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Rojansky R, Fernandez-Pol S, Wang E, Rieger KE, Novoa RA, Zehnder JL, Kunder CA, Kim YH, Khodadoust MS, Brown RA. Cutaneous T-cell lymphomas with pathogenic somatic mutations and absence of detectable clonal T-cell receptor gene rearrangement: two case reports. Diagn Pathol 2020; 15:122. [PMID: 32988392 PMCID: PMC7523289 DOI: 10.1186/s13000-020-01022-x] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Accepted: 09/02/2020] [Indexed: 12/20/2022] Open
Abstract
Background Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of extranodal non-Hodgkin lymphomas for which diagnosis can be challenging given the potential for overlap with inflammatory dermatoses. Current diagnostic criteria for CTCL incorporate clinical and histopathologic findings as well as results of T-cell receptor (TCR) gene sequencing. Molecular interrogation of TCR genes, TRG and TRB, has proven to be a critical tool for confirming diagnoses of CTCL and for disease tracking after initiation of therapy or after stem cell transplant. Methods for confirming a diagnosis of lymphoma in the absence of TCR gene clonality are lacking. We present two patients with CTCL with pathogenic somatic mutations in the absence of TRG and TRB clonality. Case presentations Case 1: A 38-year-old male had a 19-year history of a diffuse skin rash with papulosquamous, granulomatous, and verrucous features and progressive ulcerated plaques and tumors demonstrating an atypical CD4+ T-cell infiltrate with expression of cytotoxic markers CD56, TIA-1, granzyme, and perforin on histopathology. No definitive evidence for T-cell clonality was detected by conventional PCR of 6 biopsies or by next-generation sequencing (NGS) of 14 biopsies. Somatic mutational profiling of a skin biopsy revealed pathogenic mutations in PIKC3D and TERT promoter hotspots, confirming the presence of a clonal process. Case 2: A 69-year-old male with a 13-year history of progressive, diffuse hypertrophic and eroded plaques showed an atypical CD4+ T-cell infiltrate with subset expression of TIA-1 and granzyme on histopathology. No TCR clonality was detected by TCR-NGS of 6 biopsies. Somatic mutational profiling of a skin biopsy detected a pathogenic mutation in TP53, confirming the presence of a clonal process. Conclusions These cases highlight how detection of pathogenic somatic mutations can confirm a diagnosis of lymphoma in a clinically and histopathologically suspicious cutaneous lymphoid proliferation without detectable TCR clonality.
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Affiliation(s)
- Rebecca Rojansky
- Department of Pathology, Stanford Medicine, Stanford, CA, 94305, USA
| | | | - Erica Wang
- Department of Dermatology, Stanford Medicine, Stanford, CA, 94305, USA
| | - Kerri E Rieger
- Department of Pathology, Stanford Medicine, Stanford, CA, 94305, USA.,Department of Dermatology, Stanford Medicine, Stanford, CA, 94305, USA
| | - Roberto A Novoa
- Department of Pathology, Stanford Medicine, Stanford, CA, 94305, USA.,Department of Dermatology, Stanford Medicine, Stanford, CA, 94305, USA
| | - James L Zehnder
- Department of Pathology, Stanford Medicine, Stanford, CA, 94305, USA.,Division of Hematology, Department of Medicine, Stanford Medicine, Stanford, CA, 94305, USA
| | | | - Youn H Kim
- Department of Dermatology, Stanford Medicine, Stanford, CA, 94305, USA.,Division of Oncology, Department of Medicine, Stanford Medicine, Stanford, CA, 94305, USA
| | - Michael S Khodadoust
- Department of Dermatology, Stanford Medicine, Stanford, CA, 94305, USA.,Division of Oncology, Department of Medicine, Stanford Medicine, Stanford, CA, 94305, USA
| | - Ryanne A Brown
- Department of Pathology, Stanford Medicine, Stanford, CA, 94305, USA. .,Department of Dermatology, Stanford Medicine, Stanford, CA, 94305, USA. .,Department of Pathology, Veterans Affairs Palo Alto Health Care System, 3375 Hillview Ave, Room 1821, Palo Alto, CA, 94304-1204, USA.
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Fujii K, Kanekura T. Next-Generation Sequencing Technologies for Early-Stage Cutaneous T-Cell Lymphoma. Front Med (Lausanne) 2019; 6:181. [PMID: 31457014 PMCID: PMC6700355 DOI: 10.3389/fmed.2019.00181] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2019] [Accepted: 07/29/2019] [Indexed: 01/09/2023] Open
Abstract
The diagnosis of early stage cutaneous T-cell lymphoma is often difficult, particularly in mycosis fungoides (MF), because the clinical presentation, histological findings, and laboratory findings of MF resemble those of inflammatory skin diseases such as atopic dermatitis, psoriasis, and parapsoriasis en plaque. Furthermore, MF sometimes occurs with or after these inflammatory skin diseases. The current diagnostic criteria heavily rely on clinical impressions along with assessments of T cell clonality. To make a diagnosis of early-stage MF, the detection of a malignant clone is critical. T cell receptor (TCR) gene rearrangements have been detected by southern blotting or polymerase chain reaction for this purpose, but the results of these methods are insufficient. High-throughput TCR sequencing has provided insights into the complexities of the immune repertoire. Accordingly, his technique is more sensitive and specific than current methods, making it useful for the detection of early lesions and monitoring responses to therapy.
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Affiliation(s)
- Kazuyasu Fujii
- Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
| | - Takuro Kanekura
- Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
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Jeon YK, Yoon SO, Paik JH, Kim YA, Shin BK, Kim HJ, Cha HJ, Kim JE, Huh J, Ko YH, The Hematopathology Study Group of the Korean Society of Pathologists, The Molecular Pathology Study Group of Korean Society of Pathologists. Molecular Testing of Lymphoproliferative Disorders: Current Status and Perspectives. J Pathol Transl Med 2017; 51:224-241. [PMID: 28535584 PMCID: PMC5445208 DOI: 10.4132/jptm.2017.04.09] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2017] [Accepted: 04/09/2017] [Indexed: 12/13/2022] Open
Abstract
Molecular pathologic testing plays an important role for the diagnosis, prognostication and decision of treatment strategy in lymphoproliferative disease. Here, we briefly review the molecular tests currently used for lymphoproliferative disease and those which will be implicated in clinical practice in the near future. Specifically, this guideline addresses the clonality test for B- and T-cell proliferative lesions, molecular cytogenetic tests for malignant lymphoma, determination of cell-of-origin in diffuse large B-cell lymphoma, and molecular genetic alterations incorporated in the 2016 revision of the World Health Organization classification of lymphoid neoplasms. Finally, a new perspective on the next-generation sequencing for diagnostic, prognostic, and therapeutic purpose in malignant lymphoma will be summarized.
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Affiliation(s)
- Yoon Kyung Jeon
- Corresponding Author Yoon Kyung Jeon, MD, PhD Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea Tel: +82-2-2072-1347 Fax: +82-2-743-5530 E-mail:
| | - Sun Och Yoon
- Department of Pathology, Yonsei University College of Medicine, Seoul, Korea
| | - Jin Ho Paik
- Department of Pathology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea
| | - Young A Kim
- Department of Pathology, SMG-SNU Boramae Medical Center, Seoul National University College of Medicine, Seoul, Korea
| | - Bong Kyung Shin
- Department of Pathology, Korea University Guro Hospital, Korea University School of Medicine, Seoul, Korea
| | - Hyun-Jung Kim
- Department of Pathology, Inje University Sanggye Paik Hospital, Seoul, Korea
| | - Hee Jeong Cha
- Department of Pathology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea
| | - Ji Eun Kim
- Department of Pathology, SMG-SNU Boramae Medical Center, Seoul National University College of Medicine, Seoul, Korea
| | - Jooryung Huh
- Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Young-Hyeh Ko
- Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - The Hematopathology Study Group of the Korean Society of Pathologists
- Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea
- Department of Pathology, Yonsei University College of Medicine, Seoul, Korea
- Department of Pathology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea
- Department of Pathology, SMG-SNU Boramae Medical Center, Seoul National University College of Medicine, Seoul, Korea
- Department of Pathology, Korea University Guro Hospital, Korea University School of Medicine, Seoul, Korea
- Department of Pathology, Inje University Sanggye Paik Hospital, Seoul, Korea
- Department of Pathology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea
- Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
- Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - The Molecular Pathology Study Group of Korean Society of Pathologists
- Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea
- Department of Pathology, Yonsei University College of Medicine, Seoul, Korea
- Department of Pathology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea
- Department of Pathology, SMG-SNU Boramae Medical Center, Seoul National University College of Medicine, Seoul, Korea
- Department of Pathology, Korea University Guro Hospital, Korea University School of Medicine, Seoul, Korea
- Department of Pathology, Inje University Sanggye Paik Hospital, Seoul, Korea
- Department of Pathology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea
- Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
- Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
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Sholl LM, Longtine J, Kuo FC. Molecular Analysis of Genetic Markers for Non-Hodgkin Lymphomas. CURRENT PROTOCOLS IN HUMAN GENETICS 2017; 93:10.14.1-10.14.29. [PMID: 28384399 DOI: 10.1002/cphg.37] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Molecular analysis complements the clinical and histopathologic tools used to diagnose and subclassify hematologic malignancies. The presence of clonal antigen-receptor gene rearrangements can help to confirm the diagnosis of a B or T cell lymphoma and can serve as a fingerprint of that neoplasm to be used in identifying concurrent disease at disparate sites or recurrence at future time points. Certain lymphoid malignancies harbor a characteristic chromosomal translocation, a finding that may have significant implications for an individual's prognosis or response to therapy. The polymerase chain reaction (PCR) is typically used to detect antigen-receptor gene rearrangements as well as specific translocations that can be supplemented by fluorescence in situ hybridization (FISH) and karyotype analysis. © 2017 by John Wiley & Sons, Inc.
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Affiliation(s)
| | | | - Frank C Kuo
- Brigham and Women's Hospital, Boston, Massachusetts
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5
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Shan GD, Hu FL, Yang M, Chen HT, Chen WG, Wang YG, Chen LH, Li YM, Xu GQ. Clonal immunoglobulin heavy chain and T-cell receptor γ gene rearrangements in primary gastric lymphoma. World J Gastroenterol 2013; 19:5727-5731. [PMID: 24039368 PMCID: PMC3769912 DOI: 10.3748/wjg.v19.i34.5727] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/18/2013] [Revised: 07/24/2013] [Accepted: 08/06/2013] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the diagnostic value of immunoglobulin heavy chain (IgH) and T-cell receptor γ (TCR-γ) gene monoclonal rearrangements in primary gastric lymphoma (PGL).
