cell clonality
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Cancers ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 408
Author(s):  
Noemí Muñoz-García ◽  
F. Morán-Plata ◽  
Neus Villamor ◽  
Margarida Lima ◽  
Susana Barrena ◽  
...  

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.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takashi Kido ◽  
Hiroshi Ishimoto ◽  
Hiroshi Ishii ◽  
Kanako Hara ◽  
Mutsumi Ozasa ◽  
...  

AbstractDiagnosis of pulmonary lymphoma using small tissue samples is difficult and often requires surgical procedures; thus, a less invasive sampling method is desirable. We previously showed that pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma can be diagnosed by detecting MALT lymphoma translocation gene 1 (MALT1) translocations in bronchoalveolar lavage fluid (BALF) cells. Analysis of B-cell clonality based on immunoglobulin heavy chain (IGH) gene rearrangements was also reportedly useful for diagnosing pulmonary lymphoma. The aim of this prospective multicenter study was to evaluate the yet unknown diagnostic potential of combined detection of MALT1 translocations and clonality using BALF. We analyzed B- and T-cell clonality based on IGH and T-cell receptor (TCR) rearrangements together with MALT1 translocations using BALF of patients with clinically suspected pulmonary lymphomas. In total, 39 patients were evaluated and categorized into three groups: B-cell lymphoma, lymphoproliferative disorders, and other diseases. IGH rearrangement detection for B-cell lymphoma diagnosis exhibited sensitivity and specificity of 88.9% and 90.0%, respectively. TCR rearrangements were not observed in patients with B-cell lymphomas. The presence of IGH rearrangements together with the absence of TCR rearrangements indicated 96.0% specificity for the diagnosis of B-cell lymphoma. The sensitivity and specificity of MALT1 translocations for diagnosing MALT lymphoma were 28.6% and 100%, respectively. The combined detection of lymphocyte clonality and MALT1 translocations using BALF is suitable for screening and diagnosis of B-cell lymphomas. Analysis of specific genes such as MALT1 should improve the precision of B-cell lymphoma diagnosis.


2021 ◽  
Vol 99 (5-6) ◽  
pp. 353-360
Author(s):  
B. D. Chaltsev ◽  
V. I. Vasiliev ◽  
S. G. Palshina ◽  
V. R. Gorodetskiy ◽  
A. V. Torgashina ◽  
...  

Purpose of the study. To study the characteristics and frequency of lymphomas in patients with Sjogren's disease (SD) and anticentromere antibodies (ACA); to evaluate the predictors of the development of lymphoproliferative diseases (LPD) in this group of patients. Material and methods. Over the period from 1998 till 2019, 131 ACA-positive patients were under medical supervision at the Research Institute of Rheumatology named after Nasonova V.A. Isolated SD was diagnosed in 82 patients (62.6%), isolated limited form of SSc — in 12 patients (9.2%), combination of SD and limited form of SSc — in 37 patients (28.2%). Lymphoproliferative diseases (LPD) were diagnosed in 20 ACA-positive patients: in 15 — with SD, in 5 — with SD and SSc; no lymphomas were found in the group of patients with isolated SSc. All lymphomas were diagnosed on the basis of histological, immunohistochemical and PCR examination with of B-cell clonality determination in the tissue, and were classified on the base of haematopoietic and lymphoid tissue tumors classification by the World Health Organization. Further analysis included 15 ACA-positive patients with isolated SD and lymphomas. Results. In our study, 18.3% of patients with isolated ACA-positive SD were diagnosed with LPD, represented by MALT lymphomas of the salivary glands (subsequent transformation into aggressive diffuse large B-cell lymphoma (DLBCL) was noted in one patient) in most cases. The course of SD before the diagnosis of LPD was characterized by a gradual progression of dental manifestations of SD with the development of late stages of parenchymal parotitis, severe xerostomia, and significant enlargement of the salivary glands with a minimum number of systemic manifestations of the disease. Significant enlargement of salivary glands, severe infiltration of minor salivary glands, severe xerostomia, decreased level of C4-complement component, monoclonal secretion, low content of CD19+B-cells in peripheral blood, positive B-cell clonality in biopsy material were the main signs of LPD in this study. When diagnosing MALT lymphomas, a focal damage of the salivary glands with no signs of dissemination, no symptoms of B-cell intoxication, and minimal changes in laboratory assessment were found in patients with ACA-positive SD. Conclusion. The natural course of ACA-positive SD and the absence of pathogenetic therapy at an early stage contribute to the development of salivary gland lymphomas in the first 10 years of the disease. Persistent enlargement of the salivary glands in SD, especially in the presence of other predictors of lymphoproliferation, is a direct indication for biopsy followed by the research to exclude the presence of lymphoma.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2809-2809
Author(s):  
Joseph M Benoun ◽  
Fiona J Ruiz ◽  
Kate Widmann ◽  
Tiffany Jehng ◽  
Tassja Spindler ◽  
...  

