TCR-γ Gene Rearrangement Research Articles

Pityriasis lichenoides et varioliformis acuta and pityriasis lichenoides chronica: comparison of lesional T-cell subsets and investigation of viral associations

Pityriasis lichenoides (PL) exhibits a broad clinical spectrum that includes both acute and chronic forms. The precise biologic mechanisms underlying PL remain unclear. To evaluate the immunohistochemical characteristics of PL and to investigate lesional T-cell subsets and the possible role of viral infection in its pathogenesis. Samples from 10 patients with PL et varioliformis acuta (PLEVA) and 13 with PL chronica (PLC) were analyzed immunohistochemically. Epstein-Barr virus early regions were assayed by in situ hybridization and T-cell receptor-γ (TCR-γ) gene rearrangements were assayed by polymerase chain reaction (PCR). We also utilized PCR to assay for human herpesvirus-8 (HHV-8) DNA in 51 patients with PL and in 25 controls. Lymphocytes expressing CD8 and T-cell intracellular antigen-1 were more abundant in patients with PLEVA than with PLC, whereas CD4+ lymphocytes and FOXP3-positive regulatory T-cells were more abundant in PLC. HHV-8 DNA was present in 11 of 51 (21.6%) PL patients and 0 of 25 controls. A clonal TCR-γ gene rearrangement was observed in only one patient with PLEVA. Our data suggests that PL may represent an inflammatory condition induced by various triggering agents, such as HHV-8, rather than a lymphoproliferative disorder. PLEVA, characterized by an acute course with severe symptoms, may indicate a relative lack of regulatory T-cells in comparison with PLC.

EB病毒感染相关的皮肤淋巴增殖性疾病二例

：Objective To report 2cases of Epstein-Barr virus-associated cutaneous lymphoproliferative disease (LPD) and toevaluate their relationship with chronic active Epstein-Barr virus (CAEBV)infection.Methods The clinical data on, laboratory examination findings in, treatment andtherapeutic response of 2cases of LPD were analyzed. Results Both the patients had chronicintermittent fever, lymphadenopathy, recurrent lesions including papules, papulovesicles,necrosis and variola-like scar in light-exposed and unexposed areas. Pathologically, therewas a dermal infiltrate of pleomorphic lymphoid cells with the involvement of perivasculararea and some subcutaneous tissue. Immunohistochemical staining showed that most of theinfiltrating lymphoid cells were positive for CD8, PCR revealed no TCR-γ gene rearrangement, and in situhybridization for EBV was positive. The copy of EBV DNA was above the normal range in theperipheral blood from both patients. Clinical status was improved after glucocorticoidtreatment. Conclusion The biologic behavior of LPD appears to be a chronic and indolentcourse and is closely associated with CAEBV. Key words: Lymphoproliferative disorders; Herpesvirus 4, human

Diagnostic significance of TCR gene clonal rearrangement analysis in early mycosis fungoides

Mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, has various unspecific clinical and histological characteristics. Its early diagnosis is challenging. The application of T-cell receptor (TCR) gene clonal rearrangement to the diagnosis of MF has been widely studied. In this study, we used polymerase chain reaction (PCR) to investigate the diagnostic significance of detecting TCR-γ and -β gene clonal rearrangement in the early diagnosis of mycosis fungoides. PCR for TCR-γ and TCR-β gene rearrangement was performed on 19 patients with suspected early MF, 6 with typical MF, and 6 with chronic dermatitis. Of the 19 patients with suspected early MF, 13 had TCR-γ gene clonal rearrangement, whereas none had TCR-β gene clonal rearrangement. All patients with typical MF had TCR gene clonal rearrangement, in which 4 showed TCR-γ clonal rearrangement, 1 showed TCR-β gene clonal rearrangements, and 1 showed both. No patients with chronic dermatitis had TCR gene clonal rearrangement. These results indicate that TCR gene clonal rearrangement analysis is a useful tool in diagnosing early MF. TCR-γ gene is recommended to the routine analysis, whereas TCR-β gene has potential in combination toward intractable cases.

