Detection Of T-cell Clonality Research Articles

Reliable Detection of T-Cell Clonality by Flow Cytometry in Mature T-Cell Neoplasms Using TRBC1: Implementation as a Reflex Test and Comparison with PCR-Based Clonality Testing.

The T-cell receptor β constant region 1 (TRBC1) antibody can identify T-cell clonality and distinguish pathological from normal T cells. This study aims to establish optimal cutpoints for establishing monotypia and validate the diagnostic abilities of the TRBC1 antibody when used as a reflex test in conjunction with an existing T-cell antibody panel. We used 46 normal peripheral blood specimens and examined 8 patients with reactive lymphoproliferations to determine the normal biological range of TRBC1 on CD4+ and CD8+ T cells. We also evaluated 43 patient specimens that were submitted for investigation of a lymphoproliferative disorder for CD2/CD3/CD4/CD5/CD7/CD8/CD16/CD26/CD45/CD56/TCR αβ/TCR γδ, along with TRBC1 expression. The results were compared to TCR gene rearrangement patterns using polymerase chain reaction (PCR) analysis. Statistical analysis established differing cutoff points for establishing monotypia dependent on restricted TRBC1 or TRBC2 usage. Direct comparison with molecular analysis indicated that no specimen identified with the restricted expression of TRBC1 was reported as polyclonal by PCR with a concordance rate of 97% between a clonal PCR result and monotypic TRBC1 expression. Incorporation of the TRBC1 antibody using statistically derived cutoff points in a reflex setting for the evaluation of a suspected T-cell neoplasm improves the identification of clonal T-cell populations by flow cytometry and correlates well with molecular methods.

High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in Tαβ-Large Granular Lymphocytic Leukemia.

Simple SummaryTRBC1 expression analysis by flow cytometry (FCM) has been recently proved to be a useful, simple and fast approach to assessing Tαβ-cell clonality. The aim of this study was to validate the utility of this assay specifically for the diagnosis of T-cell clonality of T-large granular lymphocytic leukemias (T-LGLL), as more mature polyclonal Tαβ large granular lymphocytes (Tαβ-LGL) show broader TRBC1+/TRBC1− ratios vs. total Tαβ cells. Our results showed that a TRBC1-FCM assay is also a fast and easy method for detecting T-cell clonality in T-LGLL based on altered (increased or decreased) percentages of TRBC1+ Tαβ cells of LGL expansions (i.e., with lymphocytosis) suspected of T-LGLL, whereas in the absence of lymphocytosis (or in TαβCD4-LGLL), the detection of increased absolute cell-counts of more precisely defined subpopulations of T-LGL expressing individual TCRVβ families is required.Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor β chain (TRBC1) expression for assessing Tαβ-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαβ cells (i.e., T-LGL normal-counterpart) show broader TRBC1+/TRBC1− ratios vs. total Tαβ cells. We compared the distribution and absolute counts of TRBC1+ and TRBC1− Tαβ-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1+ or TRBC1− Tαβ-LGL ranged between 0.36 and 571 cells/μL (3.2–91% TRBC1+ cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/μL (<0.9% or >96% TRBC1+ cells). Among the distinct TCRVβ families, the CD28− effector-memory and terminal-effector polyclonal Tαβ cells ranged between 0 and 25 TRBC1+ or TRBC1− cells/μL and between 0 and 100% TRBC1+ cells, while clonal LGL ranged between 32 and 5515 TRBC1+ or TRBC1− cells/μL, representing <1.6% or >98% TRBC1+ cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαβ-LGL suspected of T-LGLL based on altered percentages of TRBC1+ Tαβ cells. However, in the absence of lymphocytosis or in the case of TαβCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαβ-LGL-expressing individual TCRVβ families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.

