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B-Lymphoblastic Lymphomas Evolving from Follicular Lymphomas Co-Express Surrogate Light Chains and Mutated Gamma Heavy Chains

2016; Elsevier BV; Volume: 186; Issue: 12 Linguagem: Inglês

10.1016/j.ajpath.2016.07.027

ISSN

1525-2191

Autores

Linda M. Slot, Robbert Hoogeboom, Laura A. Smit, Thera A.M. Wormhoudt, Bart J. Biemond, Monique E.C.M. Oud, Esther J.M. Schilder-Tol, André B. Mulder, Aldo Jongejan, Antoine H. C. van Kampen, Philip M. Kluin, Jeroen E. J. Guikema, Richard J. Bende, Carel J.M. van Noesel,

Tópico(s)

CAR-T cell therapy research

Resumo

Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT+CD20− precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization analysis showed that all four B-LBLs had acquired a MYC translocation on transformation. Comparative genomic hybridization analysis of one case demonstrated that in addition to 26 numerical aberrations that were shared between the FL and B-LBL, deletion of CDKN2A/B and 17q11, 14q32 amplification, and copy-neutral loss of heterozygosity of 9p were gained in the B-LBL cells. Whole-exome sequencing revealed mutations in FMN2, NEB, and SYNE1 and a nonsense mutation in KMT2D, all shared by the FL and B-LBL, and TNFRSF14, SMARCA2, CCND3 mutations uniquely present in the B-LBL. Remarkably, all four FL–B-LBL pairs expressed IgG. In two B-LBLs, evidence was obtained for ongoing rearrangement of IG light chain variable genes and expression of the surrogate light chain. IGHV mutation analysis showed that all FL–B-LBL pairs harbored identical or near-identical somatic mutations. From the somatic gene alterations found in the IG and non-IG genes, we conclude that the FLs and B-LBLs did not develop in parallel from early t(14;18)-positive IG-unmutated precursors, but that the B-LBLs developed from preexistent FL subclones that accumulated additional genetic damage. Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT+CD20− precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization analysis showed that all four B-LBLs had acquired a MYC translocation on transformation. Comparative genomic hybridization analysis of one case demonstrated that in addition to 26 numerical aberrations that were shared between the FL and B-LBL, deletion of CDKN2A/B and 17q11, 14q32 amplification, and copy-neutral loss of heterozygosity of 9p were gained in the B-LBL cells. Whole-exome sequencing revealed mutations in FMN2, NEB, and SYNE1 and a nonsense mutation in KMT2D, all shared by the FL and B-LBL, and TNFRSF14, SMARCA2, CCND3 mutations uniquely present in the B-LBL. Remarkably, all four FL–B-LBL pairs expressed IgG. In two B-LBLs, evidence was obtained for ongoing rearrangement of IG light chain variable genes and expression of the surrogate light chain. IGHV mutation analysis showed that all FL–B-LBL pairs harbored identical or near-identical somatic mutations. From the somatic gene alterations found in the IG and non-IG genes, we conclude that the FLs and B-LBLs did not develop in parallel from early t(14;18)-positive IG-unmutated precursors, but that the B-LBLs developed from preexistent FL subclones that accumulated additional genetic damage. Follicular lymphomas (FLs) are the prototypic germinal center (GC)-derived B-cell non-Hodgkin's lymphomas1Jaffe E. Harris R.S. Stein H. Vardiman J.W. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. IARC press, Washington2008Google Scholar and cytologically and histologically resemble normal GCs. In accordance, FLs share a gene expression profile with normal centroblasts and centrocytes2Bende R.J. Smit L.A. van Noesel C.J. Molecular pathways in follicular lymphoma.Leukemia. 