METHODS: A total of 48 patients with suspected PGL at our hospital were prospectively enrolled in this study from January 2009 to December 2011. The patients were divided into three groups (a PGL group, a gastric linitis plastica group, and a benign gastric ulcer group) based on the pathological results (gastric mucosal specimens obtained by endoscopy or surgery) and follow-up. Endoscopic ultrasonography (EUS) and EUS-guided biopsy were performed in all the patients. The tissue specimens were used for histopathological examination and for IgH and TCR-γ gene rearrangement polymerase chain reaction analyses.
RESULTS: EUS and EUS-guided biopsy were successfully performed in all 48 patients. In the PGL group (n = 21), monoclonal IgH gene rearrangements were detected in 14 (66.7%) patients. A positive result for each set of primers was found in 12 (57.1%), 8 (38.1%), and 4 (19.0%) cases using FR1/JH, FR2/JH, and FR3/JH primers, respectively. Overall, 12 (75%) patients with mucosal-associated lymphoid tissue lymphoma (n = 16) and 2 (40%) patients with diffuse large B-cell lymphoma (n = 5) were positive for monoclonal IgH gene rearrangements. No patients in the gastric linitis plastica group (n = 17) and only one (10%) patient in the benign gastric ulcer group (n = 10) were positive for a monoclonal IgH gene rearrangement. No TCR-γ gene monoclonal rearrangements were detected. The sensitivity of monoclonal IgH gene rearrangements was 66.7% for a PGL diagnosis, and the specificity was 96.4%. In the PGL group, 8 (100%) patients with stage IIE PGL (n = 8) and 6 (46.1%) patients with stage IE PGL (n = 13) were positive for monoclonal IgH gene rearrangements.
CONCLUSION: IgH gene rearrangements may be associated with PGL staging and may be useful for the diagnosis of PGL and for differentiating between PGL and gastric linitis plastica.
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6
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Sykes PJ, Morley AA. Molecular Biology Techniques in Malignant Lymphoma. J Histotechnol 2013. [DOI: 10.1179/his.1992.15.3.213] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022]
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7
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Shin S, Kim AH, Park J, Kim M, Lim J, Kim Y, Han K, Lee SA, Cho SG. Analysis of immunoglobulin and T cell receptor gene rearrangement in the bone marrow of lymphoid neoplasia using BIOMED-2 multiplex polymerase chain reaction. Int J Med Sci 2013; 10:1510-7. [PMID: 24046525 PMCID: PMC3775108 DOI: 10.7150/ijms.5342] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/05/2012] [Accepted: 08/12/2013] [Indexed: 01/22/2023] Open
Abstract
The evaluation of bone marrow (BM) involvement is important for diagnosis and staging in patients with lymphoid neoplasia. We evaluated of immunoglobulin (Ig) and/or T-cell receptor (TCR) gene rearrangements in the BM using the standardized BIOMED-2 multiplex PCR clonality assays and compared the results with microscopic findings such as histology and CD10, CD20, CD79a, CD3 and CD5 immunohistochemistry. A total of 151 samples were enrolled; 119 B cell neoplasia, 29 T cell neoplasia, and 3 Hodgkin's lymphoma. The molecular clonality assay and microscopic diagnosis were concordant in 66.9% (n=101) and discordant in 33.1 % (n=50). Ig/TCR gene clonality assay detected 43 cases of BM involvement which was not presented in the morphology. Two cases among them turned into microscopic BM involvement during a close follow up. Clonal TCR gene rearrangements were detected in 12.6% of B cell neoplasia and Ig gene rearrangement were found in 3.4% of T cell neoplasia. This molecular clonality assay is valuable particularly in diagnosing BM involvement of lymphoid neoplasia if it is morphologically uncertain. But it should be carefully interpreted because molecular clonality may be present in the reactive lymphoproliferation. Therefore, comprehensive analysis with morphologic analysis should be important to reach a final diagnosis.
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Affiliation(s)
- Soyoung Shin
- 1. Department of Laboratory Medicine, The Catholic University of Korea, College of Medicine, Seoul, Korea
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8
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Payne K, Wright P, Grant JW, Huang Y, Hamoudi R, Bacon CM, Du MQ, Liu H. BIOMED-2 PCR assays for IGK gene rearrangements are essential for B-cell clonality analysis in follicular lymphoma. Br J Haematol 2011; 155:84-92. [DOI: 10.1111/j.1365-2141.2011.08803.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
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Sholl LM, Longtine J. Molecular analysis of genetic markers for non-Hodgkin lymphomas. CURRENT PROTOCOLS IN HUMAN GENETICS 2010; Chapter 10:Unit 10.14.1-25. [PMID: 20373512 DOI: 10.1002/0471142905.hg1014s65] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Molecular analysis complements the clinical and histopathologic tools used to diagnose and subclassify hematologic malignancies. The presence of clonal antigen-receptor gene rearrangements can help to confirm the diagnosis of a B or T cell lymphoma and can serve as a fingerprint of that neoplasm to be used in identifying concurrent disease at disparate sites or recurrence at future time points. Certain lymphoid malignancies harbor a characteristic chromosomal translocation, a finding that may have significant implications for an individual's prognosis or response to therapy. The polymerase chain reaction (PCR) is typically used to detect antigen-receptor gene rearrangements as well as specific translocations that can be supplemented by fluorescence in situ hybridization (FISH) and karyotype analysis.
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10
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Oh HR, Lee MJ, Park G, Moon DS, Park YJ, Jang SJ. [A case of lambda-expressing pulmonary MALT lymphoma with dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene]. Korean J Lab Med 2009; 29:256-61. [PMID: 19571625 DOI: 10.3343/kjlm.2009.29.3.256] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
A 70-yr-old woman was hospitalized with a history of dry cough. Bronchial endoscopy and transbronchial lung biopsy were performed. However, the findings of histopathology and immunohistochemistry were not sufficient to decide whether the lesion was benign or malignant, because of the presence of crush artifacts in the biopsy specimens. We performed B-cell clonality studies using BIOMED-2 multiplex PCR (InVivoScribe Technologies, USA) to detect clonal rearrangements in the immunoglobulin gene. The results of multiplex PCR showed clonal rearrangements of both kappa and lambda immunoglobulin light chain genes. The findings of immunochemistry revealed that the lesion expressed lambda light chain, but not kappa light chain. Based on the clinical, pathologic, and molecular findings, this case was diagnosed as pulmonary MALT lymphoma. We report the first case in Korea of lambda-expressing MALT lymphoma that is shown to have dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene by multiplex PCR.
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Affiliation(s)
- Hye Ryong Oh
- Department of Laboratory Medicine, Chosun University Medical School, Dong-Gu, Gwangju, Korea
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11
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Deane M, Norton JD. Detection of Immunoglobulin Gene Rearrangement in B Cell Neoplasias by Polymerase Chain Reaction Gene Amplification. Leuk Lymphoma 2009; 5:9-22. [DOI: 10.3109/10428199109068100] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Aplicación de los protocolos de PCR BIOMED-2 en el análisis genotípico de los linfomas cutáneos primarios. ACTAS DERMO-SIFILIOGRAFICAS 2008. [DOI: 10.1016/s0001-7310(08)74757-4] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
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Longtine J, Fox E, Reynolds C, Sklar J. Molecular analysis of DNA rearrangements in leukemias and non-Hodgkin's lymphomas. CURRENT PROTOCOLS IN HUMAN GENETICS 2008; Chapter 10:Unit 10.4. [PMID: 18428241 DOI: 10.1002/0471142905.hg1004s02] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Genetic markers for leukemias and lymphomas include chromosomal translocations and antigen-receptor gene rearrangements. Clonal rearrangements of immunoglobulin or T cell receptor (TCR) genes reflect clonal proliferations of lymphocytes, a characteristic feature of lymphoid neoplasia. These rearrangements can be detected as described in this unit by Southern blot hybridization or, in many instances, the polymerase chain reaction (PCR). Specific chromosomal translocations can also serve as markers for clonality, for malignant transformation, and for various defined subtypes of hematopoietic cancers. PCR protocols are described for detection of the two most commonly assayed translocations, t(9;22) of chronic myelogenous leukemia or acute lymphoblastic leukemia, and t(14;18) of follicular lymphomas.
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Affiliation(s)
- J Longtine
- Brigham and Women's Hospital, Boston, Massachusetts, USA
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Plaza JA, Morrison C, Magro CM. Assessment of TCR-β clonality in a diverse group of cutaneous T-Cell infiltrates. J Cutan Pathol 2008; 35:358-65. [DOI: 10.1111/j.1600-0560.2007.00813.x] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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Gallardo F, Bellosillo B, Serrano S, Pujol R. Genotypic Analysis in Primary Cutaneous Lymphomas Using the Standardized Biomed-2 Polymerase Chain Reaction Protocols. ACTAS DERMO-SIFILIOGRAFICAS 2008. [DOI: 10.1016/s1578-2190(08)70328-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
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Liu H, Bench AJ, Bacon CM, Payne K, Huang Y, Scott MA, Erber WN, Grant JW, Du MQ. A practical strategy for the routine use of BIOMED-2 PCR assays for detection of B- and T-cell clonality in diagnostic haematopathology. Br J Haematol 2007; 138:31-43. [PMID: 17555445 DOI: 10.1111/j.1365-2141.2007.06618.x] [Citation(s) in RCA: 88] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
BIOMED-2 polymerase chain reaction (PCR) assays for clonality analysis of immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangements were evaluated in routine haematopathological practice where paraffin-embedded tissues constitute the majority of specimens. One hundred and twenty-five fresh/frozen and 316 paraffin specimens were analysed for DNA quality and clonality. Seventy-nine per cent of paraffin specimens yielded PCR products of over 300 bp. These specimens and all fresh/frozen specimens were analysed with the complete set of BIOMED-2 reactions for IG (8 reactions) and/or TCR (6 reactions) gene rearrangements. The rate of detection of clonality was 96% in mature B-cell neoplasms and 98% in mature T-cell neoplasms and there were no significant differences in these rates between paraffin and fresh/frozen specimens. As the value of sole use of any individual BIOMED-2 reaction in clonality detection was limited, we assessed combinations of reactions that gave the greatest sensitivity with fewest reactions and were applicable for both fresh/frozen and paraffin specimens. For IG gene rearrangements, three reactions combining one targeting the IG heavy chain framework-2 region and two targeting the IG kappa locus achieved a 91% detection rate. For TCR gene rearrangements, the two TCR gamma reactions gave a 94% detection rate. We therefore recommend this strategy as the first-line assays for routine B- and T-cell clonality analysis in diagnostic haematopathology.