Atypical fibroxanthoma with lymphomatoid reaction

Atypical fibroxanthoma (AFX) represents an uncommon skin tumor typically occurring on sun-damaged skin of the elderly. Histopathologic variants include spindled, clear cell, osteoid, osteoclastic, chondroid, pigmented, granular cell and myxoid lesions. To date, an atypical lymphoid infiltrate, including CD30-positive large cells mimicking lymphomatoid papulosis, has not been described in association with AFX. The clinical and histopathological characteristics of two AFX cases inciting an atypical lymphoid infiltrate, along with immunohistochemical profiles and T-cell receptor gamma (TCRγ) gene rearrangement results, were reviewed. Lesions in both cases occurred as solitary nodules in elderly patients. Microscopically, both lesions showed a cellular proliferation composed of pleomorphic spindle cells, associated with a prominent intralesional atypical lymphoid infiltrate. The spindle cells expressed CD10 but lacked the expression of S-100, cytokeratins and muscle markers, thereby confirming the diagnosis of AFX. CD30 highlighted a significant subset of large mononuclear cells in the lymphoid infiltrate of one case. TCRγ gene rearrangement analyses were negative for both cases. An atypical lymphoid infiltrate, including the one resembling lymphomatoid papulosis, associated with AFX has not been previously described. It is important to recognize the reactive nature of the infiltrate to avoid a misdiagnosis of lymphoma.

Significance of TCR gene clonal rearrangement analysis in diagnosis of mycosis fungoides

To investigate the significance of detecting TCR gene clonal rearrangement in the diagnosis of mycosis fungoides (MF) and to optimize the primers used for detecting the TCR gene clonal rearrangement with PCR in paraffin embedded tissues of MF. Nineteen cases of MF were enrolled into the study. A panel of 10 antibodies were used for immunophenotypic analysis and polymerase chain reaction for TCR-γ and TCR-β gene rearrangement detection in this study. TCR gene clonal rearrangements were detected in all 19 cases, in which 84.2% cases (16/19) had TCR-γ gene clonal rearrangements. The positive rates of the primers T(VG)/T(JX), V(2-5)/V(8-12)/JGT(1) and BIOMED-2-TCR-γ were 47.4%, 78.9% and 31.6%, respectively. The positive rate of V(2-5)/V(8-12)/JGT(1) was statistically significantly higher than that of T(VG)/T(JX) and BIOMED-2-TCR-γ (P < 0.05). No TCR gene clonal rearrangement was detected using the primers V(γ11)/V(γ101)/Jγ12 and V(γ11)/V(γ101)/J(p12). TCR-β gene clonal rearrangement was detected in 31.6% (6/19) cases. TCR gene clonal rearrangement analysis is a useful tool in the diagnosis of MF and TCR-γ gene is a good target gene for the detection. The primers T(VG)/T(JX), V(2-5)/V(8-12)/JGT(1) and BIOMED-2-TCR-γ can be used in clinicopathologic detection for TCR gene clonal rearrangement and V(2-5)/V(8-12)/JGT(1) may be the first choice.

Protocol for the Examination of Specimens From Patients With Non-Hodgkin Lymphoma/Lymphoid Neoplasms

Jerry W. Hussong, MD,DDS; DanielA.Arber, MD;Kyle T. Bradley,MD,MS;Michael S. Brown,MD;Chung-Che (Jeff) Chang,MD,PhD; Monica E. de Baca, MD; David W. Ellis, MBBS, FRCPA; Kathryn Foucar, MD; Eric D. Hsi, MD; Elaine S. Jaffe, MD; Michael Lill, MB, BS, FRACP, FRCPA; Stephen P. McClure, MD; L. Jeffrey Medeiros, MD; Sherrie L. Perkins, MD, PhD; for the Members of the Cancer Committee, College of American Pathologists

Common clonal origin of an acute B-lymphoblastic leukemia and a Langerhans' cell sarcoma: evidence for hematopoietic plasticity

