Clonal T-cell Receptor Gamma Rearrangements Research Articles

Increased frequency of CD45 negative T cells (T zone cells) in older Golden retriever dogs.

T zone lymphoma (TZL) is characterized by the clonal expansion of T cells lacking expression of the pan-leukocyte antigen CD45 (TZ cells). A strong breed predisposition is observed in Golden retrievers. This study aimed to confirm aberrant CD45 mRNA expression and determine if Golden retrievers without clinical lymphoma have an increased frequency of circulating TZ cells. Gene expression analysis on confirmed TZL cases showed a significant decrease in CD45 expression compared to normal dogs. Peripheral blood samples from senior dogs, 242 Golden retrievers and 42 non-Golden retrievers, without evidence of lymphoproliferative disease were assessed for the presence of TZ cells by flow cytometry. Thirty-one percent of Golden retrievers had TZ cells compared to 14% of non-Golden retrievers. Thirty-four percent of Golden retrievers with TZ cells had a clonal T cell receptor gamma (TRG) gene rearrangement. Interestingly, 20% of Golden retrievers without TZ cells also had a clonal TRG rearrangement. Golden retrievers may have an increased risk of TZL due to an increased frequency of TZ cells.

Development of XMRV producing B Cell lines from lymphomas from patients with Chronic Fatigue Syndrome

Previous studies have shown that CFS patients have an increased incidence of lympho-proliferative malignancy compared to the normal population [1]. While the incidence rate of non-Hodgkin’s lymphoma in the United States is 0.02%, nearly 5% of the CFS patients developed the disease. Additionally, development of cancer coincides with an outgrowth of gamma delta T cells with specific clonal T-cell receptor gamma rearrangements. We hypothesized that infection with XMRV and/or other viruses can trigger a dysregulated immune response which favors the development of B-cell lymphoma. In a study of 300 CFS patients, 13 developed lymphoproliferative disorders. Of those tested, 11/11 were positive for XMRV and 9/9 positive for clonal TCR gamma rearrangements. Spontaneous development of four B cells lines occurred during culture of cells from CFS patients. Three developed from B cells isolated from the peripheral blood (two of whom had B cell lymphoma) and one from a bone marrow biopsy. For all four lines, the B cells have a mature CD20+, CD23+ phenotype and produce infectious XMRV at a titer of >106 infectious units/ml. Virus production occurred despite extensive hypermutation of the proviruses in these cells by APOBEC3G.. Therefore XMRV infection may accelerate the development of B cell malignancies by either indirect chronic stimulation of the immune system and/or by direct infection of the B-cell lineage . Since viral load in peripheral blood is low, these data suggest that B cells in tissues such as spleen and lymph nodes could be an in vivo reservoir for XMRV.

Innovative technology for cancer risk analysis

After completion of the Human Genome Project, analysis of genetic and genomic variations in different pathological states became possible. The capillary system based on Sanger methods is still very expensive in terms of time, cost and professionalism required. For this reason, the National Human Genome Institute proposed an 'advanced sequencing technology development' project with the aim of sequencing a genome in 1 day for $1000. Three validated platforms are commercially available and single molecule sequencing methods have been recently introduced, which are not only competitive in time and costs, but display greater accuracy than 'past generation' sequencing. Next generation technology allows, in a single experiment, the identification of copy number variation and large rearrangements, or detection of fusion transcripts analysis thus permitting the evaluation of cancer risk at multiple levels (genomic, transcriptomic, proteomic, epigenetic).

Peripheral T-cell Lymphoma Presenting as Sclerouveitis

To report an unusual case of ocular peripheral T-cell lymphoma presenting as sclerouveitis. A 53-year-old woman presented with painful, unilateral sclerouveitis and was initially treated with antivirals and corticosteroids for what was presumed to be isolated ocular HSV infection with vasculitis. When she failed to improve the conjunctiva was biopsied and characterized by immunohistochemical and molecular methods. The literature regarding conjunctival T-cell lymphomas as well as lymphomas mimicking scleritis is reviewed. Biopsies of a scleral/conjunctival nodule from the right eye were obtained on 2 occasions, the second of which revealed the presence of intermediate to large size lymphocytes that were CD3, T-cell intracellular antigen 1 and Granzyme B positive but CD56 and Epstein Barr virus negative. Polymerase chain reaction showed clonal T-cell receptor gamma rearrangements in DNA samples isolated from biopsy tissue. Another biopsy was obtained from a mucosal lesion in the oropharynx, which was shown to contain an identical T-cell receptor gamma rearrangement. These results led to the diagnosis of peripheral T-cell lymphoma. Conjunctival T-cell lymphomas have been reported in two other patients both of which also had involvement of upper airway structures. Five other reports of lymphoma mimicking scleritis are discussed. Peripheral T-cell lymphoma should be considered in the differential diagnosis of patients who present with sclerouveitis of unknown etiology unresponsive to conventional therapy. If a conjunctival T-cell lymphoma is identified a systemic evaluation for lymphoma with particular attention to the upper airway is justified.

