TCR Gamma Chain Gene Rearrangement Research Articles

First Description Of a t(10;11)(q22;q23)/MLL-TET1 Reciprocal Translocation In a T Lymphoblastic Lymphoma With Subsequent Lineage Switch To Acute Myelomonocytic Myeloid Leukemia

TET1 genomic breakpoints and clinical features of MLL-TET1 rearrangements have been described in 13 acute leukemia cases, 11 in AML, 2 in B cell-precursor ALL. The incidence of this rare translocation was evaluated to 0.3% of MLL rearranged AML cases (5 out of 1590 MLL [Meyer C. Leukemia 2013]). Although those cases are very uncommon, their study can improve our current understanding of leukemogenesis. We report here the first t(10;11) MLL-TET1 positive case of lymphoblastic T lymphoma occurring in a 31 years old male patient, with a subsequent transformation to AML.The patient was referred for a large mediastinal mass and right pleural effusion. Mediastinal and bronchus biopsies led to the diagnosis of a precursor-T cell lymphoblastic lymphoma, expressing CD3, CD5, CD4, CD8, CD10 antigens, without any expression of CD34 or CD79.Molecular analyses of the malignant T-cells showed a clonal TCR gamma-chain gene rearrangement together with HOXA10 overexpression. FISH analysis showed a MLL breakage. The partner gene, TET1, was identified using RP11-9E13 and RP11-314J18 BACs, corresponding to the recurrent translocation t(10;11)(q22;q23). MLL-TET1 fusion transcript was detected (intron 8 of TET1 fused to exon 8 of MLL), as well as its reciprocal transcript.The patient was treated following the (GELA) LL03 protocol, and was considered in complete remission after induction and consolidation phases. Fourteen months after the diagnosis, a bone marrow examination was performed for thrombopenia (6 G/L) which revealed a myelomonocytic acute leukemia with trilineage dysplasia.At this time, The MLL-TET1 fusion transcript as well as HOXA10 overexpression was still present but the TCR rearrangement was not detected. A non-familial donor allogeneic bone marrow transplant was performed in CR after intensive chemotherapy, that was complicated by a grade IV acute graft-versus-host disease. The patient died 54 days after the transplant of bacterial sepsis leading to multi-organ failure.MLL-TET1 fusions have been described in 13 cases in the literature, mainly in AMLs (11/13 cases, mostly AML M4 or M5 cases) and in B-ALLs (2/13 cases). The case reported here presents two interesting features. Firstly, this patient harbors the first MLL-TET1 fusion reported to date in T-ALL, and secondly this case presented a lymphoid to myeloid phenotype switch during the time course of the disease.This strongly suggests that the translocation occurred very early during hematopoietic differentiation, prior to the lymphoid/myeloid commitment. As described for the Ph1 chromosome rearrangement, the t(10;11) translocation may arise in hematopoietic stem cell rather than in committed progenitors. These features are also described in 8p11 stem cell syndrome that involves FGFR1. In both cases, genetic rearrangements arise in myeloid or lymphoid neoplasms with possible subsequent transformation.TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and play a key role in active DNA demethylation. TET1 and TET2 are also the key enzymes responsible for the presence of 5hmC in mouse embryonic stem cells (ESCs) (Koh KP., Cell Stem Cell 2011), and TET1 functions to regulate the lineage differentiation potential of ESCs. In addition to its role in DNA demethylation, TET1 interacts physically with NANOG and NANOG/TET1 co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells (Costa Y., Nature 2013). Taken together, these observations enlighten possible mechanism of the lineage switch observed in this case, and may be a rationale for using demethylating agents in MLL-TET1 neoplasms. Disclosures:No relevant conflicts of interest to declare.

TCRbeta Gene Rearrangements for Detection and Monitoring of Minimal Residual Disease after Allogenic Stem-Cell Transplantation in Patients with Advanced Mycosis Fungoides and Sézary Syndrome.

