T-Cell Clonality Determination Using Polymerase ChainReaction (PCR) Amplification of the T-Cell Receptorgamma-Chain Gene and Capillary Electrophoresis ofFluorescently Labeled PCR Products

The recent development of inhibitors of key immune response proteins has revolutionized the therapy of autoimmune diseases; these immunomodulator agents include monoclonal antibodies and receptor antagonists. However, as with all therapies, these new agents are not without side effects and complications. In particular, anti-tumor necrosis factor alpha (TNFa) agents have been reported to be associated with an increased incidence of lymphoproliferative disorders, infections, and vasculitis. We evaluated the clinicopathological features of 18 cases of immunomodulator agent-related lymphoproliferative disorders (IAR-LPD) from several institutions. These included 6 cases of B-cell lymphoma, 2 cases of T-cell lymphoma, 3 cases of classical Hodgkin lymphoma, and 7 atypical lymphoid proliferations that did not fulfill diagnostic criteria for lymphoma; two of the latter regressed after discontinuation of the immunomodulator agent therapy. All eight lymphoma patients with available information had also received prior chemotherapy (methotrexate or 6-mercaptopurine). EBV was strongly associated with the B-cell and classical Hodgkin lymphomas. This case series illustrates that a broad range of lymphoid proliferations can occur after immunomodulator agent therapy and that these immunomodulator agent-related lymphoproliferative disorders have considerable overlap with other well-defined lymphoproliferative diseases associated with iatrogenic immunosuppression. Further study is warranted to evaluate how these therapies interact with other immunosuppressive agents and the underlying abnormal immune system to enhance the development of lymphomas and atypical lymphoid proliferations.

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“…12 In some cases, gene rearrangement studies had been carried out at individual institutions according to the institutional protocol.…”

Section: Gene Rearrangement Studiesmentioning

confidence: 99%

Immunomodulator agent-related lymphoproliferative disorders

Hasserjian

Chen²,

Perkins

et al. 2009

Modern Pathology

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“…Other advantages include rapid turnaround time and more efficient use of technologist resources. 12 Capillary electrophoresis has been described as a more objective and sensitive technique than gel electrophoresis in establishing clonality in peripheral TCL, 12,27,38 but this method has yet to be evaluated in the analysis of NK/TCL. In this study, we found capillary electrophoresis to be particularly useful for detecting monoclonal peaks within polyclonal backgrounds, identifying small clonal populations, and distinguishing pseudospikes from true monoclonal peaks.…”

Section: Survivalmentioning

confidence: 99%

“…2,[5][6][7][8][9] This small proportion of tumors appears to be derived from a unique subset of cytotoxic T cells, known as NK-like or NK1 þ T cell, and shows monoclonally rearranged T-cell receptor genes. [10][11][12] Hence, the term NK/T-cell lymphoma (NK/TCL) is preferred. 4,5 To date, about 200 cases of NK/TCLs have been documented in the literature, mostly from Chinese, Japanese, Korean and South American populations.…”

mentioning

confidence: 99%

“…Although frequently implicated in the pathogenesis of many human cancers, the role of p53 as a prognostic and predictive factor and its involvement in the pathogenesis of nasal-type NK/TCL is unclear. A few studies have noted a high incidence of p53 overexpression and/or mutations, 12,13 and some investigators also found an association between p53 mutation with large cell morphology and advanced disease. 14 In this study, we describe the clinicopathologic and genotypic findings of 42 cases of nasal-type NK/ TCL in Singapore, which is a multiracial society populated predominantly by Asians, and compared our findings with those of other series reported in the Eastern 8,[15][16][17][18] and Western populations.…”

mentioning

confidence: 99%

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Nasal-type extranodal natural killer/T-cell lymphomas: a clinicopathologic and genotypic study of 42 cases in Singapore