Abstract Background: Tabelecleucel (Tab-cel) is an investigational, off-the-shelf, allogenic Epstein-Barr virus (EBV)-specific T-cell immunotherapy. Tab-cel has shown clinical activity in patients with EBV + post-transplant lymphoproliferative disease (PTLD). Previously we have shown that the process for generating tab-cel from healthy donors results in a net amplification of EBV-target responsive T-cell clonality, and that upon activation, tab-cel demonstrates polyfunctionality associated with the secretion of effector and chemoattractive cytokines. Objective: We aim to comprehensively profile tab-cel through immunophenotype, TCR repertoire by analyzing overall repertoire overlap across lots, and TCR sequence homology (GLIPH 2.0 algorithm), cytokine polyfunctionality (PF), and differential gene expression patterns (GEP) between resting and TCR-MHC-driven EBV antigen stimulation states to replicate intrinsic effector responses associated with EBV + disease engagement. Methods: Immunophenotyping was performed using targeted FACS activation profiling (CD25/CD69), and 40-plex CyTOF. PF response and cytokine profiles were evaluated using the IsoLight single-cell PF strength assay. TCR repertoires were assessed using TCRβ immunoSEQ, and the GLIPH 2.0 algorithm was utilized to cluster TCRs that are predicted to bind the same MHC-restricted peptide antigen. GEP were evaluated using a custom Nanostring panel consisting of 333 T-cell lineage gene targets. Results: Baseline control activation levels of 7.9±1.3% increased specifically to 54.3±3.7% post-activation with EBV + targets (Figure 1A). Baseline PF was 0.54±0.14%, and upon EBV-specific activation, product cells demonstrated an average PF of 12.1±1.5%, demonstrating a 22.7-fold average increase (Figure 1B).The cytokine profile for activated tab-cel lots is primarily comprised of effector and chemoattractive cytokines including IFNγ and MIP1β. Baseline TCR repertoires of the initial donor peripheral T-cells are highly diverse; however, the tab-cel manufacturing process effectively amplified and enriched for EBV-specific TCRs that correspond back to a starting frequency of 2.6±0.58% of the initial donor TCR repertoire (Figure 1C). Notably, cross comparison of tab-cel-enriched TCRs against publicly available databases (VDJdb, McPas-TCR) identified previously curated EBV-specific TCRβ sequences as a component of the expanded repertoire. Additionally, using the GLIPH 2.0 clustering algorithm we were able to identify previously unannotated TCR sequences that clustered with known EBV-specific TCRβ sequences. The tab-cel post-activation GEP revealed associations with T-cell activation and polyfunctionality. The CD4/CD8 composition of a subset of tab-cel lots was analyzed: the average CD4:CD8 ratio of 0.25, with an average of CD8+ T-cells comprising 73% of the product. An extended immunophenotyping by CyTOF is currently being completed and will be reported at the time of presentation. Conclusions: In this expanded analysis we again demonstrate that the process for generating tab-cel from healthy donors enriches for known EBV-specific clones and results in a net amplification of EBV-target responsive T-cell clonality. Additionally, utilization of the GLIPH 2.0 clustering algorithm has led to the identification of novel putative EBV-specific TCR sequences that are enriched through the tab-cel manufacturing process. Upon activation, tab-cel exhibits a robust multifactorial activation signaling and demonstrates PF associated with secretion of effector and chemoattractive cytokines. Gene expression profiling of activated tab-cel lots highlights a conserved activation gene signature that is associated with post-activation PF. Lastly, these above analyses are being leveraged to perform correlation studies of immunophenotyped to post-activation PF, TCR repertoire characteristics, and GEP. These data support that the process for generating tab-cel from healthy donor PBMCs leads to the enrichment for EBV-specific T-cell clones that are capable of becoming activated upon stimulation with consistent final product characteristics. Figure 1 Figure 1. Disclosures Benoun: Atara Biotherapeutics: Current Employment. Ruiz: Atara Biotherapeutics: Current Employment. Widmann: Atara Biotherapeutics: Current Employment. Jehng: Atara Biotherapeutics: Current Employment. Spindler: Atara Biotherapeutics: Current Employment. Abraham: Atara Biotherapeutics: Current Employment. Minne: Atara Biotherapeutics: Consultancy. Tracy: Atara Biotherapeutics: Current Employment. Munson: Atara Biotherapeutics: Current Employment. Thota: Atara Biotherapeutics: Current Employment. Wang: Atara Biotherapeutics: Current Employment. Chuan: Atara Biotherapeutics: Current Employment. Yedwabnick: Atara Biotherapeutics: Current Employment. Dubovsky: Atara Biotherapeutics: Current Employment.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2404-2404
Author(s):  
Yannick Le Bris ◽  
Audrey Ménard ◽  
Anne Moreau ◽  
Nowenn Le Lan ◽  
Céline Bossard ◽  
...  