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Affiliation(s)
- Hongxiang Liu
- Department of Histopathology, Addenbrooke's Hospital, Cambridge, UK
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Fraser-Andrews EA, Mitchell T, Ferreira S, Seed PT, Russell-Jones R, Calonje E, Whittaker SJ. Molecular staging of lymph nodes from 60 patients with mycosis fungoides and Sézary syndrome: correlation with histopathology and outcome suggests prognostic relevance in mycosis fungoides. Br J Dermatol 2006; 155:756-62. [PMID: 16965425 DOI: 10.1111/j.1365-2133.2006.07428.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
BACKGROUND Histological evidence of lymph node involvement is associated with a poor prognosis in patients with cutaneous T-cell lymphoma (CTCL). OBJECTIVES To determine whether T-cell receptor (TCR) gene analysis is of prognostic relevance in CTCL. METHODS TCR gene analysis was performed on lymph node specimens from 60 patients with mycosis fungoides (MF) and Sézary syndrome (SS) using a highly sensitive polymerase chain reaction (PCR)/single-strand conformational polymorphism analysis and results were correlated with skin, overall clinical and histological lymph node stages. RESULTS The frequency with which a T-cell clone was detected in lymph node samples from patients with MF increased with skin stage, overall clinical stage and with the degree of histological involvement: six of 19 patients with uninvolved lymph nodes or limited histological involvement (LN0-2) and 13 of 14 patients with advanced histological involvement (LN3-4) had a detectable T-cell clone. In SS, 22 of 27 patients had a detectable lymph node T-cell clone. The clonal patients had a poorer prognosis than nonclonal patients (median survival from biopsy of > 72 months vs. 16 months for MF and 41.5 vs. 16.5 months for SS). Regression analysis confirmed that TCR gene analysis identifies a group of MF patients with a worse prognosis (P = 0.013). However, the molecular lymph node stage did not provide independent prognostic information in this cohort of patients in multivariate analysis. CONCLUSIONS Molecular staging in MF and SS using a PCR-based method for TCR gene analysis provides additional information to histological examination. Specifically, this study identified a group of MF patients with early lymph node involvement with a poorer prognosis. However, a larger prospective study of patients with MF and early histological lymph node involvement is required to confirm whether molecular staging of lymph nodes provides independent prognostic information in a multivariate model.
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Affiliation(s)
- E A Fraser-Andrews
- Department of Dermatopathology, St John's Institute of Dermatology, St Thomas' Hospital, London SE1 7EH, UK.
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Uemura A, Sugahara K, Nagai H, Murata K, Hasegawa H, Hirakata Y, Tsukasaki K, Yamada Y, Kamihira S. An ATL cell line with an IgH pseudo-rearranged band pattern by southern blotting: a pitfall of genetic diagnosis. ACTA ACUST UNITED AC 2005; 11:8-13. [PMID: 15790547 DOI: 10.1532/lh96.04061] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Adult T-cell leukemia (ATL), which is a mature T-cell malignancy that develops from human T-cell leukemia virus type-1 (HTLV-1)-infected T-cells, is diagnosed based on morphologic, immunophenotypic, serologic, and genetic characteristics. In particular, Southern blot hybridization (SBH) and polymerase chain reaction analyses for antigen receptor genes and the retrovirus of HTLV-1 provide a diagnostic hallmark for the clonality of leukemic cells and the causative agent of the disease. We report here a case of an ATL cell line, designated as SO4 cells, established from primary ATL cells presenting with an irrational genetic abnormality of the immunoglobulin heavy chain (IgH)-rearranged band in spite of harboring a clonally rearranged T-cell receptor gene and a clonally integrated provirus of HTLV-1 within their genomic DNA. Moreover, fluorescence in situ hybridization analysis using the IgH (14q32) dual-color break-apart probe revealed 3 pair-signals of colocalizing red and green spots, implying 2 intact and 1 amplified 14q32 regions without translocation, where the region contains the IgH gene locus. Although the exact mechanism remains to be elucidated, some alteration of a portion of the amplified 14q32 region seems to have a role in the false-positive band pattern in the SBH. The SO4 cells, in the hematology laboratory, will provide a lesson about the pitfalls of genetic tests for mature T-cell neoplasms and contribute to the genetic elucidation of leukomogenesis as an in vitro model.
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Affiliation(s)
- Akiko Uemura
- Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
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Cienava EA, Barnhart KF, Brown R, Mansell J, Dunstan R, Credille K. Morphologic, immunohistochemical, and molecular characterization of hepatosplenic T-cell lymphoma in a dog. Vet Clin Pathol 2005; 33:105-10. [PMID: 15195270 DOI: 10.1111/j.1939-165x.2004.tb00357.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
A 13-year-old neutered male Jack Russell Terrier (Parson Russell Terrier) was presented to the Texas Veterinary Medical Center with a history of lethargy, depression, vomiting, and fever. The dog had mildly regenerative anemia, severe thrombocytopenia and low antithrombin activity. Marked splenomegaly was found on physical examination and imaging studies, and malignant round cell neoplasia and marked extramedullary hematopoiesis were diagnosed on aspirates of the spleen. The dog underwent exploratory laporatomy and splenectomy. Because of a rapid decline in clinical condition postsurgery, the dog was euthanized. Splenic and hepatic biopsies were submitted for histopathologic evaluation. A neoplastic population of round cells was found throughout the splenic parenchyma and within hepatic sinusoids. The neoplastic cells stained strongly positive for CD3 (T-cell marker) and were negative for CD79a (B-cell marker) and lysozyme (histiocytic marker). A diagnosis of T-cell lymphoma was confirmed by assessment of T-cell clonality using canine-specific polymerase chain reaction-based techniques. Although expression of the gammadelta T-cell receptor was not evaluated, this case shares many similarities with a rare syndrome in humans known as hepatosplenic gammadelta T-cell lymphoma.
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Affiliation(s)
- Elizabeth A Cienava
- Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX., USA.
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21
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Thaler S, Burger AM, Schulz T, Brill B, Bittner A, Oberholzer PA, Dummer R, Schnierle BS. Establishment of a mouse xenograft model for mycosis fungoides. Exp Dermatol 2005; 13:406-12. [PMID: 15217360 DOI: 10.1111/j.0906-6705.2004.00201.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Mycosis fungoides (MF) is the most frequent variant of cutaneous T-cell lymphomas (CTCLs). MF primarily involves the skin initially with patches and plaques. In later stages, cutaneous tumors develop and tumor cells may spread to lymph nodes and finally to visceral sites. Here, we describe an animal model for MF in immune-deficient nude mice, using the CTCL cell line MyLa. Subcutaneous transplantation of MyLa cells leads to the formation of cutaneous tumors in 80% of the mice (50/60 total). Spread of tumor cells to visceral sites was detected by immunohistochemistry and polymerase chain reaction (PCR)-based detection of specific T-cell receptor-gamma rearrangement. MyLa cells were found circulating in the blood, lymph nodes, and in blood vessels of heart, kidney, lung, and liver. In lung and liver tissue, tumor cells presented perivascular invasion, but no large secondary tumors developed. The nude mouse model described here will be a valuable test system for new therapeutic approaches for the treatment of MF and opens the unique opportunity to study the disease in vivo.
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MESH Headings
- Animals
- Cell Line
- Cell Line, Tumor
- Cell Separation
- Disease Models, Animal
- Flow Cytometry
- Gene Rearrangement, T-Lymphocyte
- Immunohistochemistry
- Kinetics
- Lymph Nodes/pathology
- Lymphoma, T-Cell/pathology
- Mice
- Mice, Nude
- Mycosis Fungoides/pathology
- Neoplasm Transplantation
- Neoplastic Cells, Circulating
- Polymerase Chain Reaction
- RNA, Small Interfering/metabolism
- Receptors, Antigen, T-Cell, gamma-delta/immunology
- Skin Neoplasms/pathology
- Time Factors
- Tissue Distribution
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Affiliation(s)
- Sonja Thaler
- Institute for Biomedical Research, Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, D-60596 Frankfurt/Main, Germany
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22
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Suga M, Yamaguchi M, Ichimiya M, Yoshikawa Y, Hamamoto Y, Muto M. A rare case of the cutaneous form of adult T-cell leukaemia/lymphoma: assessment of remission by PCR for clonal T-cell receptor gamma gene rearrangements in an electron beam-irradiated cutaneous lesion. Clin Exp Dermatol 2005; 30:40-2. [PMID: 15663501 DOI: 10.1111/j.1365-2230.2004.01641.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Adult T-cell leukaemia/lymphoma is a lymphoproliferative disorder aetiologically associated with human T-cell lymphotropic virus type I infection. A cutaneous lesion often develops in the disease, and in rare cases, is even the only manifestation. Here we report a rare case of 'cutaneous' adult T-cell leukaemia/lymphoma with neither atypical cells in the peripheral blood nor lymph node involvement. All nodular lesions were completely eliminated after local electron beam irradiation (20 Gy/nodule in total). To evaluate whether or not there were residual lymphoma cells in the skin, we performed PCR to detect clonal T cell receptor gamma gene rearrangements. The sample from the nodule before irradiation showed evidence of a rearranged band, which was not detected at the same site after treatment nor in any peripheral blood. The findings suggest that this procedure is useful for the evaluation of therapeutic effects and the early detection of lymphoma recurrence.
