TCRB Gene Rearrangements Research Articles

A-209 Utility of TCR-β and TCR-γ gene rearrangement to distinguish between tumour-stage mycosis fungoides and early-stage mycosis fungoides associated with CD30+ lymphoproliferative disorders

A-209 Utility of TCR-β and TCR-γ gene rearrangement to distinguish between tumour-stage mycosis fungoides and early-stage mycosis fungoides associated with CD30+ lymphoproliferative disorders

Gamma-delta T-cell large granular lymphocytic leukemia in the setting of rheumatologic diseases.

T-cell leukemia originating from large granular lymphocytes (T-LGL leukemia) is a rare lymphoid neoplasia characterized by clonal proliferation of large granular T lymphocytes expressing αβ or γδ T-cell receptor (TCR) on the cell membrane. γδT-LGL leukemia, accounting for approximately 17% of all T-LGL leukemia cases, is associated with autoimmune diseases. However, the features of γδT-LGL leukemia in patients with rheumatologic diseases are still insufficiently characterized. In this retrospective study, 15 patients with rheumatologic disease-associated γδT-LGL leukemia were included. The patients were obtained from a single center from 2008 to 2023. Data related to clinical characteristics and rheumatologic diagnoses were collected. Immunophenotype evaluations as well as T-lymphocyte clonality (based on TCR-γ, TCR-β, and TCR-δ gene rearrangements), and signal transducer and activator of transcription (STAT) three and STAT5B mutation analyses (by next-generation sequencing) were performed on blood, bone marrow, and spleen samples. All but one patient had rheumatoid arthritis (RA). In 36% of patients, manifestations of γδT-LGL leukemia were present before or concurrently with clinical manifestations of RA. Splenomegaly was observed in 60% of patients and neutropenia (<1.5 × 109/L) was detected in 93% of cases. CD4-/CD8- and CD4-/CD8+ subtypes were detected in seven cases each. Mutations in STAT3 were detected in 80% of patients; however, STAT5B mutations were not detected. Evaluations of T-cell clonality and variant allele frequencies at STAT3 in the blood, bone marrow, and spleen tissue revealed an unusual variant of CD4-/CD8- γδT-LGL leukemia with predominant involvement of the spleen, involvement of the bone marrow to a less extent, and no tumor cells in peripheral blood. The mechanism by which γδT-LGL leukemia may induce the development of RA in some patients requires further investigation. Cases of RA-associated γδT-LGL leukemia with neutropenia and splenomegaly but no detectable tumor-associated lymphocytes in peripheral blood (the so-called splenic variant of T-LGL leukemia) are difficult to diagnose and may be misdiagnosed as Felty syndrome or hepatosplenic T-cell lymphoma.

Comparable CD8+ T-cell responses to SARS-CoV-2 vaccination in single-cell transcriptomics of recently allogeneic transplanted patients and healthy individuals.

Despite extensive research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination responses in healthy individuals, there is comparatively little known beyond antibody titers and T-cell responses in the vulnerable cohort of patients after allogeneic hematopoietic stem cell transplantation (ASCT).In this study, we assessed the serological response and performed longitudinal multimodal analyses including T-cell functionality and single-cell RNA sequencing combined with T cell receptor (TCR)/B cell receptor (BCR) profiling in the context of BNT162b2 vaccination in ASCT patients. In addition, these data were compared to publicly available data sets of healthy vaccinees.Protective antibody titers were achieved in 40% of patients. We identified a distorted B- and T-cell distribution, a reduced TCR diversity, and increased levels of exhaustion marker expression as possible causes for the poorer vaccine response rates in ASCT patients. Immunoglobulin heavy chain gene rearrangement after vaccination proved to be highly variable in ASCT patients. Changes in TCRα and TCRβ gene rearrangement after vaccination differed from patterns observed in healthy vaccinees. Crucially, ASCT patients elicited comparable proportions of SARS-CoV-2 vaccine-induced (VI) CD8+T-cells, characterized by a distinct gene expression pattern that is associated with SARS-CoV-2 specificity in healthy individuals.Our study underlines the impaired immune system and thus the lower vaccine response rates in ASCT patients. However, since protective vaccine responses and VICD8+T-cells can be induced in part of ASCT patients, our data advocate early posttransplant vaccination due to the high risk of infection in this vulnerable group.