The hierarchical organization of hematopoiesis with unidirectional lineage determination has become a questionable tenet in view of the experimental evidence of reprogramming and transdifferentiation of lineage-determined cells. Clinical examples of hematopoietic lineage plasticity are rare. Here we report on a patient who presented with an acute B-lymphoblastic leukemia and developed a Langerhans' cell sarcoma 9 years later. We provide evidence that the second neoplasm is the result of transdifferentiation. B-cell acute lymphoblastic leukemia was diagnosed in an 11-year old boy in 1996. Treatment according to the ALL-BFM-1995 protocol resulted in a complete remission. Nine years later, in 2005, Langerhans' cell sarcoma was diagnosed in a supraclavicular lymph node. Despite treatment with different chemotherapy protocols the patient had progressive disease. Finally, he received an allogeneic peripheral blood stem cell transplant and achieved a continuous remission. Molecular studies of IGH- and TCRG-gene rearrangements were performed with DNA from the Langerhans' cell sarcoma and the cryopreserved cells from the acute B-lymphoblastic leukemia. The expression of PAX5 and ID2 was analyzed with real-time reverse transcriptase polymerase chain reaction. Identical IGH-rearrangements were demonstrated in the acute B-lymphoblastic leukemia and the Langerhans' cell sarcoma. The key factors required for B-cell and dendritic cell development, PAX5 and ID2, were differentially expressed, with a strong PAX5 signal in the acute B-lymphoblastic leukemia and only a weak expression in the Langerhans' cell sarcoma, whereas ID2 showed an opposite pattern. The identical IGH-rearrangement in both neoplasms indicates transdifferentiation of the acute B-lymphoblastic leukemia into a Langerhans' cell sarcoma. Loss of PAX5 and the acquisition of ID2 suggest that these key factors are involved in the transdifferentiation from a B-cell phenotype into a Langerhans'/dendritic cell phenotype. (Clinical trial registration at: Deutsches KrebsStudienRegister, http://www.studien.de, study-ID:8).

Establishment and characterization of a novel canine B-cell line derived from a spontaneously occurring diffuse large cell lymphoma

Cell lines derived from spontaneous tumors serve as a research tool for cancer cell biology and new anti-cancer drug development. Isolation and propagation of canine lymphoma cell lines is difficult, thus only a few are available. Now we have established a new B-cell lymphoma cell line CLBL-1 from a dog with confirmed stage IV diffuse large cell lymphoma. Immunophenotyping of these CLBL-1 cells showed positive staining for CD11a, CD79αcy, CD45, CD45RA, MHC II and cells were negative for CD3, CD4, CD5, CD8, CD11d, CD14, CD21, CD34, CD56 and T-cell receptor-γδ (TCR-γδ). PCR analysis for TCR-γ and immunoglobulin heavy chain (IgH) gene rearrangements yielded a monoclonal result for the IgH gene. Furthermore, the clonality of IgH gene rearrangement was confirmed by sequencing of 16 positive bacterial clones. As canine lymphoma resembles non-Hodgkin's lymphoma (NHL) in humans in many respects, this new cell line, will promote translational and comparative lymphoma research in humans and dogs.

Better detection of Ig heavy chain and TCRγ gene rearrangement in plasma cell-free DNA from patients with non-Hodgkin Lymphoma

Plasma cell-free DNA is the soluble DNA and tumor-derived DNA in plasma which has the same mutation as the tumor cellular DNA. This study aimed at comparing the properties of plasma cell-free DNA with the biopsy's DNA in order to evaluate the clinical significance of IgH and TCRγ gene rearrangement in plasma cell-free DNA from patients with non-Hodgkin's Lymphoma. A total of 360 samples were studied. IgH (FR3A/VLJH) and TCRγ (TVG/TJX) were amplified by PCR. Results of plasma cell-free DNA were compared with biopsy's DNA and mononuclear cellular DNA respectively. Plasma cell-free DNA were successfully extracted from 288 cases of newly diagnosed, refractory and relapsed NHL in total 360 patients (80%).But nothing was found in the other 72 remittent patients. The positive percentage of IgH rearrangement in patients with B-NHL was 81% in plasma cell-free DNA and 77% in biopsy's DNA (P>0.05). As to the ratio of TCRγ rearrangement in patients with T-NHL, the former was 44%, and the latter was 39% (P>0.05). These results show tumor-derived DNA could be detected in tumor loaded plasma, even of underlying cancer patients. For NHL patients, detecting IgH and TCRγ gene rearrangement of plasma cell-free DNA has the same clinical significance as biopsy's DNA. Moreover, it's more simple, convenient and non-invasive. Lymphoma non-Hodgkin, plasma, cell-free DNA, gene rearrangement, immunoglobulin, heavy-chain gene, T-cell receptor.γ

