Hepatosplenic and Subcutaneous Panniculitis-Like γ/δ T Cell Lymphomas Are Derived from Different Vδ Subsets of γ/δ T Lymphocytes
2000; Elsevier BV; Volume: 2; Issue: 1 Linguagem: Inglês
10.1016/s1525-1578(10)60610-1
ISSN1943-7811
AutoresGrzegorz K. Przybylski, Hong Wu, William R. Macon, Janet B. Finan, Debra G. B. Leonard, Raymond E. Felgar, Joseph A. DiGiuseppe, Peter C. Nowell̀, Steven H. Swerdlow, Marshall E. Kadin, Mariusz A. Wasik, Kevin E. Salhany,
Tópico(s)T-cell and Retrovirus Studies
ResumoGamma/delta T cell lymphomas (γ/δ TCL) represent rare, often aggressive types of T cell malignancy that are clinically and pathologically diverse. Most γ/δ TCL occur as a hepatosplenic or subcutaneous type. To date, analysis of the T cell receptor δ (TCRδ) gene repertoire of hepatosplenic γ/δ TCL (γ/δ HSTCL) and subcutaneous panniculitis-like γ/δ TCL (γ/δ SPTCL) has been reported only in a limited number of cases. In this study we analyzed 11 γ/δ HSTCL and 4 γ/δ SPTCL by polymerase chain reaction and immunostaining to determine their usage of the Vδ subtypes (Vδ1–6). It is noteworthy that 10 of 11 γ/δ HSTCL expressed the Vδ1 gene. The remaining case also expressed T cell receptor δ (TCRδ) as determined by flow cytometry and TCRδ rearrangement in Southern blot. However, the Vδ gene expressed by this lymphoma could not be determined, which suggests usage of an as yet unidentified Vδ gene. In striking contrast to the γ/δ HSTCL, all 4 γ/δ SPTCL expressed the Vδ2 gene. Our data demonstrate that γ/δ HSTCL are preferentially derived from the Vδ1 subset of γ/δ T lymphocytes, whereas γ/δ SPTCL are preferentially derived from the Vδ2 subset. The pattern of Vδ gene expression in HSTCL and SPTCL corresponds to the respective, predominant γ/δ T cell subsets normally found in the spleen and skin. This finding suggests that γ/δ TCL are derived from normal γ/δ T lymphocytes which reside in the affected tissues. Furthermore, the selective, lymphoma type-specific Vδ gene segment usage may provide a molecular tool to distinguish better among various types of γ/δ TCL lymphoma particularly in the clinically advanced, widely disseminated cases. Gamma/delta T cell lymphomas (γ/δ TCL) represent rare, often aggressive types of T cell malignancy that are clinically and pathologically diverse. Most γ/δ TCL occur as a hepatosplenic or subcutaneous type. To date, analysis of the T cell receptor δ (TCRδ) gene repertoire of hepatosplenic γ/δ TCL (γ/δ HSTCL) and subcutaneous panniculitis-like γ/δ TCL (γ/δ SPTCL) has been reported only in a limited number of cases. In this study we analyzed 11 γ/δ HSTCL and 4 γ/δ SPTCL by polymerase chain reaction and immunostaining to determine their usage of the Vδ subtypes (Vδ1–6). It is noteworthy that 10 of 11 γ/δ HSTCL expressed the Vδ1 gene. The remaining case also expressed T cell receptor δ (TCRδ) as determined by flow cytometry and TCRδ rearrangement in Southern blot. However, the Vδ gene expressed by this lymphoma could not be determined, which suggests usage of an as yet unidentified Vδ gene. In striking contrast to the γ/δ HSTCL, all 4 γ/δ SPTCL expressed the Vδ2 gene. Our data demonstrate that γ/δ HSTCL are preferentially derived from the Vδ1 subset of γ/δ T lymphocytes, whereas γ/δ SPTCL are preferentially derived from the Vδ2 subset. The pattern of Vδ gene expression in HSTCL and SPTCL