What Numbers Don't Say: Immunogenetic Evidence Shows That High-Count MBL Resembles Rai 0 CLL While Low-Count MBL Does Not.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2883-2883
Author(s):  
Anna Vardi ◽  
Antonis Dagklis ◽  
Lydia Scarfò ◽  
Diane F. Jelinek ◽  
Darren J Newton ◽  
...  
Keyword(s):  
General Population ◽  
B Cell ◽  
Research Funding ◽  
Somatic Hypermutation ◽  
Molecular Genetic ◽  
High Sensitivity ◽  
Cell Receptor ◽  
World Health ◽  
High Count ◽  
Health Organization

Abstract Abstract 2883 CLL-like monoclonal B-cell lymphocytosis (MBL) shares common immunophenotypic features and cytogenetic abnormalities with CLL and is generally perceived as its premalignant state. The World Health Organization has set a consensus cut-off of 5×109/L circulating B cells to discriminate between what constitutes ‘disease’ and what not. However, the clonal size within MBL is extremely variable. High-count (HC), clinical MBL is associated with absolute lymphocytosis and progresses to CLL requiring treatment at a rate of 1–2% per year, whereas low-count (LC) MBL is found in the general population through high-sensitivity techniques and carries a risk of progression that is limited if any. Given the high frequency of CLL-like MBL in the general population, it is important to understand the underlying mechanisms and also identify biological markers endowing malignant potential that may distinguish between the different forms. To this end, we performed a detailed immunogenetic profiling of 334 CLL-like MBL cases (78 LC and 256 HC) for a total of 355 productive VDJ rearrangements (including double rearrangements), 91 from LC MBL and 264 from HC MBL. We also compared the immunoglobulin (IG) gene repertoires of MBL to 544 CLL Rai Stage 0 (CLL-0) that were part of an IG sequence dataset of 7424 CLL cases previously analyzed by our group. LC and HC MBL had distinct IG gene repertoires, with over-representation of the IGHV1–69 and IGHV4–34 genes in HC and the IGHV4–59/61 genes in LC MBL, respectively (p<0.001). The HC MBL repertoire exhibited clear similarities to CLL-0 in terms of IGHV gene usage (similar frequencies of IGHV1–69 and IGHV4–34). Regarding somatic hypermutation, no differences were identified between LC MBL versus HC MBL versus CLL-0, in that the frequency of mutated rearrangements (<98% identity to the germline) was overall similar (LC MBL: 72.5%, HC MBL: 76.1%, CLL-0: 75%). In this respect, all the aforementioned subgroups differed significantly (p<0.001) from the frequency previously reported by us in CLL where only 55% of rearrangements carried mutated IGHV genes. We finally analyzed the expression of stereotyped B cell receptor (BcR) IGs, identified through a cluster analysis of the MBL sequences together with all CLL sequences from our cohort and 5494 non-CLL IG sequences retrieved from public databases. Overall, only 6/91 (5.5%) LC MBL rearrangements could be clustered with other sequences in subsets with stereotyped BcRs. Two of these six LC MBL cases were clustered together with IG sequences from various entities, including CLL, other lymphomas and autoimmune diseases; thus, they were considered to carry ‘public’ BcR stereotypes. In contrast, HC MBL showed a significantly (p=0.0002) higher frequency of ‘CLL-specific’ BcR stereotypes versus LC MBL, with 60/264 (22.7%) HC MBL cases carrying stereotyped VH CDR3s. This frequency was comparable to the one observed in CLL-0 (20.2%). Notably, a gradation was observed in the frequency of BcR IG stereotypy depending on the absolute count of CLL-like cells, starting with 5.5% in LC MBL, raising to 22.7–20.2% in in HC MBL/CLL-0 and, finally, peaking at 30.4% in the entire CLL cohort as previously reported. Altogether, these findings suggest that rather than a true premalignant condition, LC MBL may merely reflect immune senescence or result from persistent antigen stimulation. On the other hand, HC MBL appears to be a continuum with Rai stage 0 in the evolution to clinically overt CLL, being maybe one step behind where it requires either additional genetic hits or simply extra time to cross the numerical border that discriminates it from CLL. Hence, the identification of molecular genetic markers that may predict progression of HC MBL/CLL-0 into full-fledged CLL is strongly warranted. Disclosures: Shanafelt: Genentech: Research Funding; GlaxoSmith Klein: Research Funding; Teva/Cephalon: Research Funding; Celgene: Research Funding.

