The Frequency of B- and T-Cell Gene Rearrangements and Epstein-Barr Virus in T-Cell Lymphomas
2006; Elsevier BV; Volume: 8; Issue: 4 Linguagem: Inglês
10.2353/jmoldx.2006.060016
ISSN1943-7811
AutoresBrent Tan, Roger A. Warnke, Daniel A. Arber,
Tópico(s)Eosinophilic Disorders and Syndromes
ResumoWe report on a series of 58 cases of angioimmunoblastic T-cell lymphoma (AILT) and 59 cases of peripheral T-cell lymphoma, unspecified (PTCL-NOS). Subsets of cases from both diagnostic groups were complicated by associated B-cell proliferations, and we performed B- and T-cell clonality studies and in situ hybridization for Epstein-Barr virus (EBV) to investigate the relationship between B-cell proliferation, B-cell clonality, and EBV. Using multiplex polymerase chain reaction assays based on the BIOMED-2 collaborative study, we detected TCRγ T-cell clones in 78 and 81% of AILT and PTCL-NOS cases, respectively, and IGH B-cell clones in 34 and 35% of AILT and PTCL-NOS cases, respectively. The majority of cases contained EBV-positive cells, including 50% of AILT and 57% of PTCL-NOS cases, and cases with B-cell proliferations were more often EBV-positive. Although a relatively high rate of B-cell clonality has been shown for AILT, our findings for PTCL-NOS differ from previous reports in that B-cell clonality was relatively frequent. Overall, a positive B-cell clone correlated, in part, with the presence of a B-cell proliferation but not with EBV. Our findings demonstrate that B-cell clonality is a common finding in AILT and PTCL-NOS, and its presence should not negate the diagnosis established by morphologic, immunophenotypic, and clinical findings. We report on a series of 58 cases of angioimmunoblastic T-cell lymphoma (AILT) and 59 cases of peripheral T-cell lymphoma, unspecified (PTCL-NOS). Subsets of cases from both diagnostic groups were complicated by associated B-cell proliferations, and we performed B- and T-cell clonality studies and in situ hybridization for Epstein-Barr virus (EBV) to investigate the relationship between B-cell proliferation, B-cell clonality, and EBV. Using multiplex polymerase chain reaction assays based on the BIOMED-2 collaborative study, we detected TCRγ T-cell clones in 78 and 81% of AILT and PTCL-NOS cases, respectively, and IGH B-cell clones in 34 and 35% of AILT and PTCL-NOS cases, respectively. The majority of cases contained EBV-positive cells, including 50% of AILT and 57% of PTCL-NOS cases, and cases with B-cell proliferations were more often EBV-positive. Although a relatively high rate of B-cell clonality has been shown for AILT, our findings for PTCL-NOS differ from previous reports in that B-cell clonality was relatively frequent. Overall, a positive B-cell clone correlated, in part, with the presence of a B-cell proliferation but not with EBV. Our findings demonstrate that B-cell clonality is a common finding in AILT and PTCL-NOS, and its presence should not negate the diagnosis established by morphologic, immunophenotypic, and clinical findings. Peripheral T-cell lymphoma (PTCL) is an uncommon malignancy, accounting for less than 10% of non-Hodgkin lymphomas worldwide. By current World Health Organization criteria, PTCLs are subclassified based on clinical, histologic, immunophenotypic, and genetic findings.1Jaffe ES Harris NL Stein H Vardiman JW Kleihues P Sobin LH Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon2001Google Scholar However, the diagnosis is often difficult for pathologists because PTCLs are uncommon, and the histologic findings can be nonspecific and can overlap with reactive conditions. Perhaps the most challenging aspect is the lack of good immunophenotypic markers to establish clonality for T-cell lineage neoplasms, and pathologists often rely on DNA-based studies for evidence of clonality.Given the difficulty in diagnosing PTCL, pathologists often perform both B- and T-cell clonality studies to clarify lineage and provide support for malignancy. Currently, the most commonly used method is the polymerase chain reaction (PCR), which has largely replaced Southern blot-based clonality assays. However, there are several caveats to this approach. In particular, studies evaluating large numbers of PTCLs by PCR show that simultaneous B- and T-cell clones occur relatively often (9 to 16%).2Diss TC Watts M Pan LX Burke M Linch D Isaacson PG The polymerase chain reaction in the demonstration of monoclonality in T cell lymphomas.J Clin Pathol. 