Next-Generation High Throughput Sequencing Analysis In The Search Of Infectious Pathogens In Suspect Lymphoproliferations

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3019-3019
Author(s):  
Daniela Hoehn ◽  
Jordan M. Schecter ◽  
David Hirschberg ◽  
Cadhla Firth ◽  
Craig R Street ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Genetic Material ◽  
Microbial Pathogens ◽  
Next Generation ◽  
Antigen Stimulation ◽  
T Cell Lymphomas ◽  
Infectious Etiology

Abstract Background Infectious etiologies have been established for a variety of lymphomas whereby viral, bacterial or ricketssial organisms can induce or promote lymphomagenesis either indirectly through antigen stimulation or by inflicting immune dysregulation or by directly infecting lymphocytes mimicking or co-opting signaling pathways leading to cellular transformation. EBV infection represents the most well studied pathogen that can directly infect lymphocytes, and induce neoplasia via cellular immortalization. An infectious etiology for a variety of EBV negative (EBV-) lymphoproliferative neoplasms/disorders, occurring in either immunocompromised or immunocompetent individuals has long been considered, but never been established. To investigate the possibility of novel lymphotropic pathogens as causative agents of lymphomagenesis, we used next-generation, high throughput sequencing (HTS) to analyze subsets of suspect T- and B-cell lymphomas for the presence of non-human genetic material. Methods Forty-nine lymphomas, representing 5 different entities were evaluated: 9 EBV- negative post-transplant lymphoproliferative disorders, monomorphic type (EBV- PTLD), 10 EBV-negative classical Hodgkin lymphomas (cHL), 10 peripheral T-cell lymphomas (PTCL), 10 marginal zone lymphomas (MZL) and 10 chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLL). Frozen sections of non-Hodgkin lymphoma tumor blocks were evaluated and samples with tumor representation >70% were selected for analysis. EBV status was determined by using in situ hybridization (ISH) for EBV encoded small RNAs (EBER). For next-generation sequencing, RNA (0.5 µg) was extracted from frozen tumors, DNase I digested (DNA-free; Ambion, Austin, TX) and reverse transcribed using Superscript II kit (Invitrogen) with random octamer primers (MWG, Huntsville, AL). The cDNA was RNase H treated prior to random amplification by PCR. Products of 70 bp were purified (MinElute, Qiagen) and ligated to linkers for sequencing using a GS FLX sequencer (454 Life Sciences, Branford, CT). Primer sequences were removed, followed by multiple filtering steps and sequences obtained were compared with those of known infectious agents using software available at the BLAST website (www.ncbi.nlm.nih.gov/BLAST). Results Sequencing was successful in 46 cases. Microbial sequences were detected in 5 specimens (9%). In the remaining 41 cases, including all EBV- PTLDs and cHLs no non-human genetic material was identified. Human herpes virus 4 (EBV) was detected in one PTCL harboring an EBV+ B-cell lymphoma which embodied 20% of the total tumor mass in the specimen (as evaluated by ISH). EBV sequences were not detected in 4 other PTCL exhibiting EBV+ B-cells (range 1-10% involvement by ISH). These 4 cases represented angioimmunoblastic T-cell lymphomas. Human immunodeficiency virus -1 sequences (HIV) were detected in a lung MZL occurring in a known HIV+ patient. Sequences corresponding to propionebacterium sp., tetracyclin resistant streptococcus sp. and acinetobacter sp., were identified in one case each: MZL, EBV-PTLD and CLL; and were considered contaminants, likely acquired during biopsy procurement. Conclusion No novel lymphotropic microbial pathogens were identified in non-EBV associated T- and B-cell lymphoproliferations. Our findings argue against a clonal infectious etiology, which has previously been hypothesized for subsets of the lymphomas analyzed. Inability in detecting EBV sequences in samples containing low levels of EBV infected cells, suggests that this methodology might not be suitable for investigating lymphoproliferations with low tumor burden (e.g. cHL) or those arising as a consequence of chronic antigen stimulation due to a low-frequency intratumoral microbial pathogens (e.g. MZL). Further studies in a larger cohort of lymphoproliferative neoplasms will be helpful to further validate our results. Disclosures: Schecter: Seattle Genetics: Honoraria, Research Funding.

scholarly journals T-Cell Lymphoma Clonality by Copy Number Variation Analysis of T-Cell Receptor Genes

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 340
Author(s):  
Ming Liang Oon ◽  
Jing Quan Lim ◽  
Bernett Lee ◽  
Sai Mun Leong ◽  
Gwyneth Shook-Ting Soon ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
T Cell Receptor ◽  
Copy Number ◽  
Nk Cell ◽  
Cell Receptor ◽  
T Cell Lymphomas ◽  
Number Variation ◽  
Diagnostic Applications ◽  
Cell Clonality

T-cell lymphomas arise from a single neoplastic clone and exhibit identical patterns of deletions in T-cell receptor (TCR) genes. Whole genome sequencing (WGS) data represent a treasure trove of information for the development of novel clinical applications. However, the use of WGS to identify clonal T-cell proliferations has not been systematically studied. In this study, based on WGS data, we identified monoclonal rearrangements (MRs) of T-cell receptors (TCR) genes using a novel segmentation algorithm and copy number computation. We evaluated the feasibility of this technique as a marker of T-cell clonality using T-cell lymphomas (TCL, n = 44) and extranodal NK/T-cell lymphomas (ENKTLs, n = 20), and identified 98% of TCLs with one or more TCR gene MRs, against 91% detected using PCR. TCR MRs were absent in all ENKTLs and NK cell lines. Sensitivity-wise, this platform is sufficiently competent, with MRs detected in the majority of samples with tumor content under 25% and it can also distinguish monoallelic from biallelic MRs. Understanding the copy number landscape of TCR using WGS data may engender new diagnostic applications in hematolymphoid pathology, which can be readily adapted to the analysis of B-cell receptor loci for B-cell clonality determination.

