Whole-Genome Analysis and HLA Genotyping of Enteropathy-Type T-Cell Lymphoma Reveals 2 Distinct Lymphoma Subtypes
2007; Elsevier BV; Volume: 132; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2007.03.036
ISSN1528-0012
AutoresRonald J. deLeeuw, Andreas Zettl, Erdwine Klinker, Eugenia Haralambieva, Magan Trottier, Raj Chari, Yong Ge, Randy D. Gascoyne, Andreas Chott, Hans–Konrad Müller–Hermelink, Wan L. Lam,
Tópico(s)Galectins and Cancer Biology
ResumoBackground & Aims: Enteropathy-type T-cell lymphoma (ETL) is an aggressive extranodal T-cell non-Hodgkin lymphoma assumed to arise in the setting of celiac disease. Methods: To precisely define the genetic alterations underlying the pathogenesis of ETL, 30 ETL samples were profiled for genetic copy number alterations using high-resolution whole-genome tiling path array comparative genomic hybridization. To investigate the potential association of genetic alterations in ETL with celiac disease, HLA-DQB1 genotyping was performed. Results: By array comparative genomic hybridization, 13 novel recurrent minimal regions of chromosomal alteration were identified on multiple chromosome arms. ETL is characterized by frequent complex gains of 9q31.3-qter (70% of cases), or by an almost mutually exclusive 2.5-megabase loss of 16q12.1 (23% of cases). Two distinct groups of ETL could be delineated morphologically and genetically: type 1 ETL is characterized by nonmonomorphic cytomorphology, CD56 negativity, and chromosomal gains of 1q and 5q. Type 1 ETL also appears to be linked pathogenetically to celiac disease, sharing genetic alterations and HLA-DQB1 genotype patterns with (refractory) celiac disease. Type 2 ETL shows monomorphic small- to medium-sized tumor cell morphology, frequently shows CD56 expression, MYC oncogene locus gain, and rare gains of chromosomes 1q and 5q. In contrast to type 1 ETL, type 2 ETL shows a HLA-DQB1 genotype pattern more resembling that of the normal Caucasian population. Conclusions: Contrary to current clinical classification, ETL comprises 2 morphologically, clinically, and genetically distinct lymphoma entities. In addition, type 2 ETL may not be associated with celiac disease. Background & Aims: Enteropathy-type T-cell lymphoma (ETL) is an aggressive extranodal T-cell non-Hodgkin lymphoma assumed to arise in the setting of celiac disease. Methods: To precisely define the genetic alterations underlying the pathogenesis of ETL, 30 ETL samples were profiled for genetic copy number alterations using high-resolution whole-genome tiling path array comparative genomic hybridization. To investigate the potential association of genetic alterations in ETL with celiac disease, HLA-DQB1 genotyping was performed. Results: By array comparative genomic hybridization, 13 novel recurrent minimal regions of chromosomal alteration were identified on multiple chromosome arms. ETL is characterized by frequent complex gains of 9q31.3-qter (70% of cases), or by an almost mutually exclusive 2.5-megabase loss of 16q12.1 (23% of cases). Two distinct groups of ETL could be delineated morphologically and genetically: type 1 ETL is characterized by nonmonomorphic cytomorphology, CD56 negativity, and chromosomal gains of 1q and 5q. Type 1 ETL also appears to be linked pathogenetically to celiac disease, sharing genetic alterations and HLA-DQB1 genotype patterns with (refractory) celiac disease. Type 2 ETL shows monomorphic small- to medium-sized tumor cell morphology, frequently shows CD56 expression, MYC oncogene locus gain, and rare gains of chromosomes 1q and 5q. In contrast to type 1 ETL, type 2 ETL shows a HLA-DQB1 genotype pattern more resembling that of the normal Caucasian population. Conclusions: Contrary to current clinical classification, ETL