Advanced tumor-stage mycosis fungoides (MF) and Sézary syndrome (SS) are cutaneous T-cell lymphomas (CTCL) characterized by very poor prognosis. Allogeneic haematopoietic stem cell transplantation (allo-SCT) represents an experimental treatment which has been shown to be very effective in achieving long lasting complete remission, possibly leading to cure in selected patients. However, high transplant-related mortality (TRM) limit its feasibility in the vast majority of patients with CTCL. Reduced-intensity conditioning regimens have been demonstrated to decrease TRM, allowing gradual establishment of full donor chimerism and possible graft-versus-lymphoma effect. In this setting, evaluation of minimal residual disease (MRD) is particularly useful to guide post-transplant strategies such as donor lymphocyte infusion (DLI). Detection of TCRgamma-chain gene rearrangements, owing to the relatively limited complexity of its genetic elements, is routinely used to detect MRD in T-cell malignancies. Nonetheless, due to the extensive combinatorial repertoire and the large hypervariable regions, TCRbeta represents the best target for MRD monitoring, allowing more sensitive detection of patient-specific rearrangements. With this study, we aimed to identify patient-specific TCRbeta rearrangements to monitor MRD in patients enrolled in a clinical phase II trial of reduced intensity allo-SCT for advanced stage refractory MF/SS. Skin biopsy and peripheral blood samples at diagnosis and at different time points after transplant were obtained from 6 out of 9 evaluable patients, all having achieved clinical complete remission (still enduring in 5). The BIOMED-2 multiplex TCRbeta PCR heteroduplex assay (InVivoScribe Technologies, USA) was used for identification of monoclonal TCRbeta rearrangements, clonal PCR products were directly sequenced in both directions using the complete set of V or J primers, and V, D, J segments identified using the ImMunoGeneTics database (http://imgt.cines.fr). Then, clonespecific PCR assays were performed on samples collected from every single patient and specificity tested by parallel amplification of normal polyclonal DNA samples. In all patients a monoclonal TCRbeta rearrangement has been sequenced allowing to obtain clone-specific primers for PCR assays. In 2 patients we identified the presence of a MRD at the early controls after transplant (+2 and +3 months, respectively) when both were polyclonal by standard TCRgamma rearrangement; clone-specific PCR assays for TCRbeta became negative afterwards, in concomitance with the achievement of full donor chimerism. In 3 patients polyclonality of TCRbeta was observed in all post-transplant controls. One developed chronic cutaneous GvHD and skin biopsies verified the absence of clone-specific T-lymphocytes. In another patient, all post-transplant assays performed by the standard TCRgamma-rearrangement were persistently positive, suggesting the presence of a non disease-specific monoclonality. The last of the 6 patients relapsed 52 months after transplant. Retrospective clone-specific analysis of TCRbeta rearrangements in DNA from skin biopsies unveiled the presence of molecular relapse already 24 months before, whereas standard TCRgamma assays became positive only after clinical relapse. Altogether, we observed good stability of monoclonal TCRbeta rearrangements over time. Our results suggest that the analysis of TCRbeta is more sensitive and more specific than the analysis of standard TCRgamma for detection of MRD in CTCLs, allowing earlier identification of relapses and adopatiention of pre-empatientive treatment such as DLI. With this method, we also demonstrated disappearance of MRD in concomitance with the achievement of full donor chimerism after allo-SCT. Finally, disease clone-specific TCRbeta rearrangement detection might be helpful in distinguishing cutaneous manifestations of acute GvHD from early relapse of MF. TCRbeta analyses of samples from the remaining 3 evaluable patients are ongoing and will be included in the final presentation.

Nasal/Nasal Type NK/T-Cell Lymphoma: A Brazilian Experience of 40 Cases.