Clonality, trafficking, and molecular alterations among Hprt mutant T lymphocytes isolated from control mice versus mice treated with N-ethyl-N-nitrosourea.

Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase-polymerase chain reaction based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer.

Methods for Study of Mouse T Cell Receptor α and β Gene Rearrangements.

Quantitative real-time PCR and next-generation sequencing (NGS) are invaluable techniques to analyze T cell receptor (Tcr) gene rearrangements in mouse lymphocyte populations. Although these approaches are powerful, they also have limitations that must be accounted for in experimental design and data interpretation. Here, we provide relevant background required for understanding these limitations and then outline established quantitative real-time PCR and NGS methods that can be used for analysis of mouse Tcra and Tcrb gene rearrangements in mice.

ALL-312 Ig/T-Cell Receptor Clonality Testing as a Method for Minimal Residual Disease Monitoring in T-Cell Acute Lymphoblastic Leukemia Patients

Assessment of early response to therapy by minimal residual disease (MRD) monitoring in T-cell acute lymphoblastic leukemia (T-ALL) has been proven to be a fundamental tool for guiding further therapy. Polymerase chain reaction (PCR)-based immunoglobulin (Ig)-T-cell receptor (TCR) clonality testing is a standardized technique for routine diagnosis of lymphoproliferation. It is also widely applied for MRD detection in T-ALL and is most suitable for patients without additional rearrangements detected. To present the clinical significance and utility of a molecular-based approach for post-induction MRD detection in patients with T-ALL. Single-center, descriptive, retrospective study including 11 adult patients with T-ALL treated at the University Clinic for Hematology - Skopje from January 2018 to January 2022. TCR gene analyses were performed in 11 adults with T-ALL. TCR rearrangements in bone marrow samples were analyzed by multiplex fluorescent PCR amplifications of the TCRB-Vβ-Jβ, Dβ-Jβ, TCRG-Vγ-Jy, TCRD-Vδ-Dδ-Jδ, Dδ-Dδ, Vδ-Dδ, and Dδ-Jδ regions and subsequent capillary electrophoresis of PCR products by automatic DNA analyzer, following EuroClonality/BIOMED-2 guidelines. MRD status was assessed at the end of the induction cycle. Patients received a BFM-based protocol or Hyper-CVAD/high-dose methotrexate (HDM) and cytarabine (ARA-C) regimen. TCR rearrangements were detected in all 11 patients. All patients were negative for additional molecular prognostic markers. TCRB and TCRG gene rearrangements were slightly dominant over TCRD (64.3% vs. 50%). A total of 6 adolescents and young adults (mean age: 24 years) were treated with BFM. At the end of the induction regimen, MRD negativity was obtained in 5 patients (83.3%) and was sustained up to 3 months from stem cell transplantation (SCT) in 3 patients. The other 2 patients died from CNS infiltration. Hyper-CVAD/HDM-ARA-C was administered in 5 patients (mean age: 49 years). One patient died during the induction protocol due to intracerebral hemorrhage. Among the others, CR at the end of the first cycle was achieved in 3 patients, and a total of 4 cycles were applied. Two patients proceeded to SCT. The molecular technique for testing Ig/TCR clonality is a useful tool for post-induction MRD monitoring in patients with ALL, directing the further approach and clinical decisions.

Characteristics and prognosis of children with recurrent T-cell acute lymphoblastic leukemia: a long-term follow-up report in China

This study assessed the relapse characteristics and prognosis of 145 children newly diagnosed with T-cell acute lymphoblastic leukemia (T-ALL). The overall complete response (CR) rate was 91.7% (133/145), and the overall recurrence rate was 31.6% (42/133). The recurrence rate in the intermediate-risk (IR) group and high-risk (HR) group was 15.4% and 47.1%, respectively (p < 0.001). Patients with young age, early T-cell precursor ALL, central nervous system (CNS) involvement, TCRγ gene rearrangement, karyotypic abnormalities, or absence of TCRβ gene rearrangement (p < 0.05) tended to relapse. All recurrences occurred within 36 months after diagnosis. The HR group recurred earlier than the IR group (p= 0.026). The 3-year overall survival (OS) rate was significantly lower in the HR group than in the IR group (p < 0.001). All relapsed children died within 12 months after recurrence. Early intervention may benefit children with a high risk of recurrence.