scholarly journals Research Techniques Made Simple: High-Throughput Sequencing of the T-Cell Receptor

2017 ◽  
Vol 137 (6) ◽  
pp. e131-e138 ◽  
Author(s):  
Tiago R. Matos ◽  
Menno A. de Rie ◽  
Marcel B.M. Teunissen
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cell Receptor ◽  
Research Techniques

scholarly journals A Novel Approach for Treatment of T-Cell Malignancies: Targeting T-Cell Receptor Vβ Families

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Jie Wang ◽  
Katarzyna Urbanska ◽  
Prannda Sharma ◽  
Mathilde Poussin ◽  
Reza Nejati ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Receptor ◽  
Brentuximab Vedotin ◽  
T Cell Lymphomas ◽  
T Cell Malignancies

Background: Peripheral T-cell lymphomas (PTCL) encompass a highly heterogeneous group of T-cell malignancies and are generally associated with a poor prognosis. Combination chemotherapy results in consistently poorer outcomes for T-cell lymphomas compared with B-cell lymphomas.1 There is an urgent clinical need to develop novel approaches to treatment of PTCL. While CD19- and CD20-directed immunotherapies have been successful in the treatment of B-cell malignancies, T-cell malignancies lack suitable immunotherapeutic targets. Brentuximab Vedotin, a CD30 antibody-drug conjugate, is not applicable to PTCL subtypes which do not express CD30.2 Broadly targeting pan-T cell markers is predicted to result in extensive T-cell depletion and clinically significant immune deficiency; therefore, a more tumor-specific antigen that primarily targets the malignant T-cell clone is needed. We reasoned that since malignant T cells are clonal and express the same T-cell receptor (TCR) in a given patient, and since the TCR β chain in human α/β TCRs can be grouped into 24 functional Vβ families targetable by monoclonal antibodies, immunotherapeutic targeting of TCR Vβ families would be an attractive strategy for the treatment of T-cell malignancies. Methods: We developed a flexible approach for targeting TCR Vβ families by engineering T cells to express a CD64 chimeric immune receptor (CD64-CIR), comprising a CD3ζ T cell signaling endodomain, CD28 costimulatory domain, and the high-affinity Fc gamma receptor I, CD64. T cells expressing CD64-CIR are predicted to be directed to tumor cells by Vβ-specific monoclonal antibodies that target tumor cell TCR, leading to T cell activation and induction of tumor cell death by T cell-mediated cytotoxicity. Results: This concept was first evaluated in vitro using cell lines. SupT1 T-cell lymphoblasts, which do not express a native functioning TCR, were stably transduced to express a Vβ12+ MART-1 specific TCR, resulting in a Vβ12 TCR expressing target T cell line.3 Vβ family specific cytolysis was confirmed by chromium release assays using co-culture of CD64 CIR transduced T cells with the engineered SupT1-Vβ12 cell line in the presence of Vβ12 monoclonal antibody. Percent specific lysis was calculated as (experimental - spontaneous lysis / maximal - spontaneous lysis) x 100. Controls using no antibody, Vβ8 antibody, and untransduced T cells did not show significant cytolysis (figure A). Next, the Jurkat T cell leukemic cell line, which expresses a native Vβ8 TCR, was used as targets in co-culture. Again, Vβ family target specific cytolysis was achieved in the presence of CD64 CIR T cells and Vβ8, but not Vβ12 control antibody. Having demonstrated Vβ family specific cytolysis in vitro using target T cell lines, we next evaluated TCR Vβ family targeting in vivo. Immunodeficient mice were injected with SupT1-Vβ12 or Jurkat T cells with the appropriate targeting Vβ antibody, and either CD64 CIR T cells or control untransduced T cells. The cell lines were transfected with firefly luciferase and tumor growth was measured by bioluminescence. The CD64 CIR T cells, but not untransduced T cells, in conjunction with the appropriate Vβ antibody, successfully controlled tumor growth (figure B). Our results provide proof-of-concept that TCR Vβ family specific T cell-mediated cytolysis is feasible, and informs the development of novel immunotherapies that target TCR Vβ families in T-cell malignancies. Unlike approaches that target pan-T cell antigens, this approach is not expected to cause substantial immune deficiency and could lead to a significant advance in the treatment of T-cell malignancies including PTCL. References 1. Coiffier B, Brousse N, Peuchmaur M, et al. Peripheral T-cell lymphomas have a worse prognosis than B-cell lymphomas: a prospective study of 361 immunophenotyped patients treated with the LNH-84 regimen. The GELA (Groupe d'Etude des Lymphomes Agressives). Ann Oncol Off J Eur Soc Med Oncol. 1990;1(1):45-50. 2. Horwitz SM, Advani RH, Bartlett NL, et al. Objective responses in relapsed T-cell lymphomas with single agent brentuximab vedotin. Blood. 2014;123(20):3095-3100. 3. Hughes MS, Yu YYL, Dudley ME, et al. Transfer of a TCR Gene Derived from a Patient with a Marked Antitumor Response Conveys Highly Active T-Cell Effector Functions. Hum Gene Ther. 2005;16(4):457-472. Figure Disclosures Schuster: Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria.

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