scholarly journals Prevalence of JAK2 V617F in Individuals That Meet World Health Organization Erythrocytosis Criteria for Polycythemia Vera

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1775-1775 ◽  
Author(s):  
Miguel Piris-Villaespesa ◽  
Gloria Muñoz-Martin ◽  
Ricardo Sanchez ◽  
Claudia Nuñez-Torron ◽  
Adolfo Saez-Martin ◽  
...  
Keyword(s):  
General Population ◽  
Sensitivity And Specificity ◽  
Research Funding ◽  
High Sensitivity ◽  
Jak2 V617f ◽  
World Health ◽  
Jak2 V617f Mutation ◽  
Pcr Assay ◽  
V617f Mutation ◽  
Health Organization

Abstract Introduction: The diagnostic criteria for polycythemia vera (PV) has recently been updated by the World Health Organization (WHO). The criterion for erythrocytosis has been modified downwards: hemoglobin (hb)> 16.5 g/dL or hematocrit (hto)> 49% in men and hb> 16 g/dL or hto> 48% in women. This reduction increases the potential number of patients that would be test for JAK2 V617F mutation if PV is suspected. The V617F mutation in the JAK2 gene is present in 95% of cases of PV. It is estimated that the prevalence of this mutation in the general population is around 0.2%. Our aims are to determine the prevalence of JAK2 V617F in individuals with erythrocytosis according to WHO2016 criteria and to find prognostic factors that could help to identify patients with PV. Methods: We prospectively studied all hemograms performed in our laboratory during 7 nonconsecutive days. Variables studied were hb, hto, leukocytes, neutrophils, platelets, MCV, MCH, MCHC and RDW. JAK2 V617F mutation was studied in all males that had hb> 16.5 g/dl or hto> 49% or females that had hb> 16 g/dl or hto> 48%. JAK2 V617F mutation was studied by PCR assay in which an amplification control fragment and the JAK2 mutant allele were simultaneously amplified. All positive samples were confirmed by quantitative real-time PCR in a reference laboratory. Positive results were considered when the JAK2 V617F allele ratio was ≥ 0.7. The variables collected were correlated with the result of the JAK2 test in a univariate way. The T-Student test was used for the quantitative variables and the Chi-square test for the categorical variables. For the cell count variables, the Mann-Whitney U test was used. Results: A total of 15366 HG were analyzed. 1271 (8.3%) met the inclusion criteria for erythrocytosis. JAK2 V617F was performed on 1001 samples (270 samples were not suitable for the PCR assay due to low quality). Twelve samples (1.2%) were positive for JAK2 V617F mutation. However, 5 samples were excluded due to a known diagnosis of myeloproliferative neoplasm. Therefore, finally prevalence of JAK2 V617 mutation in 996 patients that met WHO erythrocytosis criteria was 0.8% (8/996). Medians for all parameter studied for each group are shown in table 1. In order to find out parameters that could increase the incidence probabilities to identify patients with JAK2 V617F we performed an univariate analysis of the variables included, according to JAK2 mutational status. We found that patients with JAK2 V617F had higher levels of leukocytes, neutrophils, platelets and RDW than patients with negative JAK2 (p <0.001), while the levels of MCV (p = 0.033) and MCH (p = 0.015) were lower in patients with JAK2 V617F (table 2). For the variables that have been statistically significant, the area under the ROC curve (AUC) and the optimal cut-off point that maximizes sensitivity and specificity were calculated (Youden index). Of interest, neutrophils, platelets and RDW show high AUC (≥ 0.79), with high sensitivity and specificity (table 3). Finally, in order to find out the potential interest of these findings, we studied medical records of the 8 patients with JAK2 V617F mutations, finding that 4 patients (50%) had previously suffered a vascular event. Conclusion: The prevalence of JAK2 V617F mutation in subjects with elevated Hb or hto according to WHO2016 criteria is increased with respect to that of the general population. Among this group of subjects, those with JAK2 V617F show significantly different levels of leukocytes, neutrophils, platelets, MCV, CMH and RDW. Interestingly, neutrophils, platelets and RDW show high sensitivity and specificity. Therefore, it is necessary to explore combinations of these parameters and their optimal cut-off points to elaborate efficient strategies for an early diagnostic approach. Disclosures Martinez Lopez: Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau. García Gutiérrez:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Incyte: Honoraria, Research Funding.