NK/T nasal type lymphoma is a rare type of non-Hodgkin's lymphoma with a peculiar geographical distribution. Most of the reported cases come from Asian coutries and native populations from Latin America, such as Peru, Guatemala and Mexico. It is rarely reported in Brazilian population. We retrospectively studied 40 cases of Nasal/Nasal type NK/T-cell Lymphoma diagnosed at our center, between 1979 and 1999. Patient characteristics were as follows: 18 white,13 mulatto, 6 Asian and 3 black; 28 men and 12 women; median age 42.8 years (range 17–78). Patients originaly from all Brazilian geopolitical regions were present. One or more structures of the mild-facial region were involved in all cases, mainly in the nasal cavity (38 cases), ethmoidal sinus (21 cases), maxillary sinus (20 cases), palate (20 cases), nasopharingeal (13 cases), nose flap (11 cases), orbita (11 cases), sphenoidal sinus (9 cases), frontal sinus (6 cases), oropharingeal (3 cases). The most frequent symptoms were:nasal obstruction(27 cases), palate ulcer or edema(18 cases), fetid odor(14 cases), rhinorrehoea (9 cases), and orbital edema(8 cases). Considering the stage, 32 patients were in stage IE, 5 in stage IV, 1 in stage IIE and for 2, the stage was unknown. The median time from the onset of symptoms to diagnosis was 5.5 months, ranging between 1 and 17 months. The number of biopsies to confirm the diagnosis ranged between 1 and 5, with a median of 2 biopsies. Diagnosis was based on histological examination, immunohistochemistry panel(CD20, CD2, cCD3, CD43, CD45RO, CD16, CD56, granzin-B, TIA-1 and p53), in situ hybridization(ISH)forEBV and PCR for TCR gamma chain gene rearrangement. Important histological features were necrosis in almost all cases(38 cases) and angiocentricity and angioinvasion in 32 cases. There was no clear association between necrosis and angiocentricity, suggesting that other causes can be involved in the pathogenesis of the necrosis. Association between EBV and the pesence of necrosis in the histological study was present. Most of the cases showed a phenotypical and genotypical profile of NK cell lineage. Two cases presented monoclonal rearrangement for TCR gamma chain gene, characterizing T cell lineage. The only two cases of T cell lineage were also negative for EBV. Immunohistochemical reativity for p53 was observed in 24 cases. There was no association between the progression of the disease and the immunoexpression for p53. Most of the patients were treated with chemotherapy alone(14 cases) or associated with radiotherapy(20 cases). One patient was treated with radiotherapy alone and 5 patients were not treated due to a rapid progression to death. The overall median survival time was 12.6 months. The cause of death could be assessed in 23 patients;15 died due to infectious complications, and septicemia was the most frequent cause. The results of this retrospective survey confirmed that Nasal/Nasal type T/NK-cell Lymphoma is characterized by a very bad prognosis. More data are needed to know the epidemiology of this rare lymphoma in South America. The results show that EVB is also highly associated with this lymphoma in Brasilian patients.

Ocular adnexal lymphoid proliferations: Clinical, histologic, flow cytometric, and molecular analysis of forty-three cases