1995; 48: 1045-1050Crossref PubMed Scopus (130) Google Scholar3Theriault C Galoin S Valmary S Selves J Lamant L Roda D Rigal-Huguet F Brousset P Delsol G Al Saati T PCR analysis of immunoglobulin heavy chain (IgH) and TcR-gamma chain gene rearrangements in the diagnosis of lymphoproliferative disorders: results of a study of 525 cases.Mod Pathol. 2000; 13: 1269-1279Crossref PubMed Scopus (111) Google Scholar4Garcia MJ Martinez-Delgado B Granizo JJ Benitez J Rivas C IgH, TCR-gamma, and TCR-beta gene rearrangement in 80 B- and T-cell non-Hodgkin's lymphomas: study of the association between proliferation and the so-called "aberrant" patterns.Diagn Mol Pathol. 2001; 10: 69-77Crossref PubMed Scopus (31) Google Scholar5Vergier B Dubus P Kutschmar A Parrens M Ferrer J de Mascarel A Merlio JP Combined analysis of T cell receptor gamma and immunoglobulin heavy chain gene rearrangements at the single-cell level in lymphomas with dual genotype.J Pathol. 2002; 198: 171-180Crossref PubMed Scopus (26) Google Scholar6Noorali S Pervez S Moatter T Soomro IN Kazmi SU Nasir MI Smith JL Characterization of T-cell non-Hodgkin's lymphoma and its association with Epstein-Barr virus in Pakistani patients.Leuk Lymphoma. 2003; 44: 807-813Crossref PubMed Scopus (10) Google Scholar These cases, with both B- and T-cell clones, present a diagnostic dilemma for the pathologist and treating oncologist.There are numerous possible explanations for the detection of two clones in some cases, including those that are technical in nature, so-called lineage infidelity of a single clone, and the presence of two different clones in a sample.7van Dongen JJ Wolvers-Tettero IL Analysis of immunoglobulin and T cell receptor genes. Part II: possibilities and limitations in the diagnosis and management of lymphoproliferative diseases and related disorders.Clin Chim Acta. 1991; 198: 93-174Crossref PubMed Scopus (179) Google Scholar The phenomenon referred to as lineage infidelity, in this context, is the result of recombination of both T-cell receptor (TCR) and immunoglobulin (IG) genes in the same clone.7van Dongen JJ Wolvers-Tettero IL Analysis of immunoglobulin and T cell receptor genes. Part II: possibilities and limitations in the diagnosis and management of lymphoproliferative diseases and related disorders.Clin Chim Acta. 1991; 198: 93-174Crossref PubMed Scopus (179) Google Scholar This finding is more frequently seen in immature hematolymphoid neoplasms and is uncommon in mature B- and T-cell non-Hodgkin lymphomas.7van Dongen JJ Wolvers-Tettero IL Analysis of immunoglobulin and T cell receptor genes. Part II: possibilities and limitations in the diagnosis and management of lymphoproliferative diseases and related disorders.Clin Chim Acta. 1991; 198: 93-174Crossref PubMed Scopus (179) Google Scholar8Smith LJ Curtis JE Messner HA Senn JS Furthmayr H McCulloch EA Lineage infidelity in acute leukemia.Blood. 1983; 61: 1138-1145PubMed Google Scholar9Greaves MF Chan LC Furley AJ Watt SM Molgaard HV Lineage promiscuity in hemopoietic differentiation and leukemia.Blood. 1986; 67: 1-11Crossref PubMed Google Scholar10McCulloch EA Lineage infidelity or lineage promiscuity?.Leukemia. 1987; 1: 235PubMed Google Scholar In recent years, cases of PTCL with associated B-cell proliferations have been described. Many of these cases are positive for both B- and T-cell clones, and in our series of peripheral T-cell lymphoma, unspecified (PTCL-NOS), a subset of B-cell proliferations were associated with Epstein-Barr virus (EBV).11Higgins JP van de Rijn M Jones CD Zehnder JL Warnke RA Peripheral T-cell lymphoma complicated by a proliferation of large B cells.Am J Clin Pathol. 