Peripheral Thy1+ lymphocytes rearranging TCR‐γδ genes in LAT‐deficient mice

Linker for activation of T cells (LAT) is an adaptor molecule indispensable for development of alphabeta and gammadelta T lymphocytes. Surprisingly, using a new model of LAT-deficient mice we found that despite arrested thymic development, a discrete population of cells with active Lat promoter, expressing Thy1 molecules, accumulated in peripheral lymphoid organs of homozygous (Lat(Inv/Inv)) mutant mice. By measuring frequencies of TCR gene rearrangements in conjunction with a panel of cell surface Ag, we dissected two subsets of these Thy1(+) cells. Thy1(dull) cells expressed markers of NK lymphocytes and contained low frequency of TCR-gamma gene rearrangements without detectable TCR-delta rearrangements. Thy1(high) cells resembled immature CD44(+)CD25(+) thymocytes and contained high frequency of non-productive TCR-gamma and TCR-delta rearrangements, indicating that cells displaying molecular signatures of commitment toward gammadelta T-cell lineage can develop and populate lymphoid tissues of LAT-deficient mice. Phenotypically similar Thy1(high) cells were also found in lymph nodes of lymphocyte-deficient (Rag2(-/-)) mice but not in T lymphocyte proficient, heterozygous Lat(+/Inv) mice suggesting that Thy1(high) cells of LAT-deficient mice identified in this study accumulate in peripheral lymphoid organs as a result of congenital lymphopenia.

Id3 Restricts the Developmental Potential of γδ Lineage during Thymopoiesis

Most T cell progenitors develop into the alphabeta T cell lineage with the exception of a small fraction contributing to the gammadelta lineage throughout postnatal life. T cell progenitors usually commit to the alphabeta lineage upon the expression of a fully rearranged and functional TCRbeta gene, and most cells that fail to produce a functional TCRbeta-chain will die instead of adopting the alternative gammadelta T cell fate. What prevents these cells from continuing TCRgamma rearrangement and adopting the gammadelta T cell fate is not known. In this study, we show that functional loss of Id3 results in a significant increase of gammadelta T cell production from progenitor cells undergoing TCRbeta rearrangement. The enhanced gammadelta T cell development correlated with increased TCRgamma gene rearrangement involving primarily Vgamma1.1 in Id3 deficient mice. We further show that Id3 deficiency promotes gammadelta T cell production in a manner independent of TCRbeta-chain expression. Our data indicates that Id3 suppresses Vgamma1.1 rearrangement and gammadelta lineage potential among T cell progenitors that have completed TCRbeta gene rearrangement without producing a functional TCRbeta protein.

Expressions of CXCL13, CD10 and bcl-6 in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified

To study the value of immunomarkers CXCL13, CD10, bcl-6 in pathologic diagnosis of angioimmunoblastic T-cell lymphoma (AITL). One hundred and fifteen cases of AITL, 30 cases of peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) and 30 cases of reactive lymph nodes with paracortical hyperplasia (RH) encountered during the period from January, 1990 to January, 2008 were retrieved from the archival files of the Department of Pathology, West China Hospital of Sichuan University, China. The morphologic features were reviewed and compared. Immunohistochemical study was performed by SP method for CXCL13, CD10, bcl-6, CD21, CD3epsilon, CD3, CD45RO, CD20 and Ki-67. TCR-gamma gene rearrangement study was also carried out. Regressed follicles were evident in 7.8% (9/115) of AITL cases, 6.7% (2/30) of PTCL, NOS cases and 83.3% (25/30) of RH cases, respectively. A marked increase of number of arborizing venules was shown in 98.3% (113/115) of AITL cases, 63.3% (19/30) of PTCL, NOS cases and 76.7% (23/30) of RH cases, respectively. In lymph nodes with paracortical hyperplasia, the expression of CXCL13, CD10 and bcl-6 were restricted to the germinal centers. In AITL, 96.5% (111/115) of cases showed CXCL13 expression, in contrast to 26.7% (8/30) of PTCL, NOS. Expression of CD10 and bcl-6 were found in the neoplastic cells in 50.4% (58/115) and 78.3% (90/115) of AITL, and 3.3% (1/30) and 3.3% (1/30) of PTCL, NOS, respectively. Irregular meshworks of CD21-positive follicular dendritic cells were found in all the AITL cases. Clonal TCR-gamma rearrangement was detected in 83% (83/100) of the AITL cases. AITL is a type of lymphoma originated from the follicular helper T cells. Detailed morphologic assessment and use of immunohistochemical markers are essential for accurate diagnosis.