Hist-P-01 - Evaluation of the PCR-based T cell receptor-β clonality test in the diagnosis of early mycosis fungoides

<h3>Background:</h3> T-cell receptor (TCR) clonality is important for mycosis fungoides (MF) diagnosis. Routine clonality analysis is performed using a polymerase chain reaction (PCR) TCR-γ assay; yet with this method 10–50% of T-cell lymphomas escape detection. TCR-β gene rearrangement is an additional assay. Data about its efficacy is controversial. <h3>Objective:</h3> Evaluate the role of TCR-β assay in the diagnosis of early MF. <h3>Methods:</h3> A retrospective study of 61 skin biopsies, 20 from MF patients, 30 from patients suspected to have early MF, and 11 from patients with chronic inflammatory skin disease. <h3>Results:</h3> Monoclonality was detected in 16/20 (80%) MF cases; 15 (75%) with TCR-β and 12 (60%) with TCR-γ assay. Of the 30 suspected early MF cases, 14 demonstrated monoclonality, all of which with TCR-β and 5 with TCR-γ assay. None of the chronic inflammatory condition samples showed monoclonality. Therefore, TCR-β clonality assay was more sensitive in early MF than TCR-γ (83% vs. 43%, P=0.002). <h3>Limitations:</h3> A retrospective, relatively small study. <h3>Conclusion:</h3> TCR-β demonstrated a higher sensitivity rate compared with TCR-γ in early-stage MF. The combined use of the TCR-β and TCR-γ clonality tests can significantly improve the diagnosis rate of early-stage MF.

PB1684 THE FIRST‐REPORTED PATIENT WITH NATURAL KILLER‐LYMPHOBLASTIC LEUKEMIA/LYMPHOMA IN KOREA

Background:Natural killer (NK)‐lymphoblastic leukemia/lymphoma is a rare hemopoietic neoplasm that was established as an independent entity by the WHO in 2008 and in 2016, it was recategorized as a precursor lymphoid neoplasm. Its identification remains challenging because its features overlap with those of other hematological malignancies, and markers specific to NK‐cells such as CD94 and CD161 are not commonly tested. Owing to the disease's rarity, only 3 patients who meet the 2008 WHO criteria have been reported.Aims:We report the first Korean patient with NK‐lymphoblastic leukemia/lymphoma including her NGS‐based genetic profile and clinical course.Methods:.Results:A 51‐year‐old woman was referred to our institution with her WBC count 1.6 × 109/L, hemoglobin 11.9 g/dL, and platelet count 176 × 109/L. Peripheral blood smear showed a few abnormal‐looking large lymphocytes, while BM aspirate showed decreased normal hemopoietic precursors and increased blasts (63.0%). The blasts were large and had a high nuclear/cytoplasmic ratio with agranular cytoplasm, nuclei with indentation, and conspicuous nucleoli. All were negative for peroxidase and Sudan Black B but some were positive for acid alpha‐naphthyl acetate esterase. BM biopsy showed hypocellularity (10%) but increased immature cells that were positive for CD34 and terminal deoxynucleotidyl transferase (TdT) on immunohistochemistry (IHC). Flow cytometry showed that the majority of blasts were positive for HLA‐DR, CD7, CD34, and CD56 but negative for myeloid antigens (CD13, CD33, CD14, CD117, myeloperoxidase), B‐cell antigens (CD10, CD19, CD20, CD79a), and other T‐cell antigens (CD2, cytoplasmic CD3, surface CD3, CD4, CD5, CD8). The BM karyotype was 46,XX,‐7,del(7)(q35),add(11)(q24),‐12,+1mar,+2mar[10]/46,XX[10]. Multiplex reverse‐transcriptase PCR revealed negativity for all 28 tested leukemia‐causing chromosomal translocations. Targeted next‐generation sequencing of 54 genes, including BRAF, IKZF1, KRAS, MYD88, NRAS, and TP53, showed no oncogenic mutations; tests for TCRB, IGH, and IGK gene rearrangement showed no dominant clonotypes. Hence, she was diagnosed with NK‐lymphoblastic leukemia/lymphoma. Physical and imaging examinations revealed no organomegaly, lymph node enlargement, or skin lesions. The patient underwent 3 cycles of fludarabine and cyclophosphamide for 2 months and has been stable for 6 months since her diagnosis.Summary/Conclusion:The blasts of our patient was positive for CD34, TdT, HLA‐DR, CD7, and CD56 but not for other T‐cell, B‐cell, or myeloid markers; this ruled out T‐acute lymphoblastic leukemia/lymphoma (T‐ALL) and mixed‐phenotype acute leukemia. The lack of specific TCR and IG clonotypes also ruled out T‐ALL. The morphology of blasts and negativity for CD4 ruled out the possibility of blastic plasmacytoid dendritic cell neoplasm (BPDCN). Further analysis for CD4, CD56 and CD123 using IHC will be helpful to verify the differential diagnosis of NK‐lymphoblastic leukemia/lymphoma from BPDCN.