Hodgkin Lymphoma–like Posttransplantation Lymphoproliferative Disorder

2006 ◽  
Vol 130 (4) ◽  
pp. 558-560 ◽  
Author(s):  
Barbara Semakula; ◽  
Jon V. Rittenbach ◽  
Jun Wang
Keyword(s):  
B Cell ◽  
Hodgkin Lymphoma ◽  
Lymphoproliferative Disorder ◽  
Molecular Genetic ◽  
Lymphoproliferative Disorders ◽  
World Health ◽  
Solid Organ ◽  
World Health Organization Classification ◽  
Health Organization ◽  
Posttransplantation Lymphoproliferative Disorder

Abstract Posttransplantation lymphoproliferative disorders (PTLD) are a heterogeneous group of monoclonal or polyclonal lymphoproliferative lesions that occur in immunosuppressed recipients following solid organ or bone marrow transplantation, including 4 categories: (1) early lesions (reactive plasmacytic hyperplasia, and infectious-mononucleosis–like PTLD), (2) polymorphic PTLD, (3) monomorphic PTLD (including B-cell neoplasms and T-cell neoplasms), and (4) Hodgkin lymphoma (HL) and HL-like PTLD in the current World Health Organization classification. Although HL-like PTLD has been grouped with classic HL PTLD, controversy remains as to whether it is truly a form of HL or whether it should be more appropriately considered as a form of B-cell PTLD. The current available literature data indicate the presence of important immunophenotypic, molecular genetic, and clinical differences between HL PTLD and HL-like PTLD, suggesting that HL-like PTLD is in fact most often a form of B-cell PTLD. Distinction from true HL may be important for clinical management and prognosis.

Metody cytogenetyki molekularnej w różnicowaniu agresywnych B-NHL

2019 ◽  
Vol 50 (3) ◽  
pp. 109-115
Author(s):  
Beata Grygalewicz
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
World Health ◽  
High Grade ◽  
Non Hodgkin Lymphoma ◽  
Large B Cell Lymphoma ◽  
Health Organization ◽  
Large B Cell

StreszczenieB-komórkowe agresywne chłoniaki nieziarnicze (B-cell non-Hodgkin lymphoma – B-NHL) to heterogenna grupa nowotworów układu chłonnego, wywodząca się z obwodowych limfocytów B. Aberracje cytogenetyczne towarzyszące B-NHL to najczęściej translokacje onkogenów takich jak MYC, BCL2, BCL6 w okolice genowych loci dla łańcuchów ciężkich lub lekkich immunoglobulin. W niektórych przypadkach dochodzi do wystąpienia kilku wymienionych aberracji jednocześnie, tak jak w przypadkach przebiegających z równoczesną translokacją genów MYC i BCL2 (double hit), niekiedy także z obecnością rearanżacji BCL6 (triple hit). Takie chłoniaki cechuje szczególnie agresywny przebieg kliniczny. Obecnie molekularna diagnostyka cytogenetyczna przy użyciu techniki fluorescencyjnej hybrydyzacji in situ (FISH) oraz, w niektórych przypadkach, aCGH jest niezbędnym narzędziem rozpoznawania, klasyfikowania i oceny stopnia zaawansowania agresywnych, nieziarniczych chłoniaków B-komórkowych. Technika mikromacierzy CGH (aCGH) była kluczowym elementem wyróżnienia prowizorycznej grupy chłoniaków Burkitt-like z aberracją chromosomu 11q (Burkitt-like lymphoma with 11q aberration – BLL, 11q) w najnowszej klasyfikacji nowotworów układu chłonnego Światowej Organizacji Zdrowia (World Health Organization – WHO) z 2016 r. Omówione zostaną sposoby różnicowania na poziomie cytogenetycznym takich chłoniaków jak: chłoniak Burkitta (Burkitt lymphoma – BL), chłoniak rozlany z dużych komórek B (diffuse large B-cell lymphoma – DLBCL) oraz 2 nowych jednostek klasyfikacji WHO 2016, czyli chłoniaka z komórek B wysokiego stopnia złośliwości z obecnością translokacji MYC i BCL2 i/lub BCL6 (high-grade B-cell lymphoma HGBL, with MYC and BCL2 and/or BCL6 translocations) oraz chłoniaka BLL, 11q.

