Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Molecular Classification of MYC-Driven B-Cell Lymphomas by Targeted Gene Expression Profiling of Fixed Biopsy Specimens

2014; Elsevier BV; Volume: 17; Issue: 1 Linguagem: Inglês

10.1016/j.jmoldx.2014.08.006

ISSN

1943-7811

Autores

Christopher D. Carey, Daniel Gusenleitner, Bjoern Chapuy, Alexandra E. Kovach, Michael Kluk, Heather H. Sun, Rachel E. Crossland, Chris M. Bacon, Vikki Rand, Paola Dal Cin, Long P. Le, Donna Neuberg, Aliyah R. Sohani, Margaret A. Shipp, Stefano Monti, Scott J. Rodig,

Tópico(s)

CAR-T cell therapy research

Resumo

Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are aggressive tumors of mature B cells that are distinguished by a combination of histomorphological, phenotypic, and genetic features. A subset of B-cell lymphomas, however, has one or more characteristics that overlap BL and DLBCL, and are categorized as B-cell lymphoma unclassifiable, with features intermediate between BL and DLBCL (BCL-U). Molecular analyses support the concept that there is a biological continuum between BL and DLBCL that includes variable activity of MYC, an oncoprotein once thought to be only associated with BL, but now recognized as a major predictor of survival among patients with DLBCL treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). We tested whether a targeted expression profiling panel could be used to categorize tumors as BL and DLBCL, resolve the molecular heterogeneity of BCL-U, and capture MYC activity using RNA from formalin-fixed, paraffin-embedded biopsy specimens. A diagnostic molecular classifier accurately predicted pathological diagnoses of BL and DLBCL, and provided more objective subclassification for a subset of BCL-U and genetic double-hit lymphomas as molecular BL or DLBCL. A molecular classifier of MYC activity correlated with MYC IHC and stratified patients with primary DLBCL treated with R-CHOP into high- and low-risk groups. These results establish a framework for classifying and stratifying MYC-driven, aggressive, B-cell lymphomas on the basis of quantitative molecular profiling that is applicable to fixed biopsy specimens. Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are aggressive tumors of mature B cells that are distinguished by a combination of histomorphological, phenotypic, and genetic features. A subset of B-cell lymphomas, however, has one or more characteristics that overlap BL and DLBCL, and are categorized as B-cell lymphoma unclassifiable, with features intermediate between BL and DLBCL (BCL-U). Molecular analyses support the concept that there is a biological continuum between BL and DLBCL that includes variable activity of MYC, an oncoprotein once thought to be only associated with BL, but now recognized as a major predictor of survival among patients with DLBCL treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). We tested whether a targeted expression profiling panel could be used to categorize tumors as BL and DLBCL, resolve the molecular heterogeneity of BCL-U, and capture MYC activity using RNA from formalin-fixed, paraffin-embedded biopsy specimens. A diagnostic molecular classifier accurately predicted pathological diagnoses of BL and DLBCL, and provided more objective subclassification for a subset of BCL-U and genetic double-hit lymphomas as molecular BL or DLBCL. A molecular classifier of MYC activity correlated with MYC IHC and stratified patients with primary DLBCL treated with R-CHOP