QUANTITATIVE RHOA Gly17Val ALLELE-SPECIFIC POLYMERASE CHAIN REACTION AND T-CELL CLONALITY ANALYSIS IN ANGIOIMMUNOBLASTIC T-CELL LYMPHOMA

Introduction. Аngioimmunoblastic T-cell lymphoma (AITL) is a T-cell lymphoma, characterized by abundant polymorphocellular infiltrate of lymph nodes with the small number of tumor CD4 + T-cells. AITL could often be misdiagnosed as reactive processes and other lymphomas, including Hodgkin’s lymphoma. Molecular methods of determining clonality are used to confirm the diagnosis. TCR gene rearrangements detected in the lymph nodes may not coincide with those detected in the bone marrow (BM), blood or skin, that makes their interpretation difficult. Recently discovered point somatic RHOA (Ras homolog gene family, member A) Gly17Val mutation is present in 53–71 % of angioimmunoblastic T-cell lymphomas. The aim of the study was to evaluate the number of RHOA Gly17Val mutated cells in different tissues of AITL patients and to correlate it with corresponding T-cell clonality results. Materials and methods . TCRG and TCRB gene rearrangements were PCR-amplified according to BIOMED-2 standardized protocol and analyzed by capillary electrophoresis on ABI Prism 3130. Gly17Val mutation was analyzed by quantitative allele-specific (qAS) TaqMan Real-Time PCR assay. Lymph nodes (LN) and skin biopsies, blood and BM samples were studied for 40 patients with AITL. Results. The clonal TCR gene rearrangements were found in 37 (92 %) of 40 patients in LN and in 26 (96 %) of 28 patients in BM, but they were not matched in length in LN and BM in 12 (46 %) of 26 patients. RHOA (Gly17Val) mutation in LN was revealed in 60 % (24 of 40) patients. Number of cells with RHOA mutation was highest in the LN (in average 26.7 % of the total cells), while in the BM RHOA mutation were undetectable (in 7 patients), or detected in 10 patients in a low quantity (in average 2 % of the total cells). Skin, blood and BM samples with the T-cell clonality peaks that differ from those found in the LN were also negative for the presence of cells having RHOA Gly17Val mutation. Also, in four patients we showed that clonal products in BM and blood that do not coincide with LN refer to CD8+ lymphocytes. Conclusions. The results of the study demonstrate that the quantitative determination of cells with the mutation of RHOA Gly17Val must be taken into account in staging the disease and interpreting the results of T-cell clonality assay. Clonal cells with rearrangements not matching those identified for the LN should be considered reactive.

Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro.

Many promising targets for T cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing TCRs with high affinity for self-antigen are eliminated. The affinity of the remaining low avidity TCRs can be improved to increase their anti-tumor efficacy, but conventional saturation mutagenesis approaches are labor intense and the resulting TCRs may be cross-reactive. Here we report an in vitro T cell maturation and selection system on antigen-expressing feeder cells for developing high affinity antigen-specific TCRs, which takes advantage of natural Tcrb gene rearrangement to generate diversity in the length and composition of CDR3β. In vitro differentiation of progenitors transduced with a known Tcra in the presence of antigen drives differentiation of cells with a distinct agonist-selected phenotype. These cells are then purified to generate TCRβ chain libraries pre-enriched for target antigen-specificity. Several TCRβ chains were identified that paired with a transgenic TCRα chain to produce a TCR with higher affinity for target antigen compared to the parental TCR.

