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The diagnosis of cutaneous T‐cell lymphoma (CTCL) remains challenging. Demonstration of a clonal T‐cell population using T‐cell receptor (TCR) gene rearrangement studies by next‐generation sequencing (NGS) has been explored in several studies. This review summarizes the current literature on NGS‐based sequencing methods for the assessment of TCR clonality in the evaluation of atypical cutaneous lymphoid infiltrates and CTCL on behalf of the American Society of Dermatopathology Appropriate Use Criteria Committee (lymphoproliferative subgroup). PubMed was searched for relevant articles, including CTCL and NGS, for clonality from 1967 to 2022. Thirteen studies were included in the analysis. The skin was the most commonly assayed compartment with TCR NGS. Sensitivity for TCR NGS in the skin ranged between 69% and 100%, compared to 44%–72% for polymerase chain reaction (PCR)‐capillary electrophoresis. Specificity for TCR NGS in the skin ranged from 86% to 100%, compared to 77%–88% for PCR capillary electrophoresis. TCR NGS was also reported to have potential prognostic value in CTCL and can also be used to detect relapse and/or minimal residual disease after treatment. [ABSTRACT FROM AUTHOR]

Summary: Flow cytometry (FC) incorporating the T‐cell receptor β constant chain‐1 (TRBC1) has been recently proposed as a new standard in T‐cell clonality assessment. While early studies demonstrated high sensitivity in samples with conspicuous tumour burden, performance in real‐world samples, including those with low tumour burden and correlation with molecular methods has been limited. We evaluated TRBC1‐FC performance and correlated the results with high‐throughput TRB sequencing and a targeted next‐generation sequencing gene panel. Our cohort consisted of 90 evaluable samples from 57 patients. TRBC1‐FC confirmed T‐cell clonality in 37 out of 38 samples (97%) that were involved in a mature T‐cell neoplasm (MTCN). T‐cell clonality was also identified in nine samples from patients lacking a current or prior diagnosis of MTCN, consistent with the emerging entity T‐cell clonality of uncertain significance. TRBC‐FC was polyclonal in all samples and negative for disease involvement by standard pathology assessment. However, correlation with TRB sequencing in 17 of these samples identified two cases that harboured the known clonal sequence from index testing, indicating the presence of measurable residual disease not otherwise detected. Our study provides real‐world correlative validation of TRBC1‐FC, highlighting the strengths and limitations pertinent to its increasing implementation by general diagnostic laboratories. [ABSTRACT FROM AUTHOR]

Immunosequencing has emerged as a newer clinical test for assessment of T-cell clonality in the blood and skin of cutaneous T-cell lymphoma (CTCL) patients. Utilization of immunosequencing, also known as high-throughput sequencing of the T-cell receptor (HTS-TCR), enables identification and quantification of the precise genetic signature of dominant T-cell clones. Although immunosequencing is more sensitive than commonly used methods such as polymerase chain reaction (PCR) paired with capillary electrophoresis or flow cytometry, it remains underutilized for CTCL management. Nonetheless, incorporation of HTS-TCR in clinical practice offers distinct advantages compared to other molecular analyses that may improve diagnostic evaluation, prognostication, and disease monitoring in CTCL. The objective of this comprehensive review is to provide a thorough explanation of the application of immunosequencing in the context of CTCL. We describe the significance of T-cell clonality and the methods used to detect it, including a detailed comparison between PCR paired with capillary electrophoresis and HTS-TCR. The utilization of immunosequencing in the blood and skin of CTCL patients is discussed in depth, specifically outlining how HTS-TCR can assist in diagnosing CTCL, predicting outcomes, and tracking disease progression. Finally, we address the potential applications of immunosequencing in clinical management and research as well as the novel challenges it presents.

Objectives: Non‐infectious uveitis is often secondary to systemic autoimmune diseases, with Behçet's disease (BD) and Vogt‐Koyanagi‐Harada disease (VKHD) as the two most common causes. Uveitis in BD and VKHD can show similar clinical manifestations, but the underlying immunopathogenesis remains unclear. Methods: To understand immune landscapes in inflammatory eye tissues, we performed single‐cell RNA paired with T cell receptor (TCR) sequencing of immune cell infiltrates in aqueous humour from six patients with BD (N = 3) and VKHD (N = 3) uveitis patients. Results: Although T cells strongly infiltrated in both types of autoimmune uveitis, myeloid cells only significantly presented in BD uveitis but not in VKHD uveitis. Conversely, VKHD uveitis but not BD uveitis showed an overwhelming dominance by CD4+ T cells (> 80%) within the T cell population due to expansion of CD4+ T cell clusters with effector memory (Tem) phenotypes. Correspondingly, VKHD uveitis demonstrated a selective expansion of CD4+ T cell clones which were enriched in pro‐inflammatory Granzyme H+ CD4+ Tem cluster and showed TCR and Th1 pathway activation. In contrast, BD uveitis showed a preferential expansion of CD8+ T cell clones in pro‐inflammatory Granzyme H+ CD8+ Tem cluster, and pathway activation for cytoskeleton remodelling, cellular adhesion and cytotoxicity. Conclusion: Single‐cell analyses of ocular tissues reveal distinct landscapes of immune cell infiltration and T‐cell clonal expansions between VKHD and BD uveitis. Preferential involvements of pro‐inflammatory CD4+ Th1 cells in VKHD and cytotoxic CD8+ T cells in BD suggest a difference in disease immunopathogenesis and can guide precision disease management. [ABSTRACT FROM AUTHOR]

Cutaneous T-cell lymphomas are a heterogeneous group of T-cell lymphoproliferative diseases affecting the skin. Mycosis fungoides and Sezary syndrome are the most studied variants of them. The literature review includes the latest published data on the pathological processes development in mycosis fungoides and Sezary syndrome and the diagnosis of these diseases. The genomic instability features in cutaneous T-cell lymphomas are described, the existing hypotheses of the origin of these diseases are considered based on the results of T-cell receptor repertoire studying.