comprises 2 morphologically, clinically, and genetically distinct lymphoma entities. In addition, type 2 ETL may not be associated with celiac disease. Enteropathy-type T-cell lymphoma (ETL) is a primary extranodal T-cell non-Hodgkin lymphoma arising in the gastrointestinal tract that shows a differentiation of tumor cells toward the phenotype of intestinal intraepithelial T cells. ETL tends to affect multiple segments of the small intestine, most notably the jejunum, leading to intestinal ulcerations and perforations.1Isaacson P.G. Du M.Q. Gastrointestinal lymphoma: where morphology meets molecular biology.J Pathol. 2005; 205: 255-274Crossref PubMed Scopus (163) Google Scholar The clinical course of ETL is highly aggressive, with most patients dying from the disease within months of diagnosis.2Gale J. Simmonds P.D. Mead G.M. Sweetenham J.W. Wright D.H. Enteropathy-type intestinal T-cell lymphoma: clinical features and treatment of 31 patients in a single center.J Clin Oncol. 2000; 18: 795-803PubMed Google Scholar ETL shows a wide range of histologic and immunophenotypical features. Approximately 80%–90% of ETLs (subsequently referred to as nonmonomorphic or type 1 ETL) are composed of pleomorphic, anaplastic, or immunoblastic tumor cells that usually have a CD3+CD4−CD8−CD7+CD5−CD56− immunophenotype, a phenotype akin to that of the majority of normal intestinal intraepithelial T-cell receptorα/β+ T lymphocytes. About a third of these ETLs are associated with a previous clinical history of celiac disease. In 85% of these tumors there is histologic evidence of enteropathy-like alterations in the gastrointestinal mucosa adjacent to the invasive tumor. In contrast, about 10%–20% of ETLs (subsequently referred to as monomorphic or type 2 ETL) are characterized by frequent expression of CD8 and CD56, and are composed commonly of monomorphic small- to medium-sized tumor cells. These likely are derived from activated CD8+CD56+ intraepithelial T lymphocytes, which make up around 15% of intraepithelial T-cell receptorα/β+ T lymphocytes. This subtype appears to be associated rarely with a previous clinical history of celiac disease. In addition, only 50% of tumors show histologic evidence of enteropathy adjacent to the invasive neoplasm.3Chott A. Haedicke W. Mosberger I. Fodinger M. Winkler K. Mannhalter C. Muller-Hermelink H.K. Most CD56+ intestinal lymphomas are CD8+CD5-T-cell lymphomas of monomorphic small to medium size histology.Am J Pathol. 1998; 153: 1483-1490Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar We previously showed by conventional comparative genomic hybridization (CGH) that ETL is characterized by recurrent gains of 1q, 5q, 7q, and 9q, and recurrent losses of 8p, 9p, and 13q, with gains of 9q33-q34 being the most frequent alteration, occurring in 58% of cases analyzed.4Zettl A. Ott G. Makulik A. Katzenberger T. Starostik P. Eichler T. Puppe B. Bentz M. Muller-Hermelink H.K. Chott A. Chromosomal gains at 9q characterize enteropathy-type T-cell lymphoma.Am J Pathol. 2002; 161: 1635-1645Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar Similar regions of allelic imbalance were identified by Baumgartner et al,5Baumgartner A.K. Zettl A. Chott A. Ott G. Muller-Hermelink H.K. Starostik P. High frequency of genetic aberrations in enteropathy-type T-cell lymphoma.Lab Invest. 