corresponds to the respective, predominant γ/δ T cell subsets normally found in the spleen and skin. This finding suggests that γ/δ TCL are derived from normal γ/δ T lymphocytes which reside in the affected tissues. Furthermore, the selective, lymphoma type-specific Vδ gene segment usage may provide a molecular tool to distinguish better among various types of γ/δ TCL lymphoma particularly in the clinically advanced, widely disseminated cases. Similar to normal T lymphocytes, T cell lymphomas (TCL) may express two different types of T cell receptor (TCR), α/β or γ/δ. Four TCR genes (α, β, γ, and δ) are composed in their germline configuration of noncontiguous segments of variable (V), diversity (D), joining (J), and constant (C) regions. During T cell differentiation, somatic VDJ rearrangements occur and thereby generate variability of the TCR.1Gellert M Recent advances in understanding V(D)J recombination.Adv Immunol. 1997; 64: 39-64Crossref PubMed Google Scholar Only complete in-frame TCR gene rearrangements, consisting of V, D, and J regions, may form a functional TCR. Incomplete rearrangements between two D regions (D-D), between V and D region (V-D), and between D and J region (D-J) are nonfunctional. The majority of normal T lymphocytes express the α/β heterodimer; however, approximately 5% of the T cells express the γ/δ heterodimer.2Falini B Flenghi L Pileri S Pelicci P Fagioli M Martelli MF Moretta L Ciccone E Distribution of T-cells bearing different forms of the T cell receptor g/d in normal and pathological human tissues.J Immunol. 1989; 143: 2480-2488PubMed Google Scholar In contrast to α/β T lymphocytes, development of γ/δ T cells is not dependent on expression of major histocompatibility complex (MHC) I or MHC II molecules.3Correa I Bix M Liao NS Zijlstra M Jaenisch R Raulet D Most γδ T cells develop normally in β2-microglobulin-deficient mice.Proc Natl Acad Sci USA. 1992; 89: 653-657Crossref PubMed Scopus (150) Google Scholar, 4Bigby M Markowitz JS Bleicher PA Grusby MJ Simha S Siebrecht M Wagner M Nagler Anderson C Glimcher LH Most γδ T cells develop normally in the absence of MHC class II molecules.J Immunol. 1993; 151: 4465-4475PubMed Google Scholar Unlike α/β T cells, which develop almost exclusively in thymus, γ/δ T cells can be generated in extrathymic sites such as intestinal epithelium, skin, spleen, and fetal liver.5Palacios R Samaridis J Rearrangement patterns of T-cell receptor genes in the spleen of athymic (nu/nu) young mice.Immunogenetics. 1991; 33: 90-95Crossref PubMed Scopus (12) Google Scholar, 6Ota Y Kobata T Seki M Yagita H Shimada S Huang YY Takagaki Y Okumura K Extrathymic origin of Vg1/Vd6 T cells in the skin.Eur J Immunol. 1992; 22: 595-598Crossref PubMed Scopus (30) Google Scholar, 7McVay LD Carding SR Extrathymic origin of human gd T cells during fetal development.J Immunol. 1996; 157: 2873-2882PubMed Google Scholar, 8Bandeira A Itohara S Bonneville M Burlen Defranoux O Mota Santos T Coutinho A Tonegawa S Extrathymic origin of intestinal intraepithelial lymphocytes bearing T-cell antigen receptor gd.Proc Natl Acad Sci USA. 1991; 88: 43-47Crossref PubMed Scopus (275) Google Scholar The exact function of γ/δ T lymphocytes has not been fully elucidated, but some studies suggest a role for these cells in early immune responses to infections, autoimmune disorders, and cancer immune surveillance.9Haas W Pereira P Tonegawa S Gamma/delta cells.Annu Rev Immunol. 