Detection of T-Cell Clonality in Bone Marrow in Peripheral T-Cell Lymphoma, Not Otherwise Specified

Introduction: Peripheral T-cell lymphoma, unspecified (PTCL, NOS) is a tumor of mature T-lymphocytes characterized by aggressive course and low response rate to CHOP-like therapy. Bone marrow involvement in PTCL, NOS is detected by histological investigation in about 20-40% of patients and it is considered to be a poor prognostic factor. Importance of T-cell clonality examination in bone marrow as a prognostic and staging factor has been insufficiently studied.Aim: In order to evaluate the significance T-cell receptor gene γ- and β-chains (TCRG and TCRB) rearrangements for identification of bone marrow involvement; to estimate its value for staging and prognosis; to evaluate the significance of T-cell clonality persistence in bone marrow after initial therapy in patients with PTCL, NOS.Patients and methods: Bone marrow samples of 27 patients with primary PTCL, NOS (median age 62 years, range 32-77) have been obtained from 2006 till 2014 and were analyzed retrospectively. Morphological examination was performed for all bone marrow samples. Detection of T-cell clonality by revealing rearrangements of TCRB and TCRG were performed by PCR in bone marrow samples of patients before and after initial chemotherapy.Results: Bone marrow involvement was revealed by histological investigation in 22 (81%) of patients. T-cell clonality was detected by rearrangements of TCRG or TCRB in 26 (96%) patients, including 4 of 5 patients without bone marrow involvement by histology. Rearrangements of TCRG and TCRB were detected in 24 (89%) and 26 (96%) of patients, respectively. Rearrangements of TCRB were observed without rearrangements of TCRG in 2 cases. Interestingly, we detected more than 2 clonal rearrangements present simultaneously in 33% of cases. For monitoring minimal residual disease the examination of T-cell clonality in bone marrow samples was performed in 15 of 27 patients after initial therapy; all these 15 patients achieved complete remission. Persistence of T-cell clonality was observed in 14 of 15 patients after treatment, disease progression was noted in 1-3 months in all these cases.Conclusion: The examination of T-cell clonality in the bone marrow seems to be important and necessary for staging, monitoring minimal residual disease. Detection of T-cell clonality in bone marrow allows revealing its involvement even without morphological features in primary patients with PTCL, NOS. Persistence of clonal rearrangements in bone marrow after initial therapy shows continuing lesion and necessity for timely therapy escalation for patients with PTCL, NOS. DisclosuresNo relevant conflicts of interest to declare.

High Frequency of Thyroid Disorders in Patients Presenting With Neutropenia to an Outpatient Hematology Clinic STROBE-Compliant Article.

Granulopoiesis abnormalities have been described in association with thyroid disorders (TD). However, data regarding systematic evaluation of adult neutropenia and concurrent or prior TD are scarce.To investigate the frequency of TD among patients presenting with neutropenia, and the immunophenotypic and immunologic profile of neutropenic patients with concomitant thyroidopathy.Two hundred eighteen consecutive neutropenic patients were prospectively evaluated in our outpatient Hematology Clinic, with a detailed laboratory screen, including thyroid function tests, antineutrophil antibodies, blood lymphocytes immunophenotyping, and detection of T-cell clonality by PCR.Among 218 patients with neutropenia, 95 (43.6%) had TD, 65 chronic immunologic neutropenia, 20 clonal proliferation of T-large granular lymphocytes (T-LGL), 5 autoimmune disorders, and 33 other diagnoses. TD-patients had an increased frequency of recurrent infections compared with other patients (P = 0.045). The following correlations were found: negative correlation between FT3 and absolute neutrophil count (ANC) (r2 = −0.274, P = 0.007), negative correlation between TPO-Abs/TG-Abs and C4 (r2 = −0.16, P = 0.045; r2 = −0.266, P = 0.001), and CD4+ counts were inversely correlated to T4 and positively to TSH (r2 = −0.274, P = 0.024; r2 = 0.16, P = 0.045). In addition, TD-patients had significantly higher percentages of CD4+ lymphocytes (P = 0.003). Among TD-patients, 23.4% had Hashimoto thyroiditis (HT), 4.1%, Graves disease (GD), 8.2% nontoxic multinodular goiter (NTMG), 5% subclinical hypothyroidism, and 2.8% had undergone total thyroidectomy associated with nodules (TTM). Thirteen TD-patients displayed T-LGL. Patients with autoimmune thyroidopathy had an increased frequency of concomitant autoimmune manifestations (P = 0.03). Significant differences between the different thyroidopathies included: HT-patients had higher percentages of B-lymphocytes, while the opposite was evident for the TTM-subgroup (P = 0.009, 0.02); GD-patients showed an increase of the proportion of NK cells and a decrease in the percentage of TCRγδ+ lymphocytes (P = 0.001, 0.045); and NTMG-patients had significantly higher ANC (P = 0.004) compared to other thyroidopathies. Antineutrophil antibodies were found in 37.2% of TD-patients tested. Anti-TPO titers were significantly higher in patients with positive antineutrophil antibodies (P = 0.04).The frequency of TD among neutropenic patients may be higher than previously reported. The existence of antineutrophil antibodies, as well as the different distribution of lymphocyte subsets among patients with different TD, suggests both humoral and cellular mechanisms in the pathophysiology of thyroid disease-associated neutropenia.