Clonal T Cell Large Granular Lymphocyte Leukaemia of Recipient Origin after Related Mismatched Allogeneic Haematopoietic Stem Cell Transplantation for Acute Myeloid Leukaemia as a Possible Consequence of Anti-Thymocyte Globulin in Conditioning Regimen

Polyclonal or oligoclonal expansion of T cell large granular lymphocyte (T-LGL) after allogeneic haematopoietic stem cell transplantation (HSCT) has been described. A few cases of clonal T-LGL leukaemia of donor origin has been reported as a post transplantation lymphoproliferative disorder (PTLD) and described after allogeneic HSCT (Chang, Am J Clin Pathol, 2005). We report here a patient with a clonal T-LGL of recipient origin occurring after allogeneic HSCT for acute myeloid leukaemia (AML). A 56 year old male patient with an AML received an HLA mismatched (disparity on locus A) related HSCT from his sister in first complete remission. Conditioning regimen was myeloablative and combined Busilvex (12.8 mg/kg) and Cyclophosphomide (120 mg/kg) (BuCy2). Anti-Thymocyte Globulin (ATG Sangstat) was added because of the HLA disparity between donor and recipient at a dose of 15 mg/kg. Prophylaxis of Graft versus Host Disease (GVHD) combined cyclosporine and methotrexate. Complete engraftment was observed with full haematopoietic recovery and 100% donor type chimerism detected by real time quantitative PCR (RQ-PCR). A grade II acute GVHD resolved under steroid and CMV infection was treated with valganciclovir. Ten months post HSCT while all immunosuppressive treatment was discontinued, a severe neutropenia (poly morpho nuclear <0.2 × 109/l) and an hyperlymphocytosis up to 10×109/l occurred with a thrombocytopenia of 100×109/l. Relapse of AML and viral infection (CMV, HHV6, EBV, HCV, HBV, HIV, HTLV1, parvovirus B19) were eliminated. Seach for autoantibodies was negative. No chronic GVHD was diagnosed. The predominant cells in blood films were typical LGLs. Flow cytometric analysis showed a population CD3+/CD8+/CD57+/CD56− consistent with T-LGL. Bone marrow biopsy confirmed agranulocytosis and T-LGL infiltration CD3+/CD8+/CD57+/CD56−. Secondarily agranulocytosis and thrombocytopenia spontaneously resolved within one month, but hyperlymphocytosis persisted with the same T-LGL profile. Chimerism by RQ-PCR on CD3+ and CD3− peripheral blood mononuclear cells (PBMNC) population at time of profound agranulocytosis revealed that donor cells were only 8.2 % and 1.2% on CD3+ and CD3− fractions respectively. Chimerism done when cytopenia resolved became mixed with 47% and 39% donor markers on CD3+ and CD3− fractions respectively. Chimerism was then performed on selected subpopulations; donor markers were 98% on CD14+/15+ myeloid cells, 100% on CD56+ natural killer (NK) cells and 78 % on CD19+ B cells. Cell sorting isolated two T-LGL subpopulations according to CD57 expression: CD57+bright and CD57+weak. Donor markers were 0.02% on CD3+/CD8+/CD57+bright T-LGL cells and 1.2% on CD3+/CD8+/CD57+weak T-LGL cells. These results were in favor of a recipient origin of the T-LGL population without loss of the graft. The diagnosis of clonal T-LGL population was confirmed by TCRg gene rearrangement performed on the selected CD3+/CD8+/CD57+bright and CD3+/CD8+/CD57+weak population. In addition a polyclonal background was observed only in the CD3+/CD8+/CD57+weak population. These data show that T-LGL leukaemia of recipient origin can occur after allogeneic HSCT for AML and can be included as a PTLD. The role of ATG as an intensive immunosuppressive treatment cannot be excluded in the occurrence of this PTLD. In this patient no treatment of T-LGL leukaemia and a wait and watch attitude were undertaken. Eighteen months post HSCT the patient is alive and well with the same stable PTLD profile and no relapse of AML The cytolytic activity of the recipient T-LGL clone could explained the absence of AML relapse and a transient toxicity against donor haematopoiesis. In vitro cytolytic assay will be done to confirm the cytotoxic activity of T-LGL against leukaemic cells and haematopoietic stem cells.

Monoclonality and oligoclonality of T cell receptor β gene in angioimmunoblastic T cell lymphoma

Angioimmunoblastic T cell lymphoma (AILT) is an aggressive T cell lymphoma with an incidence of approximately 1–2% of all non-Hodgkin lymphoma. The detection of clonal T cell receptor (TCR) gene...