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MESH Headings
- Female
- Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor
- Humans
- Leukemia-Lymphoma, Adult T-Cell/genetics
- Leukemia-Lymphoma, Adult T-Cell/pathology
- Leukemia-Lymphoma, Adult T-Cell/radiotherapy
- Lymphoma, T-Cell, Cutaneous/genetics
- Lymphoma, T-Cell, Cutaneous/pathology
- Lymphoma, T-Cell, Cutaneous/radiotherapy
- Middle Aged
- Polymerase Chain Reaction/methods
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Affiliation(s)
- M Suga
- Department of Dermatology and Biomolecular Recognition, Yamaguchi University School of Medicine, Minami-Kogushi, Ube, Yamaguchi, Japan
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23
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Chen M, Deng A, Crowson AN, Srinivasan M, Yearsley KH, Jewell S, Morrison C, Long S, Werling R, Magro C. Assessment of T-cell Clonality via T-cell Receptor-γ Rearrangements in Cutaneous T-cell–Dominant Infiltrates Using Polymerase Chain Reaction and Single-stranded DNA Conformational Polymorphism Assay. Appl Immunohistochem Mol Morphol 2004; 12:373-9. [PMID: 15536341 DOI: 10.1097/00129039-200412000-00016] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Discerning the pathologic significance of cutaneous T-cell infiltrates can pose a diagnostic challenge for dermatopathologists. Reactive conditions such as drug-associated lymphomatoid hypersensitivity and lymphomatoid lupus erythematosus can demonstrate lymphoid atypia and a phenotype resembling cutaneous T-cell lymphoma (CTCL). Further, lymphoid dyscrasias such as pityriasis lichenoides chronica, large plaque parapsoriasis, and atypical pigmentary purpura confuse the picture because they not only mimic CTCL but also represent prelymphomatous states with inherent malignant potential. Although the emergence of a dominant clone has been considered a clue indicative of a T-cell dyscrasia, there are reports concerning the identification of monoclonality in biopsies of reactive lymphoid infiltrates. We have conducted a modified single-stranded DNA conformational polymorphism (SSCP) assay using paraffin-embedded, formalin-fixed tissue on 92 T-cell-rich biopsies to determine the relative specificity and sensitivity of this methodology. In addition, laser capture microdissection (LCM) was performed on 22 of the 92 samples to isolate the area of interest and to compare its specificity and sensitivity with those SSCP assays performed without LCM. We found that monoclonality or oligoclonality is 86% specific for preneoplastic and neoplastic states, whereas the finding of polyclonality appears to be relatively specific for a reactive process. Some cases of reversible T-cell dyscrasia produced a molecular profile mimicking lymphoma or prelymphomatous states by virtue of monoclonality or oligoclonality. Although LCM appears to improve the sensitivity for detecting preneoplastic conditions, the relative specificity appears to be the same as that encountered with routine SSCP.
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MESH Headings
- Clone Cells/physiology
- Diagnosis, Differential
- Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor/genetics
- Humans
- Leukemic Infiltration/diagnosis
- Leukemic Infiltration/genetics
- Lymphoma, T-Cell, Cutaneous/diagnosis
- Lymphoma, T-Cell, Cutaneous/genetics
- Polymerase Chain Reaction
- Polymorphism, Single-Stranded Conformational
- Precancerous Conditions/diagnosis
- Precancerous Conditions/pathology
- Receptors, Antigen, T-Cell, gamma-delta/genetics
- Skin/pathology
- Skin Diseases/diagnosis
- Skin Diseases/immunology
- Skin Diseases/pathology
- Skin Neoplasms/diagnosis
- Skin Neoplasms/pathology
- T-Lymphocytes/cytology
- T-Lymphocytes/physiology
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Affiliation(s)
- Michael Chen
- College of Medicine and Public Health, Ohio State University, Columbus, Ohio, USA
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24
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Spagnolo DV, Ellis DW, Juneja S, Leong ASY, Miliauskas J, Norris DL, Turner J. The role of molecular studies in lymphoma diagnosis: a review. Pathology 2004; 36:19-44. [PMID: 14757555 DOI: 10.1080/00313020310001648404] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Lymphoma classification is based on a multiparametric approach to diagnosis, in which clinical features, morphology, immunophenotype, karyotype and molecular characteristics are important to varying degrees. While in most cases, a diagnosis can be confidently established on the basis of morphology and immunophenotype alone, a small proportion of diagnostically difficult cases will rely on molecular studies to enable a definitive diagnosis. This review discusses the various molecular techniques available including Southern blotting (SB), polymerase chain reaction (PCR), fluorescence in situ hybridisation (FISH)--including multicolour-FISH/spectral karyotyping and comparative genomic hybridisation--and also gene expression profiling using cDNA microarray technology. Emphasis is given to the analysis of antigen receptor gene rearrangements and chromosomal translocations as they relate to lymphoma diagnosis and also in the setting of minimal residual disease (MRD) detection and monitoring. Laboratories performing these tests need to have expertise in these areas of testing, and there is a need for greater standardisation of molecular tests. It is important to know the sensitivity and specificity of each test as well as its limitations and the pitfalls in the interpretation of results. Above all, results of molecular testing should never be considered in isolation, and must always be interpreted in the context of clinical and other laboratory data.
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Affiliation(s)
- Dominic V Spagnolo
- Division of Tissue Pathology, The Western Australian Centre for Pathology and Medical Research (PathCentre), Nedlands, WA, Australia.
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25
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Lin YW, Perkins JJ, Zhang Z, Aplan PD. Distinct mechanisms lead to HPRT gene mutations in leukemic cells. Genes Chromosomes Cancer 2004; 39:311-23. [PMID: 14978792 DOI: 10.1002/gcc.20005] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
Abstract
Leukemias are considered malignant clonal disorders arising from the accumulation of mutations in hematopoietic cells; the majority of these mutations are thought to be acquired somatically. Measurement of mutation frequency (Mf) at the hypoxanthine phosphoribosyltransferase (HPRT) locus has been developed as a method for estimating genomic instability. We investigated the Mf in 16 leukemic cell lines to determine whether these cell lines showed evidence of genomic instability. Although some leukemic cell lines had markedly elevated Mfs, the Mfs at the HPRT locus in leukemic cell lines were not always higher than those of B-lymphoblastoid cell lines and T lymphocytes from normal individuals. We were able to identify the HPRT mutation for 159 of 160 individual HPRT mutants. The HPRT mutations were characterized at a molecular level and classified as either gross chromosomal rearrangements (GCRs) or point mutations, such as single-nucleotide substitutions, insertions, or deletions. With rare exceptions, individual leukemic cell lines showed either point mutations or GCR, but not both. Of note, all the cell lines that primarily showed point mutations are known to be defective in mismatch repair machinery.
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Affiliation(s)
- Ying-Wei Lin
- Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20889-510, USA.
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26
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van Dongen JJM, Langerak AW, Brüggemann M, Evans PAS, Hummel M, Lavender FL, Delabesse E, Davi F, Schuuring E, García-Sanz R, van Krieken JHJM, Droese J, González D, Bastard C, White HE, Spaargaren M, González 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 2004; 17:2257-317. [PMID: 14671650 DOI: 10.1038/sj.leu.2403202] [Citation(s) in RCA: 2366] [Impact Index Per Article: 112.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.
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Affiliation(s)
- J J M van Dongen
- Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
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27
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Esposito I, Bergmann F, Penzel R, di Mola FF, Shrikhande S, Büchler MW, Friess H, Otto HF. Oligoclonal T-cell populations in an inflammatory pseudotumor of the pancreas possibly related to autoimmune pancreatitis: an immunohistochemical and molecular analysis. Virchows Arch 2004; 444:119-26. [PMID: 14722765 DOI: 10.1007/s00428-003-0949-1] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2003] [Accepted: 11/26/2003] [Indexed: 12/31/2022]
Abstract
Inflammatory pseudotumors (IPT), also known as inflammatory myofibroblastic tumors (IMT), are benign inflammatory processes that may have an infectious etiology and are very rare in the pancreatico-biliary region. Recent studies suggest a biological distinction between IPT and IMT, the latter being a true neoplastic process. We describe a case of pancreatic IPT, originally diagnosed as malignancy, which presumably recurred 4 months after the operation. Histologically, the tumor consisted of a smooth muscle actin and CD68-positive spindle cell population and a more abundant mononuclear inflammatory cell population, primarily composed of macrophages and T-lymphocytes. Inflammatory cells were the source of connective tissue growth factor and transforming growth factor-beta1 and tended to accumulate around nerves and blood vessels, as well as around residual pancreatic parenchymal elements, where an intense angiogenetic response was detected. Comparative genomic hybridization analysis of the tumor showed no chromosomal imbalances. Polymerase chain reaction-based analysis of T-cell receptor gamma gene rearrangement revealed an oligoclonal pattern. These findings suggest that the pathogenesis of aggressive cases of IPT could be related to the development of an intense and self-maintaining immune response, with the emergence of clonal populations of T-lymphocytes. The relation of the pancreatic IPT to autoimmune pancreatitis is emphasized.
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Affiliation(s)
- Irene Esposito
- Department of Pathology, University of Heidelberg, Im Neuenheimer Feld 220, 69120, Heidelberg, Germany.
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28
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Nishie W, Yokota K, Sawamura D, Sato-Matsumura K, Tanimura S, Osawa R, Kawashima T, Yokota T, Shimizu H. Detection of circulating lymphoma cells in subcutaneous panniculitis-like T-cell lymphoma. Br J Dermatol 2003; 149:1081-2. [PMID: 14632825 DOI: 10.1111/j.1365-2133.2003.05612.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
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29
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Delabesse E, Asnafi V, Macintyre E. [Application of molecular biology techniques to malignant haematology]. Transfus Clin Biol 2003; 10:335-52. [PMID: 14572550 DOI: 10.1016/s1246-7820(03)00105-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Malignant hemopathies, although heterogeneous in their prognosis and oncogenesis, represent an interesting model for studying cancer genesis mechanisms in man through the recurrent presence of genetic abnormalities involved in oncogenesis and the availability of tumour material. Nowadays, molecular biology techniques are very much used for the diagnosis, the treatment and the follow-up of these diseases. Firstly used for research, the new techniques have completely changed our ability to characterise malignant hemopathies and to understand the cancer-inducing processes, permitting us to perform the biological assessment of patients with malignant hemopathies, the diagnosis, and to estimate and follow the outcome of patients after treatment. At a more fundamental level, the structural and functional analysis of the deregulated genes implied in leukaemia and lymphoma has improved our knowledge and understanding of oncogenic and physiologic mechanisms significantly.
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Affiliation(s)
- E Delabesse
- Laboratoire d'hématologie, hôpital Necker-Enfants Malades, 149, rue de Sèvres, 75743 Paris 15, France.