scholarly journals Immune Repertoire Analysis of Multiple Myeloma Research Samples Using NGS Characterization of Multiple B Cell Receptors in a Single Reaction

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1881-1881
Author(s):  
Geoffrey Lowman ◽  
Landon Pastushok ◽  
Karen Mochoruk ◽  
Wayne Hill ◽  
Michelle Toro ◽  
...  
Keyword(s):  
B Cell ◽  
Light Chain ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
Clonal Lineage ◽  
Repertoire Analysis ◽  
Targeted Ngs ◽  
Thermo Fisher Scientific ◽  
Fisher Scientific ◽  
Single Reaction

Abstract Introduction B cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. This issue can be especially true in MM which has a high rate of SHM. To address these issues, we have developed an assay for B cell analysis, based on Ion AmpliSeq™ technology, which enables efficient detection of IGH, IgK, and IgL chain rearrangements in a single reaction. Methods The B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IgK loci rearrangements involving Kappa deletion element and the constant region intron. To evaluate assay performance, we conducted reproducibility studies and clonality assessment using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal previously by light chain restriction via flow cytometry or IHC/ISH in tissue sections - 16 of the 45 MM samples were identified as lambda light chain restricted. For comparison, a small cohort of 12 B-ALL samples were also included in the study. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software. Results Clonality assessment of MM clinical research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains in a single reaction using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. In total, 42 of 45 samples were deemed clonal by the single tube assay based on detection for one or more receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. In two of the lambda restricted samples only a clonal lambda rearrangement was identified, showing the benefit of including primers targeting both the kappa and lambda light chains in a pan-clonality NGS assay. Both the MM and B-ALL cohorts were evaluated for biased IGHV gene usage. IGHV3-11 was observed in 5 of 45 MM and 5 of 12 B-ALL samples. IGHV4-34, typically linked to autoreactive antibodies and underrepresented in germinal center and memory B-cells, was nonetheless found in 5 of 45 MM samples surveyed. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples, as expected, contained some somatic hypermutation with 6 of 45 samples showing greater than 10% mutation rates. Automated lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Two MM samples showed no somatic hypermutation as measured using the FR3 primers contained in the BCR pan-clonality assay. These samples were also evaluated using an FR1-J targeted NGS assay, which confirmed relatively low mutation rates for these MM samples at 0.44% and 1.3%, respectively. Conclusions These results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify laboratory workflows and including analysis tools such as automated somatic hypermutation rate calculation and clonal lineage identification may open new paths for research in lymphoid cell disorders. For research use only. Disclosures Lowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Ostresh: Thermo Fisher Scientific: Current Employment. Sarda: Thermo Fisher Scientific: Current Employment. Yang: Thermo Fisher Scientific: Current Employment.

scholarly journals Signatures of B Cell Receptor Repertoire Following Pneumocystis Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Han Sun ◽  
Hu-Qin Yang ◽  
Kan Zhai ◽  
Zhao-Hui Tong
Keyword(s):  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Receptor Repertoire ◽  
Elevated Expression ◽  
Pneumocystis Infection