2007; 21: 18-29Crossref PubMed Scopus (112) Google Scholar and express somatically hypermutated B-cell antigen receptors, which in approximately 25% of cases are of the non-IgM isotype.3Aarts W.M. Bende R.J. Steenbergen E.J. Kluin P.M. Ooms E.C. Pals S.T. van Noesel C.J. Variable heavy chain gene analysis of follicular lymphomas: correlation between heavy chain isotype expression and somatic mutation load.Blood. 2000; 95: 2922-2929Crossref PubMed Google Scholar, 4Roulland S. Faroudi M. Mamessier E. Sungalee S. Salles G. Nadel B. Early steps of follicular lymphoma pathogenesis.Adv Immunol. 2011; 111: 1-46Crossref PubMed Scopus (82) Google Scholar FLs have an indolent clinical behavior with a reported median survival of 8 to 10 years after diagnosis.1Jaffe E. Harris R.S. Stein H. Vardiman J.W. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. IARC press, Washington2008Google Scholar However, 20% to 60% of FLs transform into higher-grade malignancies, an event associated with an aggressive clinical course, poor therapy responses, and short survival.5Pasqualucci L. Khiabanian H. Fangazio M. Vasishtha M. Messina M. Holmes A.B. Ouillette P. Trifonov V. Rossi D. Tabbo F. Ponzoni M. Chadburn A. Murty V.V. Bhagat G. Gaidano G. Inghirami G. Malek S.N. Rabadan R. Dalla-Favera R. Genetics of follicular lymphoma transformation.Cell Rep. 2014; 6: 130-140Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar Usually, FLs transform into other GC-type lymphomas, most often diffuse large B-cell lymphomas (DLBCLs) or, less commonly, Burkitt-like lymphomas [referred to in the latest World Health Organization classification as B-cell lymphoma unclassifiable with features intermediate between DLBCL and Burkitt lymphoma (BL)].5Pasqualucci L. Khiabanian H. Fangazio M. Vasishtha M. Messina M. Holmes A.B. Ouillette P. Trifonov V. Rossi D. Tabbo F. Ponzoni M. Chadburn A. Murty V.V. Bhagat G. Gaidano G. Inghirami G. Malek S.N. Rabadan R. Dalla-Favera R. Genetics of follicular lymphoma transformation.Cell Rep. 2014; 6: 130-140Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 6Aukema S.M. Siebert R. Schuuring E. van Imhoff G.W. Kluin-Nelemans H.C. Boerma E.J. Kluin P.M. Double-hit B-cell lymphomas.Blood. 2011; 117: 2319-2331Crossref PubMed Scopus (550) Google Scholar Infrequently, these prototypic GC-like malignancies transform into precursor B-lymphoblastic lymphomas (B-LBL).7Ning Y. Foss A. Kimball A.S. Neill N. Matz T. Schultz R. Characterization of a case of follicular lymphoma transformed into B-lymphoblastic leukemia.Mol Cytogenet. 2013; 6: 34Crossref PubMed Scopus (9) Google Scholar, 8Young K.H. Xie Q. Zhou G. Eickhoff J.C. Sanger W.G. Aoun P. Chan W.C. Transformation of follicular lymphoma to precursor B-cell lymphoblastic lymphoma with c-myc gene rearrangement as a critical event.Am J Clin Pathol. 2008; 129: 157-166Crossref PubMed Scopus (53) Google Scholar, 9Kaplan A. Samad A. Dolan M.M. Cioc A.M. Holman C.J. Schmechel S.C. Pambuccian S.E. Follicular lymphoma transformed to "double-hit" B lymphoblastic lymphoma presenting in the peritoneal fluid.Diagn Cytopathol. 2013; 41: 986-990Crossref PubMed Scopus (15) Google Scholar, 10Sun X. Gordon L.I. Peterson L.C. Transformation of follicular lymphoma to acute lymphoblastic leukemia.Arch Pathol Lab Med. 2002; 126: 997-998Crossref PubMed Google Scholar, 11Mannouji K. Tasaka T. Akiyama T. Irei I. Sano F. Matsuhashi Y. Wada H. Tohyama K. Sugihara T. Sadahira Y. Transformation from follicular lymphoma to high-grade B-cell lymphoma/leukemia with additional t(2;8)(p12;q24), with inverse expressions of c-MYC and BCL-2, and light-chain switch.Pathol Int. 2009; 59: 261-264Crossref Scopus (8) Google Scholar, 12Geyer J.T. Subramaniyam S. Jiang Y. Elemento O. Ferry J.A. de Leval L. Nakashima M.O. Liu Y.C. Martin P. Mathew S. Orazi A. Tam W. Lymphoblastic transformation of follicular lymphoma: a clinicopathologic and molecular analysis of 7 patients.Hum Pathol. 