The identification of mature T cell neoplasms by flow cytometry is often challenging, due to overlapping features with reactive T cells and limitations of currently available T cell clonality assays. The description of an antibody specific for one of two mutually exclusive T cell receptor (TCR) β-chain constant regions (TRBC1) provides an opportunity to facilitate the detection of clonal TCRαβ+ T cells based on TRBC-restriction. Here we prospectively analyzed 14 healthy controls and 63 patients with the flow cytometry protocol currently used for suspected T cell neoplasm implemented with immunostaining targeting TRBC1. Specimens were firstly classified in 3 groups based on clinical records data, laboratory findings and immunophenotypic features. T cell clonality was assessed by TCR Vβ repertoire analysis and the new rapid TRBC1 assay. Results showed that TRBC1 unimodal expression was unequivocally associated with samples presenting with immunophenotypic aberrancies. Moreover, we demonstrated that the use of TRBC1 is useful in solving uncertain cases and confirmed the high sensitivity of the method in identifying small T cell clones of uncertain significance (T-CUS). Finally, we found a high degree of concordance (97%) comparing the currently available clonality assessment methods with the proposed new method. In conclusion, our results provided real-life evidence of the utility of TRBC1 introduction in the flow cytometric clonality evaluation for the routine diagnostic work-up of T cell neoplasms.

Objective 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. Materials and Methods 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. Results 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. Conclusion 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. [ABSTRACT FROM AUTHOR]

Background: Pityriasis lichenoides (PL) is a papulosquamous disease that affects both adults and children. Previous studies have shown a subset of this entity to have clonal T-cell populations via PCR-based assays. In this study, we sought to implement next-generation sequencing (NGS) as a more sensitive and specific test to examine for T-cell clonality within the pediatric population. Methods: We identified 18 biopsy specimens from 12 pediatric patients with clinical and histopathologic findings compatible with PL. Patient demographics, clinical features, management, and histopathologic findings were reviewed. All specimens were analyzed for clonality with NGS of T-cell receptor beta (TRB) and gamma (TRG) genes. Results: Of the 12 patients, 9 (75%) had complete resolution of lesions at the time of data collection (mean follow-up 31 months). The remaining three patients significantly improved with methotrexate (with or without acitretin). Interestingly, 7 of 12 patients (58%) and 9 of 17 biopsy specimens (53%) showed evidence of T-cell clonality. Two patients showed matching TRB clones from different anatomic sites. Conclusions: T-cell clonality is a common finding in PL, probably representing a "reactive clonality" rather than a true lymphoproliferative disorder. Clonality alone cannot be used as a means to distinguish PL from lymphomatoid papulosis or cutaneous lymphoma. [ABSTRACT FROM AUTHOR]

Objectives: Flow cytometric detection of T-cell clonality is challenging, particularly in differential diagnosis of immature T-cell proliferations. Studies have shown utility of TRBC1, in conjunction with other T-cell markers, as reliable means to identify T-cell clonality by flow cytometry. One limitation of surface TRBC1 (sTRBC1) evaluation is it cannot be detected in surface CD3 (sCD3)-negative T cells, such as normal or abnormal immature T-cell precursors. Here, we assess surface and cytoplasmic TRBC1 expression patterns in the differential diagnosis of T-lymphoblastic leukemia/lymphoma (T-ALL) vs normal thymocyte expansions.Methods: Forty-three samples containing T-ALL, thymoma, normal thymus, and/or indolent T-lymphoblastic proliferation (i-TLBP), were evaluated.Results: All 24 cases with normal thymocytes or i-TLBPs revealed a characteristic and reproducible sCD3/sTRBC1 expression pattern indicative of polytypic T-cell maturation. In contrast, all 19 T-ALLs lacked this polytypic maturation pattern and were either completely negative for sCD3/sTRBC1 or showed a minor sCD3-positive subset with a monotypic TRBC1 expression pattern. Cytoplasmic TRBC1 evaluation in 9 T-ALLs demonstrated a monotypic intracellular TRBC1-positive (n = 4) or TRBC1-negative (n = 5) expression, indicative of clonality.Conclusions: Our findings demonstrate flow cytometric evaluation of surface and cytoplasmic TRBC1 expression can aid detection of T-cell clonality and differential diagnosis of immature T-cell proliferations. [ABSTRACT FROM AUTHOR]