2003; 83: 1509-1516Crossref PubMed Scopus (52) Google Scholar using a microsatellite marker study of 26 loci. In their study, monomorphic ETL was associated with a lower level of microsatellite instability, 3q27 allelic imbalances, and a trend toward lack of allelic imbalances at several loci. We therefore hypothesized that among ETL, genetically distinct tumor subtypes may be distinguishable. Thus, alternate pathogenetic pathways may be responsible for the different subtypes of ETL. To test this hypothesis we used whole-genome tiling-path array CGH to precisely delineate genetic alterations in a series of 30 histologically and clinically well-characterized ETL cases. Furthermore, to test the potential association of the tumors with celiac disease, HLA-DQB1 genotyping was performed. Because 95% of celiac disease patients carry HLA-DQ2 and most individuals who are not HLA-DQ2 positive express HLA-DQ8, celiac disease is extremely rare in patients not carrying either HLA-DQ molecule.6Green P.H. Jabri B. Coeliac disease.Lancet. 2003; 362: 383-391Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar, 7Stepniak D. Koning F. Celiac disease—sandwiched between innate and adaptive immunity.Hum Immunol. 2006; 67: 460-468Crossref PubMed Scopus (115) Google Scholar In addition, HLA-DQ homozygosity confers an increased susceptibility for celiac disease.8Al-Toma A. Goerres M.S. Meijer J.W. Pena A.S. Crusius J.B. Mulder C.J. Human leukocyte antigen-DQ2 homozygosity and the development of refractory celiac disease and enteropathy-associated T-cell lymphoma.Clin Gastroenterol Hepatol. 2006; 4: 315-319Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 9Vader W. Stepniak D. Kooy Y. Mearin L. Thompson A. van Rood J.J. Spaenij L. Koning F. The HLA-DQ2 gene dose effect in celiac disease is directly related to the magnitude and breadth of gluten-specific T cell responses.Proc Natl Acad Sci U S A. 2003; 100: 12390-12395Crossref PubMed Scopus (313) Google Scholar Therefore, although the presence of HLA-DQ2/-DQ8 cannot be used as a positive indicator of celiac disease, their absence can be used as a negative surrogate marker for celiac disease. Here, we show that ETL comprises 2 morphologically, clinically, and genetically distinct lymphoma entities, one of which, despite its current name, may not be associated with celiac disease in a substantial proportion of cases. Thirty cases of ETL were selected from the archive of the Lymph Node Reference Center at the Department of Pathology, University of Würzburg, Germany, and from the archive of the Department of Pathology, University of Vienna, Austria. All cases had been classified as ETL based on the criteria defined by the World Health Organization classification of tumors of hematopoietic and lymphoid tissues.10Isaacson P.G. Wright D. Ralkiaer E. Jaffe E.S. World Health Organization Classification of Tumours. IARC Press, Lyon2001: 208-209Google Scholar All cases were reviewed before inclusion in the study. Before the array CGH study, 18 of the 30 ETL cases had been analyzed by conventional CGH.4Zettl A. Ott G. Makulik A. Katzenberger T. Starostik P. Eichler T. Puppe B. Bentz M. Muller-Hermelink H.K. Chott A. Chromosomal gains at 9q characterize enteropathy-type T-cell lymphoma.Am J Pathol. 2002; 161: 1635-1645Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar Morphologically, 15 ETLs were classified as monomorphic small- to medium-sized type (type 2 ETL), and 15 as type 1 ETL, composed of 4 anaplastic large-cell type, 2 immunoblastic type, and 8 pleomorphic medium- to large-cell type, whereas 1 case was unclassifiable (tumorous ascites) according to the criteria previously provided by one of the authors (A.C.).11Chott A. Vesely M. Simonitsch I. Mosberger I. Hanak H. Classification of intestinal T-cell neoplasms and their differential diagnosis.Am J Clin Pathol. 