2015; 46: 260-271Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 13Fiedler W. Weh H.J. Zeller W. Fonatsch C. Hillion J. Larsen C. Wormann B. Hossfeld D.K. Translocation (14; 18) and (8; 22) in three patients with acute leukemia/lymphoma following centrocytic/centroblastic non-Hodgkin's lymphoma.Ann Hematol. 1991; 63: 282-287Crossref PubMed Scopus (32) Google Scholar, 14Gauwerky C.E. Hoxie J. Nowell P.C. Croce C.M. Pre-B-cell leukemia with a t(8; 14) and a t(14; 18) translocation is preceded by follicular lymphoma.Oncogene. 1988; 2: 431-435PubMed Google Scholar, 15Kobrin C. Cha S.C. Qin H. Raffeld M. Fend F. Quintanilla-Martinez L. Grove S. Jaffe E.S. Kwak L.W. Molecular analysis of light-chain switch and acute lymphoblastic leukemia transformation in two follicular lymphomas: implications for lymphomagenesis.Leuk Lymphoma. 2006; 47: 1523-1534Crossref PubMed Scopus (26) Google Scholar Studies on sporadic cases described so far revealed that such B-LBLs carried BCL2 translocations, indicative for a clonal relationship with the preceding FLs. Karyotype analyses showed a variety of chromosomal alterations gained in the B-LBLs, among which MYC locus translocations were most frequently observed [in 18 of 19 (95%) of the described cases].7Ning Y. Foss A. Kimball A.S. Neill N. Matz T. Schultz R. Characterization of a case of follicular lymphoma transformed into B-lymphoblastic leukemia.Mol Cytogenet. 2013; 6: 34Crossref PubMed Scopus (9) Google Scholar, 8Young K.H. Xie Q. Zhou G. Eickhoff J.C. Sanger W.G. Aoun P. Chan W.C. Transformation of follicular lymphoma to precursor B-cell lymphoblastic lymphoma with c-myc gene rearrangement as a critical event.Am J Clin Pathol. 2008; 129: 157-166Crossref PubMed Scopus (53) Google Scholar, 9Kaplan A. Samad A. Dolan M.M. Cioc A.M. Holman C.J. Schmechel S.C. Pambuccian S.E. Follicular lymphoma transformed to "double-hit" B lymphoblastic lymphoma presenting in the peritoneal fluid.Diagn Cytopathol. 2013; 41: 986-990Crossref PubMed Scopus (15) Google Scholar, 10Sun X. Gordon L.I. Peterson L.C. Transformation of follicular lymphoma to acute lymphoblastic leukemia.Arch Pathol Lab Med. 2002; 126: 997-998Crossref PubMed Google Scholar, 11Mannouji K. Tasaka T. Akiyama T. Irei I. Sano F. Matsuhashi Y. Wada H. Tohyama K. Sugihara T. Sadahira Y. Transformation from follicular lymphoma to high-grade B-cell lymphoma/leukemia with additional t(2;8)(p12;q24), with inverse expressions of c-MYC and BCL-2, and light-chain switch.Pathol Int. 2009; 59: 261-264Crossref Scopus (8) Google Scholar, 12Geyer J.T. Subramaniyam S. Jiang Y. Elemento O. Ferry J.A. de Leval L. Nakashima M.O. Liu Y.C. Martin P. Mathew S. Orazi A. Tam W. Lymphoblastic transformation of follicular lymphoma: a clinicopathologic and molecular analysis of 7 patients.Hum Pathol. 2015; 46: 260-271Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 13Fiedler W. Weh H.J. Zeller W. Fonatsch C. Hillion J. Larsen C. Wormann B. Hossfeld D.K. Translocation (14; 18) and (8; 22) in three patients with acute leukemia/lymphoma following centrocytic/centroblastic non-Hodgkin's lymphoma.Ann Hematol. 1991; 63: 282-287Crossref PubMed Scopus (32) Google Scholar, 14Gauwerky C.E. Hoxie J. Nowell P.C. Croce C.M. Pre-B-cell leukemia with a t(8; 14) and a t(14; 18) translocation is preceded by follicular lymphoma.Oncogene. 