Diagnostic significance of immunophenotyping and detection of gene rearrangement in subcutaneous panniculitis-like T-cell lymphoma

To explore the diagnostic implication of immunophenotyping and gene rearrangement in subcutaneous panniculitis-like T-cell lymphoma (SPTL). According to the selection criteria of 2005 WHO-EORTC classification for cutaneous lymphomas, 20 SPTL patients were enrolled in this study. A 10-antibody panel was used for immunophenotyping and in addition, polymerase chain reaction for TCR gamma and IgH gene rearrangement and in situ hybridization for EBER1/2 were also employed. There were 9 males and 11 female with a mean age of 29.5 years. Immunophenotypic study showed that all the patients expressed one to three T-cell associated antigens (CD2, CD3 or CD45RO), 18 patients were positive for beta F1, 18 for CD8, 20 for TIA-1 and 16 for granzyme B. None of the patients expressed CD4, CD20 and CD56. TCR gamma gene rearrangement was found in 16 of 20 cases (80.0%) and none for IgH gene rearrangement. The positive rate of EBER1/2 was 25.0% (5/20). Since the majority of SPTL patients show clonal TCR gene rearrangements, correlations among clinical presentation, histological features, immunophenotype and gene rearrangement data are considered important in confirming a diagnosis of SPTL.

A Comparative Study of Polymerase Chain Reaction Detection of Clonal T-cell Receptor Gamma Chain Gene Rearrangements Using Polyacrylamide Gel Electrophoresis versus Fluorescence Capillary Electrophoresis

Polymerase chain reaction (PCR)-based molecular techniques are useful adjunctive tools in the diagnosis of cutaneous T-cell lymphomas (CTCL). This study compares the sensitivity of PCR analysis of the T-cell receptor-gamma (TCR-gamma) gene rearrangements using conventional polyacrylamide gel electrophoresis (PCR-PAGE) and fluorescent capillary electrophoresis (PCR-FCE). A total of 22 paraffin blocks were analysed using PCR-PAGE and PCR-FCE. There were 17 cases of mycosis fungoides (MF), 4 cases of non-MF CTCL and 1 case of lymphoblastic leukaemia. Complete agreement was obtained between PCR-PAGE and PCR-FCE in 19 of the 22 cases, giving a concordance rate of 86.4%. PCR-FCE had a higher sensitivity of 77.3%, compared to 63.6% for PCR-PAGE, allowing the detection of 3 additional cases of clonal T-cell rearrangements, which had equivocal or polyclonal bands on PAGE. Two of these 3 cases were in erythrodermic MF patients. PCR-FCE also allowed the detection of matching clones in serial specimens taken from different sites and at different time intervals in patients with MF. However, matching clones from different specimens can be achieved qualitatively in PCR-PAGE by running and comparing these on the same polyacrylamide gel block. Both PCR-PAGE and PCR-FCE are useful in detecting T-cell clones in CTCL, with both methods being comparable in sensitivity and showing a high concordance rate of 86.4%. PCR-FCE has the added advantage of exhibiting semiquantitative properties, which may be important in early or erythrodermic MF cases, but the requirement for sophisticated and costly machinery limits its availability to high-capacity laboratories. The well-established PCR-PAGE method is a suitable alternative in routine clinical applications.

Characteristics of T-cell large granular lymphocyte proliferations associated with neutropenia and inflammatory arthropathy

