scholarly journals Molecular Diagnosis of T-cell Lymphoma: A correlative study of PCR-based T-cell clonality assessment and targeted NGS

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.

2020 ◽  
Vol 1 (5) ◽  
Author(s):  
Dr. Raúl Rodríguez ◽  
Dra. Esther Nimchinsky ◽  
Dr. James K Liu ◽  
Dra. Ada Baisre de León

Lymphomatoid granulomatosis (LG) is a rare, angioinvasive and angiodestructive, EBV-associated B cell lymphoproliferative disorder, which occurs in the setting of immunosuppression. We present the peculiar case of a 67-year-old lady, with systemic lupus erythematous (SLE) and lupus nephritis, on immunosuppressant therapy, who developed a new onset of seizures and was found to have multiple ring enhancing lesions on brain MRI. A biopsy of one of the lesions revealed lymphomatoid granulomatosis, grade I. DNA analysis of the neoplasm, showed T-cell receptor gene rearrangement (TRG) and no evidence of B-cell rearrangement, which is an unusual finding. On further examination several lung nodules were identified on a CT scan of the chest, a characteristic of LG. Key words: Cerebral lymphomatoid granulomatosis, T cell clonality, Epstein Bar virus, Immunodeficiency associated B-cell lymphoma


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1451-1451
Author(s):  
Chao Wang ◽  
Qiang Gong ◽  
Weiwei Zhang ◽  
Javeed Iqbal ◽  
Yang Hu ◽  
...  

Abstract Introduction: Diversity of the T-cell receptor (TCR) repertoire reflects the initial V(D)J recombination events as shaped by selection by self and foreign antigens. Next generation sequencing is a powerful method for profiling the TCR repertoire, including sequences encoding complementarity-determining region 3 (CDR3). Peripheral T-cell lymphoma (PTCL) is a group of malignancies that originate from mature T-cells. T-cell clonality of PTCL is routinely evaluated with a PCR-based method to detect TCR gamma and less frequently beta chain rearrangements using genomic DNA. However, there are limitations with this approach, chief among which is the lack of sequence information. To date, the TCR repertoire of different subtypes of PTCL remains poorly defined. Objective: The purpose of this study was to determine the utility of RNA-seq for assessing T-cell clonality and analyzing the TCR usage in PTCL samples. Methods: We analyzed RNA-seq data from 30 angioimmunoblastic T-cell lymphoma (AITL), 23 Anaplastic large cell lymphoma (ALCL), 10 PTCL-NOS, and 17 NKCL. Data from naïve T cells, TFH cells, and T-effector cells (CD4+ CD45RA− TCRβ+ PD-1lo CXCR5lo PSGL-1hi) were obtained from publicly available resources. Referenced TCR and immunoglobulin transcripts according to the International ImMunoGeneTics Information System (IMGT) database were quantified by Kallisto software. We divided the pattern of Vβ (T-cell receptor beta variable region) into three categories: monoclonal (mono- or bi-allelic), oligoclonal (3-4 dominant clones), and polyclonal. CDR3 sequences were extracted by MiXCR program. PCR of the gamma chain using genomic DNA was utilized to validate the clonality of selected cases. Single nucleotide variants (SNVs) were called from aligned RNA-seq data using Samtools and VarScan 2 programs. Results: Analysis of RNA-seq data identified preferential usage of TCR-Vβ, Dβ (diversity region), and Jβ (joining region), length diversity of CDR3, and usage of nontemplated bases. Dominant clones could be identified by transcriptome sequencing in most cases of AITL (21/30), ALCL (14/23), and PTCL-NOS (7/10). Median CDR3 length is 42 nucleotides (nt) in normal T cells, 41 nt in ALCL, 48 nt in PTCL-NOS, and 44 nt in AITL. In 30 AITL samples, 20 showed monoclonal Vβ with a single peak, and 9 showed polyclonal Vβ. One case had two dominant clones with different CDR3, only one of which was in frame, implying biallelic rearrangements. As many as 3511 clones supported by at least four reads could be detected in polyclonal cases. In monoclonal cases, the dominant clone varied between 11.8% and 92.8% of TCR with Vβ rearrangements. TRBV 20-1, which is the most commonly used segment in normal T cells, is also frequently used in the dominant clones in AITL. The monoclonal AITL cases showed mutation of TET2, RHOA, DNMT3A or IDH2 whereas most of the polyclonal cases were negative or had low VAF mutation suggesting low or absent of tumor infiltrate in the specimen sequenced. There is no obvious correlation of any of the mutations with Vβ usage. Clonal B cell expansion was noted in some AITL samples. The occurrence of a preferential TRBV9 expansion in PTCL-NOS was striking. More than half of ALCL samples (14/23) showed expression of clonal Vβ, but 3/14 dominant clones were out-of-frame. γ chain expression was very low in cells expressing TCRαβ, but both expression levels and clonality were higher in TCRγδ expressing tumors. NKCL did not express significant levels of TCR Vβ or Vγ genes. Discussion/Interpretation: Transcriptome sequencing is a useful tool for understanding the TCR repertoire in T cell lymphoma and for detecting clonality for diagnosis. Clonal, often out-of-frame, Vβ transcripts are detectable in most ALCL cases and preferential TRBV9 usage is found in PTCL-NOS. Disclosures No relevant conflicts of interest to declare.