Abstract Introduction The diagnosis of B and T cell malignancies relies on the demonstration of B-cell (BCR) or T-cell (TCR) antigen receptor clonality. This can be studied through the analysis of V(D)J rearrangements of BCR and TCR genes by PCR (van Dongen Leukemia 2003) or, more recently, by high-throughput sequencing (HTS). Amplification of a clonal population with a "primers approach" could fail in case of hybridization problems due to too fragmented DNA, somatic mutations or polymorphic variations. Here we evaluated the performance of a HTS capture system for the analysis of B and T-cell clonality in clinical samples from mature T or B malignancies. We further combined this technology to concomitant sequencing of oncogenes of interest. Patients and Methods DNA was extracted from 58 tumoral samples from fresh/frozen (FF) cells or tissues or formalin-fixed paraffin-embedded tissue (FFPE) (n=19). These samples comprised various T-cell [i.e. 1T-cell prolymphocytic leukemia, 1 T large granular lymphocytic leukemia, 2 Sézary syndrome, 4 peripheral T-cell lymphoma not otherwise specified, 14 angioimmunoblastic T-cell lymphoma] or B-cell [i.e. 14 chronic lymphocytic leukemia, 1 mantle cell lymphoma, 5 diffuse large B-cell lymphoma, 1 grey-zone lymphoma, 13 Hodgkin lymphoma, 1 Poppema, 2 Waldenström and 1 multiple myeloma] malignancies. The Biomed-2 PCR technique was used as standard for assessing the performance of TRG, IGH and IGK clonality analysis. An extensive panel of capture probes was designed (SureSelect XT HS2 DNA system, Agilent Technologies) that covered the variable (V), + diversity (D) and junction (J) segments of the IGH, IGK, TRG, TRB loci and diagnostic/theranostic genes of interest i.e. B2M, BTK, CARD11, CD28, DNMT3A, IDH2, JAK3, PLCG1, PLCG2, ROHA, SOCS1, STAT3, STAT5B, STAT6, TET2, TNFAIP3, TP53. Paired-End sequencing was performed on a MiSeq system (Illumina) in 300, 500 and 600 cycles. Analysis of clonality profiles was performed using Vidjil software and SeqOne. Results HTS runs resulted in a median total read count of 1,6M (0.7-2.9) per sample. V(D)J rearrangements were identified with a median of 1503 reads (189-6824) per sample. Five samples were excluded because less than 300 rearranged reads were obtained. The number of rearranged reads and of clonotypes identified are influenced by the number of sequencing cycles (300&lt;500 or 600) but not by the quality of DNA (FFPE vs FF). Analyses of tumoral samples with HTS versus PCR were compared. For the IGH locus (n=47), comparable PCR/HTS clonal (n=22) and polyclonal (PCL, n=20) profiles were identified. One discordant case showed a clonal PCR profile and a PCL HTS profile but the IGK was clonal. For the IGK locus (n=23), 10 clonal and 12 PCL cases were similar with both techniques. One case appeared discordant with a PCL PCR profile but a clonal HTS profile. For the TRG locus (n=31), PCR and HTS profiles were similar in 14 clonal, 5 oligoclonal and 9 PCL cases respectively. Three cases were discordant with oligoclonal PCR profiles but a clonal HTS profile. Overall in the 38 cases of B-cell malignancies, 27 and 11 cases had a concordant B-cell clonal or PCL profile with PCR and HTS. Among PCL cases, only one was discordant with a clonal HTS profile. This case and 3 other PCL cases were Hodgkin lymphomas which all disclosed another mutation (i.e. TP53, TNFAIP3, SOCS1). Of the 20 cases of T-cell malignancies, 14 displayed a clonal TRG profile with PCR and HTS. Among them, 13 showed oncogene mutations that confirmed the oncogenic nature of the clonal proliferation. Among 6 patients with a non-clonal PCR TRG profile, two cases of AITL and T-LGL had a discordant clonal TRG HTS profile and both also had specific mutations (SOCS1, RHOA and STAT3 respectively). Two other AITL samples showed a T-PCL profile with PCR and HTS but also had a mutation/CNV (RHOA, SOCS1). Conclusion A very good performance of B and T cell clonality assessment was obtained here with capture-HTS compared to Biomed-2 PCR. The combined identification of mutation/CNV allowed to confirm the malignant character in cases of clonal or PCL lymphoproliferations, while concomitantly specifying the type of lymphoproliferative disorder. The combined capture-HTS of B and T repertoires and oncogenes of diagnostic or theranostic interest thus appears as an efficient, accurate and useful approach for the diagnosis of mature B and T lymphoid malignancies in clinical practice. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi98-vi98
Author(s):  
Jasim Kada Benotmane ◽  
Jan Kückelhaus ◽  
Kevin Joseph ◽  
Jürgen Beck ◽  
Oliver Schnell ◽  
...  