Objective: This study investigated the ability of pre-transplant T-cell clonality to predict sepsis after liver transplant (LT). Summary Background Data: Sepsis is a leading cause of death in LT recipients. Currently, no biomarkers predict sepsis before clinical symptom manifestation. Methods: Between December 2013 and March 2018, our institution performed 478 LTs. After exclusions (eg, patients with marginal donor livers, autoimmune disorders, nonabdominal multi-organ, and liver retransplantations), 180 consecutive LT were enrolled. T-cell characterization was assessed within 48 hours before LT (immunoSEQ Assay, Adaptive Biotechnologies, Seattle, WA). Sepsis-2 and Sepsis-3 cases, defined by presence of acute infection plus 2 SIRS criteria, or clinical documentation of sepsis, were identified by chart review. Receiver-operating characteristic analyses determined optimal T-cell repertoire clonality for predicting post-LT sepsis. Kaplan-Meier and Cox proportional hazard modeling assessed outcome associated prognostic variables. Results: Patients with baseline T-cell repertoire clonality 0.072 were 3.82 (1.25, 11.40; P = 0.02), and 2.40 (1.00, 5.75; P = 0.049) times more likely to develop sepsis 3 and 12 months post-LT, respectively, when compared to recipients with lower (<0.072) clonality. T-cell repertoire clonality was the only predictor of sepsis 3 months post-LT in multivariate analysis (C-Statistic, 0.75). Adequate treatment resulted in equivalent survival rates between both groups: (93.4% vs 96.2%, respectively, P = 0.41) at 12 months post-LT. Conclusions: T-cell repertoire clonality is a novel biomarker predictor of sepsis before development of clinical symptoms. Early sepsis monitoring and management may reduce post-LT mortality. These findings have implications for developing sepsis-prevention protocols in transplantation and potentially other populations. [ABSTRACT FROM AUTHOR]

Background: Angioimmunoblast T-cell lymphoma (AITL) is a rare T-cell lymphoproliferative disease that is accompanied by generalized lymphadenopathy, hepatosplenomegaly, intoxication symptoms and extranodal lesions. The extranodal manifestations of the disease frequently involve various skin changes. One of the first such manifestations is maculopapular rashes observed in about half of AITL patients and usually preceding the appearance of lymphadenopathy. Other forms of skin lesions accompany the disease considerably less frequently.Aim: To characterize the range of skin changes in patients suffering from AITL, to establish a correspondence between the nature of skin changes and their histological picture.Materials and methods: 54 AITL patients were being treated at the National Research Centre for Hematology from 2000 to 2017, with the male/female ratio being 30/24. The median age was 61 (29–81) years.Results: Changes in the skin were observed in 24 (44.4 %) of 54 AITL patients, out of whom 18 (75 %) and 6 (25 %) were male and female patients, respectively. Maculopapular rash was observed in 22 (91.7 %) out of 24 patients. The morphological and molecular investigations of skin biopsy specimens exhibiting maculopapular rash demonstrated nonspecific reactive changes. Patients with maculopapular rash demonstrated an increase in the level of total (polyclonal) IgE. Specific skin lesions detected in 8 (14.8 %) cases were represented by a ‘livedo reticularis’, focal skin hyperpigmentation, erythroderma, left eyelid tumour and tumour in 3, 2, 1, 1 and 1 cases, respectively.Conclusion: Maculopapular rash frequently observed in AITL patients is a reactive process not associated with a specific skin lesion. Specific skin lesions in AITL are much less common and can be represented by various forms. In some AITL cases, skin changes of the reactive and tumour nature can be simultaneously observed.

After a history of intermittent vomiting, endoscopic biopsies of stomach and duodenum were collected from a 13-yr-old male snow leopard (Uncia uncia). On microscopic examination, monomorphic small lymphocytes expanded the duodenal mucosa and occasionally formed intraepithelial nests. Immunohistochemistry of the infiltrating small lymphocytes in the mucosa and within the epithelium had strong, perimembranous labeling for CD3e, with few CD79a-positive lymphocytes located at the base of the villi. Polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) of feline T-cell receptor gamma (TCRG) detected a monoclonal cell population. The sequence of the PCR product was 100% homologous with the feline TCRG gene. By histology, immunophenotyping, and PARR testing, a final diagnosis of enteropathy-associated T-cell lymphoma, small cell type, was made. Homology in the nucleotide sequence between U. uncia and the domestic cat (Felis catus) indicates that feline PARR testing for TCRG may be diagnostic in snow leopards. [ABSTRACT FROM AUTHOR]

Objectives: T-cell receptor (TCR) gene rearrangement studies are widely used for assessing T-cell clonality. The frequency and significance of clonal peaks restricted to TCR β (TCRB) tube C are uncertain. We retrospectively reviewed 80 TCR studies performed on bone marrow/peripheral blood.Methods: TCRB and TCR γ (TCRG) analyses were performed using BIOMED-2 primers. A peak was considered clonal or atypical if it was reproducible and 5× or more or 3× to 5× polyclonal background, respectively.Results: TCRB analysis demonstrated 12 (15%) of 80 cases with one to four isolated peaks in tube C (>3×) with polyclonal pattern in tubes A and B. TCRG analysis was monoclonal in two cases (both definite T-cell neoplasms), polyclonal in four, and oligoclonal in six. Of the 10 cases without clone in TCRG, six had autoimmune disorder and none had T-cell neoplasm.Conclusions: Peaks restricted to TCRB tube C in the TCR analysis may be misleading, as it is often not indicative of an overt T-cell neoplasm. [ABSTRACT FROM AUTHOR]