Skewed expression of natural‐killer (NK)‐associated antigens on lymphoproliferations of large granular lymphocytes (LGL)

Killer-immunoglobulin-like receptors (KIR) or C-type lectin-like receptors are heterogeneously expressed on NK cells and small subsets of T cells and might provide a new diagnostic tool for LGL lymphoproliferations (LGLL). We investigated the diagnostic impact of these cell surface molecules in T- and NK-type LGLL. Using three-color flow cytometry we examined the expression patterns of KIR (CD158a/b/e/i), CD85j, lectin-like receptors (CD94, CD161, NKG2A/D) and natural cytotoxicity receptors (NKp30/44/46) in 13 patients with LGLL (10 T-, 3 NK-LGLL) and compared them to those of the corresponding lymphocyte subsets in 20 control subjects. The presence of clonal TCR-gamma rearrangements and of Epstein Barr virus- (EBV) DNA were evaluated by PCR. All patients exhibited an altered expression of NK-associated markers. KIR were either lacking (6/13) or overexpressed (7/13). CD94 expression was significantly higher in all LGLL. NKG2A expression was significantly higher in NK-LGLL. Absence or overexpression was observed for NKG2A in T-LGLL and CD161 in most T/NK-LGLL. In NK-LGLL expression of NKp30 and NKp46 was significantly decreased, whereas CD85j was overexpressed. We consistently found a skewed expression pattern of novel NK markers as a pathological feature of LGLL. These antigens should be included in the diagnostic workup of this rare disease.

Lymphomatoid Papulosis Associated with Mycosis Fungoides: Clinicopathological and Molecular Studies of 12 Cases

The association of mycosis fungoides and a primary cutaneous CD30+ lymphoproliferative disorder has been reported and probably represents different clinical aspects of a unique T-cell monoclonal expansion. In this study, 12 patients (6 men and 6 women) presented with lymphomatoid papulosis and mycosis fungoides. A TCRgamma gene rearrangement study was performed by an automated high-resolution PCR fragment analysis method on skin biopsy specimens taken from the different clinical lesions in each patient. An indolent clinical course was observed in the majority of patients. T-cell clonality was identified in 7 of 12 lymphomatoid papulosis lesions (58%) and in 6 skin biopsies of plaque stage mycosis fungoides (50%). In each individual case, where T-cell clonality was detected, both mycosis fungoides and lymphomatoid papulosis specimens exhibited an identical peak pattern by automated high-resolution PCR fragment analysis, confirming a common clonal origin. Only one case showed a clonal TCRgamma rearrangement from the lymphomatoid papulosis lesion, which could not be demonstrated in the mycosis fungoides specimen. The demonstration of an identical clone seems to confirm that both disorders are different clinical manifestations of a unique T-cell monoclonal proliferation. Our results also seem to confirm that the association of mycosis fungoides with a primary cutaneous CD30+ lymphoproliferative disorder usually carries a favourable prognosis.

Clonality analysis by T-cell receptor gamma PCR and high-resolution electrophoresis in the diagnosis of cutaneous T-cell lymphoma (CTCL).

During T-cell maturation, T-cell receptor (TCR) gene segments rearrange, resulting in a new, unique DNA configuration. The recombined TCR gene loci display a high degree of nucleotide sequence variability. Molecular biological clonality assays focus on this cell-specific DNA pattern. The finding of an identical TCR rearrangement in a large number of T lymphocytes signals a malignant proliferation, although clonality is not always equivalent to malignancy. Thus, detection of clonal TCR gamma rearrangements by polymerase chain reaction (PCR), followed by high-resolution electrophoresis is a valuable tool in the diagnosis of cutaneous and other T-cell lymphomas. For the clonality assay described here, all rearrangements of T cells present in a given sample are amplified by a set of only three TCR gamma-PCRs. The products are investigated by either heteroduplex temperature gradient gel electrophoresis (HD-TGGE) or fluorescent fragment analysis (FFA) on a capillary DNA sequencer (or by both methods), for clonality. Both electrophoresis techniques show highly reproducible results and are comparatively easy to conduct, however, specific instruments are required. Concerning lower detection thresholds, the methods need a minimum of about 1% of clonal T-cells in mixtures with polyclonal T-cells for revealing clonality.