1999; 111: S68-S74PubMed Google Scholar Clinical follow-up information was available for all patients (Table 1). In brief, there were 19 male and 11 female patients, with the patients' ages ranging from 39 to 85 years (average, 63 y). Twenty-seven of the 30 patients already had died from the disease, with survival time ranging from 1 to 25 months after the initial diagnosis (median survival time, 3 mo). Because the specimens stemmed from a period of 20 years, very limited reliable information on pre-existing celiac disease was available. Therefore, typing for HLA-DQB1 was used as a surrogate marker for potential celiac disease association.Table 1Clinical, Morphologic, and Immunophenotypic Features of Studied CasesPatient no.AgeSexSurvival, moStageMorphologyCD3CD4CD8CD30CD56HLA-DQB1 genotype148MDOD 9NSMono sm+−+−+*0201aFour-digit high-resolution SSP-PCR result with notation of corresponding DQ specificity.*0301(DQ7)aFour-digit high-resolution SSP-PCR result with notation of corresponding DQ specificity.264MAWD 27+NSMono sm+−+−+*0301(DQ7)bTwo-digit low-resolution SSP-PCR result with notation of probably corresponding DQ specificity.*05339MDOD 5EIVMono sm+−+ND+*03(DQ8)bTwo-digit low-resolution SSP-PCR result with notation of probably corresponding DQ specificity.*06473MDOD 2NSMono sm+−+−+*0302(DQ8)aFour-digit high-resolution SSP-PCR result with notation of corresponding DQ specificity.584FDOD 1NSMono sm+−−−+*02669FDOD 1NSMono sm+−+−+*02*06751MDOD 1EIVMono sm+−−−+*0301(DQ7)aFour-digit high-resolution SSP-PCR result with notation of corresponding DQ specificity.*0303(DQ9)aFour-digit high-resolution SSP-PCR result with notation of corresponding DQ specificity.877MDOD 3NSMono sm+−+−+*04*05960MDOD 2NSMono sm+−+−+*04*061063MDOD 16EIIBMono sm+−+−+*02*03(DQ7)bTwo-digit low-resolution SSP-PCR result with notation of probably corresponding DQ specificity.1143FDOD 18EI2Mono sm−−−−+*02*051256MDOD 3NSMono sm+−+++*05*061361MDOD 5EIVMono sm+−+−+*03(DQ7)bTwo-digit low-resolution SSP-PCR result with notation of probably corresponding DQ specificity.*051468FDOD 1EIIAMono sm+−−−−*0301(DQ7)aFour-digit high-resolution SSP-PCR result with notation of corresponding DQ specificity.1550FDOD 6EIMono sm+−+−+*03(DQ8)bTwo-digit low-resolution SSP-PCR result with notation of probably corresponding DQ specificity.*051666FDOD 5EIVAnapl−−−+−*021755FDOD 2EIVAnapl+−−+−*021865FDOD 5EIVAnapl+−−+−*021971MDOD 1EIVAnapl+−−+−*02*062066FNED 80+EI2Ib+−++−*022177MDOD 7NSIb+−−+−*02*0302(DQ8)aFour-digit high-resolution SSP-PCR result with notation of corresponding DQ specificity.2266MDOD 1NSPleo m&l−+−+−*03(DQ7)bTwo-digit low-resolution SSP-PCR result with notation of probably corresponding DQ specificity.*062385MDOD 2NSPleo m&l+−++−*05*062463MDOD 25NSPleo m&l+−−+−*022572FDOD 2NSPleo m&l+−+−+*03(DQ7)bTwo-digit low-resolution SSP-PCR result with notation of probably corresponding DQ specificity.*062659MDOD 10EIVBPleo m&l+−−−+*03(DQ7)bTwo-digit low-resolution SSP-PCR result with notation of probably corresponding DQ specificity.*052745MDOD 1NSPleo m&l−−++−*02*052881FDOD 6NSPleo m&l+−++−*02*03(DQ7)bTwo-digit low-resolution SSP-PCR result with notation of probably corresponding DQ specificity.2965MDOD 7EIIPleo m&l+−++−*02*063050MNED 10+EIVUnclassified+−−+−*02NOTE. Stage according to Ann Arbor System as modified by Mussof.DOD, died of disease; NS, not staged; Mono sm, monomorphic small− to medium-sized cell; +, positive for marker; −, negative for marker; AWD, alive with disease; ND, not done; Anapl, anaplastic large cell; NED, no evidence of disease; Ib, immunoblastic; Pleo m&l, pleomorphic medium and large cell.a Four-digit high-resolution SSP-PCR result with notation of corresponding DQ specificity.b Two-digit low-resolution SSP-PCR result with notation of probably corresponding DQ specificity. Open table in a new tab NOTE. Stage according to Ann Arbor System as modified by Mussof. DOD, died of disease; NS, not staged; Mono sm, monomorphic small− to medium-sized cell; +, positive for marker; −, negative for marker; AWD, alive with disease; ND, not done; Anapl, anaplastic large cell; NED, no evidence of disease; Ib, immunoblastic; Pleo m&l, pleomorphic medium and large cell. Immunohistochemical analysis was performed for all cases on formalin-fixed paraffin-embedded tissue sections according to previously published protocols12Rudiger T. Ott G. Ott M.M. Muller-Deubert S.M. Muller-Hermelink H.K. Differential diagnosis between classic Hodgkin's lymphoma, T-cell-rich B-cell lymphoma, and paragranuloma by paraffin immunohistochemistry.Am J Surg Pathol. 