into high- and low-risk groups. These results establish a framework for classifying and stratifying MYC-driven, aggressive, B-cell lymphomas on the basis of quantitative molecular profiling that is applicable to fixed biopsy specimens. CME Accreditation Statement: This activity ("JMD 2015 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("JMD 2015 CME Program in Molecular Diagnostics") for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("JMD 2015 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2015 CME Program in Molecular Diagnostics") for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. The World Health Organization classification of tumors defines neoplastic diseases according to unique clinical and biological characteristics.1Swerdlow SH, World Health Organization. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Geneva, Switzerland, WHO Press, 2008Google Scholar Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are aggressive tumors of mature B cells categorized as individual tumor types. The reliable differentiation of BL from DLBCL is important, because these tumors are treated with distinct chemotherapeutic regimens.2Magrath I. Adde M. Shad A. Venzon D. Seibel N. Gootenberg J. Neely J. Arndt C. Nieder M. Jaffe E. Wittes R.A. Horak I.D. Adults and children with small non-cleaved-cell lymphoma have a similar excellent outcome when treated with the same chemotherapy regimen.J Clin Oncol. 1996; 14: 925-934Crossref PubMed Scopus (373) Google Scholar, 3Habermann T.M. Rituximab-CHOP versus CHOP alone or with maintenance rituximab in older patients with diffuse large B-cell lymphoma.J Clin Oncol. 2006; 24: 3121-3127Crossref PubMed Scopus (1159) Google Scholar BL is a neoplasm composed of monomorphic, intermediate-sized lymphocytes that are positive for markers of mature, germinal-center B cells and negative for the anti-apoptotic protein BCL2. Most cells (>95%) are positive for the proliferation marker, Ki-67/MIB1. The genetic hallmark of BL is a balanced translocation involving the MYC oncogene and, most commonly, the immunoglobulin heavy chain locus (IGH).1Swerdlow SH, World Health Organization. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Geneva, Switzerland, WHO Press, 2008Google Scholar, 4Hecht J.L. Aster J.C. Molecular biology of Burkitt's lymphoma.J Clin Oncol. 2000; 18: 3707-3721Crossref PubMed Scopus (340) Google Scholar Mutations in TCF3 and ID3 are also common.5Schmitz R. Young R.M. Ceribelli M. Jhavar S. Xiao W. Zhang M. et al.Burkitt lymphoma pathogenesis and therapeutic targets from structural and functional genomics.Nature. 2012; 490: 116-120Crossref PubMed Scopus (641) Google Scholar, 6Love C. Sun Z. Jima D. Li G. Zhang J. Miles R. Richards K.L. Dunphy C.H. Choi W.W.L. Srivastava G. Lugar P.L. Rizzieri D.A. Lagoo A.S. Bernal-Mizrachi L. Mann K.P. Flowers C.R. Naresh K.N. Evens A.M. Chadburn A. Gordon L.I. Czader M.B. Gill J.I. Hsi E.D. Greenough A. Moffitt A.B. McKinney M. Banerjee A. Grubor V. Levy S. Dunson D.B. Dave S.S. The genetic landscape of mutations in Burkitt lymphoma.Nat Genet. 2012; 44: 1321-1325Crossref PubMed Scopus (434) Google Scholar In contrast, DLBCL is composed of pleomorphic, large lymphoid cells and, in general, less apoptosis and a lower proliferation index than BL. DLBCLs express markers of mature B cells, with or without evidence of germinal center cell derivation, and often express BCL2. Genetically, only a small subset of DLBCLs have a MYC translocation, and mutations in TCF3 or ID3 are rare. However, mutations in genes encoding the components of the NF-κB and B-cell receptor signaling pathways are common.1Swerdlow SH, World Health Organization. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Geneva, Switzerland, WHO Press, 2008Google Scholar, 7Zhang J. Grubor V. Love C.L. Banerjee A. Richards K.L. Mieczkowski P.A. et al.Genetic heterogeneity of diffuse large B-cell lymphoma.Proc Natl Acad Sci U S A. 2013; 110: 1398-1403Crossref PubMed Scopus (421) Google Scholar, 8Morin R.D. Mungall K. Pleasance E. Mungall A.J. Goya R. Huff R. Scott D.W. Ding J. Roth A. Chiu R. Corbett R.D. Chan F.C. Mendez-Lago M. Trinh D.L. Bolger-Munro M. Taylor G. Hadj Khodabakhshi A. Ben-Neriah S. Pon J. Meissner B. Woolcock B. Farnoud N. Rogic S. Lim E. Johnson N.A. Shah S. Jones S. Steidl C. Holt R. Birol I. Moore R. Connors J.M. Gascoyne R.D. Marra M.A. Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing.Blood. 2013; 122: 1256-1265Crossref PubMed Scopus (298) Google Scholar, 9Lohr J.G. Stojanov P. Lawrence M.S. Auclair D. Chapuy B. Sougnez C. Cruz-Gordillo P. Knoechel B. Asmann Y.W. Slager S.L. Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing.Proc Natl Acad Sci U S A. 2012; 109: 3879-3884Crossref PubMed Scopus (772) Google Scholar, 10Savage K.J. Johnson N.A. Ben-Neriah S. Connors J.M. Sehn L.H. Farinha P. Horsman D.E. Gascoyne R.D. MYC gene rearrangements are associated with a poor prognosis in diffuse large B-cell lymphoma patients treated with R-CHOP chemotherapy.Blood. 2009; 114: 3533-3537Crossref PubMed Scopus (518) Google Scholar, 11Barrans S. Crouch S. Smith A. Turner K. Owen R. Patmore R. Roman E. Jack A. Rearrangement of MYC is associated with poor prognosis in patients with diffuse large B-cell lymphoma treated in the era of rituximab.J Clin Oncol. 2010; 28: 3360-3365Crossref PubMed Scopus (474) Google Scholar Most cases of BL and DLBCL are diagnosed with high confidence using traditional histopathological, immunophenotypic, and targeted genetic analyses. However, it is not uncommon to encounter tumors with one or more features overlapping BL and DLBCL. The 2008 World Health Organization Classification of Lymphoid Tumors recognized these cases with the novel diagnostic category, B-cell lymphoma unclassifiable, with features intermediate between DLBCL and BL (BCL-U).1Swerdlow SH, World Health Organization. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Geneva, Switzerland, WHO Press, 2008Google Scholar BCL-U is, by definition, a heterogeneous group, and its diagnosis requires that pathologists make subtle distinctions in histomorphological features, immunophenotype, and genetics that may not be highly reproducible. Molecular classification of aggressive B-cell lymphomas using comprehensive gene-expression profiles (GEPs) of RNA isolated from frozen tumor samples accurately differentiates BL from DLBCL and confirms that a subset of cases has transcriptional signatures intermediate between BL and DLBCL.12Dave S.S. Fu K. Wright G.W. Lam L.T. Kluin P. Boerma E.-J. Greiner T.C. Weisenburger D.D. Rosenwald A. Ott G. Molecular diagnosis of Burkitt's lymphoma.N Engl J Med. 2006; 354: 2431-2442Crossref PubMed Scopus (731) Google Scholar, 13Hummel M. Bentink S. Berger H. Klapper W. Wessendorf S. Barth T.F. Bernd H.