1993; 11: 637-685Crossref PubMed Scopus (786) Google Scholar γ/δ T cells share some features with CD8+ α/β T lymphocytes and with natural killer (NK) cells. They show MHC-dependent and MHC-independent cytotoxity, produce lymphokines, and exhibit NK-like lytic activity. γ/δ TCL represent a rare type of T cell malignancy. They comprise less than 10% of peripheral T cell lymphomas10Gaulard P Bourquelot P Kanavaros P Haioun C Le Couediac JP Divine M Goossens M Zafrani ES Farcet JP Reyes F Expression of the a/b and g/d T-cell receptors in 57 cases of peripheral T-cell lymphomas: identification of a subset of gd T-cell lymphomas.Am J Pathol. 1990; 137: 617-628PubMed Google Scholar and occur mostly at extranodal sites in hepatosplenic, subcutaneous, or intestinal form. Hepatosplenic γ/δ TCL (γ/δ HSTCL) is recognized as a provisional subset of peripheral T cell lymphoma in the Revised European-American Classification of Lymphoid Neoplasms (REAL),11Harris NL Jaffe ES Stein H Banks PM Chan JKC Cleary ML Delsol G De Wolf Peeters C Falini B Gatter KC Grogan TM Isaacson PG Knowles DM Mason DY Muller-Hermelink H-K Pileri SA Piris MA Ralfkiaer E Warnke RA A revised European-American Classification of Lymphoid Neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar although a few identified cases of α/β HSTCL appear to have similar clinicopathological characteristics. Histologically, γ/δ HSTCL is characterized by a mixture of small to medium-sized atypical lymphocytes. To date only about 40 cases of γ/δ HSTCL have been reported.10Gaulard P Bourquelot P Kanavaros P Haioun C Le Couediac JP Divine M Goossens M Zafrani ES Farcet JP Reyes F Expression of the a/b and g/d T-cell receptors in 57 cases of peripheral T-cell lymphomas: identification of a subset of gd T-cell lymphomas.Am J Pathol. 1990; 137: 617-628PubMed Google Scholar, 12Kadin ME Kamoun M Lamberg J Erythrophagocytic T γ lymphoma: a clinicopathologic entity resembling malignant histiocytosis.N Engl J Med. 1981; 304: 648-653Crossref PubMed Scopus (215) Google Scholar, 13Gaulard P Kanavaros P Farcet JP Rocha FD Haioun C Divine M Reyes F Zafrani ES Bone marrow histologic and immunohistochemical findings in peripheral. 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Patients commonly present with B symptoms and hepatosplenomegaly, but not lymphadenopathy. The disease usually follows an aggressive course with poor response to chemotherapy and short time of survival. Subcutaneous panniculitis-like TCL (SPTCL) is an uncommon form of cutaneous lymphoma, involving mainly subcutis and often mimicking lobular panniculitis.24Gonzalez CL Medeiros LJ Braziel RM Jaffe ES T-cell lymphoma involving subcutaneous tissue: a clinicopathologic entity commonly associated with hemophagocytic syndrome.Am J Surg Pathol. 1991; 15: 17-27Crossref PubMed Scopus (410) Google Scholar SPTCL also has been proposed as a provisional subset of peripheral T cell lymphoma in the REAL classification.11Harris NL Jaffe ES Stein H Banks PM Chan JKC Cleary ML Delsol G De Wolf Peeters C Falini B Gatter KC Grogan TM Isaacson PG Knowles DM Mason DY Muller-Hermelink H-K Pileri SA Piris MA Ralfkiaer E Warnke RA A revised European-American Classification of Lymphoid Neoplasms: a proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar It is sometimes associated with aggressive clinical behavior and poor prognosis, particularly when accompanied by a hemophagocytic syndrome.24Gonzalez CL Medeiros LJ Braziel RM Jaffe ES T-cell lymphoma involving subcutaneous tissue: a clinicopathologic entity commonly associated with hemophagocytic syndrome.Am J Surg Pathol. 