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30
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Magro CM, Nuovo GJ, Crowson AN. The utility of the in situ detection of T-cell receptor Beta rearrangements in cutaneous T-cell-dominant infiltrates. DIAGNOSTIC MOLECULAR PATHOLOGY : THE AMERICAN JOURNAL OF SURGICAL PATHOLOGY, PART B 2003; 12:133-41. [PMID: 12960695 DOI: 10.1097/00019606-200309000-00004] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
The diagnostic assessment of cutaneous T-cell infiltrates is problematic for dermatopathologists. A variety of conditions, including lymphomatoid hypersensitivity reactions and lymphomatoid lupus erythematosus, can demonstrate lymphoid atypia and phenotypic changes that can mimic cutaneous T-cell lymphoma (CTCL). A similar issue revolves around lymphoid dyscrasias, which includes parapsoriasis, atypical pigmentary purpura, pityriasis lichenoides chronica, indeterminate lymphocytic lobular panniculitis, and lymphomatoid papulosis, which can progress to CTCL. A reverse transcription (RT) in situ PCR assay for T-cell receptor beta rearrangements (TCRbeta) was used to assess T-cell clonality in formalin-fixed, paraffin-embedded tissues. In 7 of 8 cases of classic CTCL, the RT in situ PCR assay for TCRbeta rearrangement showed monoclonality; the other was biclonal. Further, in cases with multiple lesions over time, the same T-cell clone could be detected including in those patients whose biopsies showed large-cell transformation. Monoclonality was also demonstrated in each of 2 cases of cutaneous lymphomatoid papulosis. Demonstration of oligoclonality (and one case of biclonality) by RT in situ PCR was confined to those cases that either represented prelymphomatous conditions such as large plaque parapsoriasis or pityriasis lichenoides or lesions of drug-induced lymphomatoid hypersensitivity that all demonstrated clinical regression. In conclusion, RT in situ PCR for TCRbeta, which can be done on formalin-fixed biopsies and allows direct correlation of the molecular data with the histology, is a useful adjunctive test in the differentiation of CTCL from its mimics.
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MESH Headings
- Adult
- Aged
- Aged, 80 and over
- Biomarkers, Tumor/metabolism
- Clone Cells
- DNA, Neoplasm/analysis
- Female
- Fixatives
- Formaldehyde
- Gene Rearrangement, beta-Chain T-Cell Antigen Receptor/genetics
- Genes, T-Cell Receptor/genetics
- Humans
- Immunoenzyme Techniques
- Leukemic Infiltration
- Lymphoma, T-Cell, Cutaneous/genetics
- Lymphoma, T-Cell, Cutaneous/metabolism
- Lymphoma, T-Cell, Cutaneous/pathology
- Male
- Middle Aged
- Paraffin Embedding
- Reverse Transcriptase Polymerase Chain Reaction/methods
- Skin/metabolism
- Skin/pathology
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Affiliation(s)
- Cynthia M Magro
- Department of Pathology, Ohio State University, Columbus, OH, USA
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31
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Mognato M, Bortoletto E, Ferraro P, Baggio L, Cherubini R, Canova S, Russo A, Celotti L. Genetic damage induced by in vitro irradiation of human G0 lymphocytes with low-energy protons (28 keV/microm): HPRT mutations and chromosome aberrations. Radiat Res 2003; 160:52-60. [PMID: 12816523 DOI: 10.1667/0033-7587(2003)160[0052:gdibiv]2.0.co;2] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Cell survival, mutations and chromosomal effects were studied in primary human lymphocytes exposed in G0 phase to a proton beam with an incident energy of 0.88 MeV (incident LET of 28 keV/microm) in the dose range 0.125-2 Gy. The curves for survival and mutations at the hypoxanthine-guanine phosphoribosyl transferase locus were obtained by fitting the experimental data to linear and linear-quadratic equations, respectively. In the dose interval 0-1.5 Gy, the alpha parameters of the curves were 0.42/Gy and 3.6 x 10(-6) mutants/Gy, respectively. The mutation types at the HPRT locus were analyzed by multiplex-PCR in 94 irradiated and 41 nonirradiated clones derived from T lymphocytes from five healthy donors. All clones showed a normal multiplex-PCR pattern and were classified as point mutations. Chromosome aberration data were fitted as a linear function of dose (alpha = 0.62 aberrations per cell Gy(-1)). By irradiating G0 lymphocytes from a single subject with 28 keV/microm protons and gamma rays, an RBE of 6.07 was obtained for chromosome aberrations. An overinvolvement of chromosome 9 relative to chromosome 7 was found in chromosome breaks after chromosome painting analysis.
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32
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Sigurdardottir M, Sigurdsson H, Barkardottir RB, Kristjansdottir S, Agnarsson BA. Lymphoid tumours of the ocular adnexa: a morphologic and genotypic study of 15 cases. ACTA OPHTHALMOLOGICA SCANDINAVICA 2003; 81:299-303. [PMID: 12780412 DOI: 10.1034/j.1600-0420.2003.00067.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
PURPOSE To examine all lymphoproliferative lesions of the ocular adnexa diagnosed in Iceland during 1983-2000 and to determine whether polymerase chain reaction (PCR) methods to determine clonality are helpful in characterizing these lesions. METHODS All patients diagnosed with lymphoproliferative lesions in the ocular adnexa in the years 1983-2000 were included in the study. Polymerase chain reaction studies for clonality were performed on these lesions. RESULTS Fifteen cases were identified. Seven were classified as inflammatory pseudotumour, one as lymphoid hyperplasia, four as atypical lymphoid hyperplasia and three as lymphoma. Of 12 cases examined by PCR, three were monoclonal for B-cells (one lymphoma, one inflammatory pseudotumour and one atypical lymphoid hyperplasia) while the remaining lesions (including two lymphomas) appeared polyclonal. CONCLUSION The results of this study suggest that analysis of clonality by PCR methods may be of limited use in classifying lymphoproliferative lesions of the ocular adnexa as benign or malignant. These results underscore the importance of using several techniques when determining clonality.
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33
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Lawnicki LC, Rubocki RJ, Chan WC, Lytle DM, Greiner TC. The distribution of gene segments in T-cell receptor gamma gene rearrangements demonstrates the need for multiple primer sets. J Mol Diagn 2003; 5:82-7. [PMID: 12707372 PMCID: PMC1907316 DOI: 10.1016/s1525-1578(10)60456-4] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Limited data exist regarding the distribution of gene segments used in T-cell receptor gamma gene rearrangements (TCR gamma GR) in T-cell lymphoproliferative disorders. The reported efficacy of TCR gamma GR protocols ranges from 60% to greater than 90%. Laboratories reporting a lower detection rate tend to use a limited set of primers. The goal of our study was to provide TCR gamma GR data to demonstrate the molecular biological basis for needing multiple primer sets targeting all gene segments. Sixty cases with a confirmed histological diagnosis of a T-cell lymphoproliferative disorder and TCR gamma GR were identified in our lymphoma registry from 1995 to 2001. DNA was obtained from fresh/frozen tissue, cell lysates, or paraffin-embedded tissue. Variable (V gamma) region gene segments were identified using denaturing gradient gel electrophoresis, which was used to select the cases in the study. Capillary electrophoresis using fluorescent-labeled joining (J gamma) region primers was performed to identify J gamma segments. Sixty cases contained a total of 98 TCR gamma GR, as some cases have more than one rearrangement. The most frequent gene segment combination involved the V gamma 1-8 and J gamma 1/2 segments. If a single primer set directed at these two segments were used for clinical diagnosis, that pair of primers would only diagnose 67% of cases as positive for TCR gamma GR. Our gene segment distribution data emphasize the importance of using a comprehensive set of V gamma and J gamma primers for an optimal detection rate of TCR gamma GR. Protocols with limited numbers of primers should be reconsidered.
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Affiliation(s)
- Lyle C Lawnicki
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska 68198, USA
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34
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Szatrowski TP, Dodge RK, Reynolds C, Westbrook CA, Frankel SR, Sklar J, Stewart CC, Hurd DD, Kolitz JE, Velez-Garcia E, Stone RM, Bloomfield CD, Schiffer CA, Larson RA. Lineage specific treatment of adult patients with acute lymphoblastic leukemia in first remission with anti-B4-blocked ricin or high-dose cytarabine: Cancer and Leukemia Group B Study 9311. Cancer 2003; 97:1471-80. [PMID: 12627512 DOI: 10.1002/cncr.11219] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
BACKGROUND Anti-B4-blocked ricin is an immunotoxin comprised of an anti-CD19 murine monoclonal antibody (B4) conjugated to blocked ricin, which has cytotoxic activity in patients with lymphoid malignancies. METHODS Adults with untreated acute lymphoblastic leukemia (ALL) were treated with a previously developed and tested chemotherapeutic regimen. Patients with CD19 positive ALL were given anti-B4-blocked ricin as 2 7-day continuous infusions 1 week apart. Patients with CD19 negative ALL received high-dose cytarabine. Serial polymerase chain reaction (PCR) assays of BCR-ABL, immunoglobulin heavy chain (IGH), and T-cell receptor (TCR) genes were used to measure the impact of lineage specific intensification treatment on minimal residual disease. RESULTS Eighty-two adults were enrolled, and 78 were eligible. The median age was 34 years (range, 17-81 years). Sixty-six patients (85%) achieved complete remission. Forty-six patients received the anti-B4-blocked ricin, which generally was well tolerated; 80% were able to receive both courses. The most common toxicity was asymptomatic transient elevation of liver function tests in 72% of patients. Lymphopenia occurred in 46% of patients. Two patients developed antibodies to the anti-B4-blocked ricin. Molecular monitoring before and after the experimental course of intensification did not show a consistent change in the number of leukemia cells remaining, and the immediate posttreatment PCR studies did not correlate with remission duration. CONCLUSIONS Intensification therapy with anti-B4-blocked ricin is feasible for patients with CD19 positive ALL, although there is little evidence of an additional clinical benefit from the anti-B4-blocked ricin. Cancer 2003;97:1471-80.
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Affiliation(s)
- Ted P Szatrowski
- Weill Medical College of Cornell University, New York, New York, USA
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35
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Kempf W, Haeffner AC, Zepter K, Sander CA, Flaig MJ, Mueller B, Panizzon RG, Hardmeier T, Adams V, Burg G. Angiolymphoid hyperplasia with eosinophilia: evidence for a T-cell lymphoproliferative origin. Hum Pathol 2002; 33:1023-9. [PMID: 12395376 DOI: 10.1053/hupa.2002.128247] [Citation(s) in RCA: 71] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Angiolymphoid hyperplasia with eosinophilia (ALHE) is commonly regarded an angioproliferative process characterized by the presence of prominent, bizarrely shaped blood vessels. These vessels are accompanied by an inflammatory infiltrate that is thought to be a reactive component. Both the cell of origin and the pathogenesis of ALHE remain controversial. To define the histogenesis of this disorder, we analyzed the phenotypic and genotypic profile of the inflammatory infiltrate in ALHE by immunohistochemistry and T-cell receptor gene rearrangement by polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis, as well as automated high-resolution PCR fragment analysis. Five of 7 ALHE patients displayed a clonal T-cell population and proliferative T-cell activity in lesional tissue. Most of these cases followed a protracted and therapy-reluctant course with recurrences. These data suggest that ALHE or a subset of ALHE cases harboring a clonal T-cell population may represent a T-cell lymphoproliferative disorder of a benign or low-grade malignant nature.