1998; 22: 1184-1191Crossref PubMed Scopus (108) Google Scholar (Table 1). Immunostains included markers CD2 (Novocastra, Newcastle, UK; dilution 1:20), CD3 (Dako, Copenhagen, Denmark; 1:400), CD4 (Novocastra; 1:10), CD8 (Dako; 1:30), CD30 (Dako; 1:80), CD56 (Sanbio, Uden, Netherlands; 1:200), and TIA1 (Coulter; Hialeah, FL; 1:800). In all cases, tumor tissue sections were screened for tumor cell content and only cases that contained greater than 60% neoplastic cells were included in this study. DNA was extracted as previously described.13Zettl A. Rudiger T. Konrad M.A. Chott A. Simonitsch-Klupp I. Sonnen R. Muller-Hermelink H.K. Ott G. Genomic profiling of peripheral T-cell lymphoma, unspecified, and anaplastic large T-cell lymphoma delineates novel recurrent chromosomal alterations.Am J Pathol. 2004; 164: 1837-1848Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar In 28 cases, DNA was extracted from formalin-fixed, paraffin-embedded tissue, whereas in 2 cases fresh material was available (cases 15 and 30). In all but 1 case, the material stemmed from the initial surgical resection specimen. In the 1 case, tumorous ascites from a patient with known ETL was used for DNA extraction (case 30). Bacterial artificial chromosome (BAC) array CGH was conducted as previously described.14Baldwin C. Garnis C. Zhang L. Rosin M.P. Lam W.L. Multiple microalterations detected at high frequency in oral cancer.Cancer Res. 2005; 65: 7561-7567PubMed Google Scholar, 15de Leeuw R.J. Davies J.J. Rosenwald A. Bebb G. Gascoyne R.D. Dyer M.J. Staudt L.M. Martinez-Climent J.A. Lam W.L. Comprehensive whole genome array CGH profiling of mantle cell lymphoma model genomes.Hum Mol Genet. 2004; 13: 1827-1837Crossref PubMed Scopus (108) Google Scholar, 16Garnis C. Lockwood W.W. Vucic E. Ge Y. Girard L. Minna J.D. Gazdar A.F. Lam S. Macaulay C. Lam W.L. High resolution analysis of non-small cell lung cancer cell lines by whole genome tiling path array CGH.Int J Cancer. 2006; 118: 1556-1564Crossref PubMed Scopus (111) Google Scholar, 17Ishkanian A.S. Malloff C.A. Watson S.K. DeLeeuw R.J. Chi B. Coe B.P. Snijders A. Albertson D.G. Pinkel D. Marra M.A. Ling V. MacAulay C. Lam W.L. A tiling resolution DNA microarray with complete coverage of the human genome.Nat Genet. 2004; 36: 299-303Crossref PubMed Scopus (531) Google Scholar Briefly, for BAC array CGH, 300 ng of sample or pooled reference male DNA (Novagen, Mississauga, Ontario) was labeled with cyanine-3 deoxycytidine triphosphate or cyanine-5 deoxycytidine triphosphate (Perkin Elmer Life Sciences Inc., Boston, MA), respectively. Labeled probe was purified, precipitated with Cot-1 DNA (Invitrogen, Burlington, Ontario), and resuspended in hybridization buffer.14Baldwin C. Garnis C. Zhang L. Rosin M.P. Lam W.L. Multiple microalterations detected at high frequency in oral cancer.Cancer Res. 2005; 65: 7561-7567PubMed Google Scholar, 15de Leeuw R.J. Davies J.J. Rosenwald A. Bebb G. Gascoyne R.D. Dyer M.J. Staudt L.M. Martinez-Climent J.A. Lam W.L. Comprehensive whole genome array CGH profiling of mantle cell lymphoma model genomes.Hum Mol Genet. 