1991; 15: 17-27Crossref PubMed Scopus (410) Google Scholar, 25Mehregan DA Su WP Kurtin PJ Subcutaneous T-cell lymphoma: a clinical, histopathologic, and immunohistochemical study of six cases.J Cutan Pathol. 1994; 21: 110-117Crossref PubMed Scopus (64) Google Scholar, 26Salhany KE Macon WR Choi JK Elenitsas R Lessin SR Felgar RE Wilson DM Przybylski GK Lister J Wasik MA Swerdlow SH Subcutaneous panniculitis-like T-cell lymphoma: clinicopathologic, immunophenotypic, and genotypic analysis of a/b and g/d subtypes.Am J Surg Pathol. 1998; 22: 881-893Crossref PubMed Scopus (297) Google Scholar Based on TCR expression, SPTCL can be divided into α/β and γ/δ SPTCL subsets, which are not recognized as distinct entities in the REAL classification. To date only a few γ/δ SPTCL cases have been reported.26Salhany KE Macon WR Choi JK Elenitsas R Lessin SR Felgar RE Wilson DM Przybylski GK Lister J Wasik MA Swerdlow SH Subcutaneous panniculitis-like T-cell lymphoma: clinicopathologic, immunophenotypic, and genotypic analysis of a/b and g/d subtypes.Am J Surg Pathol. 1998; 22: 881-893Crossref PubMed Scopus (297) Google Scholar, 27Burg G Dummer R Wilhelm M Nestle F Ott MM Feller A Hefner H Lanz U Schwinn A Wiede J A subcutaneous delta-positive T-cell lymphoma that produces interferon γ.N Engl J Med. 1991; 325: 1078-1081Crossref PubMed Scopus (145) Google Scholar, 28Fujita M Miyachi Y Furukawa F Toichi E Furukawa I Nakajima N Imamura S A case of cutaneous T-cell lymphoma expressing γδ T-cell receptors.J Am Acad Dermatol. 1993; 28: 355-360Abstract Full Text PDF PubMed Scopus (27) Google Scholar, 29Avinoach I Halevy S Argov S Sacks M Gamma/delta T-cell lymphoma involving the subcutaneous tissue, and associated with a hemophagocytic syndrome.Am J Dermatopathol. 1994; 16: 426-433Crossref PubMed Scopus (78) Google Scholar, 30Munn SE McGregor JM Jones A Amlot P Rustin MH Russell Jones R Whittaker S Clinical and pathological heterogeneity in cutaneous γ-δ T-cell lymphoma: a report of three cases and a review of the literature.Br J Dermatol. 1996; 135: 976-981Crossref PubMed Scopus (58) Google Scholar, 31Arnulf B Copie Bergman C Delfau Larue MH Lavergne Slove A Bosq J Wechsler J Wassef M Matuchansky C Epardeau B Stern M Bagot M Reyes F Gaulard P Nonhepatosplenic gd T-cell lymphoma: a subset of cytotoxic lymphomas with mucosal or skin localization.Blood. 1998; 91: 1723-1731PubMed Google Scholar The TCRδ gene consists of at least six Vδ gene segments.32Takihara Y Tkachuk D Michalopoulos E Champagne E Reimann J Minden M Mak TW Sequence and organization of the diversity, joining, and constant region genes of the human T-cell delta-chain locus.Proc Natl Acad Sci USA. 1988; 85: 6097-6101Crossref PubMed Scopus (125) Google Scholar, 33Takihara Y Reimann J Michalopoulos E Ciccone E Moretta L Mak TW Diversity and structure of human T cell receptor delta chain genes in peripheral blood γ/δ-bearing T lymphocytes.J Exp Med. 1989; 169: 393-405Crossref PubMed Scopus (119) Google Scholar A detailed analysis revealed that over 95% of the γ/δ T cells express either Vδ1 or Vδ2 gene.2Falini B Flenghi L Pileri S Pelicci P Fagioli M Martelli MF Moretta L Ciccone E Distribution of T-cells bearing different forms of the T cell receptor g/d in normal and pathological human tissues.J Immunol. 