T-cell clonality is detected in a high frequency among patients with incidental lymphocytosis by PCR assays for TCR gene rearrangements

AimsClonal expansion of lymphocytes may account for asymptomatic lymphocytosis. Unlike monoclonal B lymphocytosis, the detection of T-cell clones is difficult using immunophenotype-based assays. This study aimed to evaluate PCR-based clonality...

Analysis of T-cell receptor-γ gene rearrangements using heteroduplex analysis by high-resolution microcapillary electrophoresis

Determination of T-cell clonality has an important additional value for diagnosis of T-cell lymphomas. Various molecular methods have been developed, including polymerase chain reaction (PCR) of T-cell receptor γ (TCRγ). The detection of PCR products usually relies commonly on either GeneScan (GS) analysis or heteroduplex (HD) analysis by polyacrylamide gel electrophoresis (PAGE). These techniques have their disadvantages, being relatively time-consuming and laborious or requiring expensive equipment. Here, we propose an alternative method that combines multiplex PCR and HD analysis by microcapillary electrophoresis (ME) on the Agilent 2100 Bioanalyzer. The sensitivity of the method was determined with clonal PEER T-cell line DNA dilution in polyclonal DNA and was evaluated as 1–5%. Fifty-three samples from patients with T-cell lymphoproliferative disorders were analyzed by HD analysis using ME and GS analyses. Comparison of the two techniques showed them to be highly concordant (93% similarity). The rate of clonality detection by GS analysis was higher than HD analysis by ME, but none of the discordant patients (n=5) has yet developed lymphoma. HD analysis by ME to reveal TCRγ gene rearrangements in clinical specimens was consistent with clinical data and the outcome of patients. Detection of T-cell clonality by HD analysis with ME is sensitive, practical, safe and represents a potential alternative to PAGE and GS analysis.

Molecular detection of circulating Sezary cells in patients with mycosis fungoides: could it predict future development of secondary Sezary syndrome? A single-institution experience

While the majority of patients with early-stage mycosis fungoides (MF) have an excellent prognosis, a few cases progress to secondary Sezary syndrome (sSS), which carries a dismal clinical outcome. We retrospectively analyzed 135 cases of MF/SS and correlated molecular detection of T-cell clones in the skin and blood with other clinicopathologic findings. When stratified by the diagnoses, patients with MF demonstrated a 26.5% (31/117) positive rate for a blood T-cell clone, of which 50% (10/20) had an identical T-cell clone in the skin. Follow-up evaluation showed conversion into sSS or leukemic phase in 50% (5/10) of cases with a positive blood T-cell clone (estimated mean interval 41.8 months) in comparison to no cases in the group without a clone (0/31). Interestingly, 4/5 cases of sSS had an identical T-cell clone in the skin, while the remaining case did not have the test performed on skin for clonal comparison. Kaplan–Meier survival analysis demonstrated a poor clinical outcome in the group with a blood T-cell clone, in comparison with the group without, in overall survival (p < 0.0001) and progression-free survival (p < 0.0001; HR = 22.6). These findings suggest that molecular detection of a blood T-cell clone may have a role in predicting sSS. Due to amplification of non-neoplastic T-cell expansion in a significant number of cases, comparison of blood T-cell clones with skin may have confirmatory value.