B cells play vital roles in host defense against Pneumocystis infection. However, the features of the B cell receptor (BCR) repertoire in disease progression remain unclear. Here, we integrated single-cell RNA sequencing and single-cell BCR sequencing of immune cells from mouse lungs in an uninfected state and 1–4 weeks post-infection in order to illustrate the dynamic nature of B cell responses during Pneumocystis infection. We identified continuously increased plasma cells and an elevated ratio of (IgA + IgG) to (IgD + IgM) after infection. Moreover, Pneumocystis infection was associated with an increasing naïve B subset characterized by elevated expression of the transcription factor ATF3. The proportion of clonal expanded cells progressively increased, while BCR diversity decreased. Plasma cells exhibited higher levels of somatic hypermutation than naïve B cells. Biased usage of V(D)J genes was observed, and the usage frequency of IGHV9-3 rose. Overall, these results present a detailed atlas of B cell transcriptional changes and BCR repertoire features in the context of Pneumocystis infection, which provides valuable information for finding diagnostic biomarkers and developing potential immunotherapeutic targets.

scholarly journals Impaired memory B-cell recall responses in the elderly following recurrent influenza vaccination

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0254421
Author(s):  
Rodrigo B. Abreu ◽  
Greg A. Kirchenbaum ◽  
Giuseppe A. Sautto ◽  
Emily F. Clutter ◽  
Ted M. Ross
Keyword(s):  
Influenza Virus ◽  
High Risk ◽  
Antibody Response ◽  
B Cell ◽  
Influenza A ◽  
The Elderly ◽  
World Health ◽  
Memory B Cell ◽  
Virus Vaccination ◽  
Health Organization

Influenza is a highly contagious viral respiratory disease that affects million of people worldwide each year. Annual vaccination is recommended by the World Health Organization with the goal of reducing influenza severity and limiting transmission through elicitation of antibodies targeting the hemagglutinin (HA) glycoprotein. The antibody response elicited by current seasonal influenza virus vaccines is predominantly strain-specific, but pre-existing influenza virus immunity can greatly impact the serological antibody response to vaccination. However, it remains unclear how B cell memory is shaped by recurrent annual vaccination over the course of multiple seasons, especially in high-risk elderly populations. Here, we systematically profiled the B cell response in young adult (18–34 year old) and elderly (65+ year old) vaccine recipients that received annual split inactivated influenza virus vaccination for 3 consecutive seasons. Specifically, the antibody serological and memory B-cell compartments were profiled for reactivity against current and historical influenza A virus strains. Moreover, multiparametric analysis and antibody landscape profiling revealed a transient increase in strain-specific antibodies in the elderly, but with an impaired recall response of pre-existing memory B-cells, plasmablast (PB) differentiation and long-lasting serological changes. This study thoroughly profiles and compares the immune response to recurrent influenza virus vaccination in young and elderly participants unveiling the pitfalls of current influenza virus vaccines in high-risk populations.

Management for Different Glioma Subtypes: Are All Low-Grade Gliomas Created Equal?

2019 ◽  
pp. 133-145 ◽  
Author(s):  
Martin C. Tom ◽  
Daniel P. Cahill ◽  
Jan C. Buckner ◽  
Jörg Dietrich ◽  
Michael W. Parsons ◽  
...  
Keyword(s):  
Molecular Genetic ◽  
World Health ◽  
Low Grade ◽  
Lower Grade ◽  
Future Directions ◽  
Molecular Alterations ◽  
Lower Grade Glioma ◽  
Grade 3 ◽  
Health Organization

Following the identification of key molecular alterations that provided superior prognostication and led to the updated 2016 World Health Organization (WHO) Central Nervous System (CNS) Tumor Classification, the understanding of glioma behavior has rapidly evolved. Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are present in the majority of adult grade 2 and 3 gliomas, and when used in conjunction with 1p/19q codeletion for classification, the prognostic distinction between grade 2 versus grade 3 is diminished. As such, the previously often used term of “low-grade glioma,” which referred to grade 2 gliomas, has now been replaced by the phrase “lower-grade glioma” to encompass both grade 2 and 3 tumors. Additional molecular characterization is ongoing to even further classify this heterogeneous group of tumors. With such a colossal shift in the understanding of lower-grade gliomas, management of disease is being redefined in the setting of emerging molecular-genetic biomarkers. In this article, we review recent progress and future directions regarding the surgical, radiotherapeutic, chemotherapeutic, and long-term management of adult lower-grade gliomas.