1988; 2: 431-435PubMed Google Scholar, 15Kobrin C. Cha S.C. Qin H. Raffeld M. Fend F. Quintanilla-Martinez L. Grove S. Jaffe E.S. Kwak L.W. Molecular analysis of light-chain switch and acute lymphoblastic leukemia transformation in two follicular lymphomas: implications for lymphomagenesis.Leuk Lymphoma. 2006; 47: 1523-1534Crossref PubMed Scopus (26) Google Scholar Lymphomas with two recurrent translocations, one of which being MYC, are referred to as double-hit-B-cell lymphomas and have a dismal prognosis.6Aukema S.M. Siebert R. Schuuring E. van Imhoff G.W. Kluin-Nelemans H.C. Boerma E.J. Kluin P.M. Double-hit B-cell lymphomas.Blood. 2011; 117: 2319-2331Crossref PubMed Scopus (550) Google Scholar Here, we analyzed four FLs that transformed into B-LBLs. We demonstrate that these B-LBLs phenotypically and functionally display key features of early B cells and that, in addition to MYC translocations, also BCL6 gene translocation may accompany this infrequent mode of FL progression. Genome-wide copy number variation analysis and whole-exome sequencing (WES) of one of the transformed FLs demonstrated that a limited number of genomic alterations were gained in the B-LBLs, including deletions of the tumor suppressor genes CDKN2A/B and NF-1 and mutations in TNFRSF14, SMARCA2, and CCND3. The clinicopathologic characteristics of the four patients with a FL that transformed into a B-LBL are shown in Table 1. This study was conducted in accordance with the ethical standards in our institutional medical committee on human experimentation, as well as in agreement with the Helsinki Declaration of 1975, revised in 1983.Table 1Four Patients with a FL that Has Transformed into a B-LBLPatientSexAge, yearsDiagnosisYears from FL diagnosisLocationTF1aF57FL0Lymph nodeTF1bB-LBL2EpicardiumTF2aF51FL0Lymph nodeTF2bB-LBL1MouthTF2cB-LBL2MouthTF3aF24FL0Lymph nodeTF3bFL2Lymph nodeB-LBL2Lymph nodeTF4aM54FL0Lymph nodeTF4bDLBCL1Abdominal wallTF4cB-LBL1RetroperitoneumIn TF1b, TF2b, and TF4b the preexisting FL was no longer present, whereas in TF3b both the FL and B-LBL were detected. The biopsies of TF2b and TF2c were both taken from the mucosa of the jaw.Patients TF1, TF2, TF3, and TF4 all died within 2 years after FL diagnosis.F, female; M, male; B-LBL, precursor B-lymphoblastic lymphoma; FL, follicular lymphoma. Open table in a new tab In TF1b, TF2b, and TF4b the preexisting FL was no longer present, whereas in TF3b both the FL and B-LBL were detected. The biopsies of TF2b and TF2c were both taken from the mucosa of the jaw. Patients TF1, TF2, TF3, and TF4 all died within 2 years after FL diagnosis. F, female; M, male; B-LBL, precursor B-lymphoblastic lymphoma; FL, follicular lymphoma. A FL grade I was diagnosed, which was treated with chlorambucil. After 1 year a B-LBL was diagnosed, and the patient was treated with high-dose chemotherapy consisting of iphosphamide, etoposide, methotrexate, and two consolidation cycles with cytarabine, etoposide, and methotrexate. Complete remission was reached. A few weeks later progression of the disease occurred, and no further treatment was undertaken. The patient died subsequently. A FL grade I was diagnosed, which was not treated. Two years later a B-LBL was diagnosed, which was treated with daunorubicin, vincristine, prednisone, asparaginase, and methotrexate. Three months later the B-LBL reoccurred. The patient was treated with dexamethasone, but died a few weeks later. A FL grade I was diagnosed. Two years later, laboratory evaluation showed a leukocyte count of 10.7 × 109/L with 38% atypical lymphocytes. Immunophenotyping demonstrated two lymphocytic populations, that is, one population of mature monoclonal B cells and a distinct population of lymphoid blasts. Combined chemotherapy with cytarabine, etoposide, and