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Affiliation(s)
- Werner Kempf
- Department of Dermatology, University Hospital Zurich, Switzerland
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36
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Schwab C, Willers J, Niederer E, Ludwig E, Kündig T, Grob P, Burg G, Dummer R. The use of anti-T-cell receptor-Vbeta antibodies for the estimation of treatment success and phenotypic characterization of clonal T-cell populations in cutaneous T-cell lymphomas. Br J Haematol 2002; 118:1019-26. [PMID: 12199780 DOI: 10.1046/j.1365-2141.2002.03726.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Sézary syndrome and Mycosis fungoides are the most common forms of cutaneous T-cell lymphomas. To assess the response to different therapies especially in Sézary syndrome, it is helpful to monitor the percentage of circulating tumour cells in the blood. The use of T-cell receptor (TCR)-Vbeta specific monoclonal antibodies provides a suitable tool for detecting Sézary cells. In this study, we analysed the levels of clonal CD4+Vbeta+ cells of seven patients with various treatment modalities using flow cytometry and investigated the immunophenotype of the clonal cells by double staining with a panel of antibodies recognizing lymphatic surface markers. Additionally, a polymerase chain reaction-denaturing gradient gel electrophoresis assay was performed on clonal CD4+Vbeta2+ cells, showing that these cells carry a Vgamma10/11, JgammaP1/2 TCR rearrangement. Follow-up studies revealed close association of the Vbeta+ clone developmentwith the clinical response to different therapiesinsixpatients. Intwo cases, the CD4+Vbeta+ cells decreased accompanied by partial regression or even complete remission. In four cases, a stable or increasing clonal CD4+Vbeta+ population reflected well a stable or progressing course of the disease. Double staining of Vbeta+ cells revealed the following pattern, CD3+, CD5+, CD7+, CD28+, CD80-, CD86+ and human leucocyte antigen (HLA) class I+. In contrast, HLA-DR was heterogeneously expressed. We conclude that identification and monitoring of CD4+Vbeta+ clonal T cells by fluorescence-activated cell sorting with double staining is a suitable method to assess clinical responses to different therapies.
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MESH Headings
- Aged
- Antibodies, Monoclonal
- CD4-Positive T-Lymphocytes/immunology
- Female
- Flow Cytometry
- Gene Rearrangement, beta-Chain T-Cell Antigen Receptor
- Humans
- Immunophenotyping
- Lymphocyte Count
- Lymphoma, T-Cell, Cutaneous/drug therapy
- Lymphoma, T-Cell, Cutaneous/genetics
- Lymphoma, T-Cell, Cutaneous/immunology
- Male
- Middle Aged
- Peptide Fragments/immunology
- Polymerase Chain Reaction
- Receptors, Antigen, T-Cell, alpha-beta/immunology
- Treatment Outcome
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Affiliation(s)
- Cornelia Schwab
- Department of Dermatology, University Hospital of Zurich, Zurich, Switzerland
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37
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Greiner TC, Rubocki RJ. Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements. J Mol Diagn 2002; 4:137-43. [PMID: 12169674 PMCID: PMC1906981 DOI: 10.1016/s1525-1578(10)60694-0] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
We describe the use of fluorescent-labeled primers to analyze T-cell receptor gamma gene rearrangements (TCR gamma GR) using capillary electrophoresis in the ABI Prism 310 Genetic Analyzer. We also compare the performance with denaturing gradient gel electrophoresis (DGGE). In a single multiplex polymerase chain reaction (PCR) we amplified TCR gamma GR with primers for all known groups of variable region genes, and joining region genes described in lymphoid neoplasms. Ten reactive samples, followed by five cell lines and 25 tumor samples with 41 individual TCR gamma GR (due to many biallelic rearrangements) previously identified by DGGE, were analyzed to validate the technique. The capillary electrophoresis protocol has 92% concordance for both TCR clonal status (23 of 25) and 95% concordance in the number of individual TCR gamma GR (38 of 41) identified by DGGE. The reproducible sensitivity for detecting TCR gamma GR diluted in reactive lymphoid DNA is 2% in clinical applications. Discrimination of predominant rearrangements requires a minimum ratio of two times the height of the normal distribution of polyclonal peaks. Capillary electrophoresis can provide results within 60 minutes for each specimen after PCR is complete. Capillary electrophoresis provides a faster result than sequence-based separation methods and gives an archival electronic record. Fluorescent labeling allows the identification of both the variable and joining gene segments used in a TCR gamma GR. The effectiveness of capillary electrophoresis is similar to DGGE.
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Affiliation(s)
- Timothy C Greiner
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-3135, USA.
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38
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Crisi GM, Emanuel JR, Johnson C, Crotty P, Costa J, Tallini G. Semireannealing, single-stranded conformational polymorphism: a novel and effective tool for the diagnosis of T-cell clonality. DIAGNOSTIC MOLECULAR PATHOLOGY : THE AMERICAN JOURNAL OF SURGICAL PATHOLOGY, PART B 2002; 11:67-74. [PMID: 12045709 DOI: 10.1097/00019606-200206000-00002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Single-stranded conformational polymorphism (SSCP) is often used for the diagnosis of T-cell clonality in lymphoproliferative disorders. We introduce a semireannealing SSCP (SR-SSCP) protocol that is rapid, reproducible, and effective. By denaturing and reannealing the polymerase chain reaction (PCR) product before high-resolution polyacrylamide gel electrophoresis, it is possible to generate a diagnostic fingerprint for each case with clonal T-cell receptor-gamma (TCR-gamma) gene rearrangement detected after PCR with TCR-gamma specific consensus primers. Discrete and distinct denatured single-stranded DNA band profiles characterize the rearranged TCR-gamma clones. In the same gel, the clone size may be estimated in the reannealed double-stranded PCR DNA and can be assessed down to the 2% clonal T-cell population level. Eighty-four cases, including 37 T-cell neoplasms, 29 B-cell neoplasms, and 18 reactive lymph node samples were analyzed by SR-SSCP. Clonal TCR-gamma rearrangement was diagnosed in 32 out of 37 T-cell neoplasms but in none of the B-cell tumors or reactive lymph node samples corresponding to sensitivity and specificity of 86.5% and 100%, respectively. We compare the results of SR-SSCP to those obtained by capillary electrophoresis and direct sequence analysis with 100% correlation. This novel method is applicable to any system for identification and quantitation of microheterogeneity in PCR products.
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MESH Headings
- Base Sequence
- Clone Cells/pathology
- DNA, Neoplasm/analysis
- Electrophoresis, Capillary/methods
- Electrophoresis, Polyacrylamide Gel
- Gene Rearrangement
- Humans
- Lymph Nodes/pathology
- Lymphoma, B-Cell/genetics
- Lymphoma, B-Cell/pathology
- Lymphoma, T-Cell/genetics
- Lymphoma, T-Cell/pathology
- Molecular Sequence Data
- Nucleic Acid Conformation
- Polymerase Chain Reaction/methods
- Polymorphism, Single-Stranded Conformational
- Pseudolymphoma/genetics
- Pseudolymphoma/pathology
- Receptors, Antigen, T-Cell, gamma-delta/genetics
- Sequence Alignment
- Sequence Analysis, DNA
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Affiliation(s)
- Giovanna Maria Crisi
- Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA
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39
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Szczepański T, Flohr T, van der Velden VHJ, Bartram CR, van Dongen JJM. Molecular monitoring of residual disease using antigen receptor genes in childhood acute lymphoblastic leukaemia. Best Pract Res Clin Haematol 2002; 15:37-57. [PMID: 11987915 DOI: 10.1053/beha.2002.0184] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are assumed to be unique 'fingerprint-like' sequences for each acute lymphoblastic leukaemia (ALL). Various clonal Ig/TCR gene rearrangements can be identified at diagnosis in virtually all childhood ALL patients, representing molecular targets for detection of minimal residual disease (MRD) during follow-up analysis. The usage of at least two MRD-PCR targets per patient generally ensures high sensitivity (</=1:10(4) normal cells) and prevents false-negative results owing to ongoing or secondary rearrangements.MRD monitoring in childhood ALL employing Ig/TCR gene rearrangements as PCR targets has significant prognostic value. This is particularly powerful for evaluation of early treatment response and consequently can be used for improved therapy stratification. Prolonged continuous MRD monitoring might be important for patients at intermediate or high risk of relapse. MRD monitoring in second complete remission identifies patients with excellent drug sensitivity and predicts outcome after stem cell transplantation.
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Affiliation(s)
- Tomasz Szczepański
- Department of Immunology, University Hospital, Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands
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40
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Plonquet A, Gherardi RK, Créange A, Antoine JC, Benyahia B, Grisold W, Drlicek M, Dreyfus P, Honnorat J, Khouatra C, Rouard H, Authier FJ, Farcet JP, Delattre JY, Delfau-Larue MH. Oligoclonal T-cells in blood and target tissues of patients with anti-Hu syndrome. J Neuroimmunol 2002; 122:100-5. [PMID: 11777548 DOI: 10.1016/s0165-5728(01)00452-0] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
T-cell clones of unknown significance (TCUS), assessed by monoclonal or oligoclonal T-cell patterns in PCR-DGGE, were detected in blood of 7/9 patients with anti-Hu syndrome. Clonal patterns were also detected in 2/2 neoplastic lymph nodes, and in 2/2 inflamed dorsal root ganglia from three patients. Only some T-cell clones found in target tissues were also detected in blood or non-target tissues, and likely corresponded to TCUS. In one patient, an identical T-cell clone was found in both neoplastic lymph node tissue and dorsal root ganglia, but not in blood. Dorsal root-infiltrating lymphocytes were cytotoxic CD8(+) TIA-1(+) T-cells. They were often found in close contact to sensory neurons, most of which expressed MHC-1. Taken together, these data support a direct effector role of cytotoxic CD8(+) T-cells, the same clones being likely operative in sensory neuron damage and immune-mediated tumor growth control.