Purpose To describe the clinical features, histologic findings, flow cytometric immunophenotypes, and molecular profiles of ocular adnexal lymphoid proliferations. Study design Prospective noncomparative case series. Participants Forty-three patients suspected of having ocular adnexal lymphoid proliferations were biopsied and prospectively evaluated. Methods Provisional diagnoses were made on the basis of routine histology and immunohistochemistry for B and T cells. Results of flow cytometric immunophenotyping (FCI) and molecular assessment using polymerase chain reaction for immunoglobulin heavy chain (IgH) and TCR gamma chain gene rearrangement and bcl-2/IgH translocation were then incorporated into a final diagnosis. Demographic and clinical outcome data were collected. Main outcome measures Final diagnosis based on histology, flow cytometry, and polymerase chain reaction. Results Forty-three cases were studied. Final diagnoses included 17 lymphomas, 18 chronic inflammations, 4 reactive lymphoid hyperplasias, and 4 atypical lymphoid infiltrates. Preliminary evaluation accurately categorized all 43 cases as either lymphoma or nonlymphoma. FCI permitted more precise subclassification of the lymphomas according to the Revised European American Lymphoma (REAL) system of nomenclature as follows: eight marginal zone B cell (mucosa-associated lymphoid tissue type), three mantle cell, two follicular, three large cell, and one lymphoplasmacytoid lymphoma. FCI showed a clonal B cell proliferation in 94% (16 of 17) of the lymphomas; FCI identified a clonal B cell population in 4% (1 of 25) of cases of nonlymphomas. Molecular evidence of clonality was identified in 88% (15 of 17) of lymphomas, 39% (7 of 18) of chronic inflammations, and 50% (4 of 8) of reactive lymphoid hyperplasias and atypical lymphoid infiltrates. Conclusions The histologic diagnosis of ocular adnexal lymphoid lesions is highly accurate when determined by an experienced pathologist. FCI refines the histologic diagnosis and classification. Results of molecular studies should be interpreted in conjunction with clinical, histologic, and immunophenotyping findings.

Evaluation of T Cell Receptor Testing in Lymphoid Neoplasms: Results of a Multicenter Study of 29 Extracted DNA and Paraffin-Embedded Samples

To evaluate current diagnostic methods used for the evaluation of T cell receptor (TCR) gene rearrangements, 24 different laboratories analyzed 29 lymphoid neoplasm samples of extracted DNA and paraffin-embedded tissue and were asked to complete a technical questionnaire related to the testing. Participating laboratories performed Southern blot and polymerase chain reaction (PCR) testing for rearrangements of the TCRbeta chain gene and PCR for the TCRgamma chain gene rearrangements. Of 14 laboratories performing TCRbeta Southern blot analysis, there was complete agreement in 10 of 14 cases, with some false negative results obtained in 4 cases. No false positive results were obtained by Southern blot analysis. TCRbeta PCR analysis was only performed by two laboratories, and only 47.1% of positive samples were detected. Twenty-one laboratory results were obtained for TCRgamma PCR. This method showed an overall detection rate of 77.9% for T cell gene rearrangements with a 4.1% false positive rate, as compared to both TCRgamma Southern blot analysis results and immunophenotyping. The detection rate for TCRgamma PCR, however, significantly differed when extracted DNA samples from frozen tissue were compared to paraffin-embedded tissue (85.4% versus 65.9%; P = 0.0005). Significant differences in true positive results were obtained when laboratories using primers directed against multiple TCRgamma variable regions (V1-8 plus one to three other primer sets) were compared to laboratories that used only a single set of TCR primers directed against the V1-8 (P < 0.0001). Other technical factors significantly affecting results were also identified. These findings provide useful data on the current state of diagnostic TCR testing, highlight the risk of false negative results for TCR testing directed against only portions of the TCRgamma gene, and identify limitations of testing of paraffin-embedded tissues in some laboratories.

Clonal T-cell receptor gamma-chain gene rearrangement by PCR-based GeneScan analysis in advanced cutaneous T-cell lymphoma: a critical evaluation.