Kikuchi-Fujimoto disease (KFD) is a benign self-limiting disorder that frequently leads to swelling of cervical lymph nodes in young women. It has a characteristic histologic appearance with sharply demarcated foci containing apoptotic debris, histiocytes, and proliferating large T-cells. Since in the past years, core needle biopsies have been increasingly used for diagnostic work-up, a small biopsy of the pathognomonic proliferating T-cell foci may lead to misinterpretation as a large T-cell neoplasia. The aim of the present study therefore was to analyze how frequently clonal T-cell receptor (TCR) amplificates may be obtained in KFD using a commonly used TCR gamma rearrangement clonality assay. In 88 KFD cases, TCR gamma clonality assays could be successfully applied. Clonal peaks of TCR gamma in front of a polyclonal background were observed in 15 cases (18%). The investigated clinical parameters (age, gender, extent of infiltration of the lymph node, percentage of proliferative compartment) did not differ between patients with detectable TCR gamma clones from those patients who had polyclonal TCR gamma results. Our study therefore demonstrates that clonal TCR gamma amplificates may be obtained in any type of KFD and that an over-interpretation of clonal T-cell proliferates in diagnostically equivocal material should be avoided., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background Hydroa vacciniforme ( HV) is associated with Epstein-Barr virus ( EBV) infection and a risk of transformation to lymphoma. Methods We retrospectively analyzed six HV cases for EBV association and transformation to HV-like T-cell lymphoma. Clinicopathologic features were reviewed and cases were assessed for EBV-encoded RNA ( EBER) by in situ hybridization, double staining with immunohistochemistry and EBER and for T-cell clonality. Results The male-to-female ratio was 5:1, with a median age at diagnosis of 18.5 years. All patients initially had recurrent vesicles, necrotic ulcers or scars on sun-exposed areas. Symptoms were present before diagnosis between 2 weeks to 10 years. The mean follow-up time was 106.3 months. Four patients (67%) were EBV-positive. All four EBV-positive and one EBV-negative patients had relapsing clinical course. Double staining proved EBV infection in T-cells. Moreover, one EBV-positive patient developed HV-like T-cell lymphoma with hemophagocytosis after 209 months of recurrent papulovesicular eruptions and eventually died. T-cell clonality was successfully performed in four HV patients and all showed polyclonal results; the transformed HV-like T-cell lymphoma was monoclonal. Conclusions In EBV endemic areas, HV is frequently (67%) associated with EBV infection, but transformation to HV-like T-cell lymphoma seems to be uncommon (17%) and bear a dismal outcome. [ABSTRACT FROM AUTHOR]

Currently, arsenic trioxide therapy is widely used for the treatment of acute promyelocytic leukemia (APL), relapsed and refractory adult T-cell leukemia/lymphoma and myelodysplastic syndrome. Regarding the broad antitumor activity of arsenic, certain studies have been undertaken to test its efficacy in treating acute T-cell lymphoblastic leukemia (T-ALL) cell lines and patients; however, to the best of our knowledge, no reports document that arsenic is able to induce the remission of T-ALL patients. The present study reports the case of young male patient diagnosed with T-ALL, with no significant response to common chemotherapy regimens, who finally achieved complete remission without minimal residual disease (as detected by flow cytometry) due to arsenic treatment. This result is encouraging, and the present study has shown that malignant TCRαβ+ cell clones can be detected at the molecular level using reverse transcription-polymerase chain reaction (PCR) combined with the GeneScan technique. The result is mainly based on the T-cell receptor (TCR) Vβ1 clone (a 190-base pair PCR product that with the same complementarity determining region 3 length can be detected for all samples collected during various statuses) and on undetectable TCR Vγ subfamily members, at the time of disease diagnosis. It is important to analyze the dynamically changing TCR pool in leukemia patients during therapy. Although the molecular mechanism through which arsenic contributes to malignant clone elimination remains unclear in the case presented, the use of arsenic is expected to be effective for clinically treating refractory and relapsed T-ALL patients. [ABSTRACT FROM AUTHOR]

Autoimmune hemolytic anemia (AIHA) is a rare blood disease associated with the production of auto-antibodies and autoimmune hemolysis. A critical role of B-cells in the development of AIHA has been demonstrated before. Here, we present the analysis of the clonal T-cell populations in patients with AIHA. Thirty-three patients with AIHA were included in this study. Thirteen patients with other anemias, 14 patients with other autoimmune conditions (SLE – 6, RA – 8) and 20 healthy donors were included in the study as a control group. The clonality of T-cell was evaluated by the assessment of the T-cell receptor gamma and beta chain gene rearrangements (TCRG and TCRB). The incidence of T-cell monoclonality detected in patients with AIHA was significantly higher compared to the control group. The persistence of T-cell clones did not correlate with the level of hemoglobin and other signs of remission or relapse and did not disappear after the therapy and clinical improvement (observation period was between 1 and 10 years). There was no correlation between the T-cell clonality and the gender, age, splenectomy, duration or severity of the disease. Fractionation of T-lymphocytes (CD4+, CD8+, CD4+25+) revealed that the monoclonal T-cells belonged to the CD8+ sub-population. We assume that besides a possible causative role of the T-cell clones in AIHA to autoimmune process, these clones do not directly participate in the development and maintenance of hemolysis. Most of the AIHA patients (48.5%) demonstrated a T-cell monoclonality, which requires monitoring and should be distinguished from T-cell tumors. [ABSTRACT FROM AUTHOR]