-W. Cogliatti S.B. Dierlamm J. Feller A.C. A biologic definition of Burkitt's lymphoma from transcriptional and genomic profiling.N Engl J Med. 2006; 354: 2419-2430Crossref PubMed Scopus (810) Google Scholar However, the pathological diagnoses corresponding to these biologically intermediate tumors have been inconsistent.13Hummel M. Bentink S. Berger H. Klapper W. Wessendorf S. Barth T.F. Bernd H.-W. Cogliatti S.B. Dierlamm J. Feller A.C. A biologic definition of Burkitt's lymphoma from transcriptional and genomic profiling.N Engl J Med. 2006; 354: 2419-2430Crossref PubMed Scopus (810) Google Scholar Complicating the evaluation of aggressive lymphomas is the recognition that high MYC expression and biological activity, once thought to be only associated with BL, are major, independent predictors of poor clinical outcome among patients with primary DLBCL treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone).14Johnson N.A. Slack G.W. Savage K.J. Connors J.M. Ben-Neriah S. Rogic S. Scott D.W. Tan K.L. Steidl C. Sehn L.H. Chan W.C. Iqbal J. Meyer P.N. Lenz G. Wright G. Rimsza L.M. Valentino C. Brunhoeber P. Grogan T.M. Braziel R.M. Cook J.R. Tubbs R.R. Weisenburger D.D. Campo E. Rosenwald A. Ott G. Delabie J. Holcroft C. Jaffe E.S. Staudt L.M. Gascoyne R.D. Concurrent expression of MYC and BCL2 in diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone.J Clin Oncol. 2012; 30: 3452-3459Crossref PubMed Scopus (726) Google Scholar, 15Kluk M.J. Chapuy B. Sinha P. Roy A. Cin P.D. Neuberg D.S. Monti S. Pinkus G.S. Shipp M.A. Rodig S.J. Immunohistochemical detection of MYC-driven diffuse large B-cell lymphomas.PLoS One. 2012; 7: e33813Crossref PubMed Scopus (137) Google Scholar, 16Zhou K. Xu D. Cao Y. Wang J. Yang Y. Huang M. C-MYC aberrations as prognostic factors in diffuse large B-cell lymphoma: a meta-analysis of epidemiological studies.PLoS One. 2014; 9: e95020Crossref PubMed Scopus (55) Google Scholar, 17Cook J.R. Goldman B. Tubbs R.R. Rimsza L. Leblanc M. Stiff P. Fisher R. Clinical significance of MYC expression and/or "high-grade" morphology in non-Burkitt, diffuse aggressive B-cell lymphomas: a SWOG S9704 correlative study.Am J Surg Pathol. 2014; 38: 494-501Crossref PubMed Scopus (42) Google Scholar, 18Perry A.M. Alvarado-Bernal Y. Laurini J.A. Smith L.M. Slack G.W. Tan K.L. Sehn L.H. Fu K. Aoun P. Greiner T.C. Chan W.C. Bierman P.J. Bociek R.G. Armitage J.O. Vose J.M. Gascoyne R.D. Weisenburger D.D. MYC and BCL2 protein expression predicts survival in patients with diffuse large B-cell lymphoma treated with rituximab.Br J Haematol. 2014; 165: 382-391Crossref PubMed Scopus (145) Google Scholar In some series, the prognostic value of MYC is enhanced among tumors that co-express BCL2.14Johnson N.A. Slack G.W. Savage K.J. Connors J.M. Ben-Neriah S. Rogic S. Scott D.W. Tan K.L. Steidl C. Sehn L.H. Chan W.C. Iqbal J. Meyer P.N. Lenz G. Wright G. Rimsza L.M. Valentino C. Brunhoeber P. Grogan T.M. Braziel R.M. Cook J.R. Tubbs R.R. Weisenburger D.D. Campo E. Rosenwald A. Ott G. Delabie J. Holcroft C. Jaffe E.S. Staudt L.M. Gascoyne R.D. Concurrent expression of MYC and BCL2 in diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone.J Clin Oncol. 2012; 30: 3452-3459Crossref PubMed Scopus (726) Google Scholar, 19Green T.M. Young K.H. Visco C. Xu-Monette Z.Y. Orazi A. Go R.S. Nielsen O. Gadeberg O.V. Mourits-Andersen T. Frederiksen M. Pedersen L.M. Moller M.B. Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone.J Clin Oncol. 