Detection of clonal T-cell receptor gamma (TCRgamma)-chain gene rearrangement is a promising approach to distinguish between cutaneous T-cell lymphomas (CTCLs) and reactive T-cell infiltrates. Despite the improved sensitivity by using the polymerase chain reaction (PCR) rather than Southern blot analysis, monoclonality could be demonstrated in only 53-90 per cent of CTCL biopsies in recent studies. In the present study, formalin-fixed, paraffin-embedded specimens of 21 selected patients with clear-cut advanced-stage CTCL were analysed using a semi-nested TCRgamma PCR with newly developed consensus primer pairs. Detection of PCR products was done by GeneScan analysis (GSA); this technique is advantageous due to its sensitivity and accuracy in the detection and size determination of PCR products and it is easier to interpret than direct read-outs from TGGE or DGGE gels. In serial dilution experiments, TCRgamma-PCR-GSA allowed the detection of clonal, rearranged T-cells with a high in vitro sensitivity against a polyclonal background (1-6 per cent). Despite the selection of clear-cut, advanced-stage CTCL cases, however, dominant clonal TCRgamma-chain gene rearrangement was found in only 16 of the 21 patients analysed, indicating an overall clinical sensitivity of 76 per cent. Specificity was evaluated using biopsy specimens from 21 control patients suffering from long-standing psoriasis (n=13) and eczema (n=8). Surprisingly, GeneScan profiles showing apparently single dominant peaks were detected in 14 per cent of these skin lesions, but these profiles turned out to be pseudo-monoclonal by repeated determinations. In conclusion, TCRgamma-PCR-GSA does not suffice reliably to exclude malignancy, due to its limited clinical sensitivity, but with precautions taken to detect pseudo-monoclonality and to secure specificity, TCRgamma-PCR-GSA is a valuable instrument in the diagnosis of CTCL.

Prognostic Significance of a Polymerase Chain Reaction–Detectable Dominant T-Lymphocyte Clone in Cutaneous Lesions of Patients With Mycosis Fungoides

Although mycosis fungoides (MF) is considered to be an indolent lymphoma, survival is highly influenced by TNM stage. At diagnosis, most MF patients present with early stage disease and a high probability of long-term survival. Treatment is generally directed towards skin lesions, and achievement and duration of complete responses are variable. A dominant T-cell clone is detectable in the cutaneous lesions of 60% of patients. The aim of this study was to determine whether the presence of a T-cell clonal population influences the clinical course of the disease after topical therapy. Cutaneous biopsies from 68 patients were histologically diagnosed as MF and T-cell clonality was analyzed by in vitro amplification of TCR-gamma chain gene rearrangements (polymerase chain reaction gamma [PCRgamma]). After a median follow-up of 48 months, response to treatment was clinically assessed. Age, sex, duration of symptoms before diagnosis, type of cutaneous lesions (T stage), TNM stage, and PCRgamma were evaluated as predictive factors of response to treatment in univariate and multivariate analyses. Univariate analysis demonstrated that T1 cutaneous lesions (P = .05) and PCRgamma negativity (P = .007) were associated with a higher complete remission rate. Using multivariate analysis, T stage (relative risk, 3.13; P = .06) and PCRgamma (relative risk, 4.4; P = .01) remained independent significant predictive parameters of response. In conclusion, T stage and cutaneous PCRgamma at diagnosis are the two predictive parameters of treatment response for MF. Therefore, the cutaneous PCRgamma findings should be considered in the analysis of future therapeutic trials.

Clonal Disease in Early Cutaneous T-Cell Lymphoma

Our experience with 185 cases of T-cell dominated skin infiltrates demonstrates that the PCR-based method for detection of TCR gamma chain gene rearrangement in combination with temperature gradient electrophoresis can routinely be used for the demonstration of clonal T cells in formalin-fixed and paraffin-embedded biopsies of lesional skin. In contrast to Southern blot analysis, the amplification by PCR is nonradioactive, is not time consuming (approximately 3 days), can be performed using frozen or paraffin-embedded tissue, and allows additional molecular biologic analyses, such as sequencing. Furthermore, it offers the possibility to design patient-specific primers for monitoring of the disease activity. It also has to be concluded from our study that all available clinical, histologic, cytologic, immunophenotypical, and rearrangement studies have to be considered in order to establish the correct diagnosis.