methotrexate was started. Complete remission was established after two additional courses of vincristine, dexamethasone, and adriamycin. Consolidation therapy consisted of cytarabine and asparaginase. Shortly thereafter the disease relapsed. Local radiotherapy was given. The patient died a few weeks later. A FL was diagnosed, which was treated with eight cycles of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. A year later, positron emission tomography–computed tomography scan showed lesions in the abdominal wall and in the retroperitoneum, which pathologically turned out to be a DLBCL and a B-LBL, respectively. Rituximab combined with dexamethasone, high-dose cytarabine and cisplatin treatment was started, but eventually the patient died several months later. Immunohistochemical stainings were performed on acetone-fixed cryostat sections and formalin-fixed paraffin-embedded tissue using Powervision+ detection system (Immunovision Technologies, Daly City, CA). Monoclonal antibodies used were CD20 (B-Ly1), CD21-L (DRC-1), BCL2 (124), BCL6 (PG-B6P), CD79a (HM57), CD19 (HD37), Igλ (A193), Igκ (A191) (Dako, Glostrup, Denmark); CD10 (CALLA) and PAX5 (24) [Becton and Dickinson (BD), Erembodegem-Aalst, Belgium]; and CD3 (SP7; Labvision, Neomarkers, Fremont, CA). Terminal deoxynucleotidyl transferase (TdT) was stained with a polyclonal serum (Klinipath, Duiven, the Netherlands). RNA was isolated using TRIzol reagent (Invitrogen, Breda, the Netherlands) and cDNA was synthesized.3Aarts W.M. Bende R.J. Steenbergen E.J. Kluin P.M. Ooms E.C. Pals S.T. van Noesel C.J. Variable heavy chain gene analysis of follicular lymphomas: correlation between heavy chain isotype expression and somatic mutation load.Blood. 2000; 95: 2922-2929Crossref PubMed Google Scholar Immunoglobulin heavy chain variable region (IGHV) transcripts were amplified by PCR.16Aarts W.M. Willemze R. Bende R.J. Meijer C.J. Pals S.T. van Noesel C.J. VH gene analysis of primary cutaneous B-cell lymphomas: evidence for ongoing somatic hypermutation and isotype switching.Blood. 1998; 92: 3857-3864Crossref PubMed Google Scholar, 17van Dongen J.J. Langerak A.W. Bruggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. Garcia-Sanz R. van Krieken J.H. Droese J. Gonzalez D. Bastard C. White H.E. Spaargaren M. Gonzalez M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. 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 (2505) Google Scholar In some experiments, IGHV-PCR products were cloned in pTOPO-TA-vectors and sequenced. IGHV genes were identified using V-Quest version 3.3 (http://www.imgt.org/IMGT_vquest/vquest?livret=0&Option=humanIg).18Brochet X. Lefranc M.P. Giudicelli V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis.Nucleic Acids Res. 2008; 36: W503-W508Crossref PubMed Scopus (834) Google Scholar VpreB and Lambda5 were amplified with the respective primer pairs: 5′-CTCATGCACTTTGTCTACTG-3′/5′-CTGGCTCTTGTCTGATTGTGAG-3′ and 5′-CTCCTGTCCTGCTCATGCTG-3′/5′-GTACACACCGATGTCATGGTCG-3′. The BCL2 PCR was performed on DNA, as described.19Fenton J.A. Vaandrager J.W. Aarts W.M. Bende R.J. Heering K. van Dijk M. Morgan G. van Noesel C.J. Schuuring E. Kluin P.M. Follicular lymphoma with a novel t(14;18) breakpoint involving the immunoglobulin heavy chain switch mu region indicates an origin from germinal center B cells.Blood. 