2012; 30: 3460-3467Crossref PubMed Scopus (537) Google Scholar, 20Horn H. Ziepert M. Becher C. Barth T.F.E. Bernd H.W. Feller A.C. Klapper W. Hummel M. Stein H. Hansmann M.L. Schmelter C. Moller P. Cogliatti S. Pfreundschuh M. Schmitz N. Trumper L. Siebert R. Loeffler M. Rosenwald A. Ott G. German High-Grade Non-Hodgkin Lymphoma Study GroupMYC status in concert with BCL2 and BCL6 expression predicts outcome in diffuse large B-cell lymphoma.Blood. 2013; 121: 2253-2263Crossref PubMed Scopus (422) Google Scholar, 21Hu S. Xu-Monette Z.Y. Tzankov A. Green T. Wu L. Balasubramanyam A. et al.MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program.Blood. 2013; 121: 4021-4031Crossref PubMed Scopus (542) Google Scholar Indeed, recent evidence suggests that high co-expression of MYC and BCL2 in tumor cells provides a biological basis for the inferior outcome among patients with the activated B-cell (ABC) type DLBCL when treated with standard chemotherapy.21Hu S. Xu-Monette Z.Y. Tzankov A. Green T. Wu L. Balasubramanyam A. et al.MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program.Blood. 2013; 121: 4021-4031Crossref PubMed Scopus (542) Google Scholar DLBCL with high MYC activity cannot be identified with certainty by morphological or genetic studies alone.15Kluk M.J. Chapuy B. Sinha P. Roy A. Cin P.D. Neuberg D.S. Monti S. Pinkus G.S. Shipp M.A. Rodig S.J. Immunohistochemical detection of MYC-driven diffuse large B-cell lymphomas.PLoS One. 2012; 7: e33813Crossref PubMed Scopus (137) Google Scholar The detection of MYC in fixed tumor biopsy specimens by immunohistochemistry (IHC) has the potential to identify DLBCLs with high MYC protein that corresponds to high MYC biological activity.15Kluk M.J. Chapuy B. Sinha P. Roy A. Cin P.D. Neuberg D.S. Monti S. Pinkus G.S. Shipp M.A. Rodig S.J. Immunohistochemical detection of MYC-driven diffuse large B-cell lymphomas.PLoS One. 2012; 7: e33813Crossref PubMed Scopus (137) Google Scholar However, IHC methods are difficult to standardize between institutions, and the interpretation of IHC staining is subjective.22de Jong D. Rosenwald A. Chhanabhai M. Gaulard P. Klapper W. Lee A. Sander B. Thorns C. Campo E. Molina T. Norton A. Hagenbeek A. Horning S. Lister A. Raemaekers J. Gascoyne R.D. Salles G. Weller E. Immunohistochemical prognostic markers in diffuse large B-cell lymphoma: validation of tissue microarray as a prerequisite for broad clinical applications–a study from the Lunenburg Lymphoma Biomarker Consortium.J Clin Oncol. 2007; 25: 805-812Crossref PubMed Scopus (261) Google Scholar These data highlight a need for quantitative methods that capture the phenotypic, genetic, and molecular heterogeneity of aggressive B-cell lymphomas in clinical practice. Molecular classification on the basis of the unique GEPs of BL, DLBCL, and MYC-driven B-cell lymphomas has the potential to satisfy this need, but, until recently, GEP has not been amenable to formalin-fixed, paraffin-embedded (FFPE) tissues.23Rimsza L.M. Wright G. Schwartz M. Chan W.C. Jaffe E.S. Gascoyne R.D. Campo E. Rosenwald A. Ott G. Cook J.R. Tubbs R.R. Braziel R.M. Delabie J. Miller T.P. Staudt L.M. Accurate classification of diffuse large B-cell lymphoma into germinal center and activated B-cell subtypes using a nuclease protection assay on formalin-fixed, paraffin-embedded tissues.Clin Cancer Res. 2011; 17: 3727-3732Crossref PubMed Scopus (60) Google Scholar, 24Scott D.W. Wright G.W. Williams P.M. Lih C.-J. Walsh W. Jaffe E.S. Rosenwald A. Campo E. Chan W.C. Connors J.M. Smeland E.B. Mottok A. Braziel R.M. Ott G. Delabie J. Tubbs R.R. Cook J.R. Weisenburger D.D. Greiner T.C. Glinsmann-Gibson B.J. Fu K. Staudt L.M. Gascoyne R.D. Rimsza L.M. Determining cell-of-origin subtypes of diffuse large B-cell lymphoma using gene expression in formalin-fixed paraffin embedded tissue.Blood. 