A 9-year old boy presented with a 4-month history of a truncal monomorphic eruption with self-healing papulonecrotic lesions. A skin biopsy revealed a dermal infiltrate of CD4, CD8 and CD30-positive T-cells, consistent with lymphomatoid papulosis. He responded to 4 months of treatment with narrowband UVB phototherapy (311 nm) which stabilised his disease. Five years later he presented with an acute onset of nausea and vomiting, dizziness, headache and ataxia. Magnetic resonance imaging of the brain revealed a lesion in the cerebellum and stereotactic resection was undertaken. Histology showed CD4, CD8 and CD30-positive T-cells similar to his skin lesions, with a monoclonal T-cell receptor ( TCR) gamma gene rearrangement. Subsequent analysis of the skin detected a monoclonal band of the same size as the cerebellar lesion. Treatment was initiated for a primary central nervous system ( CNS) lymphoma but ceased after one course of high-dose methotrexate. Opinion on the pathology was divided as to whether the cerebellar lesion represented an atypical reactive T-cell lymphoproliferative response or a T-cell lymphoma. On follow-up 2 years later, the patient remains clinically and radiologically clear, making CNS lymphoma unlikely. [ABSTRACT FROM AUTHOR]

Mycosis fungoides (MF) and primary cutaneous CD30-positive T-cell lymphoproliferative disorders (CD30LPD) are the most frequent primary cutaneous T-cell lymphomas. Our objective was to study pan-T-cell antigens and PD-1 expression in a large cohort of MF and CD30LPD with a special interest in antigen losses as a diagnostic tool. We retrospectively reviewed 160 consecutive samples from 153 patients over a 3 year period, including 104 with MF and 49 with CD30LPD. As controls, benign inflammatory dermatoses (BID, n=19) were studied. A semi-quantitative evaluation of CD2, CD3, CD4, CD5, CD7, CD8 expression was performed. PD-1 and double stainings (CD3+CD7 and PD-1+CD7) were performed in a subset of MF cases. CD8+ MF was frequent (23%) and CD7 was the most frequently lost antigen in both MF (45%) and CD30LPD (86%), while no significant T-cell antigen loss was observed in BID. CD7 loss was less frequent in folliculotropic MF (p<0.001). PD-1 was variably expressed in MF with no differences with BID. The CD3+/CD7- and PD-1+/CD7- neoplastic lymphocytes were highlighted by the use of chromogenic double staining experiments in MF with a CD7 loss identified with single staining. Multiple pan T-cell antigen losses were mostly seen in CD30LPD with CD2 being the most frequently preserved marker (90%). While PD-1 does not discriminate between MF and BID, CD7 is frequently lost in MF infiltrates as well as other pan-T-cell antigens in CD30LPD, which can be used as routine markers for diagnosis. We recommend the use of CD7 in addition to CD3, CD4 and CD8 as a minimal immunohistochemical panel for MF assessment, and the use of double stainings for CD3 and CD7 in difficult cases., (Copyright © 2022 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Although Sézary syndrome (SS) represents an advanced stage of cutaneous T-cell lymphoma, this diagnosis presents a challenge even for the most experienced dermatologic clinicians. SS is characterized clinically by erythroderma, but can also be identified in the presence of specific histologic and peripheral blood findings. Erythrodermic cutaneous T-cell lymphoma can mimic a number of nonmalignant disorders with erythroderma, including pityriasis rubra pilaris, psoriasis, atopic dermatitis, and graft-versus-host disease. The diagnosis is made even more challenging because the histology of SS is often nonspecific and rarely pathognomonic. As a result, peripheral blood studies in patients with erythroderma are frequently informative in the diagnosis of SS. Peripheral blood abnormalities including elevated CD4/CD8 ratio, aberrant CD26, CD27 and CD7 expression, and T-cell clonality can all be used to help arrive at a diagnosis. This review evaluates current data on the usefulness and limitations of specific peripheral blood markers detected by flow cytometry and T-cell receptor gene rearrangement polymerase chain reaction. [Copyright &y& Elsevier]

Flow cytometric T-cell receptor Vβ-repertoire analysis (TCR-Vβ-R) is a sensitive method to detect T-cell clonality; however, its implementation in low-cellularity specimens has not been established. We developed a strategy to use TCR-Vβ in cerebrospinal fluid (CSF) and fine-needle aspirate (FNA) specimens. Initially, full TCR-Vβ-R was evaluated in diagnostic/screening specimens from 8 patients with T-cell neoplasia to determine tumor-specific TCR-Vβ protein expression. Subsequently, an abbreviated, patient-specific TCR-Vβ-R evaluation was performed in 17 paucicellular specimens from the patients (8 CSF, 9 FNA) for staging and monitoring of minimal residual disease (MRD). A single cocktail containing 3 anti-Vβ antibodies (1 tumor-specific and 2 negative controls) in combination with other antibodies chosen to help gate on atypical T cells is highly sensitive and specific for detecting low-level neoplastic T-cell involvement in paucicellular specimens. This TCR-Vβ-R strategy is valuable in staging and evaluating MRD in patients with T-cell non-Hodgkin lymphoma. [ABSTRACT FROM AUTHOR]

