Detection of Clonal T-Cell Receptor γ Gene Rearrangements in Early Mycosis Fungoides/Sezary Syndrome by Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR/DGGE)

Wood, Gary S.; Tung, Rosnn M.; Heaffner, Andreas C.; Crooks, Carol; Liao, Shaoyi; Orozco, Rachaci; Veelken, Hendrik; Kadin, Marshall E.; Koh, Howard K.; Heald, Peter; Barnhill, Raymond L.; Sklar, Jeffrey

doi:10.1111/1523-1747.ep12389114

Cited by 340 publications

(204 citation statements)

References 30 publications

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“…One of the main difficulties of PCRbased clonality detection is discrimination between monoclonal and polyclonal PCR products. This problem can be solved by further analyzing the PCR products in various ways, ranging from high resolution polyacrylamide gel electrophoresis (fingerprinting) and gene scanning [25][26][27][28] to DGGE [17][18][19][20] and heteroduplex analysis. [21][22][23] In heteroduplex analysis hetero-and homoduplexes, resulting from denaturation and renaturation of PCR products, are separated in non-denaturing polyacrylamide gels based on their conformation.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the sensitivities that we reach under conditions that guarantee reliable clonality assessment, are largely comparable to those mentioned by other authors who used serial dilutions in polyclonal MNC DNA and performed high resolution non-denaturing PAGE 34 or DGGE. [17][18][19][20] Sensitivities of around 5% are clinically relevant for initial diagnosis, but they are certainly not sufficient for detection of minimal residual disease (MRD) in ALL patients which requires sensitivities of 10 −4 to 10 −5 . 35 It has been suggested that sensitivities of 10 −2 to 10 −3 as determined by PCR might be predictive for slow remission upon chemotherapy, 36 but such sensitivities cannot easily be reached via heteroduplex analysis of PCR products, unless most of the polyclonal T cells are depleted before DNA or RNA extraction.…”

Section: Figurementioning

confidence: 99%

“…The latter implies that PCR analysis is not sufficient for clonality assessment, unless it is followed by analyses that discern between PCR products derived from polyclonal and monoclonal T cell populations. Methods that have been applied to solve this PCR 'background problem' include: direct sequencing of the PCR products, 13,14 single-strand conformation polymorphism (SSCP)-based analysis, 15,16 denaturing gradient gel electrophoresis (DGGE), [17][18][19][20] heteroduplex analysis, [21][22][23] temperature gradient gel electrophoresis (TGGE), 24 and gene scanning analysis. [25][26][27][28] From these techniques, heteroduplex analysis is probably the simplest, fastest and cheapest method for analysis of PCR products of rearranged TCR genes.…”

Section: Introductionmentioning

confidence: 99%

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Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations

et al. 1997

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Molecular analysis of T cell receptor (TCR) genes is frequently used to prove or exclude clonality and thereby support the diagnosis of suspect T cell proliferations. PCR techniques are more and more being used for molecular clonality studies. The main disadvantage of the PCR-based detection of clonal TCR gene rearrangements, is the risk of false-positive results due to 'background' amplification of similar rearrangements in polyclonal reactive T lymphocytes. Therefore, PCR-based clonality assessment should include analyses that discern between PCR products derived from monoclonal and polyclonal cell populations. One such method is heteroduplex analysis, in which homo-and heteroduplexes resulting from denaturation (at 94°C) and renaturation (at lower temperatures) of PCR products, are separated in non-denaturing polyacrylamide gels based on their conformation. After denaturation/renaturation, PCR products of clonally rearranged TCR genes give rise to homoduplexes, whereas in case of polyclonal cells heteroduplexes with heterogeneous junctions are formed. We studied heteroduplex PCR analysis of TCR gene rearrangements with respect to the time and temperature of renaturation and the size of the PCR products. Variation in time did not have much influence, but higher renaturation temperatures (Ͼ30°C) clearly showed better duplex formation. Nevertheless, distinction between monoclonal and polyclonal samples was found to be more reliable at a renaturation temperature of 4°C, using relatively short PCR products. To determine the sensitivity of heteroduplex analysis with renaturation at 4°C, (c)DNA of T cell malignancies with proven clonal rearrangements was serially diluted in (c)DNA of polyclonal mononuclear peripheral blood cells and amplified using V and C primers (TCRB genes) or V and J primers (TCRG and TCRD genes). Clonal TCRB and TCRD gene rearrangements could be detected with a sensitivity of at least 5%, whereas the sensitivity for TCRG genes was somewhat lower (10-15%). The latter could be improved by use of V␥ member primers instead of V␥ family primers. We conclude from our results that heteroduplex PCR analysis of TCR gene rearrangements is a simple, rapid and cheap alternative to Southern blot analysis for detection of clonally rearranged TCR genes.

