInitial Tumor, %D0, %7 mo, %11 mo, %CD79BNM_000626.3c.587A>G ; p.(Tyr196Cys)34465247IRF4NM_002460.3c.166C>T ; p.(His56Thr)1SNV targeted by aSHM located at DGYW/WRCH AID target sites at diagnosis.20///c.178C>A ; p.(Gln60Lys)1SNV targeted by aSHM located at DGYW/WRCH AID target sites at diagnosis.///53c.312C>A ; p.(Ser104Arg)1SNV targeted by aSHM located at DGYW/WRCH AID target sites at diagnosis.//100100MYD88NM_002468.4c.794T>C ; p.(Leu265Pro)36554950PIM1NM_001243186.1c.367G>C ; p.(Glu123Gln)43515250c.370C>T ; p.(Pro124Ser)31484850c.475C>T ; p.(His159Tyr)26334337c.497C>T ; p.(Ser166Phe)1SNV targeted by aSHM located at DGYW/WRCH AID target sites at diagnosis.55959696c.658C>G ; p.(Leu220Val)36529694c.676G>A ; p.(Glu226Lys)//100100Abbreviations: AID, activation-induced cytidine deaminase; aSHM, aberrant somatic hypermutation; D0, day 0; DLBCL, diffuse large B-cell lymphoma; SNV, single nucleotide variation; WES, whole-exome sequencing.This shows the 36 genes involved in lymphomagenesis on the basis of literature data and WES of relapsed/refractory DLBCL.1 SNV targeted by aSHM located at DGYW/WRCH AID target sites at diagnosis. Open table in a new tab Supplementary Table S3List of Antibodies Used for Flow Cytometry and ImmunohistochemistryAntibodySupplierClonepHDilutionAntibodies for flow cytometry Mouse anti-humanCD19-BV421BD Biosciences (Le pont de Claix, France)HIB19Not applicable1/50 Mouse anti-human CD20-PEBD Biosciences (Le pont de Claix, France)2H7Not applicable1/50 Mouse anti-human CD45-PECy5.5BD Biosciences (Le pont de Claix, France)J.33Not applicable1/50Antibodies for Immunohistochemistry Mouse monoclonal anti-HLA-ABCAbcam (Paris, France)EMR8-561/100 Mouse monoclonal anti-human CD20Agilent (Les Ullis, France)L.2691/300 Mouse monoclonal anti-human MUM1Agilent (Les Ullis, France)MUM1p91/100 Mouse monoclonal anti-human BCL2Agilent (Les Ullis, France)12461/200 Rabbit monoclonal anti-C-MYCAbcam (Paris, France)Y6991/50 Rabbit polyclonal Anti-IgMRoche (Rotkreuz, Switzerland)6Ready to use Rabbit polyclonal Anti-IgGRoche (Rotkreuz, Switzerland)6Ready to use Mouse monoclonal anti-Ki-67Agilent (Les Ullis, France)MIB-191/300 Mouse monoclonal anti-BCL6Agilent (Les Ullis, France)PG-B6p91/25 Mouse monoclonal anti-human CD10Thermo Fisher Scientific (Illkirch-Graffenstaden, France)56C691/20Abbreviations: BV, brilliant violet; Cy, cyanine; PE, phycoerythrin. Open table in a new tab Abbreviation: mFISH, multicolor FISH. Abbreviations: AID, activation-induced cytidine deaminase; aSHM, aberrant somatic hypermutation; D0, day 0; DLBCL, diffuse large B-cell lymphoma; SNV, single nucleotide variation; WES, whole-exome sequencing. This shows the 36 genes involved in lymphomagenesis on the basis of literature data and WES of relapsed/refractory DLBCL. Abbreviations: BV, brilliant violet; Cy, cyanine; PE, phycoerythrin.