2014; 123: 1214-1217Crossref PubMed Scopus (438) Google Scholar, 25Masqué-Soler N. Szczepanowski M. Kohler C.W. Spang R. Klapper W. Molecular classification of mature aggressive B-cell lymphoma using digital multiplexed gene expression on formalin-fixed paraffin-embedded biopsy specimens.Blood. 2013; 122: 1985-1986Crossref PubMed Scopus (54) Google Scholar, 26Linton K. Howarth C. Wappett M. Newton G. Lachel C. Iqbal J. Pepper S. Byers R. Chan W.J. Radford J. Microarray gene expression analysis of fixed archival tissue permits molecular classification and identification of potential therapeutic targets in diffuse large B-cell lymphoma.J Mol Diagn. 2012; 14: 223-232Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Herein, we report a method of targeted expression profiling, followed by a two-stage molecular classifier of aggressive mature B-cell lymphomas that is applicable to FFPE biopsy specimens. This study was performed with approval from the Institutional Review Boards (IRBs) of Brigham and Women's Hospital (Boston, MA; IRB number 2010P002736) and Massachusetts General Hospital (Boston, MA; IRB number 2007P001458). For each case, one or both of the corresponding pathologists of this study (S.J.R. and A.R.S.) reviewed H&E-stained slides and the original diagnostic reports to ensure that the final diagnosis fulfilled 2008 World Health Organization diagnostic criteria. The training set (n = 41) comprises 12 BLs and 29 DLBCLs (one additional DLBCL later failed analytical quality control). The BLs were selected on the basis of the quality of available tissue and include all BL subtypes, as well as pediatric and adult patients (median age of diagnosis, 30.5 years; range, 3 to 62 years) (Supplemental Table S1). The DLBCLs were selected from a previously published larger series of adult patients15Kluk M.J. Chapuy B. Sinha P. Roy A. Cin P.D. Neuberg D.S. Monti S. Pinkus G.S. Shipp M.A. Rodig S.J. Immunohistochemical detection of MYC-driven diffuse large B-cell lymphomas.PLoS One. 2012; 7: e33813Crossref PubMed Scopus (137) Google Scholar who had all been diagnosed as having DLBCL not otherwise specified. Previously, MYC IHC-High was defined as >50% expression in tumor cells, and MYC IHC-Low was defined as ≤50% expression in tumor cells.15Kluk M.J. Chapuy B. Sinha P. Roy A. Cin P.D. Neuberg D.S. Monti S. Pinkus G.S. Shipp M.A. Rodig S.J. Immunohistochemical detection of MYC-driven diffuse large B-cell lymphomas.PLoS One. 2012; 7: e33813Crossref PubMed Scopus (137) Google Scholar For training, cases were deliberately selected to represent the extremes of MYC IHC-High (median, 70%; n = 13) and MYC IHC-Low (median, 20% to 30%; n = 16) to assist development of the MYC activity classifier. DLBCLs were not selected with regard to cell of origin (COO27Alizadeh A.A. Eisen M.B. Davis R.E. Ma C. Lossos I.S. Rosenwald A. Boldrick J.C. Sabet H. Tran T. Yu X. Powell J.I. Yang L. Marti G.E. Moore T. Hudson J. Lu L. Lewis D.B. Tibshirani R. Sherlock G. Chan W.C. Greiner T.C. Weisenburger D.D. Armitage J.O. Warnke R. Levy R. Wilson W. Grever M.R. Byrd J.C. Botstein D. Brown P.O. Staudt L.M. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (8087) Google Scholar) subtype, but previously classified using GEP as 10 ABC-type (34.5%), 13 germinal center B-cell type (GBC; 44.8%), 5 type 3 (17.2%), and 1 unclassified (3.4%) (Supplemental Table S2). The test set (n = 55) comprised 9 BLs (all adult patients, 8 sporadic and 1 immunodeficiency associated), 41 DLBCLs, and 5 BCL-Us (Supplemental Tables S1, S2, and S3). Four additional cases failed analytical quality control. Eight of these tumors were genetic double-hit lymphomas (DHLs), for the purposes of this study defined as the combination of a MYC rearrangement and either a BCL2 or BCL6 rearrangement, and these were divided into three tumors with a pathological diagnosis of DLBCL and five tumors with a pathological diagnosis of BCL-U (Supplemental Table S3). The DLBCLs included these three DHLs, which were characterized by a combination of MYC and BCL2 rearrangements, as well as one single-hit lymphoma (SHL), characterized by a MYC rearrangement in isolation. The DLBCLs were chosen on the basis of the quality of available biopsy material and to represent a full range of MYC IHC expression. DLBCLs for the test set were not selected on the basis of COO subtype.27Alizadeh A.A. Eisen M.B. Davis R.E. Ma C. Lossos I.S. Rosenwald A. Boldrick J.C. Sabet H. Tran T. Yu X. Powell J.I. Yang L. Marti G.E. Moore T. Hudson J. Lu L. Lewis D.B. Tibshirani R. Sherlock G. Chan W.C. Greiner T.C. Weisenburger D.D. Armitage J.O. Warnke R. Levy R. Wilson W. Grever M.R. Byrd J.C. Botstein D. Brown P.O. Staudt L.M. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (8087) Google Scholar COO classification data, using GEP (if available) and/or Han's IHC criteria,28Hans C.P. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray.Blood. 2004; 103: 275-282Crossref PubMed Scopus (3260) Google Scholar, 29Bogusz A.M. Baxter R.H.G. Currie T. Sinha P. Sohani A.R. Kutok J.L. Rodig S.J. Quantitative immunofluorescence reveals the signature of active B-cell receptor signaling in diffuse large B-cell lymphoma.Clin Cancer Res. 2012; 18: 6122-6135Crossref PubMed Scopus (36) Google Scholar showed a distribution of 16 ABC/non-GCB types (39%), 17 GCB-types (41.5%), 3 type 3 (7.3%), and 5 unclassified (12.2%) (Supplemental Table S2). The five BCL-Us were selected on the basis of available cases and were all DHLs. Four of the five BCL-Us were characterized by a combination of MYC and BCL2 rearrangements and the remaining case had concurrent MYC and BCL6 rearrangements. Patients included in an outcome cohort (outcome series; n = 40, 22 patients from the training set and 18 from the test set) were derived from a single institution (Brigham and Women's Hospital). All had confirmed primary DLBCLs and received standard immunochemotherapy (R-CHOP), as previously reported.15Kluk M.J. Chapuy B. Sinha P. Roy A. Cin P.D. Neuberg D.S. Monti S. Pinkus G.S. Shipp M.A. Rodig S.J. Immunohistochemical detection of MYC-driven diffuse large B-cell lymphomas.PLoS One. 2012; 7: e33813Crossref PubMed Scopus (137) Google Scholar All clinical data were collected before, and independent of, the reference and index tests reported in this study. MYC IHC was performed on 96 tumors using a rabbit monoclonal antibody (clone Y69, catalog number ab32072; Epitomics/Abcam, Burlingame, CA), as described.15Kluk M.J. Chapuy B. Sinha P. Roy A. Cin P.D. Neuberg D.S. Monti S. Pinkus G.S. Shipp M.A. Rodig S.J. Immunohistochemical detection of MYC-driven diffuse large B-cell lymphomas.PLoS One. 2012; 7: e33813Crossref PubMed Scopus (137) Google Scholar The status of the MYC locus was determined by fluorescence in situ hybridization