2000; 114: 236-247Crossref PubMed Scopus (2) Google Scholar Others have reported similar B-cell proliferations in PCTL-NOS as well as angioimmunoblastic T-cell lymphoma (AILT), and many of these cases are EBV-positive and exhibit B-cell clones.12Lome-Maldonado C Canioni D Hermine O Delabesse E Damotte D Raffoux E Gaulard P Macintyre E Brousse N Angio-immunoblastic T cell lymphoma (AILD-TL) rich in large B cells and associated with Epstein-Barr virus infection. A different subtype of AILD-TL?.Leukemia. 2002; 16: 2134-2141Crossref PubMed Scopus (42) Google Scholar13Xu Y McKenna RW Hoang MP Collins RH Kroft SH Composite angioimmunoblastic T-cell lymphoma and diffuse large B cell lymphoma: a case report and review of the literature.Am J Clin Pathol. 2002; 118: 848-854Crossref PubMed Scopus (55) Google Scholar14Zettl A Lee SS Rudiger T Starostik P Marino M Kirchner T Ott M Muller-Hermelink HK Ott G Epstein-Barr virus-associated B-cell lymphoproliferative disorders in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, unspecified.Am J Clin Pathol. 2002; 117: 368-379Crossref PubMed Scopus (161) Google ScholarWe hypothesized that the relative frequency of B-cell clones in PTCL may be because of associated B-cell proliferations and/or EBV. To address this issue, we performed PCR-based B- and T-cell clonality studies on a total of 117 cases of PTCL. The series is composed of 58 cases of AILT, 12 of which show an associated B-cell proliferation, and 59 cases of PTCL-NOS, 11 of which show an associated B-cell proliferation. In addition, we performed in situ hybridization for EBV to characterize the relationship between the B-cell proliferation, EBV, and underlying T-cell lymphoma.In this article, we describe the results of these studies, which include a high overall frequency of B-cell clones in both AILT (34%) and PTCL-NOS (35%) that correlates, in part, with an associated B-cell proliferation but not with EBV. Interestingly, cases without demonstrable B-cell proliferations also exhibited B-cell clonality, although with less frequency than cases with B-cell proliferations. This finding suggests that additional factors may contribute to B-cell clonality.Materials and MethodsCasesFifty-eight cases of AILT and fifty-nine cases of PTCL-NOS were selected from the Laboratory of Hematopathology, Stanford University Medical Center, Stanford, CA. The cases were received between January 1, 2000, and April 4, 2005, and a diagnosis was rendered based on criteria established by the World Health Organization.1Jaffe ES Harris NL Stein H Vardiman JW Kleihues P Sobin LH Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon2001Google Scholar AILT and PTCL-NOS cases were further subclassified as to whether they showed an associated-B-cell proliferation. Briefly, a B-cell proliferation was defined as a readily identifiable infiltrate of B cells complicating a PTCL as described by Higgins and colleagues.11Higgins JP van de Rijn M Jones CD Zehnder JL Warnke RA Peripheral T-cell lymphoma complicated by a proliferation of large B cells.Am J Clin Pathol. 2000; 114: 236-247Crossref PubMed Scopus (2) Google Scholar Cases were selected based on availability of paraffin-embedded tissue and/or residual DNA remaining from clonality studies performed for the original diagnosis. Use of tissue for this study was approved by the Stanford University Panel on Medical Human Subjects [protocol ID 79034; institutional review board number 348 (panel 1)].Histology and ImmunohistochemistryHistologic sections were prepared from formalin-fixed, paraffin-embedded tissue by cutting 3- to 4-μm-thick sections and staining with hematoxylin and eosin. Sections were stained for immunohistochemistry on a Ventana BenchMark instrument (Ventana Medical Systems, Tucson, AZ) using the biotin-avidin technique in which diaminobenzidine was used as a chromogen.15Bindl JM Warnke RA Advantages of detecting monoclonal antibody binding to tissue sections with biotin and avidin reagents in Coplin jars.Am J Clin Pathol. 