Abstract: Background: Hemophagocytic lymphohistiocytosis (HLH) is a serious immune disorder. Epstein–Barr virus (EBV) is the most common trigger of secondary HLH. T-cell receptor (TCR) gene rearrangement is detectable in half of patients with EBV-associated HLH using Southern blotting and conventional polymerase chain reaction (PCR) analyses. However, its clinical significance is unclear. The impact of TCR gene clonality on the outcome of patients with EBV-HLH should be evaluated using more sensitive methods. In this study, we used BIOMED-2 multiplex PCR and GeneScan analysis to evaluate TCR gene rearrangement in childhood EBV-HLH. Methods: Six children with EBV-HLH were enrolled in this study. EBV load in blood was quantified by real-time PCR. TCR gene rearrangement was analyzed by BIOMED-2 multiplex PCR. Results: All 6 patients showed monoclonal peaks in TCRβ and/or TCRγ at diagnosis. Serial monitoring of one patient disclosed a change in the rearrangement pattern of the TCR genes in response to immunochemotherapy. Conclusion: These findings suggest that BIOMED-2 multiplex PCR, a highly sensitive method for detecting T-cell clonality, is useful for predicting the therapeutic response in childhood EBV-HLH. [Copyright &y& Elsevier]

Flow cytometric T-cell receptor (TCR)-V(β) repertoire analysis (TCR-V(β)-R) is a sensitive method for detection of T-cell clonality; however, no uniform approach exists to define clonality in neoplastic T cells. TCR-V(β)-R was evaluated in patients with a diagnosis of T-cell neoplasia in initial diagnostic specimens from 41 patients and for minimal residual disease (MRD) monitoring in 61 sequential samples from 14 patients with mature T-cell neoplasia. Gating strategies and criteria for detection of T-cell clonality were determined. In all 41 initial specimens, T-cell clonality was demonstrated via TCR-V(β)-R. The frequency of V(β) usage was consistent with random neoplastic transformation of TCR-V(β) subsets. MRD was successfully detected in follow-up samples from all 14 patients evaluated, Furthermore, MRD after therapy was quantitated in 48 peripheral blood specimens. TCR-V(β)-R analysis is a sensitive method for detection of T-cell clonality and is useful for diagnosis and MRD detection in multiple specimen types. [ABSTRACT FROM AUTHOR]

Angiolymphoid hyperplasia with eosinophilia (ALHE) is a rare, benign entity of unknown pathogenesis. It often presents as painful or pruritic intradermal or subcutaneous red to brown papules or nodules on the head and neck of young adults. A 38-year-old man had a gradually enlarging and mild pruritic plaque on the scrotum for half a year. Pathological findings showed dermal proliferation of anomalous blood vessels lined by plump endothelium with a significant perivascular inflammatory infiltrate composed of lymphocytes, histiocytes, scattered plasma cells and many eosinophils. They were consistent with the diagnosis of ALHE. In addition, the inflammatory infiltrate was analyzed by immunohistochemistry and T-cell receptor (TCR) gene rearrangement. They were mostly CD3+ T cells and a monoclonal T-cell population. To the best of our knowledge, this is the first case of ALHE affecting the scrotum to be reported in the published work. We present this case to expand the anatomical distribution of this rare tumor. The molecular study of our case supports that ALHE might be a low-grade T-cell lymphoproliferative disorder. [ABSTRACT FROM AUTHOR]

Extracorporeal photopheresis (ECP) is a leukapheresis-based therapy that uses 8-methoxypsoralen and ultraviolet A irradiation. Used alone or in combination with biological agents, ECP is an established and effective therapy for advanced cutaneous T-cell lymphoma. ECP has also shown promising efficacy in a number of other severe and difficult-to-treat conditions, including systemic sclerosis, graft-versus-host disease, prevention and treatment of rejection in solid organ transplantation, and Crohn disease. Furthermore, the use of ECP in some of these conditions may allow a significant reduction in the use of systemic steroids and other immunosuppressants, reducing long-term morbidity and mortality. The accumulated experience shows ECP to be well tolerated, with no clinically significant side effects. Progress is also being made in the search for understanding of the mechanisms of action of ECP, which will ultimately facilitate improvements in the use of this therapy. [Copyright &y& Elsevier]

The toxicity of benzene to lymphocytes is well known; however, little is known about the variety of T-cells that are rearranged after benzene exposure. In order to further understand the relative toxicity of benzene on T-cells, we investigated the features of the complementarity determining region 3 (CDR3) of T cells from benzene exposed workers. CDR3 of the T-cell receptor variable β-chain (TCRVβ) subfamily genes were amplified using reverse transcriptase-polymerase chain reaction. The polymerase chain reaction products were further subjected to genescan analysis to evaluate the clonality of T cells. Results revealed 2–12 Vβ subfamilies identified in 16 benzene-exposed workers. The number of detectable Vβ subfamilies present in the benzene-exposed group was significantly lower than in the control group (p<0·05). The most frequently used Vβ genes were Vβ3, Vβ21, Vβ6, and Vβ16. We observed a skewed distribution and clonal expansion of TCR Vβ subfamilies in benzene-exposed workers. [ABSTRACT FROM AUTHOR]