2004; 13: 1827-1837Crossref PubMed Scopus (108) Google Scholar, 16Garnis C. Lockwood W.W. Vucic E. Ge Y. Girard L. Minna J.D. Gazdar A.F. Lam S. Macaulay C. Lam W.L. High resolution analysis of non-small cell lung cancer cell lines by whole genome tiling path array CGH.Int J Cancer. 2006; 118: 1556-1564Crossref PubMed Scopus (111) Google Scholar, 17Ishkanian A.S. Malloff C.A. Watson S.K. DeLeeuw R.J. Chi B. Coe B.P. Snijders A. Albertson D.G. Pinkel D. Marra M.A. Ling V. MacAulay C. Lam W.L. A tiling resolution DNA microarray with complete coverage of the human genome.Nat Genet. 2004; 36: 299-303Crossref PubMed Scopus (531) Google Scholar Probe mixtures then were denatured, allowed to block at 45°C, and applied to tiling-path arrays composed of 26,819 duplicate spotted BAC clones (53,638 elements) selected from the previously described submegabase resolution tiling set to give optimal genome coverage (available at: http://www.bccrc.ca/arraycgh/).17Ishkanian A.S. Malloff C.A. Watson S.K. DeLeeuw R.J. Chi B. Coe B.P. Snijders A. Albertson D.G. Pinkel D. Marra M.A. Ling V. MacAulay C. Lam W.L. A tiling resolution DNA microarray with complete coverage of the human genome.Nat Genet. 2004; 36: 299-303Crossref PubMed Scopus (531) Google Scholar, 18Watson S.K. Deleeuw R.J. Horsman D.E. Squire J.A. Lam W.L. Cytogenetically balanced translocations are associated with focal copy number alterations.Hum Genet. 2007; 120: 795-805Crossref PubMed Scopus (36) Google Scholar, 19Wong K.K. Dosanjh N.S. Kimm L.R. Cheng Z. Horsman D.E. MacAulay C. Ng R.T. Brown C.J. Eichler E.E. Lam W.L. A comprehensive analysis of common copy number variations in the human genome.Am J Human Genet. 2007; 80 (dR): 91-104Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar These arrays were batch quality-controlled by hybridization and analysis of cell-line material with known alterations. The arrays were hybridized at 45°C for approximately 40 hours, washed with 0.1× standard saline citrate/0.1% sodium dodecyl sulfate, and rinsed with 0.1× standard saline citrate. Imaging and analysis were conducted as previously described.14Baldwin C. Garnis C. Zhang L. Rosin M.P. Lam W.L. Multiple microalterations detected at high frequency in oral cancer.Cancer Res. 2005; 65: 7561-7567PubMed Google Scholar, 15de Leeuw R.J. Davies J.J. Rosenwald A. Bebb G. Gascoyne R.D. Dyer M.J. Staudt L.M. Martinez-Climent J.A. Lam W.L. Comprehensive whole genome array CGH profiling of mantle cell lymphoma model genomes.Hum Mol Genet. 2004; 13: 1827-1837Crossref PubMed Scopus (108) Google Scholar, 16Garnis C. Lockwood W.W. Vucic E. Ge Y. Girard L. Minna J.D. Gazdar A.F. Lam S. Macaulay C. Lam W.L. High resolution analysis of non-small cell lung cancer cell lines by whole genome tiling path array CGH.Int J Cancer. 2006; 118: 1556-1564Crossref PubMed Scopus (111) Google Scholar, 17Ishkanian A.S. Malloff C.A. Watson S.K. DeLeeuw R.J. Chi B. Coe B.P. Snijders A. Albertson D.G. Pinkel D. Marra M.A. Ling V. MacAulay C. Lam W.L. A tiling resolution DNA microarray with complete coverage of the human genome.Nat Genet. 2004; 36: 299-303Crossref PubMed Scopus (531) Google Scholar Briefly, a charged-couple device camera system was used to capture the cyanine-3 and cyanine-5 channels of hybridized arrays (Applied Precision, Issaquah, WA). The images then were analyzed using SoftWoRx analysis software (Applied Precision). The resultant data were normalized using a stepwise normalization algorithm.20Khojasteh M. Lam W.L. Ward R.K. MacAulay C. A stepwise framework for the normalization of array CGH data.BMC Bioinformatics. 2005; 6: 274Crossref PubMed Scopus (78) Google Scholar Custom software SeeGH was used to visualize all data.21Chi B. DeLeeuw R.J. Coe B.P. MacAulay C. Lam W.L. SeeGH—a software tool for visualization of whole genome array comparative genomic hybridization data.BMC Bioinformatics. 