et al. 2004

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We studied the clinicopathologic features of 42 cases of nasal-type extranodal natural killer (NK)/T-cell lymphoma in Singapore and compared our findings with other series reported in the Asian and Western populations. A panel of immunohistochemical stains, which included CD2, CD3, CD4, CD8, CD56, T-cell intracellular Antigen-1 and granzyme B, and in situ hybridization for Epstein-Barr virus encoded RNA (EBER) were performed. Polymerase chain reaction for T-cell receptor-gamma gene rearrangement using both gel and capillary electrophoresis were evaluated to determine the proportion of tumors which are of true T-cell lineage. We also studied the functional status of the overexpressed p53 protein in these lymphomas by correlating p53 expression with its downstream target protein, p21. In all, 31 out of 42 cases presented in the upper aerodigestive tract. The other sites of involvement included gastrointestinal tract, skin, soft tissue, testis, liver, spleen, bone marrow and brain. The tumors displayed characteristic morphologic features. In situ hybridization for EBER was detected in 41 out of 42 cases (97.6%). The only significant adverse prognostic factor identified was an International Prognostic Index of two or more. A significantly higher proportion of the tumors (27%), compared to previous studies, demonstrated monoclonal T-cell receptor-gamma gene rearrangement. There was, however, no difference in survival or clinicopathologic features between the true NK-cell tumors and their T-cell counterparts. Overexpression of p53 was present in 40% of the cases, but no significant difference in survival rate was detected in patients with p53 overexpression and there was no association between p53 overexpression with large cell morphology, and advanced stage of disease. These findings suggest that molecular aberrations other than those of the p53 pathway may be operative in the pathogenesis of this malignancy.

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“…Since monitoring of disease relapse typically depends on evaluation of blood or bone marrow samples, it is reassuring to note a relatively low frequency and heights of pseudospikes in the normal blood samples we have analyzed, suggesting that the distinction between normal and malignancy can be more clearly defined in these tissue types. We read with great interest the recently published paper by Sprouse et al 25 They describe an alternate method for approaching the same problem of quantifying in TCR-PCR assays the ratio of clonal to polyclonal components in samples containing both. Additional experiments may need to be performed to compare the two approaches directly.…”

Section: Discussionmentioning

confidence: 99%

Pseudo-Spikes Are Common in Histologically Benign Lymphoid Tissues

Lee¹,

Berg²,

Racke³

et al. 2000

The Journal of Molecular Diagnostics

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T cell receptor gene rearrangement is a classic marker of T cell clonality and is a useful adjunct in the diagnosis of T cell lymphomas and leukemias. Rearranged V-J gene segments amplified by polymerase chain reaction (PCR) are traditionally analyzed by polyacrylamide gel electrophoresis. We and others have analyzed TCR-␥ PCR products using capillary gel electrophoresis, which produces single nucleotide resolution and provides improved diagnostic sensitivity over conventional methods. However, with this marked increase in resolution and sensitivity, it is necessary to re-define normal variation of TCR-␥ gene rearrangement in control tissues to allow appropriate interpretation of monoclonality if present. Using DNA capillary gel electrophoresis, we examined the spectrum of normal patterns for TCR-␥ in a variety of T-cell-rich, histologically benign tissue types, including spleen, lymph node, tonsil, and blood, and compared this with the patterns in T cell lymphoma samples. We defined relative peak heights as h 1 /h 2 , where h 1 represents the peak height of the largest peak above the normally distributed population, and h 2 represents the peak height of the normally distributed curve. We found spikes in almost 20% of histologically benign samples with relative peak heights that were more than 0.5 and up to 1.5. We designated these as pseudo-spikes, because they may be mistaken for monoclonal spikes. In contrast, the relative peak height of the T cell lymphoma samples that showed clonal rearrangement was much higher than that of the pseudo-spikes, being at least 2 in 11/11 and at least 3 in 10/11 cases. Our data suggest that peaks with relative height of at least 3 represent a true clonal population in diagnostic samples. Peaks with relative heights of less than 1.5 may be insignificant, while peaks with relative heights between 1.5 to 3 may warrant further evaluation. Although capillary gel electrophoresis is superior in assessing T cell clonality, caution must be exercised when interpreting results, because pseudo-spikes appear to be common in benign tissues with lymphoid populations T cells, the mediators of cellular immunity, bear T cell receptors (TCR) on their surfaces that recognize and bind to specific antigens. These receptors are unique to each T cell clone, and demonstrate great diversity through rearrangement of germline gene segments during maturation.

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T-Cell Clonality Determination Using Polymerase ChainReaction (PCR) Amplification of the T-Cell Receptorgamma-Chain Gene and Capillary Electrophoresis ofFluorescently Labeled PCR Products

Cited by 48 publications

References 33 publications

Immunomodulator agent-related lymphoproliferative disorders

Immunomodulator agent-related lymphoproliferative disorders

Nasal-type extranodal natural killer/T-cell lymphomas: a clinicopathologic and genotypic study of 42 cases in Singapore

Pseudo-Spikes Are Common in Histologically Benign Lymphoid Tissues

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