Evolution of Clinical and Molecular Responses to Bexarotene Treatment in Cutaneous T-Cell Lymphoma

Background: The mechanisms of action of bexarotene are not well understood. Methods: A retrospective study on patients with cutaneous T-cell lymphoma (CTCL) treated with bexarotene was performed to see if bexarotene could act on the dominant T-cell clones. Thirty-five patients were included. Twenty-three were treated with bexarotene for more than 3 months (300 mg/m²). In 7 patients, phototherapy was given with bexarotene. Results: Dominant T-cell clones were observed in 11 patients in peripheral blood and in 19 patients in skin. Our results demonstrate no significant evolution of T-cell clones either in skin or peripheral blood. Furthermore, the detection of T-cell clones in peripheral blood before starting bexarotene was significantly associated with the progression of the disease. UV therapy given with bexarotene significantly improved therapeutic response without any correlation with T-cell clones. Conclusion: This is the first study on the evolution of the T-cell clone in blood and skin in CTCL patients during bexarotene therapy.

Flow cytometric assessment of TCR-VBETA expression in the evaluation of peripheral blood involvement by mature T-cell leukaemias

Background Mature T-cell leukaemias have always been considered difficult and challenging to diagnose by flow cytometry, owing to a paucity of immunophenotyping markers that are sensitive for detection of a clonal T-cell population. Demonstration of the neoplastic nature of T-cell proliferations traditionally has relied on morphological criteria and molecular tests. TCR gamma gene rearrangement studies have been used as the only reliable test to prove clonal T-cell receptor. This notion is changing now. Aim To validate the current commercially available kit IOTest TCR Vβ Repertoire Kit (#IM3497, Beckman Coulter) for routine use in the clinical flow cytometry laboratory. Methods EDTA antigoagulated blood specimens: 10 controls and 10 patients with known or suspected T-cell lymphoproliferative disorders were tested for the expression of 24 TCR Vβ antigens with TCR Vβ Repertoire Kit. All specimens were subjected to four-colour immunophenotyping on FC 500 cytometer. Correlation was performed with TCR gene rearrangement by PCR using Cohen’s Unweighted Kappa score. Results All 13 patients had clonal T-cell population detected by flow cytometry. PCR confirmed T-cell clonality in all 13 cases studied– Kappa score 1. No false positive or negative results were observed in our settings. Conclusion Detection of T-cell clonality is no longer restricted to molecular techniques or research laboratories. It is now possible to prove T-cell clonality by means of flow cytometry, based on abnormal distribution of TCR Vβ antigens. Apart from the very high cost, the assay is easy to perform and interpret, with a turnaround time of approximately 2 hours. This test represents a powerful tool for rapid diagnosis of peripheral T-cell lymphoma before the disease becomes a medical emergency and, in some instances, might eliminate the need for further genetic testing.

Distinguishing angioimmunoblastic T‐cell lymphoma from peripheral T‐cell lymphoma, unspecified, using morphology, immunophenotype and molecular genetics

To identify distinguishing histological, immunophenotypic and molecular genetic features between angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma (PTL). Nodal T-cell lymphomas examined (n =137), included AITL (n = 89), PTL (n = 22), anaplastic large cell lymphoma (n = 16) and 'AITL/PTL indeterminate' (n = 10) with overlapping features between AITL and PTL, showing morphology typical of AITL but lacking follicular dendritic cell expansion. Immunohistochemistry for CD3, CD20, CD21 and CD10, in situ hybridization for Epstein-Barr virus encoded RNA (EBER) and polymerase chain reaction for T-cell and B-cell clonality analysis were performed. Of the AITLs, 74/89 showed typical morphology, whereas 15/89 showed hyperplastic follicles. AITL and 'AITL/PTL indeterminate' showed a polymorphous infiltrate and prominent vascularity in all cases. In both groups, CD10 was present in the majority and clear cells and EBER positivity were specific (but not universal) features lacking in PTL. Detection of T-cell clonality was significantly higher in AITL (90%) compared with PTLu (59%). Clear cells and EBV infection (when present) are useful distinguishing features and CD10 a sensitive and specific marker of AITL. Hyperplastic follicles are present in a significant minority of AITL. AITL/PTL indeterminate probably falls within the spectrum of AITL rather than PTL.