1986; 85: 490-493PubMed Google Scholar All cases were stained with at least one B- and one T-cell marker. The following monoclonal antibodies were used: 4C7 (anti-CD5; Vision Bio-System, Norwell, MA), 56C6 (anti-CD10; Vision Bio-System), L26 (anti-CD20; DAKO, Carpinteria, CA), IF8 (anti-CD21; DAKO), IB12 (anti-CD23; Vision Bio-System), JCV117 (anti-CD79a; DAKO), and B-B4 (anti-CD138; Serotec, Raleigh, NC). Polyclonal antibodies directed against the following antigens were used: CD3 (Cell Marque, Hot Springs, AR) and κ and λ immunoglobulin light chains (DAKO). Antigen retrieval was achieved through automated heat pretreatment for 4C7, 56C6, L26, IB12, JCV117, B-B4, and antibody against CD3 (Ventana Medical Systems). Antigen retrieval was achieved through automated protease pretreatment for IF8 and antibodies against immunoglobulin light chains (Ventana Medical Systems).Isolation of DNADNA was obtained from formalin-fixed, paraffin-embedded tissue blocks by cutting four to eight 20-μm-thick sections followed by deparaffinization by extracting three times in 1.0 ml of either xylene or Histoclear (National Diagnostics, Atlanta, GA). The extracted tissue was washed two times in 1.0 ml of 100% ethanol and then dried at 65°C. The tissue was resuspended in 2 vol (30 to 250 μl) of a mixture of 200 mmol/L potassium chloride, 40 mmol/L Tris [Tris(hydroxymethyl)aminomethane]-hydrochloride (pH 8.5), 0.1% sodium dodecyl sulfate, and 0.6 mg/ml proteinase K and incubated at 55 to 65°C overnight. The sample was then boiled for 8 minutes and centrifuged at 15,000 × g for 10 minutes, and the DNA-containing supernatant was collected. To evaluate the yield of DNA, a 5-μl aliquot of supernatant was separated by electrophoresis on a 0.8% agarose gel (Invitrogen, Carlsbad, CA), stained with ethidium bromide, and visualized by ultraviolet illumination. Based on an estimate of DNA concentration, the crude extract was diluted with an appropriate volume of reagent-grade water (Teknova, Hollister, CA) and used directly in a PCR. Typically, a 1:10 to 1:20 dilution was prepared, and 5 μl was used for the PCR.B-Cell and T-Cell Clonality StudiesCases were evaluated for B- and T-cell clonality using two commercially available PCR-based kits that detect clonal rearrangements in IGH and TCRγ, respectively (InVivoScribe Technologies, San Diego, CA). These multiplex PCRs are based on a European collaborative study (BIOMED-2 Concerted Action).16van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA 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 (2470) Google Scholar The PCRs were performed according to the manufacturers' protocols. Briefly, a 5-μl aliquot of DNA sample was added to 45 μl of each reaction mixture and 1.25 U of AmpliTaq Gold (Applied Biosystems, Foster City, CA). Amplification was performed in a Perkin-Elmer 9700 thermocycler (Perkin-Elmer Applied Biosystems, Foster City, CA) by initially heating at 95°C for 7 minutes, followed by 35 cycles of 95°C for 45 seconds, 60°C for 45 seconds, and 72°C for 90 seconds. The final step was incubation at 72°C for 10 minutes.After amplification, 1 μl of PCR product was added to 10 μl of HI-Deionized Formamide (Applied Biosystems) and 1 μl of ROX-250 internal size standard (Applied Biosystems). The mixture was then denatured at 95°C for 5 minutes, chilled on ice for 5 minutes, and resolved by capillary electrophoresis on an ABI 3100 instrument using performance-optimized polymer-4 (POP-4; Applied Biosystems). The data were stored electronically and analyzed using GeneScan analysis software (Applied Biosystems). Printed electropherograms were reviewed independently by two separate pathologists (B.T.T. and D.A.A.) who were blinded to the identity of specimens. Criteria for assigning a clone were similar to those used for clinical specimens at the Stanford molecular pathology laboratory. Briefly, a clone was required to yield one or two distinct peaks within the expected size range for a given primer set. In some cases, a polyclonal background was present in addition to the distinct peak(s). To meet criteria for a clone, the height of at least one distinct peak had to exceed that of the polyclonal background by at least twofold. When a primer set yielded three or more distinct peaks, the results were classified as oligoclonal, regardless of whether a polyclonal background was present or absent. Cases with a clonal peak demonstrated by one or multiple