1989; 143: 2480-2488PubMed Google Scholar, 9Haas W Pereira P Tonegawa S Gamma/delta cells.Annu Rev Immunol. 1993; 11: 637-685Crossref PubMed Scopus (786) Google Scholar Interestingly, normal γ/δ T lymphocytes present in spleen, thymus, and intestinal epithelium predominantly express the Vδ1 gene, whereas the majority of γ/δ T cells in peripheral blood, tonsils, and skin express the Vδ2 gene.2Falini B Flenghi L Pileri S Pelicci P Fagioli M Martelli MF Moretta L Ciccone E Distribution of T-cells bearing different forms of the T cell receptor g/d in normal and pathological human tissues.J Immunol. 1989; 143: 2480-2488PubMed Google Scholar The reason for this dichotomy in the Vδ gene usage repertoire remains unclear. Vδ gene usage by γ/δ TCL has not been studied so far in the great detail, and the small number of the reported cases does not allow any definitive conclusions in regard to Vδ usage by the specific subtype of γ/δ TCL. In this study we analyzed the Vδ usage in 11 hepatosplenic and 4 subcutaneous γ/δ TCL using polymerase chain reaction (PCR) and flow cytometry. Preferential usage of Vδ1 gene was found in γ/δ HSTCL (10/11 cases) and of Vδ2 gene in γ/δ SPTCL (4/4 cases). Biological and diagnostic implications of this finding are discussed. We investigated 15 patients with γ/δ TCL of hepatosplenic (11 cases) and subcutaneous (4 cases) type in this study. The cases were derived from files of the participating institutions: the University of Pennsylvania, Vanderbilt University, Johns Hopkins University, the University of Pittsburgh, and Harvard University. Data on cases 1–3, 12, and 13 including Vδ subtype expression were previously reported in part.20Salhany KE Feldman M Kahn MJ Peritt D Schretzenmair RD Wilson DM DiPaola RS Glick AD Kant JA Nowell PC Kamoun M Hepatosplenic gammadelta T-cell lymphoma: ultrastructural, immunophenotypic, and functional evidence for cytotoxic T lymphocyte differentiation.Hum Pathol. 1997; 28: 674-685Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 26Salhany KE Macon WR Choi JK Elenitsas R Lessin SR Felgar RE Wilson DM Przybylski GK Lister J Wasik MA Swerdlow SH Subcutaneous panniculitis-like T-cell lymphoma: clinicopathologic, immunophenotypic, and genotypic analysis of a/b and g/d subtypes.Am J Surg Pathol. 1998; 22: 881-893Crossref PubMed Scopus (297) Google Scholar Metaphase cytogenetic analysis was performed by standard trypsin Giemsa banding using unstimulated cell cultures of spleen or bone marrow cells. Slides for FISH were prepared from spleen or peripheral blood mononuclear cells according to a standard method. In brief, liquid nitrogen-stored, dimethyl sulfoxide-preserved frozen cells from three patients were cultured overnight, cytospun onto slides at a concentration of 104 cells/slide, air-dried, fixed in Carnoy's for 20 minutes, and air-dried overnight. The cytospins were analyzed with VYSIS (Downers Grove, IL) CEP 8 probe to enumerate chromosome 8 centromeres or combination of CEP 7 and LSI D7S486 probes to detect simultaneously chromosome 7 centromere and band 7q31 