scholarly journals Mechanisms behind Functional Avidity Maturation in T Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Marina Rode von Essen ◽  
Martin Kongsbak ◽  
Carsten Geisler
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
Affinity Maturation ◽  
Functional Avidity ◽  
Cell Clones ◽  
Genes Encoding ◽  
Avidity Maturation

During an immune response antigen-primed B-cells increase their antigen responsiveness by affinity maturation mediated by somatic hypermutation of the genes encoding the antigen-specific B-cell receptor (BCR) and by selection of higher-affinity B cell clones. Unlike the BCR, the T-cell receptor (TCR) cannot undergo affinity maturation. Nevertheless, antigen-primed T cells significantly increase their antigen responsiveness compared to antigen-inexperienced (naïve) T cells in a process called functional avidity maturation. This paper covers studies that describe differences in T-cell antigen responsiveness during T-cell differentiation along with examples of the mechanisms behind functional avidity maturation in T cells.

scholarly journals High-Grade B-Cell Neoplasm with Surface Light Chain Restriction and Tdt Coexpression Evolved in aMYC-Rearranged Diffuse Large B-Cell Lymphoma: A Dilemma in Classification

2017 ◽  
Vol 2017 ◽  
pp. 1-9
Author(s):  
Dina Sameh Soliman ◽  
Ahmad Al-Sabbagh ◽  
Feryal Ibrahim ◽  
Ruba Y. Taha ◽  
Zafar Nawaz ◽  
...  
Keyword(s):  
B Cell ◽  
Metabolic Response ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
World Health ◽  
Cell Phenotype ◽  
High Grade ◽  
Large B Cell Lymphoma ◽  
Health Organization ◽  
Large B Cell

According to World Health Organization (WHO) classification (2008), B-cell neoplasms are classified into precursor B-cell or a mature B-cell phenotype and this classification was also kept in the latest WHO revision (2016). We are reporting a male patient in his fifties, with tonsillar swelling diagnosed as diffuse large B-cell lymphoma (DLBCL), germinal center. He received 6 cycles of RCHOP and showed complete metabolic response. Two months later, he presented with severe CNS symptoms. Flow cytometry on bone marrow (BM) showed infiltration by CD10-positive Kappa-restricted B-cells with loss of CD20 and CD19, and downregulation of CD79b. Moreover, the malignant population showed Tdt expression. BM Cytogenetics revealed t(8;14)(q24;q32) within a complex karyotype. Retrospectively, MYC and Tdt immunostains performed on original diagnostic tissue and came negative for Tdt and positive for MYC. It has been rarely reported that mature B-cell neoplasms present with features of immaturity; however the significance of Tdt acquisition during disease course was not addressed before. What is unique in this case is that the emerging disease has acquired an immaturity marker while retaining some features of the original mature clone. No definitive WHO category would adopt high-grade neoplasms that exhibit significant overlapping features between mature and immature phenotypes.

First Clinical Application of Talen Engineered Universal CAR19 T Cells in B-ALL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2046-2046 ◽  
Author(s):  
Waseem Qasim ◽  
Persis Jal Amrolia ◽  
Sujith Samarasinghe ◽  
Sara Ghorashian ◽  
Hong Zhan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Receptor ◽  
Research Funding ◽  
Cell Receptor ◽  
Receptor Expression ◽  
Molecular Remission ◽  
Equity Ownership