primer sets were scored as positive. Cases with only polyclonal and/or oligoclonal results were scored as negative.Control specimens included clonal DNA isolated from cell lines (InVivoScribe), polyclonal DNA isolated from human tonsil specimens (InVivoScribe), a sensitivity control composed of clonal DNA diluted in polyclonal DNA, and a blank composed of reagent-grade water (Teknova). Multiple polyclonal DNA specimens, each isolated from different patients (InVivoScribe), consistently yielded polyclonal signals and no clonal signals. For a run of clonality studies to be accepted into the data set, a positive clone had to be detectable from the sensitivity control, which was prepared at 2 or 3% (expressed as μg of clonal DNA/μg of polyclonal DNA) for TCRγ or IGH assays, respectively. Individual DNA specimens had to yield detectable products of at least 300 nucleotides from the control amplification tube, which contained primers to amplify HLA-DQ. Samples yielding inadequate amplification were purified using a QIAamp DNA blood mini kit (Qiagen, Valencia, CA), and the purified DNA typically yielded adequate amplification. In our validation of the InVivoScribe TCRγ and IGH kits, we were able to detect clones in 80 to 90% of known B- and T-cell neoplasms, and amplification of the provided polyclonal control DNA (InVivoScribe) or DNA isolated from paraffin-embedded reactive tonsil specimens in our laboratory yielded only polyclonal results.Sequencing of PCR ProductsEight cases yielding clonal Vγ10 rearrangements by GeneScan analysis were reamplified from the original DNA sample using a TCRγ clonality kit that uses primers of identical sequence to those used for GeneScan analysis, but the primers lack a 5′-fluorescent label, which would otherwise interfere with subcloning (InVivoScribe). The amplification products were separated by electrophoresis on a 2% low melting point agarose gel (Invitrogen), visualized by ethidium bromide staining, excised from the gel, and purified using a QIAquick gel extraction kit (Qiagen). The purified products were ligated to a plasmid vector using a TOPO TA cloning kit for sequencing (Invitrogen), and the ligation products were transformed into Escherichia coli. From each transformation, three bacterial colonies were picked, bacterial cultures were grown, and plasmid DNA was prepared using a PureLink quick plasmid miniprep kit (Invitrogen). The DNA was sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) with a bacteriophage T7 primer (Invitrogen). The resulting sequences were compared to those of Vγ10 listed on the international ImMunoGeneTics website (http://imgt.cines.fr/as accessed on January 23, 2006).In Situ Hybridization for EBVEBV-encoded small RNA (EBER) was detected from 3- to 4-μm-thick, formalin-fixed, paraffin-embedded tissue sections by in situ hybridization using a Ventana BenchMark instrument running a standardized program incorporating deparaffinization, hybridization to the Inform EBER probe cocktail, and staining with ISH iVIEW nitro blue tetrazolium (Ventana Medical Systems). Cases were evaluated for EBV-labeled cells under visible light microscopy, and the number of cells with blue-colored nuclei was visually estimated at 0, 10 to 100, or >100 per medium-power field using a ×15 ocular and ×20 objective lens on an Olympus BX50 microscope (Olympus, Tokyo, Japan). The minimum number of labeled nuclei required for a nonzero score was three per section, which is more than that typically seen in a lymph node or tonsil removed from a healthy individual without evidence of immunodeficiency (B.T.T. and R.A.W., unpublished observations). In approximately half of the cases studied for EBV (depending on availability of sample), a control hybridization was performed with an oligo-deoxythymidine probe to detect polyadenylated mRNA. In addition, an external positive control consisting of an EBV-positive malignancy (NK/T-cell lymphoma or nasopharyngeal carcinoma) was run with each set of hybridizations.Statistical AnalysisThe data were analyzed using the software program JMP-IN version 5.1 (SAS Institute, Cary, NC). Age was entered as a continuous