on the chromosome's long arm. The staining was performed as recommended by the probe manufacturer. Flow cytometry and frozen section or paraffin immunohistochemistry were used for immunophenotyping of the lymphomas. All cases were studied by flow cytometry and/or frozen section immunohistochemistry with a set of standard anti-T cell and anti-NK cell antibodies as previously described.20Salhany KE Feldman M Kahn MJ Peritt D Schretzenmair RD Wilson DM DiPaola RS Glick AD Kant JA Nowell PC Kamoun M Hepatosplenic gammadelta T-cell lymphoma: ultrastructural, immunophenotypic, and functional evidence for cytotoxic T lymphocyte differentiation.Hum Pathol. 1997; 28: 674-685Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 26Salhany KE Macon WR Choi JK Elenitsas R Lessin SR Felgar RE Wilson DM Przybylski GK Lister J Wasik MA Swerdlow SH Subcutaneous panniculitis-like T-cell lymphoma: clinicopathologic, immunophenotypic, and genotypic analysis of a/b and g/d subtypes.Am J Surg Pathol. 1998; 22: 881-893Crossref PubMed Scopus (297) Google Scholar To confirm the γδ phenotype, we used antibodies to the α/β and γ/δ TCR (Endogen, Woburn, MA).20Salhany KE Feldman M Kahn MJ Peritt D Schretzenmair RD Wilson DM DiPaola RS Glick AD Kant JA Nowell PC Kamoun M Hepatosplenic gammadelta T-cell lymphoma: ultrastructural, immunophenotypic, and functional evidence for cytotoxic T lymphocyte differentiation.Hum Pathol. 1997; 28: 674-685Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 26Salhany KE Macon WR Choi JK Elenitsas R Lessin SR Felgar RE Wilson DM Przybylski GK Lister J Wasik MA Swerdlow SH Subcutaneous panniculitis-like T-cell lymphoma: clinicopathologic, immunophenotypic, and genotypic analysis of a/b and g/d subtypes.Am J Surg Pathol. 1998; 22: 881-893Crossref PubMed Scopus (297) Google Scholar Vδ subtype expression was also determined by flow cytometry or frozen section immunohistochemistry using commercially available monoclonal antibodies specific for different V regions of the TCRδ chain (Vδ1 and Vδ2, Endogen; Vδ3, Immunotech, Westbrook, ME). Southern blot analysis for TCRδ gene rearrangements was performed on genomic DNA extracted from frozen tumor in five cases of γ/δ HSTCL. The DNA was treated with restriction enzymes Eco RI, Hin dIII and Bam HI, transferred to a nylon membrane and hybridized to a TCRδ gene probe TCRDJ1 (Dako Corp., Carpinteria, CA), which corresponds to the Jδ1 exon and its 3′ flanking region. A nonoverlapping 3.0-kb probe, which corresponds to the Jδ2 exon and its 5′ flanking region (pjk 3.0s, kindly provided by Dr. Carlo Croce, Philadelphia, PA)34Edwards RH Wasik MA Finan J Rodriguez R Moore J Kamoun M Rennert H Bird J Nowell PC Salhany KE Evidence for hematopoietic progenitor cell involvement in acute promyelocytic leukemia.Am J Clin Pathol. 1999; 112: 819-827PubMed Google Scholar was also used in one case that did not show a TCRδ rearrangement using the TCRDJ1 probe. PCR of 50 μl total volume was performed in a Trio-Thermoblock (Biometra, Goettingen, Germany) with 0.1 μg of genomic DNA, 10 pmol of each primer, 5 nmol each dATP, dCTP, dGTP, dTTP (Perkin Elmer-Cetus, Norwalk, CT), 1.5 U Taq polymerase, and PCR Buffer (Perkin Elmer-Cetus) including 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, and 0,001% (w/v) gelatin. After 3′ denaturation at 94°C, 35 PCR cycles were performed, each cycle consisting of denaturation at 94°C, annealing at 60°C, and extension at 72° (for 1 minute each), followed by a final 7-minute extension at 72°C. Oligonucleotide primers used for PCR were previously described26Salhany KE Macon WR Choi JK Elenitsas R Lessin SR Felgar RE Wilson DM Przybylski GK Lister J Wasik MA Swerdlow SH Subcutaneous panniculitis-like T-cell lymphoma: clinicopathologic, immunophenotypic, and genotypic analysis of a/b and g/d subtypes.Am J Surg Pathol. 