We report a case of an invasive ductal breast carcinoma with significant heterogeneity: a HER-2+ tumor component was densely infiltrated by T-cells, whereas the HER2− tumor component, including two axillary lymph node metastases, showed much fewer tumor infiltrating lymphocytes. Array comparative genomic hybridization of dissected tumor cells from both components revealed many shared chromosomal aberrations but also unique alterations of the HER2+ tumor cell population besides HER2 amplification. We found a clonally dominated T-cell receptor rearrangement of the tumor infiltrating lymphocytes in the HER2+, but not in the HER2− tumor component. Thus, in this case HER2 overexpression is associated with a marked infiltration by T-cells suggesting a specific T-cell response against the HER2+ tumor cell population. [ABSTRACT FROM AUTHOR]

A letter to the editor regarding the clinicopathological features shown by myelodysplastic syndrome (MDS) with pure red cell aplasia is presented.

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 TCRγ 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 TCRγ 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. [ABSTRACT FROM AUTHOR]

The clonality of T-cell populations mediating acute and chronic rejection (AR and CR, respectively) of kidney allografts was ascertained by investigating the diversity of TCRBV genes expressed by allograft-infiltrating T cells. Both oligoclonality and polyclonality cases were found in biopsy specimens of AR as well as CR. These results indicated that the T-cell clonality in each specimen did not correlate directly with the mode of rejection. When AR and CR specimens were compared, however, the CR specimen group was significantly more polyclonal (or less oligoclonal) than the AR group. This result may reflect the higher chance of epitope spreading in the more slowly progressing CR than in AR. [ABSTRACT FROM AUTHOR]

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Mycosis fungoides constitutes more than 60% of primary cutaneous T-cell lymphomas. The course of disease is progradient: the gradual changes of macular, plaque and tumor stages are noted. The evaluation of the spreading process is critically important for the prognosis determination and therapy choosing. T-cell clonality detection by rearranging genes of the gamma chain of the T-cell receptor in the blood and bone marrow aspirates can be detected at the early stages. It is known to be a prognostically unfavorable factor for diagnostics in peripheral blood, while the value of the rearranging presence of T-cell receptor gene in the bone marrow aspirates has not been fully studied. To evaluate the frequency and prognostic value of the T-cell clonality detection by the gamma chain of the T-cell receptor in the bone marrow in patients with mycosis fungoides/Sézary syndrome (MF/SS). The patients with mycosis fungoides/Sézary syndrome were observed at the National Research Centre of Hematology during the period from 2000 to 2015. 45 patients diagnosed with mycosis fungoides/Sézary syndrome (MF/SS) corresponded to inclusion criteria. Men/women 1:1. The age of the participants is varied from 20 to 81 years. The diagnosis of mycosis fungoides was made in 36 cases (80%) and the diagnosis of Sézary syndrome was made in 9 cases (20%) respectively. The early stages were diagnosed in 13 patients (29%), the later stages were diagnosed in 32 patients. T-cell clonality by the gamma chain rearrangement of the T-cell receptor was detected in the bone marrow in 15 out of 45 patients (33%). 13 cases were the patients with the late disease stages and clonality at the early Ib stage was detected only in 2 cases. The morphological bone marrow involvement was identified in 5 patients, but in two cases T-cell clonality was not detected. The median survival of patients with morphologically confirmed bone marrow damage was 3 months. The median survival of patients with defined T-cell clonality in the bone marrow was 21 months. The median survival with no molecular damages of bone marrow was 41 months. T-cell clonality by gamma chain rearranging of the T-cell receptor in the bone marrow in patients with mycosis fungoides/Sézary syndrome is determined in a third of patients mainly at the late MF stages. This is an unfavorable prognostic factor. [ABSTRACT FROM AUTHOR]

In recent years, the evolution of treatments has made it possible to significantly improve the outcomes of patients with non-small cell lung cancer (NSCLC). In particular, while molecular targeted therapies are effective in specific patient sub-groups, immune checkpoint inhibitors (ICIs) have greatly influenced the outcomes of a large proportion of NSCLC patients. While nivolumab activity was initially assessed irrespective of predictive biomarkers, subsequent pivotal studies involving other PD-1/PD-L1 inhibitors in pre-treated advanced NSCLC (atezolizumab within the OAK study and pembrolizumab in the Keynote 010 study) reported the first correlations between clinical outcomes and PD-L1 expression. However, PD-L1 could not be sufficient on its own to select patients who may benefit from immunotherapy. Many studies have tried to discover more precise markers that are derived from tumor tissue or from peripheral blood. This review aims to analyze any characteristics of the immunogram that could be used as a predictive biomarker for response to ICIs. Furthermore, we describe the most important genetic alteration that might predict the activity of immunotherapy. [ABSTRACT FROM AUTHOR]