numerical parameter, and all other data were entered as categorical parameters. Differences in age between categorically defined groups were evaluated by t-test and f-test, and the lower P value was recorded. Correlations between categorical parameters were evaluated by Pearson's χ2 test and likelihood ratio test, and the lower P value was recorded. Only P values <0.15 were reported, except for some correlations with EBV, which were based on a lower number of cases.ResultsCasesWe collected 58 cases of AILT and 59 cases of PTCL-NOS from the Stanford hematopathology laboratory. Each case corresponded to a different patient, and all patients were adults. The mean ages of patients with AILT and PTCL-NOS were 64 and 62 years, respectively. Men slightly outnumbered women, accounting for 60 and 61% of patients with AILT and PTCL-NOS, respectively. Comparison of the AILT versus PTCL-NOS groups yielded no statistically significant difference in patient age or sex. The majority of specimens were from lymph nodes, and cases with involvement limited to skin, central nervous system, bone marrow, or body fluid/cavity were excluded. There were a total of 11 extranodal specimens, including five lung, two liver, one mediastinum, one spleen, one stomach, and one subcutaneous tissue specimen. The subcutaneous specimen was in the AILT group, and other extranodal specimens were in the PTCL-NOS group.All cases of AILT showed the histologic features of an atypical mixed infiltrate within a background of prominent arborizing vessels. In Figure 1, A–F, representative histologic, immunohistochemical, and EBV in situ studies are shown for a case of AILT with an EBV-associated large B-cell proliferation. For 56 of the 58 total AILT cases, a network of extrafollicular dendritic cells was demonstrated by immunohistochemical staining for CD21 and/or CD23. In the remaining two cases, the diagnosis was rendered based on characteristic morphology and staining demonstrating T-cell lineage. A stain for CD10 was performed in 33 cases, demonstrating scattered CD10-positive cells unassociated with follicles in 20 cases, negative results in seven cases, and equivocal results in six cases. Eleven cases of AILT were complicated by an associated proliferation of B cells with seven composed of large B cells and four composed of small B cells. Two large B-cell proliferations and one small B-cell proliferation were extensive and met criteria for B-cell lymphoma based on the sheet-like architecture of the B-cell proliferation. One additional case showed an associated proliferation of plasma cells and was considered in the subgroup of cases with B-cell proliferations. The B-lineage proliferations were demonstrated by stains for various antigens including CD20, CD79a, CD138, and κ and λ immunoglobulin light chains.The PTCL-NOS group was composed of cases that met criteria for peripheral T-cell lymphoma, unspecified, by the current World Health Organization classification.1Jaffe ES Harris NL Stein H Vardiman JW Kleihues P Sobin LH Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon2001Google Scholar These criteria are exclusionary in nature, and cases with morphologic, immunophenotypic, or clinical features diagnostic or suggestive of specific subtypes of PTCL were excluded from the PTCL-NOS group. In Figure 1, G and H, representative histologic and EBV in situ studies are shown for a case of PTCL-NOS. For 21 of the 59 total PTCL-NOS cases, an immunohistochemical stain for CD21 was performed and was negative in all 21 cases. A stain for CD10 was performed in 12 cases, demonstrating scattered CD10-positive cells in three cases, negative results in five cases, and equivocal results in four cases. Eleven cases of PTCL-NOS were complicated by an associated proliferation of B cells with seven composed of large B cells, three composed of small B cells, and one composed of mixed small and large B cells. One large B-cell proliferation and one small B-cell proliferation were classified as B-cell lymphoma based on the sheet-like architecture of the B-cell proliferation. The B-cell proliferations were demonstrated by stains for various antigens