Abstract Chimeric antigen receptor (CAR)19 T-cells exhibit powerful anti-leukemic effects in patients with B cell malignancies. However, the complexity of production of patient bespoke T cell products is a major barrier to the broader application of this approach. We are investigating a novel strategy to enable "off-the-shelf"' therapy with mismatched donor CAR19 T cells. Transcription activator-like effector nucleases (TALEN)s can be used to overcome HLA barriers by eliminating the risk of graft-versus-host disease (GvHD) through disruption of T cell receptor expression, and by simultaneously targeting CD52, cells can be rendered insensitive to the lymphodepleting agent Alemtuzumab. Administration of Alemtuzumab can then be exploited to prevent host-mediated rejection of HLA mismatched CAR19 T cells. We manufactured a bank of such cells from volunteer donor T cells under GMP conditions on behalf of Cellectis S.A for final stage validation studies using a third generation self inactivating lentiviral vector encoding a 4g7 CAR19 (CD19 scFv- 41BB- CD3ζ) linked to RQR8, an abbreviated sort/suicide gene encoding both CD34 and CD20 epitopes. Cells were then electroporated with two pairs of TALEN mRNA for multiplex targeting of both the T cell receptor alpha constant chain locus, and the CD52 gene locus. Following ex-vivo expansion, cells still expressing TCR were depleted using CliniMacs alpha/beta TCR depletion, yielding a T cell product with <1% TCR expression, 85% of which expressed CAR19, and 64% becoming CD52 negative. This universal CAR19 (UCART19) cell bank has been characterized in detail, including sterility, molecular and cytometric analyses and human/murine functional studies ahead of submissions for regulatory approvals and Phase 1 testing in trials for relapsed B cell leukaemia. In the interim we received a request for therapy on a compassionate basis for an infant with refractory relapsed B-ALL, and with the agreement of Cellectis, we treated this first patient under UK special therapy regulations. An 11 month girl with high risk CD19+infant ALL (t(11;19) rearrangement) relapsed in bone marrow 3 months after a myeloablative 8/10 mismatched unrelated donor transplant. Leukaemic blasts expressed CD19 but were CD52negative. Her disease progressed despite treatment with Blinatumomab (70% blasts in marrow) and we were unable to generate donor-derived CAR19 T cells on an existing study. Following institutional ethics review, detailed counseling, and parental consent, the patient received cytoreduction with Vincristine, Dexamethasone and Asparaginase followed by lymphodepleting conditioning with Fludarabine 90mg/m2, Cyclophosphamide 1.5g/m2 and Alemtuzumab 1mg/kg. Immediately prior to infusion of UCART19 cells, the bone marrow showed persisting disease (0.5% FISH positive). She received a single dose (4.5x106/kg) of UCART19 T cells without any significant toxicity. To date there has been no significant perturbation of cytokine levels in peripheral blood, and no indication of cytokine release syndrome. Although profoundly lymphopenic, UCART19 T cells were detectable by qPCR in the circulation by day 14 and at increased levels in both blood (VCN 0.35) and marrow (VCN 0.22) on day 28. The patient exhibited signs of count recovery and the bone marrow, while hypoplastic, was in cytogenetic and molecular remission. Chimerism was 90% donor, and a clearly demarcated population (7%) of third party cells indicated persistence of UCART19. A residual persistence of 3% recipient cells in the marrow suggests that leukemic clearance was not mediated by transplant mediated alloreactivity. Within the short period of follow up available, our intervention comprising lymphodepletion and infusion of UCART19 T cells has induced molecular remission where all other treatments had failed. This first-in-man application of TALEN engineered cells provides early proof of concept evidence for a ready-made T cell strategy that will now be tested in early phase clinical trials. Disclosures Qasim: CATAPULT: Research Funding; CELLMEDICA: Research Funding; CALIMMUNE: Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding; CELLECTIS: Research Funding. Off Label Use: UCART19 T Cells are an unlicensed investigational medicinal product and in this case were used under MHRA special licence arrangements. Stafford:CELLECTIS: Research Funding. Peggs:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership. Thrasher:CATAPULT: Patents & Royalties, Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding. Pule:AUTOLUS: Employment, Equity Ownership, Research Funding; CELLECTIS: Research Funding; AMGEN: Honoraria; UCLB: Patents & Royalties.

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