2004; 5: 13Crossref PubMed Scopus (75) Google Scholar The software program aCGH-Smooth was used to determine breakpoints and relative levels of copy number.22Jong K. Marchiori E. Meijer G. Vaart A.V. Ylstra B. Breakpoint identification and smoothing of array comparative genomic hybridization data.Bioinformatics. 2004; 20: 3636-3637Crossref PubMed Scopus (149) Google Scholar Deletion, loss, normal, gain, and amplification levels were classified as less than −0.8, −0.8 to −0.2, −0.2 to +0.2, +0.2 to +0.8, and greater than +.8, respectively, as previously determined for this platform by replicate analysis of normal vs normal DNA samples.15de Leeuw R.J. Davies J.J. Rosenwald A. Bebb G. Gascoyne R.D. Dyer M.J. Staudt L.M. Martinez-Climent J.A. Lam W.L. Comprehensive whole genome array CGH profiling of mantle cell lymphoma model genomes.Hum Mol Genet. 2004; 13: 1827-1837Crossref PubMed Scopus (108) Google Scholar Regions of copy number alteration first were determined by aCGH-Smooth analysis and then compared with the raw normalized data for verification. HLA-DQB1 genotyping was performed at the Department of Transfusion Medicine, University of Würzburg. Tumor DNA was tested first with low-resolution molecular techniques (2-digit notation) using commercially available kits (Dynal AllSet+ sequence specific primer [SSP] DQ low resolution [Invitrogen, Karlsruhe, Germany] or Olerup SSP HLA-DQ [Qiagen, Düsseldorf, Germany]) to identify HLA-DQ2 (HLA-DQB1*02 group or alleles) and HLA-DQ8. To distinguish those DQB1*03 alleles that correspond to the DQ8 specifically (DQB1*0302, 0310) from the others or to control low-resolution results, high-resolution SSP polymerase chain reaction (Olerup SSP DQB1*03 or Olerup SSP DQB1 high-resolution kit) was performed. Homozygous results were confirmed by testing in a second low-resolution technique using the Dynal SSO DRB1* kit. HLA typing was limited to HLA-DQB1 without further typing for HLA-DQA1. About 95% of celiac disease patients carry HLA-DQ2 (DQA1*0501/DQB1*0201), most individuals that are not HLA-DQ2 positive express HLA-DQ8 (DQA1*0501/DQB1*0302).7Stepniak D. Koning F. Celiac disease—sandwiched between innate and adaptive immunity.Hum Immunol. 2006; 67: 460-468Crossref PubMed Scopus (115) Google Scholar The HLA-DQ2 heterodimer can be formed by 5 combinations23Koning F. Celiac disease: caught between a rock and a hard place.Gastroenterology. 2005; 129: 1294-1301Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar: DQA1*0501 + DQB1*0201 (the most frequent HLA-DQ haplotype found in CD), DQA1*0501 + DQB1*0202, DQA1*0505 + DQB1*0201, and DQA1*0505 + DQB1*0202, encoded in cis or trans.24Sollid L.M. Coeliac disease: dissecting a complex inflammatory disorder.Nat Rev Immunol. 2002; 2: 647-655Crossref PubMed Scopus (827) Google Scholar The fifth possible HLA-DQ2 heterodimer, DQA1*0201 + DQB1*0202, is not associated with celiac disease. Thus, by limiting HLA typing to HLA-DQB1, the actual frequency of HLA-DQ2 may be overestimated slightly. Celiac disease is extremely rare in patients not carrying either HLA-DQ molecule.6Green P.H. Jabri B. Coeliac disease.Lancet. 2003; 362: 383-391Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar In addition, homozygosity for HLA-DQ2 is associated with at least a 5-fold higher risk of disease development than DQ2 heterozygosity.8Al-Toma A. Goerres M.S. Meijer J.W. Pena A.S. Crusius J.B. Mulder C.J. Human leukocyte antigen-DQ2 homozygosity and the development of refractory celiac disease and enteropathy-associated T-cell lymphoma.Clin Gastroenterol Hepatol. 