2002; 99: 716-718Crossref PubMed Scopus (27) Google Scholar Chromosomal aberrations were investigated in 4-μm thick formalin-fixed paraffin-embedded tissue sections by fluorescence in situ hybridization (FISH). A segregation-detection assay was used to detect breaks in BCL2 (probe Y5407), BCL6 (probe Y5408), and MYC (probe Y5410) (Dako). Lymph node suspensions of TF3b were stained with CD19-allophycocyanin, CD10-peridinin-chlorophyll protein complex-cyanine 5.5, and CD20-fluorescein isothiocyanate monoclonal antibodies (BD). Cells were sorted in small CD19+CD10+CD20lo cells, small CD19+CD10+CD20hi cells, small CD19+CD10−CD20hi cells, and large CD19+CD10+CD20lo cells by using a FACSAria (BD). Genomic DNA was isolated using DNeasy kit (Qiagen, Hilden, Germany) and analyzed for copy number variation using HumanCytoSNP-12 BeadChips (Illumina, San Diego, CA). Processing of DNA samples, hybridization, staining, and scanning of the BeadChips were performed according to the Illumina Infinium II protocol at the array facility of ServiceXS (Leiden, the Netherlands). Data were analyzed using Illumina GenomeStudio software version 2009.2. The LogR ratio and the B allele frequency data were processed into the OverUnder plugin as described previously,20Attiyeh E.F. Diskin S.J. Attiyeh M.A. Mossé Y.P. Hou C. Jackson E.M. Kim C. Glessner J. Hakonarson H. Biegel J.A. Maris J.M. Genomic copy number determination in cancer cells from single nucleotide polymorphism microarrays based on quantitative genotyping corrected for aneuploidy.Genome Res. 2009; 19: 276-283Crossref PubMed Scopus (60) Google Scholar and copy numbers were calculated. Because of lack of normal tissue, it cannot be excluded that some of the copy number variation are germline polymorphisms. Genomic DNA was amplified using the illustra GenomiPhi V2 DNA amplification kit (GE Health Care, Little Chalfont, United Kingdom). Unamplified DNA of the B-LBL isolated from blood was used to control for amplification errors. Fragmentation was performed with the Covaris sonicator (GC Biotec, Alphen aan de Rijn, the Netherlands). Library preparation was performed using Solid chemistry. An exome capture was performed with whole-exome capture EZ version 3 of Nimblegen (Roche, Almere, the Netherlands) with platform-specific adaptors and blockers. Clonal amplification of the captured library was performed by emulsion PCR (Life Technologies, Carlsbad, CA). Finally, samples were sequenced on a Solid 5500 xl sequencer. Obtained data were processed according to the NGS pipeline described by Houten et al.21Houten S.M. Denis S. Te Brinke H. Jongejan A. van Kampen A.H. Bradley E.J. Baas F. Hennekam R.C. Millington D.S. Young S.P. Frazier D.M. Gucsavas-Calikoglu M. Wanders R.J. Mitochondrial NADP(H) deficiency due to a mutation in NADK2 causes dienoyl-CoA reductase deficiency with hyperlysinemia.Hum Mol Genet. 2014; 23: 5009-5016Crossref PubMed Scopus (43) Google Scholar Because of the lack of normal tissue, only differential variants between FL and B-LBL could be determined. In addition, in our WES data we also checked for mutations in sets of 92 and 40 genes, previously found to be mutated in WES data of FLs and B-cell acute lymphoblastic leukemias (B-ALLs), respectively (Supplemental Table S1).5Pasqualucci L. Khiabanian H. Fangazio M. Vasishtha M. Messina M. Holmes A.B. Ouillette P. Trifonov V. Rossi D. Tabbo F. Ponzoni M. Chadburn A. Murty V.V. Bhagat G. Gaidano G. Inghirami G. Malek S.N. Rabadan R. Dalla-Favera R. Genetics of follicular lymphoma transformation.Cell Rep. 2014; 6: 130-140Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 22Morin R.D. Mendez-Lago M. Mungall A.J. Goya R. Mungall K.L. Corbett R.D. et al.Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma.Nature. 2011; 476: 298-303Crossref PubMed Scopus (1222) Google Scholar, 23Green M.R. Gentles A.J. Nair R.V. Irish J.M. Kihira S. Liu C.L. Kela I. Hopmans E.S. Myklebust J.H. Ji H. Plevritis S.K. Levy R. Alizadeh A.A. Hierarchy in somatic mutations arising during genomic evolution and progression of follicular lymphoma.Blood. 