2013 ◽  
Vol 66 (9) ◽  
pp. 749-752 ◽  
Author(s):  
Fenglin Cao ◽  
Shuye Wang ◽  
Hui Zhao ◽  
Jin Zhou ◽  
Guisheng Yang ◽  
...  

2020 ◽  
Author(s):  
Vadim R. Gorodetskiy ◽  
Yulia V. Sidorova ◽  
Natalia A. Kupryshina ◽  
Vladimir I. Vasilyev ◽  
Natalya A. Probatova ◽  
...  

Abstract Objectives Approximately 15% of patients with T-cell large granular lymphocytic leukemia (T-LGLL) have rheumatoid arthritis (RA). RA-associated T-LGLL with low large granular lymphocyte counts (aleukemic presentation) and Felty's syndrome (FS) have indistinguishable clinical presentations. These disorders are distinguished by T-cell clonality which is observed in T-LGLL but not in FS. Activating somatic mutations in the signal transducer and activator of transcription 3 (STAT3) and 5 (STAT5b) genes are involved in T-LGLL pathogenesis; however, the prevalence of these mutations in FS is unknown.Methods Based on the rearrangements of T-cell receptor (TCR) gamma and beta genes according to the BIOMED-2 protocol, we examined T-cell clonality in 81 patients with RA and unexplained neutropenia. We stratified these patients by the presence or absence of T-cell clonality, respectively, into 2 groups: RA-associated T-LGLL (56 patients) and FS (25 patients). Allele-specific TaqMan Real-Time polymerase chain reaction assay was employed to detect point somatic mutations in STAT3 and STAT5b genes in each group.Results Mutations of the STAT3 gene were detected in none of the 24 cases of FS and in 22 of 56 cases of RA-associated T-LGLL (39%) (p < 0.001). No mutation of the STAT5b gene was detected in any of the patients in each group.Conclusions Although further data are needed, our results suggest that activating somatic mutations in STAT3 and STAT5b genes are not involved in the pathogenesis of FS.


Acta Naturae ◽  
2015 ◽  
Vol 7 (3) ◽  
pp. 116-125 ◽  
Author(s):  
Yu. V. Sidorova ◽  
N. G. Chernova ◽  
N. V. Ryzhikova ◽  
S. Yu. Smirnova ◽  
M. N. Sinicina ◽  
...  

Aim: To assess the feasibility and informative value of T-cell clonality testing in peripheral T-cell lymphoma (PTCL). Patients and methods: 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). Results: 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. Conclusion: 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.


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.


2015 ◽  
Vol 73 (2) ◽  
pp. 228-236.e2 ◽  
Author(s):  
Kari E. Sufficool ◽  
Christina M. Lockwood ◽  
Haley J. Abel ◽  
Ian S. Hagemann ◽  
Jonathan A. Schumacher ◽  
...  

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