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Affiliation(s)
- A Plonquet
- INSERM E0011, "Système neuromusculaire et inflammation", Faculté de Médecine Paris XII, 94010, Créteil, France
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41
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Merz H, Lange K, Gaiser T, Müller A, Kapp U, Bittner C, Harder S, Siebert R, Bentz M, Binder T, Diehl V, Feller AC. Characterization of a novel human anaplastic large cell lymphoma cell line tumorigenic in SCID mice. Leuk Lymphoma 2002; 43:165-72. [PMID: 11908723 DOI: 10.1080/10428190210193] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
L82, a novel anaplastic large cell lymphoma (ALCL) cell line was established from the pleural effusion of a 24-year-old patient with recurrent ALCL. L82 cells showed the typical morphologic features of ALCL cells with irregular, often indented, nuclear profiles, prominent nucleoli, and abundant cytoplasm. The immunoprofile of L82 corresponds to that seen typically in primary ALCL cells, with positivity for CD30, EMA, CD3, CD4, CD25, CD71, TIA1, and granzyme B; the cells were negative for EBV-related antigens. Cytogenetic analysis showed a complex, near triploid karyotype with 72-77 chromosomes, including the ALCL specific translocation t(2;5)(p23;q35). Chromosomal analysis revealed a number of secondary structural alterations including amplification of 7q21-31, 1q, and 6p, and gain of chromosomal material in 8q (affecting the c-myc gene). The rearrangement of the T-cell receptor-gamma locus shows that L82 is clonally derived from T-lineage lymphoid cells. mRNAs for interleukin 7 (IL-7), IL-8, IL-9, IL-10, TNF-beta, and for the IL-7 and IL-9 receptor were found. These data show that the T-helper cell (Th)1/Th2 balance was polarized to Th2. L82 were inoculated intraperitoneally into 4 week-old SCID mice and produced a disseminated tumor within 4-6 weeks. Morphological, immunohistochemical, and molecular genetic investigation confirmed that the xenograft and the original ALCL tumor were identical. SCID mice xenografted with the human ALCL cell line, L82, provide a useful model system for the investigation of the biology of ALCL and of new therapeutic approaches, such as specific immunotherapy.
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MESH Headings
- Adult
- Animals
- Chromosome Aberrations
- Cytogenetic Analysis
- Cytokines/analysis
- Disease Models, Animal
- Female
- Humans
- Immunophenotyping
- Lymphoma, Large B-Cell, Diffuse/genetics
- Lymphoma, Large B-Cell, Diffuse/immunology
- Lymphoma, Large B-Cell, Diffuse/pathology
- Lymphoma, T-Cell/genetics
- Lymphoma, T-Cell/immunology
- Lymphoma, T-Cell/pathology
- Mice
- Mice, SCID
- Pleural Effusion, Malignant/pathology
- Polyploidy
- T-Lymphocytes, Helper-Inducer/metabolism
- Transplantation, Heterologous
- Tumor Cells, Cultured/pathology
- Tumor Cells, Cultured/transplantation
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Affiliation(s)
- Hartmut Merz
- Department of Pathology, Medical University of Luebeck, Germany.
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42
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Berg KD, Brinster NK, Huhn KM, Goggins MG, Jones RJ, Makary A, Murphy KM, Griffin CA, Rosenblum-Vos LS, Borowitz MJ, Nousari HC, Eshleman JR. Transmission of a T-cell lymphoma by allogeneic bone marrow transplantation. N Engl J Med 2001; 345:1458-63. [PMID: 11794194 DOI: 10.1056/nejmoa010041] [Citation(s) in RCA: 69] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
MESH Headings
- Adult
- Bone Marrow Transplantation/adverse effects
- Disease Transmission, Infectious
- Female
- Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor
- Humans
- Lymphoma, Large-Cell, Anaplastic/complications
- Lymphoma, Large-Cell, Anaplastic/drug therapy
- Lymphoma, Large-Cell, Anaplastic/therapy
- Lymphoma, T-Cell/etiology
- Lymphoma, T-Cell/genetics
- Microsatellite Repeats
- Middle Aged
- Panniculitis/etiology
- Panniculitis/genetics
- Polymerase Chain Reaction
- Sequence Analysis, DNA
- Transplantation, Homologous
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Affiliation(s)
- K D Berg
- Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
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43
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zur Stadt U, Rischewski J, Schneppenheim R, Kabisch H. Denaturing HPLC for Identification of Clonal T-Cell Receptor γ Rearrangements in Newly Diagnosed Acute Lymphoblastic Leukemia. Clin Chem 2001. [DOI: 10.1093/clinchem/47.11.2003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022]
Abstract
Abstract
Background: Denaturing HPLC (DHPLC) can be used to screen DNA for known and unknown mutations. We describe a novel, HPLC-based method for discrimination among polyclonal, oligoclonal, and/or clonal T-cell receptor γ (TCR-γ) rearrangements in samples from children with newly diagnosed acute lymphoblastic leukemia.
Methods: TCR rearrangements were PCR amplified from initial leukemic samples and, after heteroduplex-induction, the clonality status of each product was evaluated. To attain this, we used DHPLC on a high-resolution micropellicular matrix. Running conditions were established by melting-curve analysis of known clonal and polyclonal products and melting-point prediction software. Elution profiles were studied at 50 °C (native) and, to achieve optimal separation, at different column temperatures between 56 and 64 °C.
Results: For VγI-Jγ1.3/2.3 rearrangements, an analysis temperature of 60 °C with a linear triethylammoniumacetate—acetonitrile gradient separated clonal bands from the polyclonal background amplification. Less than 15% clonal PCR product was detectable in mixtures of initial leukemic cell DNA and polyclonal DNA. Biallelic rearrangements produced two sharp peaks. Clonality of PCR products from 100 initial leukemic samples was completely identified in all investigated cases.
Conclusions: Heteroduplex analysis with standardized DHPLC conditions simplifies the detection of unknown clonal or polyclonal TCR rearrangements in newly diagnosed leukemias. Clonal targets for detection of minimal residual disease are available after a short, automated analysis of PCR amplified rearrangements.
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Affiliation(s)
- Udo zur Stadt
- Department of Pediatric Hematology and Oncology, University Children’s Hospital, Martinistrasse 52, D-20246 Hamburg, Germany
| | - Johannes Rischewski
- Department of Pediatric Hematology and Oncology, University Children’s Hospital, Martinistrasse 52, D-20246 Hamburg, Germany
| | - Reinhard Schneppenheim
- Department of Pediatric Hematology and Oncology, University Children’s Hospital, Martinistrasse 52, D-20246 Hamburg, Germany
| | - Hartmut Kabisch
- Department of Pediatric Hematology and Oncology, University Children’s Hospital, Martinistrasse 52, D-20246 Hamburg, Germany
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44
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Fraser-Andrews EA, Russell-Jones R, Woolford AJ, Wolstencroft RA, Dean AJ, Whittaker SJ. Diagnostic and prognostic importance of T-cell receptor gene analysis in patients with Sézary syndrome. Cancer 2001; 92:1745-52. [PMID: 11745245 DOI: 10.1002/1097-0142(20011001)92:7<1745::aid-cncr1689>3.0.co;2-0] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
BACKGROUND Sézary syndrome (SS) is characterized by erythroderma, peripheral lymphadenopathy, and circulating Sézary cells and is clinically heterogeneous. METHODS T-cell receptor (TCR) gene analysis was performed using DNA extracted from peripheral blood mononuclear cells from 74 patients, and the results were correlated with a variety of other diagnostic parameters and patient outcomes. RESULTS Two groups were identified: 66 patients with clonal TCR gene rearrangement (clonal patients) detected with Southern blot analysis and/or polymerase chain reaction/single-strand conformational polymorphism analysis and 8 patients with no clonal rearrangement detected (nonclonal patients) using either technique. Clonal patients were compared with nonclonal patients. The following median blood parameters were significantly greater in the clonal group: total white cell count (13.7 10(9)/L vs. 9.6 10(9)/L), lymphocyte count (4.9 10(9)/L vs. 2.2 10(9)/L), absolute Sézary count (3.22 10(9)/L vs. 0.99 10(9)/L), CD4 count (3.17 10(9)/L vs. 1.36 10(9)/L), and CD4:CD8 ratio (15.86 vs. 3.21). An expanded population of T-cells of a specific TCR variable beta subset was detected in 7 of 36 clonal patients and in 1 of 4 nonclonal patients. Cytogenetic analysis of peripheral blood from 1 nonclonal patient and 6 clonal patients was normal. The median survival from the time of diagnosis was 45 months in the clonal group, and 40 of 49 deaths were cutaneous T-cell lymphoma (CTCL)-related, whereas 3 deaths in the nonclonal group were unrelated to CTCL (P < 0.01; log-rank test). Multivariate proportional hazards analysis showed that the absolute Sézary count and lymph node status were independent prognostic variables (P = 0.016 and P = 0.036, respectively). CONCLUSIONS TCR gene analysis defines a distinct clinicopathologic group of patients with SS. Clonal patients have a poor prognosis and are likely to die from leukemia/lymphoma, whereas nonclonal patients may have a reactive, inflammatory T-cell disorder. The authors suggest that the definitive diagnostic criteria for patients with SS should include the presence of a clonal TCR gene rearrangement.
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Affiliation(s)
- E A Fraser-Andrews
- Skin Tumor Unit, St. John's Institute of Dermatology, St. Thomas' Hospital, Lambeth Palace Road, London, SE1 7EH, United Kingdom.
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45
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Zhu D, Kadin ME, Samoszuk M. Detection of clonal T-cell receptor-gamma gene rearrangement by PCR/temporal temperature gradient gel electrophoresis. Am J Clin Pathol 2001; 116:527-34. [PMID: 11601137 DOI: 10.1309/8kmv-t6bm-g9pc-ek24] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Abstract
Limited combinatorial and junctional diversity in TCR-gamma gene rearrangement can result in amplification products that are difficult to interpret when analyzed by conventional gel electrophoresis methods that separate DNA based on size (polymerase chain reaction [PCR]/polyacrylamide gel electrophoresis [PAGE]). We describe a simple approach to the detection of clonal TCR-gamma gene rearrangement using temporal temperature gradient gel electrophoresis (TTGE) that uses a gradual and uniform increase in the temperature of a constant denaturing gel to resolve different DNA molecules based on base pair composition. We tested 42 clinical specimens (30 blood specimens and 12 formalin-fixed paraffin-embedded tissues) for T-cell clonality by PCR/PAGE and PCR/TTGE. Concordant results were obtained in only 22 specimens (52%). Of the 20 discordant cases, 18 samples were positive by TTGE and negative by PAGE. For all of the discordant cases, the TTGE yielded results that correlated better with the clinical data than did the PAGE method. We conclude that PCR/TTGE is more accurate and easier to perform than current methods for detecting clonal populations of T cells.