PAXX and Xlf interplay revealed by impaired CNS development and immunodeficiency of double KO mice.

The repair of DNA double-stranded breaks (DNAdsb) through non-homologous end joining (NHEJ) is a prerequisite for the proper development of the central nervous system and the adaptive immune system. Yet, mice with Xlf or PAXX loss of function are viable and present with very mild immune phenotypes, although their lymphoid cells are sensitive to ionizing radiation attesting for the role of these factors in NHEJ. In contrast, we show here that mice defective for both Xlf and PAXX are embryonically lethal owing to a massive apoptosis of post-mitotic neurons, a situation reminiscent to XRCC4 or DNA Ligase IV KO conditions. The development of the adaptive immune system in Xlf-/-PAXX-/- E18.5 embryos is severely affected with the block of B- and T-cell maturation at the stage of IgH and TCRβ gene rearrangements, respectively. This damaging phenotype highlights the functional nexus between Xlf and PAXX, which is critical for the completion of NHEJ-dependent mechanisms during mouse development.

Increased Frequencies of PD-1+ CD8+ Marrow-Infiltrating Lymphocytes Associated with Highly Clonal T-Lymphocyte Expansions in Relapsed and Refractory AML Patients but Not Healthy Adults

Immune checkpoint therapy, particularly inhibition of the PD-1 axis, has shown remarkable efficacy in multiple solid tumor types and some hematological malignancies. Clinical results using anti-PD1 immunotherapy in AML have not yet been reported. Immune checkpoint expression levels and magnitude and/or repertoire of tumor-infiltrating lymphocytes have all been proposed as potential predictive biomarkers of response to such targeted immunotherapy. We therefore examined the marrow infiltrating T-lymphocyte compartment of Relapsed/Refractory Acute Myeloid Leukemia (RR-AML) patients undergoing salvage chemotherapy and that of healthy donors (HD) to determine baseline data for PD-1 expression and T-lymphocyte clonality.Nine adult RR-AML patients recruited to the clinical trial NCT02527447 (Myeloid Malignancies Section, NHLBI, NIH) were analyzed. Cryopreserved bone marrow aspirate mononuclear cells (BMMCs) from these patients prior to salvage chemotherapy (Day 0) and HD (n=9) were assessed using two custom ten-color flow cytometry panels. Frequencies of naive (TN), central memory (TCM), effector memory (TEM), terminal effector (TEMRA), stem cell memory (TSCM), and activated T-lymphocyte subsets, and the expression of PD-1, TIM-3, CTLA-4, 4-1BB on CD8+ T-lymphocyte subpopulations were calculated. DNA extracted from bone marrow of 5 of the RR-AML patients and 5 HD was used for sequencing of the CDR3 region of TCRB gene to evaluate TCR repertoire (ImmunoSEQ, Adaptive Biotechnologies).Immunohistochemistry of pre-treatment bone marrow biopsy sections revealed 10-40% CD3+ cells, typically scattered with focal small aggregations. Flow cytometry of marrow aspirate demonstrated more activated CD8+ CD69+ T-lymphocytes in the marrow of RR-AML patients compared with HD (19.8% vs. 9.4%, p=0.031). Significantly more CD8+ TEMRA T-lymphocytes (60.8% vs. 45.2%, p=0.040) and a trend towards fewer CD8+ TEM T-lymphocytes (19.0% vs. 32.1%, p=0.062) were observed in RR-AML versus HD. An increased proportion of total CD8+ T-lymphocytes in RR-AML had PD-1 expression compared to HD (22.0% vs. 9.2%, p=0.008). This pattern held true for every CD8+ sub-population in AML versus HD: TN (14.0% vs. 4.0%, p=0.014); TCM (24.2% vs. 7.3%, p=0.003); TEMRA (19.6% vs. 10.5%, p=0.060) and TEM (37.3% vs. 16.2%, p=0.004) (Figure 1a). There were no significant differences in frequencies of CD8+ T-lymphocyte subsets or PD-1 expression between Day 0 and Day 28 in AML patients (in 4 patients tested). Furthermore, there was negligible PD-1 co-expression with other immune checkpoint markers and no differences in 4-1BB or CTLA4 expression in AML versus HD. However, we noted significantly higher TIM-3 expression on TN and TCM CD8+ T-lymphocytes in AML (6.5% vs. 2.2%, p=0.001, and 4.5% vs. 0.7%, p=0.021, respectively).Sequencing of productive TCRB gene rearrangements revealed significantly higher average marrow T-cell clonality in AML versus HD (0.232 vs. 0.086, respectively, n=5 AML, n=5 HD). When considering the top 10 most frequent clonotypes for each AML patient and HD, frequencies were found to be much higher in AML than HD (Figure 1b), consistent with large clonal T-lymphocyte expansions in the marrow of RR-AML patients. Furthermore, we identified several TCR clones that were found in AML patients but never seen in HD. Sequencing of an additional cohort of 20 newly diagnosed adult AML patients however discovered that only a subset (25%) of those patients had T cell clonal expansions above the upper limit of that seen in HD.AML is already known to be an immunogenic malignancy, as demonstrated by the measurable efficacy of allogeneic transplantation and donor lymphocyte infusion. Our data demonstrate that the bone marrow tumor microenvironment in RR-AML is enriched for PD-1+ CD8+ marrow-infiltrating lymphocytes, and that large clonal expansions of T-lymphocytes can be found in the bone marrow in these patients. This is suggestive that RR-AML patients, for whom cytotoxic chemotherapy is often suboptimal, may benefit from immune checkpoint blockade therapies, particularly PD-1 axis inhibition, to enable improved immunologic control of AML by autologous T-lymphocytes already resident in the tumor microenvironment. Based in part on these data, a clinical trial of anti-PD-1 immunotherapy in combination with a hypomethylating agent for treatment of relapsed and refractory AML patients will open at our institution. [Display omitted] DisclosuresHourigan:Sellas Life Sciences Group: Research Funding.