2006; 4: 315-319Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 9Vader W. Stepniak D. Kooy Y. Mearin L. Thompson A. van Rood J.J. Spaenij L. Koning F. The HLA-DQ2 gene dose effect in celiac disease is directly related to the magnitude and breadth of gluten-specific T cell responses.Proc Natl Acad Sci U S A. 2003; 100: 12390-12395Crossref PubMed Scopus (313) Google Scholar The correlations of all recurrent genetic events to each other and to all clinical features were calculated using a 2-tailed Fisher exact test for categoric variables (Supplementary Table 1). Data for principal component analysis were derived by determining the mean value for all array elements within the annotated regions of interest (Supplementary Table 2). Principal component analysis is a classic statistical method used to reduce the dimensionality of the data set to discover potential classifications.25Hoglund M. Gisselsson D. Sall T. Mitelman F. Coping with complexity multivariate analysis of tumor karyotypes.Cancer Genet Cytogenet. 2002; 135: 103-109Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Analysis of the samples was performed using the Statistics Toolbox (version 5.1) of MATLAB (version 7.1) (The MathWorks Inc., Natick, MA). The immunophenotypical features of the cases are summarized in Table 1. Briefly, the majority of ETL cases (26 of 30) were CD3+/CD4−. Of the remaining 4 cases, 3 were CD3−/CD4− and 1 was CD3−/CD4+. Seventeen ETL cases were CD8+, of which 10 were of monomorphic small- to medium-sized tumor cell morphology. Anaplastic large-cell morphology correlated with CD30 positivity (P = .037) and CD8 negativity (P = .026). Fifteen ETL cases were CD56+, 13 of which were monomorphic, whereas the remaining 2 were pleomorphic. In our study, CD56 expression correlated well with monomorphic cytomorphology (P < .0001), which is in agreement with a previous report by Chott et al.3Chott A. Haedicke W. Mosberger I. Fodinger M. Winkler K. Mannhalter C. Muller-Hermelink H.K. Most CD56+ intestinal lymphomas are CD8+CD5-T-cell lymphomas of monomorphic small to medium size histology.Am J Pathol. 1998; 153: 1483-1490Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar Thirty cases of ETL were analyzed by CGH to BAC arrays to determine genetic alterations associated with clinical and immunophenotypic features. The majority of samples assayed showed multiple genetic alterations throughout the genome (Figure 1). In fact, all regions of the genome were altered in at least 2 cases, except 2p11-p13, 4q13-q26, 6q14-q16, and 11p12-p15. The raw data used to generate the 30 array CGH profiles have been made publicly available at http://www.bccrc.ca/cg/ArrayCGH_Group.html. Scanning tumor genomes at tiling-resolution revealed a total of 750 copy number alterations (median per case, 22; range, 7–59), with 416 losses (median, 12; range, 1–43) and 334 gains (median, 10.5; range, 3–23) (Figure 2). From these, regions of alteration (ROA) were identified based on the frequency and exclusivity of gains or losses. Detailed descriptions of all ROA are listed in Figure 3.Figure 2Summary of chromosomal imbalances detected by tiling-path array CGH in 30 ETL samples. Green lines on the left side of the ideogram indicate loss of chromosomal material; red lines on the right side indicate a gain of chromosomal material. High-frequency small losses at 7q34 and 14q11.2 are caused by TCR gene rearrangement. 15q11.2 contains a natural copy number polymorphism.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Recurrent gains and losses in ETL. Chromosome 9q gains were found in all morphologic subtypes of ETL. However, 9q31.3-33.2 gains were predominantly absent in ETL with 16q12.1 losses (P < .01). Gains of 1q32.2-q41 and 5q34-q35.2 and those of 7q11.23-q21.3 and 8q13.3-q21.11 tended to occur within the same samples (P < .01). Compared with nonmonomo
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