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Section: Discussionmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations

et al. 1997

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“…[46][47][48][49] Clonal TCR␥ gene rearrangement was detectable in the skin and/or the PBMC in 1/1 ATL, 3/3 SS, 1/2 MF, and strikingly only 2/6 pleomorphic CTCL (including one indeterminate MF/pCTCL). This may be due to partial infiltration by the malignant cells in some of the biopsies used for DNA extraction, since the PCR technique for TCR detection has a sensitivity of approximately 5%.…”

Section: Discussionmentioning

confidence: 99%

“…47 Regarding the pleomorphic CTCL, a clonal TCR positivity was found in only two cases of six but, to our knowledge, very few molecular data have been published on this rare form of CTCL. [47][48][49] Our series emphasizes the need for a comparison of clinical, histopathological, hematological, virological and molecular studies to make a proper diagnosis of CTCL, especially since in tropical areas, differential diagnosis between MF and SS on one hand and smoldering ATL on the other hand is sometimes very difficult.…”

Section: Discussionmentioning

confidence: 99%

Cutaneous T cell lymphomas: mycosis fungoides, Sezary syndrome and HTLV-I-associated adult T cell leukemia (ATL) in Mali, West Africa: a clinical, pathological and immunovirological study of 14 cases and a review of the African ATL cases

et al. 1998

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Cutaneous T cell lymphomas (CTCL) are rare lymphoproliferative diseases, which are frequently suspected to be of viral origin. As very few data were available concerning cutaneous T cell lymphomas in tropical Africa, we undertook a clinical, histopathological, immunological and viro-molecular study of patients with a clinical diagnosis of cutaneous lymphoma, in Bamako, Mali. While prior to this study, no case of CTCL had been reported in this country, 14 patients (five women, nine men; mean age 58 years) with a diagnosis of cutaneous lymphoma were seen over a period of 30 months (1992-1994) in the only dermatological department in Mali. Clinically, the most frequent pattern was an infiltrated erythrodermia similar to Sezary syndrome. Nodular lesions and/or plaques were rarely observed. All these cutaneous tumors were T cell lymphoproliferations, only one expressing the CD8 + antigen. A comprehensive analysis of all the available data permitted characterization of three cases of adult T cell leukemia/lymphoma (ATL) associated with HTLV-I (one definitive case, of leukemic type, with demonstration of clonal integration of HTLV-I proviral genome and two probable ATL cases), three cases of Sezary syndrome (SS), two cases of mycosis fungoides (MF) and five cases of pleomorphic cutaneous lymphoma. In one case, the differentiation between MF and pleomorphic cutaneous lymphoma could not be established. HTLV-I serological and/or molecular markers were restricted to the three ATL cases. From the unique definitive ATL case, a T cell line was established from culture of peripheral blood mononuclear cells and sequence analysis of the env gene and the U3-LTR region demonstrated that the virus present in this patient belonged to the cosmopolitan subtype A. Thus, we report here the first evidence of HTLV-I infection and associated ATL in Mali. This is the second ATL case described for the whole Sahelian region (one ATL of the lymphoma type was reported previously in a Mauritanian patient). Furthermore, we demonstrate that the main types of CTCL described in Europe and North America are also present in this African area and that the prevalence of these diseases is greatly underestimated in such regions. Furthermore, no association was observed between HTLV-I/II infection and SS, MF or pleomorphic cutaneous lymphoma in Mali in contrast to other studies.

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Analysis of T-Cell Receptor Gene Rearrangement in Lymph Nodes of Patients With Mycosis Fungoides

Kern¹,

Kidd²,

Moe³

et al. 1998

Arch Dermatol

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Detection of Clonal T-Cell Receptor γ Gene Rearrangements in Early Mycosis Fungoides/Sezary Syndrome by Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR/DGGE)

Cited by 340 publications

References 30 publications

Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations

Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations

Cutaneous T cell lymphomas: mycosis fungoides, Sezary syndrome and HTLV-I-associated adult T cell leukemia (ATL) in Mali, West Africa: a clinical, pathological and immunovirological study of 14 cases and a review of the African ATL cases

Analysis of T-Cell Receptor Gene Rearrangement in Lymph Nodes of Patients With Mycosis Fungoides

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