1998; 22: 881-893Crossref PubMed Scopus (297) Google Scholar, 35Yokota S Hansen-Hagge TE Ludwig WD Reiter A Raghavachar A Kleihauer E Bartram CR Use of polymerase chain reaction to monitor minimal residual disease in acute lymphoblastic leukemia patients.Blood. 1991; 77: 331-339PubMed Google Scholar, 36Przybylski G Oettle H Ludwig WD Siegert W Schmidt CA Molecular characterization of illegitimate tcrδ gene rearrangements in acute myeloid leukemia.Br J Haematol. 1994; 87: 301-307Crossref PubMed Scopus (22) Google Scholar, 37Schmidt CA Przybylski G Tietze A Oettle H Siegert W Ludwig WD Acute myeloid and T-cell acute lymphoblastic leukaemia with aberrant antigen expression exhibit similar TCR? gene rearrangements.Br J Haematol. 1996; 92: 929-936Crossref PubMed Scopus (17) Google Scholar and are listed in Table 1. Their specificity was confirmed by sequencing of their products36Przybylski G Oettle H Ludwig WD Siegert W Schmidt CA Molecular characterization of illegitimate tcrδ gene rearrangements in acute myeloid leukemia.Br J Haematol. 1994; 87: 301-307Crossref PubMed Scopus (22) Google Scholar, 37Schmidt CA Przybylski G Tietze A Oettle H Siegert W Ludwig WD Acute myeloid and T-cell acute lymphoblastic leukaemia with aberrant antigen expression exhibit similar TCR? gene rearrangements.Br J Haematol. 1996; 92: 929-936Crossref PubMed Scopus (17) Google Scholar (and data not shown) and nested PCR which yielded products of expected molecular weight (data not shown). To exclude DNA contamination, negative controls were included, and to avoid false negative results an internal positive control was run in each reaction. Amplification of recombinase activating gene (RAG1) served as such a positive control. PCR products were visualized by 2% agarose gel electrophoresis containing ethidium bromide. Under these experimental conditions, lymphoma-derived, clonal TCRδ gene rearrangements, which are present in the large proportion of cells in the samples, are seen as distinct, well-defined bands in the gel. Because the incidence of a specific Vδ rearrangement is low in normal reactive polyclonal T cells, TCRδ genes from reactive T cells present in the samples are amplified, but not visible as distinct bands.Table 1List of PrimersPrimersSource5′ primers Vδ15′-ACT CAA GCC CAG TCA TCA GTYokota et al35Yokota S Hansen-Hagge TE Ludwig WD Reiter A Raghavachar A Kleihauer E Bartram CR Use of polymerase chain reaction to monitor minimal residual disease in acute lymphoblastic leukemia patients.Blood. 1991; 77: 331-339PubMed Google Scholar Vδ1b5′-GCA AAG TAC TTT TGT GCT CTT GYokota et al35Yokota S Hansen-Hagge TE Ludwig WD Reiter A Raghavachar A Kleihauer E Bartram CR Use of polymerase chain reaction to monitor minimal residual disease in acute lymphoblastic leukemia patients.Blood. 