Multiple Clonal TCR Gene Rearrangements Are Typical in Peripheral T-Cell Lymphoma Not Otherwise Specified

Introduction: Peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) is a heterogeneous subgroup of PTCL with no consistent immunophenotypic, genetic and clinical features. PTCL-NOS represents less than 29% of all T-cell lymphomas in adults and often has an aggressive course with extranodal and systemic involvement. This study is focused on the application of clonality testing methods for PTCL-NOS diagnosis and monitoring.Patients and Methods: Biopsies, blood and bone marrow samples were tested for 30 patients with PTCL-NOS: the ratio of male/female was 15/15, median age was 56 years (32-75), and 22 of 30 patients had IV stage of the disease. TCRG and TCRB gene rearrangements were evaluated for T cell clonality analysis. We used PCR-based methods (BIOMED-2 standardized clonality protocol) followed by GeneScan analysis on the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems).Results: Clonal TCR gene rearrangements were found in 29 of 30 patients (97%): TCRG in 27 of 30 (90%) and TCRB in 29 of 30 (97%). Primary biopsies or bone marrow samples showed oligoclonal pattern of rearrangements (more than 3 clonal peaks in TCRG or TCRB GeneScan profiles) in 13 of 30 patients (43%). New clonal rearrangements in affected tissues (skin, tonsil, CSF, lymph nodes and etc.) during disease progression were found in 15 of 24 (63%) patients. Various representations of clonal rearrangements in different tissues indicate the presence of several tumor clones in the majority of cases (19 of 30 patients; 63%). The presence of several clones correlated with the number of tissues examined (rs = 0.6, p <0.0005). The detection rate of additional clonal rearrangements was 90% (9 of 10 patients) when 3 and more tissues were tested. The number of clonal rearrangements was not correlated with age (p = 0.43) or the stage of disease (p = 0.29). One can speculate that the presence of multiple clones in the PTCL-NOS could reflect genetic instability of this tumor.Conclusion: Oligoclonality, clonal evolution and the appearance of new clones in the progression of the disease are typical features of PTCL-NOS. Multiple clonal rearrangements complicate diagnostics and assessment of minimal residual disease in this disease. DisclosuresNo relevant conflicts of interest to declare.