2013; 121: 1604-1611Crossref PubMed Scopus (220) Google Scholar, 24Zhang J. Mullighan C.G. Harvey R.C. Wu G. Chen X. Edmonson M. Buetow K.H. Carroll W.L. Chen I.M. Devidas M. Gerhard D.S. Loh M.L. Reaman G.H. Relling M.V. Camitta B.M. Bowman W.P. Smith M.A. Willman C.L. Downing J.R. Hunger S.P. Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group.Blood. 2011; 118: 3080-3087Crossref PubMed Scopus (206) Google Scholar, 25Okosun J. Bodor C. Wang J. Araf S. Yang C.Y. Pan C. Boller S. Cittaro D. Bozek M. Iqbal S. Matthews J. Wrench D. Marzec J. Tawana K. Popov N. O'Riain C. O'Shea D. Carlotti E. Davies A. Lawrie C.H. Matolcsy A. Calaminici M. Norton A. Byers R.J. Mein C. Stupka E. Lister T.A. Lenz G. Montoto S. Gribben J.G. Fan Y. Grosschedl R. Chelala C. Fitzgibbon J. Integrated genomic analysis identifies recurrent mutations and evolution patterns driving the initiation and progression of follicular lymphoma.Nat Genet. 2014; 46: 176-181Crossref PubMed Scopus (505) Google Scholar, 26Green M.R. Kihira S. Liu C.L. Nair R.V. Salari R. Gentles A.J. Irish J. Stehr H. Vicente-Duenas C. Romero-Camarero I. Sanchez-Garcia I. Plevritis S.K. Arber D.A. Batzoglou S. Levy R. Alizadeh A.A. Mutations in early follicular lymphoma progenitors are associated with suppressed antigen presentation.Proc Natl Acad Sci U S A. 2015; 112: E1116-E1125Crossref PubMed Scopus (240) Google Scholar Eleven mutations, in 10 genes identified in TF3b, were validated by Sanger sequencing in TF3b, and, in addition, these 11 mutations were examined in TF1a, TF2a, and TF2c. Moreover, enhancer of zeste homolog 2 (EZH2), which is frequently mutated in FL, was screened for mutations in TF1a and TF3b. Before transformation, the four FLs (TF1a to TF4a) displayed a follicular growth pattern with a characteristic phenotype, that is, CD20+, CD79a+, BCL2+ cells co-expressing the GC markers CD10 and BCL6 that expanded in networks of follicular dendritic cells (Tables 1 and 2). After B-LBL transformation, the follicular architecture of TF1, TF2, and TF4 was lost. The tumor cells of all four cases still expressed CD79a, CD10, and BCL2 but were devoid of CD20. In addition, all B-LBLs expressed TdT. In the TF2b B-LBL approximately 10% of the tumor cells stained positive for TdT. However, no TdT expression was observed in the TF2c biopsy. In TF3b, the follicular growth pattern was in part retained and alternated with sheets of lymphoblastic cells (Figure 1). To analyze the molecular features of the entities in TF3b, CD19+ cells of lymph node suspensions underwent fluorescence-activated cell sorting based on size and expression of CD20 and CD10, in: i) small CD19+CD10+CD20lo cells, ii) small CD19+CD10+CD20hi cells, iii) small CD19+CD10−CD20hi cells, and iv) large CD19+CD10+CD20lo cells. Both CD20lo fractions expressed TdT, as determined by RT-PCR, demonstrating that these fractions represent the B-LBL (Supplemental Figure S1). Of note, the FL of TF4 transformed into a DLBCL before transforming into B-LBL (Table 1).Table 2Immunophenotypes of Four Lymphomas before and after ProgressionPatientDiagnosisCD20FDCCD10BCL6BCL2TdTPax5CD79aCD19IgλIgκTF1aFL+++++−++++−TF1bB-LBL−ND+−++++++−TF2aFL+++++−++NDNDNDTF2bB-LBL−ND++++++++−TF2cB-LBL−ND+++−++++−TF3aFL+++++−++NDNDNDTF3bFL+++++−+++NDNDB-LBL−−+−+++++NDNDTF4aFL+ND+++−+++NDNDTF4bDLBCL−−+−+−++NDNDNDTF4cB-LBL−−+−++++NDNDNDProtein expression of CD20, CD21-L, CD10, BCL6, BCL2, TdT, Pax5, CD79a, CD19, and Igλ, as determined by IHC on formalin-fixed paraffin-embedded and frozen sections.BCL, B-cell lymphoma; B-LBL, precursor B-cell lymphoblastic lymphoma; FDC, follicular dendritic cells (as determined by CD21-L