including CD20, CD79a, and κ and λ immunoglobulin light chains.T-Cell Clonality StudiesWe performed T-cell clonality studies using a multiplex PCR developed in a European BIOMED-2 collaborative study that detects clonal rearrangements in the T-cell receptor γ chain gene (TCRγ).16van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA 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 (2470) Google Scholar Data from a representative TCRγ clonality study are shown in Figure 2, and the results for all cases are summarized in Table 1. The proportion of cases with a positive T-cell clone was 78 and 81% for total AILT and total PTCL-NOS cases, respectively. These figures included cases with associated B-cell proliferations, and when considered separately, 75% of AILT cases with B-cell proliferations and 73% of PTCL-NOS cases with B-cell proliferations were positive for T-cell clonality. There was no statistically significant correlation between T-cell clonality and diagnostic group, presence or absence of a B-cell proliferation, or the results of B-cell clonality or EBV studies. Interestingly, a positive T-cell clone correlated with older age (P = 0.045) and male sex (P = 0.009).Figure 2Electropherogram of PCR for TCRγ rearrangements. The x axis indicates DNA length in nucleotides. The y axis indicates relative fluorescence units. Shown is electrophoresis of products from one multiplex tube, which contains primers to amplify Vγ1-8 and/or Vγ10 rearrangements, with expected product sizes above the x axis. The case was interpreted as positive with a clonal peak detected by Vγ10 primers (arrow) and polyclonal signals detected by Vγ1-8 primers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1T-Cell Clonality by PCR for TCRγ by DiagnosisNumber of positive cases detected by specific primer sets (%)DiagnosisNumber of cases studiedNumber of positive cases (%)Vγ1-8Vγ9Vγ10Vγ11Vγ9/11*Vγ9/11 indicates a clonal PCR product within a range of sizes that is indeterminate between Vγ9 and Vγ11.AILT Without B-cell proliferation4636 (78)23 (50)18 (39)22 (48)4 (9)6 (13) With B-cell proliferation†One case yielded oligoclonal Vγ10 and clonal Vγ9/11 products and was scored as positive.129 (75)6 (50)2 (17)6 (50)0 (0)2 (17) Total5845 (78)29 (50)20 (34)28 (48)4 (7)8 (14)PTCL-NOS Without B-cell proliferation‡One case yielded oligoclonal Vγ1-8 products only and was scored as negative.4840 (83)29 (60)20 (42)21 (44)4 (8)2 (4) With B-cell proliferation118 (73)8 (73)4 (36)6 (55)0 (0)1 (9) Total5948 (81)37 (63)24 (41)27 (46)4 (7)3 (5)* Vγ9/11 indicates a clonal PCR product within a range of sizes that is indeterminate between Vγ9 and Vγ11.† One case yielded oligoclonal Vγ10 and clonal Vγ9/11 products and was scored as positive.‡ One case yielded oligoclonal Vγ1-8 products only and was scored as negative. Open table in a new tab Also listed in Table 1 are the frequencies of clonal detection for individual primer sets, which targeted rearrangements between specific variable genes (Vγ) and the joining segment (Jγ) of TCRγ. For a given case, clonality was often demonstrable by more than one primer set, as summarized in Table 2. There was no statistically significant difference in variable gene distribution between AILT and PTCL-NOS cases. Overall, rearrangements in Vγ1-8 were most common, detected in 50% and 63% of total AILT and PTCL-NOS cases, respectively. In addition, rearrangements in Vγ10 were observed frequently, occurring in 48% and 46% of total AILT and PTCL-NOS cases, respectively. Of the 93 total T-cell clones, 66 (71%) were detected by Vγ1-8 primers and 55 (59%) were detected by Vγ10 primers. The majority of Vγ10 rearrangements were seen in combination with Vγ1-8 and/or Vγ9 rearrangements, and cases with only Vγ10 rearrangements were uncommon, occurring in three AILT and three PTCL-NOS cases. Clonal rearrangements involving Vγ10 are reportedly uncommon in PTCL.17Theodorou I Raphael M Bigorgne C Fourcade C Lahet C Cochet G Lefranc MP Gaulard P Farcet JP Recombination pattern of the TCR gamma locus in human peripheral T-cell lymphomas.J Pathol. 1994; 1
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