Co-Expression of NUP98-HOXD13 and Mutant IDH2 Triggers an Early T-Cell Precursor-like Leukemia in Mice

Background and hypothesis: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, comprising of B lineage ALL and T-cell lineage ALL (about 85% and 15%, respectively). In most cases, treatment regimens for ALL patients have proved efficient and prognosis is usually encouraging. However, a recently described subgroup of T-cell ALL patients have been found to have a poor prognosis when treated with conventional therapy. These higher risk T-cell leukemias have a cell of origin thought to resemble an early T-cell precursor (ETP), which retains the capability to differentiate into both myeloid or T-lineage cells, and are therefore termed ETP ALL. ETP ALL is characterized by lack of or low expression of typical T-cell surface markers, aberrant expression of myeloid and hematopoietic stem cell (HSC) surface markers and a gene expression signature resembling that of murine ETP cells. Recently, ETP ALL patients were found to have a unique mutational landscape that includes acquired mutations in genes typically associated with myeloid differentiation. The ambiguous lineage attributes of ETP ALL may explain the ineffectiveness of current therapies, stressing the need for establishing pre-clinical animal models. Here we show that co-expression of Nup98-Hoxd13 (NHD13) fusion gene and a mutant IDH2 gene (IDH2 R140Q) triggers an ETP-like leukemia in mice. Expression of NHD13 has been reported in MDS and AML patients, and NHD13 mice have been previously shown to develop MDS, AML, and T-cell ALL. Mutations in IDH1/2 genes occur in several cancers, including AML; the most common IDH mutation in AML patients is the IDH2 R140Q. However, expression of mutant IDH1/2 in mice has been reported to induce an expansion of the HSC compartment but was not sufficient for establishment of leukemia. Targeted sequencing of NHD13 mice has identified recurrent mutations in IDH1, suggesting that these two aberrations may collaborate to cause leukemia.Study Design and Methods: IDH2 R140Q mice were crossed with NHD13 mice and the progeny were monitored for survival. Classification of leukemic samples was done using flow cytometry and PCR analysis of TCRβ gene rearrangements. Global expression and mutational patterns of IDH2/NHD13 leukemias were analyzed using gene chip arrays and whole exome sequencing (WES).Results and conclusions: Transgenic IDH2/NHD13 mice showed decreased survival compared to all control groups. Detailed analysis of surface marker expression revealed that most double Tg cases (10/11 tested) developed leukemias resembling ETP (cKit+CD44+CD25-) or double negative (DN) 2 cells (cKit+CD44+CD25+). PCR analysis of the TCRβ locus revealed that unlike NHD13 T-cell leukemias, IDH2/NHD13 leukemias do not have clonal VDJ rearrangements. However, most of IDH2/NHD13 leukemias have clonal or oligoclonal DJ rearrangements of the TCRβ locus, suggesting that these leukemias emerged during an early stage in T cell development. Expression analysis revealed that the IDH2/NHD13 leukemia expression signature is enriched in genes that are upregulated in ETP or DN2 cells. In addition, we found that the IDH2/NHD13 leukemia expression signature is also enriched in genes uniquely expressed in ETP ALL patients. Finally, WES showed correspondence between genes mutated in the mouse ETP-like leukemia and genes recurrently mutated in ETP ALL patients. These results indicate that IDH2/NHD13 leukemias resemble ETP ALL in terms of gene expression, immunophenotype, and cooperative mutations.Relevance and importance: The poor prognosis of ETP ALL may be related to incomplete effectiveness of treatment strategies, emphasizing the need for novel therapies. IDH2/NHD13 mice can serve as an excellent pre-clinical model to study ETP ALL and to test potential treatments. DisclosuresAplan:NIH Office of Technology Transfer: Patents & Royalties.