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Affiliation(s)
- D Zhu
- Nichols Institute, Quest Diagnostics, 33608 Ortega Highway, San Juan Capistrano, CA, USA
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46
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Vega F, Medeiros LJ, Jones D, Abruzzo LV, Lai R, Manning J, Dunmire V, Luthra R. A novel four-color PCR assay to assess T-cell receptor gamma gene rearrangements in lymphoproliferative lesions. Am J Clin Pathol 2001; 116:17-24. [PMID: 11447747 DOI: 10.1309/5wfq-n12e-dt05-ux1t] [Citation(s) in RCA: 69] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Abstract
We describe a novel 4-color polymerase chain reaction (PCR) assay combined with GeneScan analysis to assess for T-cell receptor gamma chain gene (TCRgamma) rearrangements and evaluate its usefulness in 86 lymphoproliferative lesions. In this assay, each variable region (Vgamma) family primer is 5' end-labeled with a different fluorescent dye, allowing determination of the Vgamma family involved in each TCRgamma rearrangement. PCR products were analyzed by capillary electrophoresis. We detected clonal TCRgamma rearrangements in 60 (98%) of 61 T-cell lymphomas, 2 (15%) of 13 B-cell lymphomas, and 3 (25%) of 12 reactive lesions. These results compared favorably with conventional PCR methods using denaturing gradient gel electrophoresis, which revealed clonal TCRgamma rearrangements in 37 (90%) of 41 T-cell lymphomas, 1 (25%) of 4 B-cell lymphomas, and 2 (25%) of 8 reactive lesions. This 4-color PCR assay is at least equivalent to conventional PCR methods and is convenient, allows accurate size determination of TCRgamma rearrangements, and identifies the specific Vgamma family involved, providing more specific information about TCRgamma rearrangement.
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Affiliation(s)
- F Vega
- Division of Pathology and Laboratory Medicine, University of Texas M.D. Anderson Cancer Center, 8515 Fannin, Houston, TX 77030-4095, USA
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47
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Gutzmer R, Mommert S, Kiehl P, Wittmann M, Kapp A, Werfel T. Detection of clonal T cell receptor gamma gene rearrangements in cutaneous T cell lymphoma by LightCycler-polymerase chain reaction. J Invest Dermatol 2001; 116:926-32. [PMID: 11407983 DOI: 10.1046/j.1523-1747.2001.01344.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Cutaneous T cell lymphoma is thought to be characterized by a monoclonal T cell infiltrate in the skin that can be detected by polymerase chain reaction-based amplification of T cell receptor gamma gene rearrangements. We sought to establish a new, simple, and fast LightCycler-based real-time polymerase chain reaction assay for the detection of monoclonality in cutaneous T cell lymphoma, which was suitable for routine laboratory application. Monoclonal T cell receptor gamma gene rearrangements were detected by polymerase chain reaction with consensus primers using: (i) a thermocycler followed by polyacrylamide gel electrophoresis; (ii) a Light Cycler followed by melting curve analysis; and (iii) a LightCycler and subsequent polyacrylamide gel electrophoresis. The detection limit of monoclonal Jurkat T cells diluted in polyclonal peripheral blood mononuclear cells was: (i) 1--3% by thermocycler--polymerase chain reaction and polyacrylamide gel electrophoresis; (ii) 10% by LightCycler--polymerase chain reaction and melting curve analysis; and (iii) 1% by LightCycler--polymerase chain reaction and polyacrylamide gel electrophoresis. In skin biopsies of 22 cutaneous T cell lymphoma patients, a monoclonal or biclonal T cell infiltrate was detected in: (i) 15 of 22 (68%) by thermocycler--polymerase chain reaction and polyacrylamide gel electrophoresis; (ii) 13 of 22 (59%) by LightCycler--polymerase chain reaction and melting curve analysis; and (iii) 16 of 22 (72%) by LightCycler--polymerase chain reaction and polyacrylamide gel electrophoresis. All three techniques revealed negative results in skin biopsies from 26 patients with benign dermatitis. In conclusion, LightCycler--polymerase chain reaction and melting curve analysis is a fast, simple and specific method to detect monoclonal T cell infiltrates in cutaneous T cell lymphoma. Sensitivity of LightCycler--polymerase chain reaction and polyacrylamide gel electrophoresis is slightly higher compared with sensitivity of thermocycler--polymerase chain reaction and polyacrylamide gel electrophoresis. Melting curve analysis, however, is less sensitive compared with polyacrylamide gel electrophoresis, and in case of negative results of the melting curve analysis, it is recommended to resolve LightCycler--polymerase chain reaction samples by gel electrophoresis.
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Affiliation(s)
- R Gutzmer
- Department of Dermatology and Allergology, Hannover Medical University, Hannover, Germany
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Kluin PM, Feller A, Gaulard P, Jaffe ES, Meijer CJ, Müller-Hermelink HK, Pileri S. Peripheral T/NK-cell lymphoma: a report of the IXth Workshop of the European Association for Haematopathology. Histopathology 2001; 38:250-70. [PMID: 11260307 DOI: 10.1046/j.1365-2559.2001.01058.x] [Citation(s) in RCA: 72] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
AIMS In April 1998, The European Association for Haematopathology organized the IXth workshop on peripheral T-cell and NK-cell lymphomas and leukaemias. The workshop focused on unusual subtypes of these rare malignancies, allowing evaluation of the recently published WHO classification of neoplastic diseases of the lymphoid tissues. METHODS AND RESULTS One-hundred and three cases were centrally immunophenotyped and hybridized for EBER1/2 of Epstein--Barr virus. All cases were reviewed by a panel of experienced haematopathologists and classified according to the new WHO classification for lymphoid neoplasms. Three cases were considered as precursor T-cell and 95 cases as peripheral T/NK-cell lymphoma/leukaemia. Although the cases represented a selected series of unusual cases, the following conclusions could be made: (i) Most lymphomas except the hepatosplenic gamma/delta T-cell lymphomas showed a rather broad morphological spectrum, with differences both between and within individual tumours. (ii) This heterogeneity was also reflected by the immunophenotype, for instance a variable expression of CD30 was found in many enteropathy type T-cell lymphomas. (iii) Exceptions in phenotype were regularly found in almost all categories, indicating that phenotype should not be the final determining factor in classification. (iv) The great majority of T-cell lymphomas expressed the alpha/beta T-cell receptor, with the exception of all but one hepatosplenic T-cell lymphomas and a few other extranodal peripheral T cell lymphomas. (v) Malignancies of precursor cells, blastic NK-cell lymphoma/leukaemia, adult T-cell lymphoma/leukaemia and most AIL-type T-cell lymphomas did not express cytotoxic molecules such as TIA1 and granzyme-B. In contrast, all five aggressive NK/T-cell lymphomas/leukaemias, a single case of large granular lymphocyte leukaemia and 40 of 47 primary extranodal lymphoma/leukaemias expressed these molecules. In hepatosplenic gamma/delta T-cell lymphoma, five of six cases showed expression of TIA1 but not of granzyme-B. (vi) Seven tumours developed after organ-transplant, four cases being EBV-positive. No distinct phenotype could be attributed to these cases. CONCLUSIONS Most peripheral T/NK cell lymphomas could be categorized as distinct entities as described in the recently proposed WHO classification for lymphoid neoplasms.
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MESH Headings
- Adult
- Child, Preschool
- Female
- Humans
- Immunoblastic Lymphadenopathy/genetics
- Immunoblastic Lymphadenopathy/immunology
- Immunoblastic Lymphadenopathy/pathology
- Immunochemistry
- Immunophenotyping
- Intestinal Neoplasms/genetics
- Intestinal Neoplasms/immunology
- Intestinal Neoplasms/pathology
- Killer Cells, Natural/immunology
- Lymphoma, Large B-Cell, Diffuse/genetics
- Lymphoma, Large B-Cell, Diffuse/immunology
- Lymphoma, Large B-Cell, Diffuse/pathology
- Lymphoma, Non-Hodgkin/classification
- Lymphoma, Non-Hodgkin/genetics
- Lymphoma, Non-Hodgkin/immunology
- Lymphoma, Non-Hodgkin/pathology
- Male
- Middle Aged
- RNA, Viral/genetics
- Skin Neoplasms/genetics
- Skin Neoplasms/immunology
- Skin Neoplasms/pathology
- T-Lymphocytes/immunology
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Affiliation(s)
- P M Kluin
- Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
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Mognato M, Ferraro P, Canova S, Sordi G, Russo A, Cherubini R, Celotti L. Analysis of mutational effects at the HPRT locus in human G(0) phase lymphocytes irradiated in vitro with gamma rays. Mutat Res 2001; 474:147-58. [PMID: 11239972 DOI: 10.1016/s0027-5107(01)00061-6] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
The mutational effects of ionising radiation at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus were studied in human peripheral blood G(0) phase lymphocytes irradiated in vitro with gamma rays. The presence of radiation induced mutants was assessed by selecting the HPRT mutants every week on the basis of 6-thioguanine resistance up to 1 month after irradiation. A dose-related increase of 14.25x10(-6) mutants/Gy was measured after an expression time of 7 days. After 2 weeks from culture starting the fraction of clonable cells in irradiated and control cell populations decreased, limiting the measurements of mutant frequency. The mutational spectrum of the HPRT gene was determined by PCR analyses in a total of 99 mutant clones derived from irradiated lymphocytes. The independent origin of mutant clones carrying the same mutation was assessed by analysing the TCR gamma gene rearrangements. The results showed a dose-related increase of deletion mutants up to 3Gy, whereas point mutation frequency increased only up to 2Gy. Two preferentially deleted regions were identified; one involving the HPRT exon 3, and another one the 3'-terminal and the 3'-flanking region of the gene. One complex mutation involving a non-contiguous deletion of exons 2-5 and 7/8 was observed among the mutants isolated after 3Gy irradiation.
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Affiliation(s)
- M Mognato
- Dipartimento di Biologia, Università degli Studi di Padova, via U. Bassi 58B, 35121, Padova, Italy
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Wilson VL, Wade KR, Yin X, Albertini RJ. Temporal delineation of sequential HPRT mutations arising in vivo in a T-cell clone with a mutator phenotype. Mutat Res 2001; 473:181-99. [PMID: 11166036 DOI: 10.1016/s0027-5107(00)00148-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Recurrent mutations in vivo in T-lymphocytes identify clonally restricted genomic instabilities in some individuals. Cell-based assays allow initial recognition of clones with mutator phenotypes, but genotypic selection is required to determine frequencies and temporal sequences of potentially independent mutational events isolated only as complex changes in the same allele. The present work illustrates how two single-base insertions in the HPRT gene recovered only as a double event in a cell-based assay were shown to arise as separate in vivo mutations, being individually present at frequencies of < or =10(-4) and < or =10(-5), respectively, in peripheral blood. Full characterizations of mutator clones will allow elucidation of the earliest events in the emergence of genomic instability in human somatic cells.
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Affiliation(s)
- V L Wilson
- Institute of Environmental Studies and the Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
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