Abstract The diversity to T cell responses and clonality in spatially heterogeneous glioblastoma is of paramount importance to explore underlying mechanisms of anti-tumor immunity. Spatial transcriptomics, a novel technology to map the transcriptional architecture, is technically limited to discover T cell receptor (TCR) sequences as the 3' approach lacks sufficient coverage. Here, we established SPTCR-seq, a method to capture TCR sequences followed by long-read sequencing to enable full-length TCR reconstruction. We performed 10X Visium spatial transcriptomics on 9 primary and recurrent glioblastoma with both 3’-sequencing and SPTCR-seq. For SPTCR-seq, we target enriched T cell receptor sequences by capturing by hybridization followed by Oxford-Nanopore long-read sequencing. The on-target rate was above 80% for captured TCR genes and spatial barcode was successfully aligned in more than 60%. IgBlast and MixCR were used to reconstruct the TCR and map T cell clonality. Within our recent developed spatial transcriptomic analysis framework (SPATA2), we build a novel toolbox, SPATA-Immunology, which enables integration of stRNA-sequencing data and spatially resolved TCR sequencing. Our data showed that clonal evolution of T cells is limited to regional areas underpinned by significant spatial autocorrelation coefficient (0.6-0.95, padj&lt; 0.001). In the surrounding tumor cell spots, the recently described transcriptional program “reactive immune” (RI), was significantly enriched. Using spotlight, a computational approach to project scRNA-sequencing into the spatial space, we found a local enrichment of CD163 positive macrophages exclusively in areas of large T cell clonality. Imaging mass cytometry of a consecutive section confirmed the spatial confluence of T-cell infiltration and CD163-positive macrophages. Through DeepTCR we uncovered potential epitopes which correlate with T cell clonality and might help to discover novel targets for CART therapy. Spatial profiling of TCR sequences through SPTCR-seq is a powerful tool to investigate anti-tumor immunity in glioblastoma and allows to discover general and personalized targets for immunotherapy.


Author(s):  
Shyam S. Raghavan ◽  
Jennifer Y. Wang ◽  
Alejandro A. Gru ◽  
Ann L. Marqueling ◽  
Joyce M.C. Teng ◽  
...  

Author(s):  
Charlotte Syrykh ◽  
Pauline Gorez ◽  
Sarah Péricart ◽  
David Grand ◽  
Fréderic Escudié ◽  
...  

Immunomorphological diagnosis of T-cell lymphomas (TCL) may be challenging, especially on needle biopsies. Multiplex polymerase chain reaction (PCR) assays to assess T-cell receptor (TCR) gene rearrangements are now widely used to detect T-cell clones and provide diagnostic support. However, PCR assays only detect 80% of TCL, and clonal lymphocyte populations may also appear in non-neoplastic conditions. More recently, targeted next-generation sequencing (t-NGS) technologies have been deployed to improve lymphoma classification. To the best of our knowledge, the comparison of these techniques' performance in TCL diagnosis has not been reported yet. In this study, 82 TCL samples and 25 non-neoplastic T-cell infiltrates were divided into two cohorts (test and validation) and analyzed with both multiplex PCR and t-NGS to investigate TCR gene rearrangements and somatic mutations, respectively. The detection of mutations appeared to be more specific (100.0%) than T-cell clonality assessment (41.7% to 45.5%), whereas no differences were observed in terms of sensitivity (95.1% to 97.4%). Furthermore, t-NGS provided a reliable basis for TCL diagnosis in samples with partially degraded DNA that was impossible to assess with PCR. Finally, although multiplex PCR assays appeared to be less specific than t-NGS, both techniques remain complementary, as PCR recovered some t-NGS negative cases.


2021 ◽  
pp. ji2100404
Author(s):  
Lauren E. Higdon ◽  
Steven Schaffert ◽  
Huang Huang ◽  
Maria E. Montez-Rath ◽  
Marc Lucia ◽  
...  

2021 ◽  
Vol Publish Ahead of Print ◽  
Author(s):  
Woo Cheal Cho ◽  
Pavandeep Gill ◽  
Priyadharsini Nagarajan ◽  
Phyu P. Aung ◽  
Carlos A. Torres-Cabala ◽  
...  

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