Heteroduplex analysis of T-cell receptor gamma gene rearrangement as an adjuvant diagnostic tool in skin biopsies for erythroderma.

Erythroderma, defined as red skin covering most of the body surface often accompanied or followed by exfoliation, is the clinical manifestation of at least six different underlying etiologies with allergic or irritant contact dermatitis, atopic/asteotic dermatitis, pityriasis rubra pilaris (PRP), psoriasis, and seborrheic dermatitis accounting for the majority of cases. Approximately 10% of cases are due to adverse drug reactions with roughly another 10% due to cutaneous T-cell lymphoma (CTCL), predominantly mycosis fungoides, or leukemia. It is clear from multiple studies that the clinical diagnosis of the underlying entity is often difficult, as these diseases can present in a very similar fashion. A skin biopsy is usually employed in this setting as a diagnostic tool. However, the histopathologic diagnosis of the underlying cause is complicated by the subtlety of the distinguishing histologic features. In this situation, an ancillary technique demonstrating the presence of a monoclonal T-cell proliferation could help to rule in or out CTCL in cases that clinically and histopathologically do not allow a definitive diagnosis. We retrospectively studied 25 biopsies from sixteen patients who presented to the Stanford Dermatology Clinic with erythroderma. We examined the specimens morphologically and analyzed the gamma chain of the T-cell receptor (TCR- gamma) by polymerase chain reaction (PCR) followed by heteroduplex analysis for clonality. We then correlated the results of our PCR and heteroduplex analyses with the patients' clinical outcomes. Four biopsies, from three patients, contained clonal TCR-gamma rearrangements; the four biopsies, all of which were equivocal histologically, correlated to diagnoses of mycosis fungoides (MF) or Sézary syndrome (SS). Twenty-one biopsies contained polyclonal T-cell populations. Eighteen of these biopsies represent patients with inflammatory dermatoses. Three of these biopsies, all of which were taken from a single patient, correlate to a diagnosis of MF. TCR-gamma PCR heteroduplex analysis seems to represent an important adjuvant diagnostic tool that, used in conjunction with histopathology and clinical history, could help to clarify the underlying etiology of erythroderma.

Polymerase chain reaction analysis of T-cell receptor gamma gene rearrangements in cutaneous B-cell lymphomas.

PCR analyses of T-cell receptor (TCR) gamma gene rearrangements in B-lymphoid neoplasms have shown lineage infidelity and double rearrangements involving both immunoglobulin heavy chain (igH) and TCRgamma genes. In order to investigate if this event is also a feature of cutaneous B-cell malignancies, we tested for clonal TCRgamma rearrangements a panel of immunophenotypically and genotypically well characterized cutaneous B cell lymphomas (CBCL). Fifteen samples of frozen CBCL biopsies were selected for the study. Diagnoses were established by routine histology and immunohistochemistry. Each of these cases displayed clonal igH gene rearrangement. Polymerase chain reaction (PCR) analysis of the TCRgamma rearrangements followed by high-resolution polyacrylamide gel electrophoresis was utilized for detection of clonal TCRgamma rearrangements. In our investigation, none of the cases of CBCL investigated by PCR showed the presence of clonal TCRgamma gene rearrangements. These data indicate that double rearrangements involving both igH and TCRgamma genes are not a feature of CBCL and confirm the B cell lineage specificity of this group of cutaneous lymphoid neoplasms.

Multiplex PCR for rapid detection of T-cell receptor-gamma chain gene rearrangements in patients with lymphoproliferative diseases.

We describe a multiplex polymerase chain reaction (PCR) method suitable for the detection of all T-cell receptor (TCR) gamma-chain gene rearrangements in patients with lymphoproliferative diseases. 40 patients with various lymphoproliferative disorders and 40 healthy individuals were tested. Clonal TCR gamma rearrangements were identified in all patients with malignant disorders, and in one of 10 cases with established reactive lymphocytosis but not in normal controls. In all individuals testing positive, the patient's specific V and J segment involved in the rearrangement could be determined by simply splitting the multiplex primer mix. Our data show that the multiplex PCR technique enables rapid, simple and sensitive screening for clonal TCR gamma chain gene rearrangements.