A patient with marked chronic eosinophilia and episodic angioedema (Gleich's syndrome) with a concomitant clonal proliferation of helper T-lymphocytes is described. This association has not previously been documented. The symptoms and clinical course differ markedly from the idiopathic hypereosinophilic syndrome, including an absence of end organ involvement. However, the disease appears to have a similar pathogenesis, with elevated levels of the eosinophil-specific cytokine interleukin-5 (IL-5) produced by the T-cell clone. [ABSTRACT FROM AUTHOR]

Background: Flow cytometric detection of T-cell clonality is challenging. The current available methodology for T-cell receptor (TCR) Vβ repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T-cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant β chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells., Methods: Here, we used a single TRBC1 antibody, in conjunction with other T-cell associated markers, to detect T-cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T-cell neoplasm and 97 patients with no T-cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1., Results: We examined TRBC1 expression on neoplastic T-cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T-cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T-cell neoplasms, and arbitrarily dissected T-cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets., Conclusions: Single TRBC1 antibody detection of T-cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories., (© 2020 International Clinical Cytometry Society.)

Background: The diagnosis of T-cell neoplasms is often challenging, due to overlapping features with reactive T-cells and limitations of currently available T-cell clonality assays. The description of an antibody specific for one of two mutually exclusive T-cell receptor (TCR) β-chain constant regions (TRBC1) provide an opportunity to facilitate the detection of clonal TCRαβ T-cells based on TRBC-restriction., Methods: Twenty patients with mature T-cell neoplasms and 44 patients without evidence of T-cell neoplasia were studied. Peripheral blood (51), bone marrow (10), and lymph node (3) specimens were evaluated by 9-color flow cytometry including TRBC1 (CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1 and/or CD2/CD3/CD4/CD5/CD7/CD8/CD26/CD45/TRBC1). Monophasic TRBC1 expression on any immunophenotypically distinct CD4-positive or CD8-positive/TCRγδ-negative T-cell subset was considered indicative of clonality., Results: Monophasic (clonal) TRBC1 expression was identified on immunophenotypically abnormal T-cells from all 20 patients with T-cell malignancies (100% sensitivity), including 17 cases with either >97% or <3% TRBC1-positive events, and three cases with monophasic homogenous TRBC1-dim expression. All immunophenotypically distinct CD4-positive and CD8-positive/TCRγδ-negative T-cell subsets from 44 patients without T-cell malignancies showed the expected mixture of TRBC1-positive and TRBC-1-negative subpopulations (non-clonal), except for seven patients (16%) with very small CD8-positive T-cell subsets exhibiting a monophasic (clonal) pattern., Conclusion: Inclusion of a single anti-TRBC1 antibody into a diagnostic T-cell flow cytometry panel facilitates the rapid identification of T-cell neoplasms, in addition to small monotypic CD8-positive subsets of uncertain significance. © 2019 International Clinical Cytometry Society., (© 2019 International Clinical Cytometry Society.)

Background T-cell receptor (TCR) clonality assessment is a principal diagnostic test in the management of mycosis fungoides (MF). However, current polymerase chain reaction–based methods may produce ambiguous results, often because of low abundance of clonal T lymphocytes, resulting in weak clonal peaks that cannot be size-resolved by contemporary capillary electrophoresis (CE). Objective We sought to determine if next-generation sequencing (NGS)-based detection has increased sensitivity for T-cell clonality over CE-based detection in MF. Methods Clonality was determined by an NGS-based method in which the TCR-γ variable region was polymerase chain reaction amplified and the products sequenced to establish the identity of rearranged variable and joining regions. Results Of the 35 MF cases tested, 29 (85%) showed a clonal T-cell rearrangement by NGS, compared with 15 (44%) by standard CE detection. Three patients with MF had follow-up testing that showed identical, clonal TCR sequences in subsequent skin biopsy specimens. Limitations Clonal T-cell populations have been described in benign conditions; evidence of clonality alone, by any method, is not sufficient for diagnosis. Conclusion TCR clonality assessment by NGS has superior sensitivity compared with CE-based detection. Further, NGS enables tracking of specific clones across multiple time points for more accurate identification of recurrent MF. [ABSTRACT FROM AUTHOR]

Objectives: Analysis of T-cell clonality using the IO Test Beta Mark (Beckman Coulter, Fullerton, CA) flow cytometry assay is cumbersome owing to the required eight-part analyses that limit its clinical utility. Here, we describe a simple modification that may improve its clinical utility., Methods: By simply combining all T-cell receptor (TCR) Vβ antibodies into one reagent tube, we show that T-cell repertoire analysis can be efficiently performed with an increased potential to closely link the aberrant immunophenotype of a neoplastic T-cell clone to its apparent clonal TCR Vβ restriction. We review our clinical experience and include flow cytometry sorting studies in conjunction with TRG gene rearrangement analysis to demonstrate the feasibility of this approach., Results: We show in representative cases that this modified approach permits evaluation of TCR Vβ clonality in T-cell neoplasms without a priori knowledge of the TCR Vβ isoform restriction and that this approach is suitable for samples containing a minor population of neoplastic T cells, hypocellular samples such as skin biopsy specimens and body fluids, and fine-needle aspirations. We estimate a false-positive rate for identifying a clonal population for this approach to be less than 6.2%., Conclusions: Our modified approach of TCR Vβ analysis could improve routine clinical evaluation for abnormal T-cell populations by flow cytometry., (© American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)