Clonal Rearrangements and Malignant Clones in Peripheral T-cell Lymphoma

To assess the feasibility and informative value of T-cell clonality testing in peripheral T-cell lymphoma (PTCL). Biopsies of involved sites, blood, and bone marrow samples from 30 PTCL patients are included in the study. Rearranged TCRG and TCRB gene fragments were PCR-amplified according to the BIOMED-2 protocol and analyzed by capillary electrophoresis on ABI PRISM 3130 (Applied Biosystems). TCRG and TCRB gene clonality assay was valuable in confirming diagnosis in 97% of PTCL patients. T-cell clonality assay performed on blood or bone marrow samples reaffirmed lymphoma in 93% of cases, whereas morphological methods were informative in 73% of cases only. We observed multiple TCRG and TCRB gene rearrangements, loss of certain clones in the course of the disease, as well as acquisition of new clones in 63% of PTCL cases, which can be attributed to the genetic instability of the tumor. TCRG and TCRB gene clonality assay is beneficial for the diagnosis of PTCL. However, the presence of multiple clonal rearrangements should be considered. Clonal evolution in PTCL, particularly acquisition of new clones, should not be treated as a second tumor. Multiple TCRG and TCRB gene rearrangements may interfere with minimal residual disease monitoring in PTCL.

Donor CD4 T Cell Diversity Determines Virus Reactivation in Patients After HLA-Matched Allogeneic Stem Cell Transplantation

Delayed reconstitution of the T cell compartment in recipients of allogeneic stem cell grafts is associated with an increase of reactivation of latent viruses. Thereby, the transplanted T cell repertoire appears to be one of the factors that affect T cell reconstitution. Therefore, we studied the T cell receptor beta (TCRβ) gene rearrangements of flow cytometry–sorted CD4+ and CD8+ T cells from the peripheral blood of 23 allogeneic donors before G-CSF administration and on the day of apheresis. For this purpose, TCRβ rearrangements were amplified by multiplex PCR followed by high-throughput amplicon sequencing. Overall, CD4+ T cells displayed a significantly higher TCRβ diversity compared to CD8+ T cells irrespective of G-CSF administration. In line, no significant impact of G-CSF treatment on the TCR Vβ repertoire usage was found. However, correlation of the donor T cell repertoire with clinical outcomes of the recipient revealed that a higher CD4+ TCRβ diversity after G-CSF treatment is associated with lower reactivation of cytomegalovirus and Epstein–Barr virus. By contrast, no protecting correlation was observed for CD8+ T cells. In essence, our deep TCRβ analysis identifies the importance of the CD4+ T cell compartment for the control of latent viruses after allogeneic stem cell transplantation.

Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint.

RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoHtg) mice expressed three times more RhoH protein than wild-type mice, β-selection, positive, and negative selection in the thymus from RhoHtg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4+CD8+ (DP) thymocytes, indicating that overexpression of RhoH could bypass β-selection without TCRβ gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.

Developmental regulation of p53-dependent radiation-induced thymocyte apoptosis in mice

The production of T cell receptor αβ(+) (TCRαβ(+) ) T lymphocytes in the thymus is a tightly regulated process that can be monitored by the regulated expression of several surface molecules, including CD4, CD8, cKit, CD25 and the TCR itself, after TCR genes have been assembled from discrete V, D (for TCR-β) and J gene segments by a site-directed genetic recombination. Thymocyte differentiation is the result of a delicate balance between cell death and survival: developing thymocytes die unless they receive a positive signal to proceed to the next stage. This equilibrium is altered in response to various physiological or physical stresses such as ionizing radiation, which induces a massive p53-dependent apoptosis of CD4(+) CD8(+) double-positive (DP) thymocytes. Interestingly, these cells are actively rearranging their TCR-α chain genes. To unravel an eventual link between V(D)J recombination activity and thymocyte radio-sensitivity, we analysed the dynamics of thymocyte apoptosis and regeneration following exposure of wild-type and p53-deficient mice to different doses of γ-radiation. p53-dependent radio-sensitivity was already found to be high in immature CD4(-) CD8(-) (double-negative, DN) cKit(+) CD25(+) thymocytes, where TCR-β gene rearrangement is initiated. However, TCR-αβ(-) CD8(+) immature single-positive thymocytes, an actively cycling intermediate population between the DN and DP stages, are the most radio-sensitive cells in the thymus, even though their apoptosis is only partially p53-dependent. Within the DP population, TCR-αβ(+) thymocytes that completed TCR-α gene recombination are more radio-resistant than their TCR-αβ(-) progenitors. Finally, we found no correlation between p53 activation and thymocyte sensitivity to radiation-induced apoptosis.

Clinical Spectrum of SCID: The Key is in the Thymus?

Clinical Spectrum of SCID: The Key is in the Thymus?