Cutaneous T-Cell Infiltrates: Analysis of T-Cell Receptor γ Gene Rearrangement by Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis

In cutaneous T-cell infiltrates, the demonstration of a clonal T-cell receptor (TCR) gene rearrangement has been considered helpful to distinguish Cutaneous T-cell lymphomas from reactive lymphoproliferation. Hence, a polymerase chain reaction (PCR) method using GC-clamp primers and denaturing gradient gel electrophoresis has been developed in our laboratory to analyze the TCR gamma locus configuration. Two hundred eleven cutaneous samples from 155 patients were analyzed. A detectable clonal TCR gamma rearrangement was significantly associated with cutaneous T-cell lymphomas as defined by morphologic and immunologic criteria. A clonal TCR gamma rearrangement was also detected frequently in lymphomatoid papulosis, never in reactive lymphocytic infiltrates and B-cell lymphomas, and rarely in parapsoriasis en plaque and cutaneous lymphoid hyperplasia. Forty five patients had both a cutaneous and a peripheral blood sample. Fifteen had a detectable clonal rearrangement in the two samples and 22 were negative. Six patients had a positive skin sample and a negative blood sample, whereas two patients had a positive blood sample and a negative skin sample. Four lymph node samples were analyzed and the PCR results were the same as in the skin. Finally, 21 patients had sequential samples of recurrent skin lesions. The PCR results were concordant in all and, when detectable, the clonal TCR gamma rearrangement remained unchanged in a given patient. Because of its simplicity and accuracy, the newly designed PCR procedure improves the monitoring of diagnosis, staging, and follow-up in cutaneous T-cell infiltrates.

Changing antigen receptor gene rearrangements in a case of early pre-B cell leukemia: evidence for a tumor progenitor cell with stem cell features and implications for monitoring residual disease

A case of acute lymphoblastic leukemia (ALL) was encountered in which the two clonal gamma T-cell receptor gene (TCR gamma) rearrangements found in bone marrow (BM) samples at relapse both differed from the single clonal TCR gamma rearrangement present in BM obtained at diagnosis 5 years previously. In contrast, two clonal Ig heavy chain gene (IgH) rearrangements present at relapse were identical to those present at diagnosis. Comparison of the DNA sequences of the relapse TCR gamma rearrangements with that of the diagnostic TCR gamma rearrangement indicated that they must have been generated de novo from TCR gamma loci in germline configuration. By polymerase chain reaction using clonotypic N-region oligonucleotide primers (N-PCR), cells bearing the diagnosis or relapse TCR gamma rearrangements were undetectable in the sample from the opposite time point. Two BM samples obtained at different times in clinical remission were both devoid of detectable residual tumor when analyzed by N-PCR, indicating a depth of remission of less than 1 tumor cell per 4 x 10(5) BM mononuclear cells. The tumor cells showed a primitive phenotype: T-cell antigen-negative, CALLA/CD10-negative, CD20-negative, CD19-positive, and positive for the myeloid marker My9. This case, which appears to represent a tumor arising from a progenitor cell with both early B-lineage and certain stem cell features, has implications for monitoring residual ALL and possibly also for treatment of the disease.

Changing Antigen Receptor Gene Rearrangements in a Case of Early Pre-B Cell Leukemia: Evidence for a Tumor Progenitor Cell With Stem Cell Features and Implications for Monitoring Residual Disease

A case of acute lymphoblastic leukemia (ALL) was encountered in which the two clonal gamma T-cell receptor gene (TCR gamma) rearrangements found in bone marrow (BM) samples at relapse both differed from the single clonal TCR gamma rearrangement present in BM obtained at diagnosis 5 years previously. In contrast, two clonal Ig heavy chain gene (IgH) rearrangements present at relapse were identical to those present at diagnosis. Comparison of the DNA sequences of the relapse TCR gamma rearrangements with that of the diagnostic TCR gamma rearrangement indicated that they must have been generated de novo from TCR gamma loci in germline configuration. By polymerase chain reaction using clonotypic N-region oligonucleotide primers (N-PCR), cells bearing the diagnosis or relapse TCR gamma rearrangements were undetectable in the sample from the opposite time point. Two BM samples obtained at different times in clinical remission were both devoid of detectable residual tumor when analyzed by N-PCR, indicating a depth of remission of less than 1 tumor cell per 4 x 10(5) BM mononuclear cells. The tumor cells showed a primitive phenotype: T-cell antigen-negative, CALLA/CD10-negative, CD20-negative, CD19-positive, and positive for the myeloid marker My9. This case, which appears to represent a tumor arising from a progenitor cell with both early B-lineage and certain stem cell features, has implications for monitoring residual ALL and possibly also for treatment of the disease.