staining); FL, follicular lymphoma; LN, lymph node; ND, not determined due to lack of appropriate patient material; TdT, terminal deoxynucleotidyl transferase. Open table in a new tab Protein expression of CD20, CD21-L, CD10, BCL6, BCL2, TdT, Pax5, CD79a, CD19, and Igλ, as determined by IHC on formalin-fixed paraffin-embedded and frozen sections. BCL, B-cell lymphoma; B-LBL, precursor B-cell lymphoblastic lymphoma; FDC, follicular dendritic cells (as determined by CD21-L staining); FL, follicular lymphoma; LN, lymph node; ND, not determined due to lack of appropriate patient material; TdT, terminal deoxynucleotidyl transferase. IGHV gene analysis showed that in all patients the FL and B-LBL were of the same clonal origin, harboring identical IGHV rearrangements (Table 3). In TF3b, all four fractions undergoing fluorescence-activated cell sorting, including the small CD19+CD10−CD20hi cells, harbored an identical IGHV rearrangement, suggesting that this population is either a CD10− subclone of the FL or represents a stage intermediate between the FL and B-LBL. In accordance with their GC phenotype, all four FLs harbored somatically mutated IGHV. Intriguingly, all four lymphomas expressed IgG, as determined by RT-PCR, instead of the commonly found IgM in FL4Roulland S. Faroudi M. Mamessier E. Sungalee S. Salles G. Nadel B. Early steps of follicular lymphoma pathogenesis.Adv Immunol. 2011; 111: 1-46Crossref PubMed Scopus (82) Google Scholar (Table 3). Because of the poor DNA quality, the IGHV of the TF3a FL could only be investigated from framework 2. We were unable to determine the IGHV of the TF4b DLBCL due to lack of material.Table 3IGHV and IGLV Rearrangements of the Four FLs and in Their Ensuing B-LBLPatientDiagnosisSubclassIGHV rearrangementMut no. (shared with FL)IGLV rearrangementTF1aFLIgGIGHV4-61/NA/IGHJ5a22IGLV2-18/IGLJ2/3aTF1bB-LBLIgGIGHV4-61/NA/IGHJ5a27 (19)IGLV3-21/IGLJ3bTF2aFLIgGIGHV3-20/NA/IGHJ6b23IGLV1-36/IGLJ3bTF2bB-LBLIgGIGHV3-20/NA/IGHJ6b23 (23)PolyclonalTF3aFLNDIGHV1-69/IGHD1-26/IGHJ6c10∗IGHV SHM was determined starting from FR1 in all instances, except for TF3a, in which SHMs were determined starting from FR2, and TF4, in which SHM could not be determined in the first part of FR1.NDTF3b†Lymph node suspensions of TF3b were sorted in four fractions (Supplemental Figure S1 and text).FL (CD10hi)IgGIGHV1-69/IGHD1-26/IGHJ6c17IGLV4-69/IGLJ2/3aFL (CD10low)IgGIGHV1-69/IGHD1-26/IGHJ6c15IGLV4-69/IGLJ2/3aB-LBL (S)IgGIGHV1-69/IGHD1-26/IGHJ6c15 (14)IGLV4-69/IGLJ2/3aB-LBL (L)IgGIGHV1-69/IGHD1-26/IGHJ6c15 (14)IGLV4-69/IGLJ2/3aTF4aFLIgGIGHV3-73/IGHD1-7/IGHJ5*0221NDTF4bDLBCLNDNDNDNDTF4cB-LBLIgGIGHV3-73/NA/IGHJ5*0225 (18)NDB-LBL, precursor B-cell lymphoblastic lymphoma; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; FR, framework; L, large; Mut, mutation; NA, not applicable; ND, not determined; S, small; SHM, somatic hypermutation.∗ IGHV SHM was determined starting from FR1 in all instances, except for TF3a, in which SHMs were determined starting from FR2, and TF4, in which SHM could not be determined in the first part of FR1.† Lymph node suspensions of TF3b were sorted in four fractions (Supplemental Figure S1 and text). Open table in a new tab B-LBL, precursor B-cell lymphoblastic lymphoma; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; FR, framework; L, large; Mut, mutation; NA, not applicable; ND, not determined; S, small; SHM, somatic hypermutation. All four FL/B-LBL pairs harbored high numbers of shared somatic IGHV mutations (Figure 2), further underscoring their clonal relationship. Comparison of individual molecular IGHV clones of TF1 and TF3 showed intraclonal

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