IntroductionThe purpose of this study was to analyze the data of patients with T-cell large granular lymphocyte (T-LGL) lymphocytosis associated with inflammatory arthropathy or with no arthritis symptoms.MethodsClinical, serological as well as histopathological, immuhistochemical, and flow cytometric evaluations of blood/bone marrow of 21 patients with T-LGL lymphocytosis were performed. The bone marrow samples were also investigated for T-cell receptor (TCR) and immunoglobulin (IG) gene rearrangements by polymerase chain reaction with heteroduplex analysis.ResultsNeutropenia was observed in 21 patients, splenomegaly in 10, autoimmune diseases such as rheumatoid arthritis (RA) in 9, unclassified arthritis resembling RA in 2, and autoimmune thyroiditis in 5 patients. T-LGL leukemia was recognized in 19 cases. Features of Felty syndrome were observed in all RA patients, representing a spectrum of T-LGL proliferations from reactive polyclonal through transitional between reactive and monoclonal to T-LGL leukemia. Bone marrow trephines from T-LGL leukemia patients showed interstitial clusters and intrasinusoidal linear infiltrations of CD3+/CD8+/CD57+/granzyme B+ lymphocytes, reactive lymphoid nodules, and decreased or normal granulocyte precursor count with left-shifted maturation. In three-color flow cytometry (FCM), T-LGL leukemia cells demonstrated CD2, CD3, and CD8 expression as well as a combination of CD16, CD56, or CD57. Abnormalities of other T-cell antigen expressions (especially CD5, CD7, and CD43) were also detected. In patients with polyclonal T-LGL lymphocytosis, T cells were dispersed in the bone marrow and the expression of pan-T-cell antigens in FCM was normal. Molecular studies revealed TCRB and TCRG gene rearrangements in 13 patients and TCRB, TCRG, and TCRD in 4 patients. The most frequently rearranged regions of variable genes were Vβ-Jβ1, Jβ2 and Vγ If Vγ10-Jγ. Moreover, in 4 patients, additional rearrangements of IG kappa and lambda variable genes of B cells were also observed.ConclusionRA and neutropenia patients represented a continuous spectrum of T-LGL proliferations, although monoclonal expansions were most frequently observed. The histopathological pattern and immunophenotype of bone marrow infiltration as well as molecular characteristics were similar in T-LGL leukemia patients with and without arthritis.

TCR Gene Rearrangement in B-Cell Chronic Lymphocytic Leukaemia.

Background: Chronic lymphocytic leukaemia (B-CLL) is a heterogenous B cell disorder. However, clonal/oligoclonal T cell expansion has also been detected at a low frequency in B-CLL using a range of methodologies including Southern blotting and flow cytometric analysis. The underlying cause of this phenomenon is unclear and it has even been suggested that this clonal T cell receptor (TCR) gene rearrangement occurred in the leukaemic cells, leading to the concept of lineage infidelity. Other suggestions include that the observed T cell clonality may simply be related to the normal aging process or that it may result from the immune response to malignant cells or viral infection. During normal T cell maturation in the thymus, the TCR genes undergo a complex process of V(D)J segment rearrangement to produce a wide repertoire of antigen specificity. As it is an early event in T cell development, TCRγ gene rearrangement is commonly used as a marker of clonality. The TCRβ gene is also routinely investigated due to its greater combinatorial diversity. Lack of sufficiently sensitive detection methodology is often a limiting factor in the discrimination between clonal and polyclonal T-cell populations.Aims: The aim of this study was to use high resolution heteroduplex gel and fluorescence GeneScan analysis to screen for TCRγ and TCRβ gene rearrangements respectively in a cohort of B-CLL patients and to ascertain if the clonal rearrangement occurred in B cells or T cells.Methods: B and T cells were purified from peripheral blood from 34 B-CLL patients using a CD19+ or CD3+ magnetic bead system (autoMACS). DNA was extracted from purified B or T cells and the TCRγ and TCRβ genes were amplified by BIOMED-2 multiplex PCR assays (InVivoScribe Technologies). Heteroduplex and GeneScan analysis were then performed to detect clonal TCR gene rearrangments.Results: Clonal TCR gene rearrangements were detected in11/34 (32%) of cases in purified T cell fractions. No clonal TCR gene rearrangements were found in purified B cell fractions. Clonal TCRβ rearrangements were found in 10/34 (29%) and clonal TCRγ in 8/34 (23%). In most instances a weak clonal pattern was observed. Clonal TCR rearrangements were detected in 7 males and 4 females. Five patients had mutated IGHV genes, while the remaining 6 possessed unmutated IGHV genes. Seven patients presented with more advanced clinical stage (Binet B or C), and 8 patients received treatment.Summary/conclusions: These results are in agreement with previous work demonstrating that T-cell clonal/oligoclonal expansions occur frequently in B-CLL patients. However this is the first report to demonstrate that the clonal expansions occur in T cells and not in B cells. In most instances a weak clonal pattern was observed which was probably due to minor clonal T-cell populations. It is possible that T-cell clonality may be associated with a poorer prognosis in this disease category. Of interest, we found that 8/11(73%) patients in our B-CLL sub-group with T-cell expansion also had advanced stage disease and/or unfavourable molecular (IGHV gene usage/mutational status) markers. We are currently investigating the possibility of a specific antigenic stimulus resulting in this clonal T-cell expansion.

Study on Clinical and Laboratory Characteristics of Chronic Eosinophilic Leukemia CEL/Hypereosinophilic Syndrome (CEL/HES).