Double-negative (CD4- CD8-) T cells from adult T-cell leukemia patients also have poor expression of the T-cell receptor alpha beta/CD3 complex

We present four patients with adult T-cell leukemia (ATL) derived from a novel T-cell subset (CD4-, CD8- [double-negative, DN], T-cell receptor [TCR] alpha beta+). In the ATL cells of these patients, neither gene nor surface expression of CD4 and CD8 antigens was detected. Clinical and laboratory data showed no difference between DN-ATL and CD4+ATL patients. In contrast to typical CD4+ATL cells, DN-ATL cells were shown to express the protein and messenger RNA (mRNA) for S100 beta in immunocytochemical assay and the reverse-transcription polymerase chain reaction assay. The mean fluorescence intensity of the TCR/CD3 complex was extremely low in all four DN-ATL patients as well as in typical CD4+ ATL. All four patients had TCR beta and gamma chain gene rearrangements, with deletion of TCR delta chain gene and mRNA expression for TCR alpha, beta, and CD3 delta but not for TCR gamma and delta chain genes. Thus, CD4- CD8- TCR alpha beta T cells are also a target for human T-cell lymphotrophic virus type I-induced leukemogenesis. In addition, expression of the TCR alpha beta/CD3 complex on the DN-ATL cells was further diminished by the addition of anti-CD3 or anti-TCR alpha beta monoclonal antibody. These results suggest that the decreased expression of the TCR alpha beta/CD3 complex by ATL cells plays a key role in the development of ATL, irrespective of CD4 expression.

Double-Negative (CD4– CD8–) T Cells From Adult T-Cell Leukemia Patients Also Have Poor Expression of the T-Cell Receptor αβ/CO3 Complex

Abstract We present four patients with adult T-cell leukemia (ATL) derived from a novel T-cell subset (CD4-, CD8- [double-negative, DN], T-cell receptor [TCR] alpha beta+). In the ATL cells of these patients, neither gene nor surface expression of CD4 and CD8 antigens was detected. Clinical and laboratory data showed no difference between DN- ATL and CD4+ATL patients. In contrast to typical CD4+ATL cells, DN-ATL cells were shown to express the protein and messenger RNA (mRNA) for S100 beta in immunocytochemical assay and the reverse-transcription polymerase chain reaction assay. The mean fluorescence intensity of the TCR/CD3 complex was extremely low in all four DN-ATL patients as well as in typical CD4+ ATL. All four patients had TCR beta and gamma chain gene rearrangements, with deletion of TCR delta chain gene and mRNA expression for TCR alpha, beta, and CD3 delta but not for TCR gamma and delta chain genes. Thus, CD4- CD8- TCR alpha beta T cells are also a target for human T-cell lymphotrophic virus type I-induced leukemogenesis. In addition, expression of the TCR alpha beta/CD3 complex on the DN-ATL cells was further diminished by the addition of anti-CD3 or anti-TCR alpha beta monoclonal antibody. These results suggest that the decreased expression of the TCR alpha beta/CD3 complex by ATL cells plays a key role in the development of ATL, irrespective of CD4 expression.

Coding joint formation of endogenous T cell receptor genes in lymphoid cells from scid mice: unusual P-nucleotide additions in VJ-coding joints

The mouse mutation scid adversely affects the process of VDJ recombination. Attempts to form coding joints, that is, to joint V or D to J gene segments generally fail in developing scid lymphocytes. It has been proposed that the scid mutation results a defective VDJ recombinase system. Here we describe five scid T cell lymphomas containing one or two TcR gamma coding joints each, even though the majority of the multiple TcR gamma chain gene rearrangements and all TcR beta rearrangements in these cells were abnormal with the deletions typically found in scid lymphoid cells. One of the five T cell lymphomas was shown to have an active VDJ recombinase system; however, this activity was defective indicating that the scid phenotype has been retained. We conclude that the scid VDJ recombinase system has not completely lost the ability to form coding joints. P-nucleotide additions of unusual length or composition were found at the junctional border in five of the eight TcR gamma coding joints. This might reflect a defect in the activity of a component of the VDJ recombinase system involved in the generation of P-nucleotide additions. In one of the observed rearrangements, a V gamma 5-J gamma 3 coding joint was formed. This establishes the transcriptional orientation of J gamma 3-C gamma 3 and confirms a previously proposed organization of the TcR gamma genes.