1991; 77: 331-339PubMed Google Scholar Vδ25′-GAG TCA TGT CAG CCA TTG AGYokota et al35Yokota S Hansen-Hagge TE Ludwig WD Reiter A Raghavachar A Kleihauer E Bartram CR Use of polymerase chain reaction to monitor minimal residual disease in acute lymphoblastic leukemia patients.Blood. 1991; 77: 331-339PubMed Google Scholar Vδ2b5′-GCA CCA TCA GAG AGA GAT GAYokota et al35Yokota S Hansen-Hagge TE Ludwig WD Reiter A Raghavachar A Kleihauer E Bartram CR Use of polymerase chain reaction to monitor minimal residual disease in acute lymphoblastic leukemia patients.Blood. 1991; 77: 331-339PubMed Google Scholar Vδ35′-ACA GCA GAT CAG AAG GTG CAPrzybylski et al36Przybylski G Oettle H Ludwig WD Siegert W Schmidt CA Molecular characterization of illegitimate tcrδ gene rearrangements in acute myeloid leukemia.Br J Haematol. 1994; 87: 301-307Crossref PubMed Scopus (22) Google Scholar Vδ45′-CCA GTG ATC CAA GTT ATG GTCPrzybylski et al36Przybylski G Oettle H Ludwig WD Siegert W Schmidt CA Molecular characterization of illegitimate tcrδ gene rearrangements in acute myeloid leukemia.Br J Haematol. 1994; 87: 301-307Crossref PubMed Scopus (22) Google Scholar Vδ55′-CTG AAG GTC CTA CAT TCC TGPrzybylski et al36Przybylski G Oettle H Ludwig WD Siegert W Schmidt CA Molecular characterization of illegitimate tcrδ gene rearrangements in acute myeloid leukemia.Br J Haematol. 1994; 87: 301-307Crossref PubMed Scopus (22) Google Scholar Vδ65′-TAT CAT GGA TTC CCA GCC TGPrzybylski et al36Przybylski G Oettle H Ludwig WD Siegert W Schmidt CA Molecular characterization of illegitimate tcrδ gene rearrangements in acute myeloid leukemia.Br J Haematol. 1994; 87: 301-307Crossref PubMed Scopus (22) Google Scholar Dδ25′-AGA GGG TTT TTA TAC TGA TGTSchmidt et al37Schmidt CA Przybylski G Tietze A Oettle H Siegert W Ludwig WD Acute myeloid and T-cell acute lymphoblastic leukaemia with aberrant antigen expression exhibit similar TCR? gene rearrangements.Br J Haematol. 1996; 92: 929-936Crossref PubMed Scopus (17) Google Scholar3′ primers Jδ15′-GAG TTA CTT ACT TGG TTC CACYokota et al35Yokota S Hansen-Hagge TE Ludwig WD Reiter A Raghavachar A Kleihauer E Bartram CR Use of polymerase chain reaction to monitor minimal residual disease in acute lymphoblastic leukemia patients.Blood. 1991; 77: 331-339PubMed Google Scholar Dδ35′-AGG GAA ATG GCA CTT TTG CCYokota et al35Yokota S Hansen-Hagge TE Ludwig WD Reiter A Raghavachar A Kleihauer E Bartram CR Use of polymerase chain reaction to monitor minimal residual disease in acute lymphoblastic leukemia patients.Blood. 1991; 77: 331-339PubMed Google ScholarReference primers RAG1(5′)5′-GCC ATG AAG AGC AGT GAA TTASalhany et al26Salhany KE Macon WR Choi JK Elenitsas R Lessin SR Felgar RE Wilson DM Przybylski GK Lister J Wasik MA Swerdlow SH Subcutaneous panniculitis-like T-cell lymphoma: clinicopathologic, immunophenotypic, and genotypic analysis of a/b and g/d subtypes.Am J Surg Pathol. 1998; 22: 881-893Crossref PubMed Scopus (297) Google Scholar RAG1(3′)5′-AGG AAT TAA CTC ACA AAC TGCSalhany et al26Salhany KE Macon WR Choi JK Elenitsas R Lessin SR Felgar RE Wilson DM Przybylski GK Lister J Wasik MA Swerdlow SH Subcutaneous panniculitis-like T-cell lymphoma: clinicopathologic, immunophenotypic, and genotypic analysis of a/b and g/d subtypes.Am J Surg Pathol. 1998; 22: 881-893Crossref PubMed Scopus (297) Google Scholar RAG2(5′)5′-TTG GC
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