An approach to the sensitivity of temperature-gradient gel electrophoresis in the detection of clonally expanded T-cells in cutaneous T-cell lymphoma.

Detection of T-cell clonality by polymerase chain reaction (PCR) and high-resolution electrophoresis facilitates differentiation of early stages of cutaneous T-cell lymphoma (CTCL) from benign T-cell-rich dermatoses. However, data regarding the sensitivity of the various electrophoresis techniques differ remarkably. In the present study, the capacity of heteroduplex (HD)-loaded temperature-gradient gel electrophoresis (TGGE) to detect clonally expanded T-cells was assessed systematically and modifications to the procedure were defined. Using our standard protocol, HD-TGGE detected clonal T-cell receptor (TCR)-gamma PCR products, generated from the Jurkat cell line, down to a total of 2 ng/microL (14 ng) DNA. However, slowly migrating single strands of the clonal PCR product reduced the amount of the clonality indicating homoduplices. To overcome this single-strand formation, thus decreasing the detection limit, the urea concentration in the gel and the temperature ramp for the HD-formation were altered, as well as the temperature gradient in the gel. Application of the modified protocol resulted in a tenfold lower detection limit of 0.15 ng/microL (1.05 ng) DNA in the clonal band. The sensitivity of the adapted HD-TGGE was investigated by dilution experiments using the well established T-cell lines Jurkat, Molt-4, MyLa and SeAx. By these approaches clonal PCR products diluted in nonclonal PCR products were detectable down to concentrations of 5-10%. Comparably, in the case of mixtures of clonal in nonclonal DNA the detection limit reached 5-10% clonal DNA. However, by dilution of clonal cells in nonclonal peripheral blood mononuclear cells, which corresponds to in vivo conditions, a lower detection limit of approximately 1-5% was observed.

A rapid polymerase chain reaction-based technique for detecting clonal T-cell receptor gene rearrangements in cutaneous T-cell lymphomas of both the alpha beta and gamma delta varieties.

T-cell receptor-gamma gene rearrangements provide specific clonal markers for a variety of lymphoid malignancies. T-cell receptor gene rearrangements in patients with cutaneous T-cell lymphoma were examined using conventional Southern blot analysis and a newly developed polymerase chain reaction (PCR)-based technique. The oligoprimers amplified a rearranged V gamma and J gamma segment (including the N region) of the T-cell receptor-gamma gene, and products were resolved using high-resolution nondenaturing polyacrylamide gel electrophoresis. Our results demonstrated concordance between the two techniques in 10 patients with cutaneous T-cell lymphomas (including nine cases of C beta and one case of delta 2 TCR gene rearrangements) and 10 negative controls. In the present study, we have shown that this PCR-based method provides a highly sensitive, specific technique for the detection of T-cell clones of both the alpha beta and gamma delta varieties and could be used in both fresh and formalin-fixed, paraffin-embedded tissues. It is estimated that this PCR-based technique is 10 to 50 times more sensitive than conventional Southern blot analysis in the detection of small T-cell clones.

Detection of T-cell clonality in paraffin-embedded tissues.

In lymph node diagnosis, difficulties are frequently encountered with the differential diagnosis of reactive and neoplastic T-cell proliferations. Immunohistochemistry is of limited use, and fresh frozen material for DNA studies is not available in many cases. We have established a polymerase chain reaction (PCR) technique to amplify rearranged T-cell receptor (TCR)-gamma sequences from paraffin-embedded material. Our method differs from other techniques previously described in that it uses four sets of family-specific variable (V)gamma primers. Clonality of the investigated T cells is reflected not only by the varying lengths of amplified products but also by differences in the relative amount of rearranged V gamma families. Preliminary studies indicate that this approach can provide information about the presence of predominant T-cell clones within the sample and thus help to classify the lymph node lesion. With this technique we were able to confirm clonality in seven of 12 T-cell lymphomas in paraffin-embedded tissues.