Hypereosinophilic syndrome (HES) was a group of diseases associated with persistent eosinophilia without unknown causes, companied with tissue and organ impairments. In 2001, WHO classification of hematopoietic and lymphoid neoplasms classified chronic eosinophilic leukemia (CEL) / HES into the category of chronic myeloproliferative disease, and proposed that the principal basis for the identification of CEL and HES was whether to have the evidence of eosinophils clonal proliferation: CEL had the evidence of eosinophils clonal proliferation, while HES was lack of the evidence of eosinophils clonal proliferation. In this study, 20 cases of CEL/ HES patients were analyzed retrospectively, and nested RT-PCR was used to detect FIP1L1-PDGFRA (F/P) fusion gene; Allele-specific PCR (ASP) conjoint sequencing analysis was used to detect JAK2 V617F, and PCR-RFLP was adopted to detect the mutation status of JAK2 V617F; and PCR is applied to detect TCRγ rearrangement. The clinical and laboratory characteristics of CEL and HES were compared. The ratio of male and female in the 20 cases of patients was 19:1, and the median age was 33 (20–57). F/P detection was positive for 12 cases, and the sequencing confirmed that FIP1L1 break point was at intron 10–12, while PDGFRA break point was at exon 12. There was 1 case of patient found that had JAK2 V617F, and the mutation status analysis showed that it was the mutation on heterozygote. 6 cases were detected having TCRγ gene rearrangement, of which 4 cases were CEL patients, and other 2 cases were HES patients. Most of CEL patients had respiratory symptoms in the early stage, easily companied with circulatory systematic impairment and nervous systematic symptoms. The incidence of splenomegaly of CEL patients was obviously higher than that of HES patients (92.5% vs 42.5%, p=0.031), so did the incidence of anemia and myelofibrosis. There was no difference in EO, WBC, PLT and EO% in peripheral blood as well as bone-marrow eosinophils percentage and bone-marrow primitive cells' percentage between two groups. Abnormal morphology of eosinophils was often found in CEL patients, with the main manifestation of eosinopenia, basopenia and plasma vacuoles. Our data showed that1.Eosinophilia is often caught by male, mainly by the young;2.CEL patients have the main manifestation of the circulatory systematic, respiratory and gastrointestinal symptoms, and have a high incidence of anemia and thrombocytopenia. The routine examination has a little significance for the identification o CEL and HES, while the bone marrow smears morphological examination has a certain help for the diagnosis of CEL;3.Some HES patients have JAK2 V617F mutation, and further studies on the effect of JAK2 V617F mutation on the pathogenesis of HES can contribute to the diagnosis of such patients in the future and the development of the new targeted drug therapy;4.Some CEL patients have TCRγ rearrangement, while the relationship of CEL and TCRγ needs a further study.

Immunoglobulin and T-Cell Receptor (Ig/TCR) Gene Rearrangements in Chinese Adult Acute Lymphoblastic Leukemia Patients.

To determine the feasibility of Ig/TCR gene rearrangements as patient-specific targets for the detection of minimal residual disease in Chinese ALL patients, forty adult patients with B-lineage and T cell acute lymphoblastic leukemia(ALL) were enrolled in this study. The frequency and pattern of IGH, IGK, TCRB, TCRG and TCRD gene rearrangement were analyzed by BIOMED-2 multiplex PCR protocol. All DNA samples were obtained from the bone marrow of these patients. IGH, IGK, TCRB, TCRG and TCRD rearrangements were found in 86%,14%,27%,77%,59% of adult patients with B-lineage ALL, respectively. While in T-ALL, clonal TCRB, TCRG and TCRD rearrangements were detected in 83%,78%,39% of patients. There are several features in the pattern of Ig/TCR gene rearrangements in Chinese adult ALL patients. Firstly, IGK rearrangement in adult B-lineage ALL had a lower frequency than in children's pre-B-ALL (14% vs 41%) in reported literature. Secondly, the frequency of TCRG Vrfl-Jr1.3, Vrfl-Jr1.1 and Vr9-Jr1.3 rearrangement was 65%,6%,29% in adult patients with B-lineage ALL, while 60%,27%,13% was the frequency of those in T-ALL, respectively. In conclusion, our data have shown that several special patterns in Ig/TCR gene rearrangements in Chinese adult ALL patients, may provide a guidance for individual treatment protocol designation and MRD-based risk stratification.