analysis for 96 tumors using a Vysis LSI MYC break-apart probe set (catalog number 05-J91-001), as described.15Kluk M.J. Chapuy B. Sinha P. Roy A. Cin P.D. Neuberg D.S. Monti S. Pinkus G.S. Shipp M.A. Rodig S.J. Immunohistochemical detection of MYC-driven diffuse large B-cell lymphomas.PLoS One. 2012; 7: e33813Crossref PubMed Scopus (137) Google Scholar Fluorescence in situ hybridization analyses were performed on indicated cases using the BCL2-IgH dual-fusion (catalog number 05-J71-001) and BCL6-IgH break-apart (catalog number 01N23-020; Abbott Laboratories, Abbott Park, IL) probe sets, respectively, following manufacturer's recommendations. For a few cases, a karyotype was obtained as part of the original diagnostic evaluation.15Kluk M.J. Chapuy B. Sinha P. Roy A. Cin P.D. Neuberg D.S. Monti S. Pinkus G.S. Shipp M.A. Rodig S.J. Immunohistochemical detection of MYC-driven diffuse large B-cell lymphomas.PLoS One. 2012; 7: e33813Crossref PubMed Scopus (137) Google Scholar FFPE tissue blocks were divided into sections immediately before the RNA extraction. For each block, the initial section (10 μm thick) was discarded and three subsequent sections (10 μm thick) were taken for analysis. If the estimated surface area of lesional tissue was <5 mm2, an extra section (10 μm thick) was taken. Total RNA was isolated using the Qiagen RNeasy kit (catalog number 73504; Qiagen, Hilden, Germany) and quantified using a Nanodrop spectrophotometer (NanoDrop Products, Thermo Scientific, Wilmington, DE). RNA was diluted to 150 to 200 ng/5 μL, aliquoted, and stored at −80°C until use. For the multiplexed, digital gene expression analysis, 150 to 200 ng of RNA for each sample was hybridized with 20 μL of reporter probes/reaction buffer and 5 μL of capture probes at 65°C for 20 hours. The hybridized samples were then processed on the NanoString nCounter preparation station for 2.5 hours and expression data were subsequently generated on the NanoString nCounter digital analyzer (NanoString Technologies, Seattle, WA) using the 600 fields of view setting over 4 hours.30Geiss G.K. Bumgarner R.E. Birditt B. Dahl T. Dowidar N. Dunaway D.L. Fell H.P. Ferree S. George R.D. Grogan T. James J.J. Maysuria M. Mitton J.D. Oliveri P. Osborn J.L. Peng T. Ratcliffe A.L. Webster P.J. Davidson E.H. Hood L. Direct multiplexed measurement of gene expression with color-coded probe pairs.Nat Biotechnol. 2008; 26: 317-325Crossref PubMed Scopus (1581) Google Scholar In total, tumors from 96 patients were profiled, with a further 5 tumors (5%) failing analytical quality control. Candidate gene targets were initially selected from published GEPs of BL and DLBCL,12Dave S.S. Fu K. Wright G.W. Lam L.T. Kluin P. Boerma E.-J. Greiner T.C. Weisenburger D.D. Rosenwald A. Ott G. Molecular diagnosis of Burkitt's lymphoma.N Engl J Med. 2006; 354: 2431-2442Crossref PubMed Scopus (731) Google Scholar, 13Hummel M. Bentink S. Berger H. Klapper W. Wessendorf S. Barth T.F. Bernd H.-W. Cogliatti S.B. Dierlamm J. Feller A.C. A biologic definition of Burkitt's lymphoma from transcriptional and genomic profiling.N Engl J Med. 2006; 354: 2419-2430Crossref PubMed Scopus (810) Google Scholar with preference given to genes within the transcription factor 3 (TCF3)/inhibitor of DNA binding 3 (ID3) signaling pathway,5Schmitz R. Young R.M. Ceribelli M. Jhavar S. Xiao W. Zhang M. et al.Burkitt lymphoma pathogenesis and therapeutic targets from structural and functional genomics.Nature. 20

Referência(s)