Molecular Immunoprofiling the T Cell Repertoire after Rituximab Administration Reveals Frequent Oligoclonality Albeit with Different Patterns Depending on the Clinical Context

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5792-5792
Author(s):  
Apostolia Papalexandri ◽  
Michail Iskas ◽  
Evangelia Stalika ◽  
Maria Karypidou ◽  
Barbara Tachynopoulou ◽  
...  
Keyword(s):  
T Cell ◽  
Research Funding ◽  
Late Onset ◽  
Sequence Data ◽  
Lymphocytic Leukemia ◽  
Cytotoxic T Cell ◽  
Cell Populations ◽  
Gene Repertoire ◽  
Group A ◽  
Group B

Abstract In patients with B lymphoid malignancies, depletion of B cells by Rituximab, an anti-CD20 humanized monoclonal antibody, can induce changes in the subset composition, activation and function of T cells. The spectrum of resultant immune-mediated sequelae encompasses organ-specific manifestations (e.g. pneumonitis, gastritis) as well as Rituximab-associated late onset neutropenia (R-LON). Although the pathogenesis of these clinical syndromes is not fully elucidated, evidence suggests that at least a fraction of cases may develop in a setting of expanded cytotoxic T cell populations with a large granular lymphocyte (LGL) phenotype (CD3+CD8+CD57+). Similar cytotoxic T cell expansions can be observed after Rituximab administration in other clinical settings e.g. allogeneic transplantation (allo-HCT), where selective restriction of the T-cell receptor (TR) gene repertoire is probably driven by multifactorial mechanisms. Here, we sought to obtain more insight into this phenomenon by molecular immunoprofiling of the TR gene repertoire in two groups of patients who received Rituximab: (i) Group A: patients (n=10) with chronic lymphocytic leukemia (CLL) treated with fludarabine-cyclophosphamide-rituximab (FCR); and, Group B: patients (n=14) who underwent allo-HCT for hematologic malignancies and received Rituximab either as pre-emptive treatment for EBV reactivation or against refractory cGvHD. Each patient included in the study received a mean of 6 cycles of Rituximab (range, 1-14). TR repertoire analysis was performed 11-88 (median, 36) and 5-24 months (median, 5) after the first Rituximab administration in Group A and Group B, respectively. TR beta gene rearrangements were PCR amplified on genomic DNA isolated from bone marrow samples using the BIOMED2 protocol and subjected to classic subcloning/Sanger sequencing. Sequence data were analyzed using the IMGT/V-QUEST tool. A total of 579 productive TRBV-TRBD-TRBJ rearrangements were analysed, 291 for Group A, 288 for Group B (6-91/case, median=20). Among the 46 TRBV functional genes identified, 3 accounted for >25% of cases in both groups: (i) TRBV27 (13% in both Groups A and B); ii) TRBV19-1 (13% in group A, 7% in group B); and, (iii) TRBV6-1 (7% and 6%, respectively). Clusters of identical (>=2) rearrangements corresponding to clonotypes were identified in all patients. Oligoclonality with immunodominant clonotypes (>12% of the repertoire) accounting for over 30% of the analyzed sequences was more frequent in Group A (7/10 cases) versus Group B (5/10 cases); however, larger clonotype expansions were seen in group B. Longitudinal analysis was performed in 3 patients with oligoclonality, 1 from group A and 2 from Group B: in the Group A patient, immunodominant clonotypes disappeared, while both patients in Group B retained the oligoclonal repertoire. Lymphocyte subpopulation analysis by flow cytometry was performed in 6 patients of each group. T-LGL proliferations (defined as CD4/CD8 abnormal ratio and CD3+CD8+CD57+ >30%) were found more often in Group B (3/6 cases in Group A versus 6/6 in Group B). They were related to oligoclonal TR gene repertoire in Group A (3/3) but not in Group B patients (3/6 cases). However, true expansions could be considered only in group B patients, since CD8+ lymphocytes >1.0*109/l were seen in all 6 Group B versus only 1/6 group A cases. Self-limiting R-LON was observed in 8 patients (4 in each group), but no association of oligoclonality to R-LON could be found. In conclusion, we report frequent development of oligoclonal T cell populations after Rituximab treatment in two different clinical contexts. The Groups analyzed differed with respect to the extent of oligoclonality, suggesting that the precise clinical setting determines the amplitude of TR repertoire skewing after Ritximab. Sustained oligoclonal cytotoxic expansions were recognised more often among allo-HCT patients, presenting with a highly restricted TR gene repertoire and likely reflecting strong antigenic stimulation by viruses and/or cGVHD aggravated by T cell imbalances induced by Rituximab. Disclosures Stamatopoulos: Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Late Onset Neutropenia Develops Selectively in Only a Subset of Patients with T Large Granular Lymphocyte Proliferation After Rituximab Treatment for Lymphoma,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3675-3675
Author(s):  
Apostolia Papalexandri ◽  
Michalis Iskas ◽  
Evangelia Stalika ◽  
Leonidas Marinos ◽  
Barbara Tachynopoulou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Late Onset ◽  
Autologous Transplantation ◽  
Lymphocyte Subset ◽  
Large Granular Lymphocyte ◽  
Cytotoxic T Cell ◽  
Group A ◽  
Group B

Abstract Abstract 3675 Emerging evidence suggests that B cells can actively modulate T cell immune responses by presenting antigen, providing co-stimulation and secreting cytokines. This has prompted investigation whether B cell depletion by monoclonal antibodies, including Rituximab, can alter the subset composition, activation or function of T cells. Rituximab-associated late onset neutropenia (R-LON) is increasingly recognized as a long-term adverse event of Rituximab. Although the etiology of R-LON is not fully elucidated, the syndrome seems to be multifactorial and likely caused by immune-mediated mechanisms. We have previously shown that at least a proportion of R-LON may develop in a setting of expanded cytotoxic T cell populations in peripheral blood (PB) with a large granular lymphocyte (LGL) phenotype (CD3+CD8+CD57+). Here, we extend our observations regarding PB lymphocyte subset composition in a cohort of 107 Rituximab-treated patients with available results from PB flow cytometry analysis performed at roughly similar intervals after the initial Rituximab administration. The present cohort included 107 patients, aged 16–83 (median 60), who received Rituximab for the treatment of chronic lymphocytic leukemia (CLL) (29), diffuse large B cell lymhoma (DLBCL) (20), marginal zone lymphomas (15), follicular lymhoma (FL) (15), mantle cell lymphoma (MCL) (8) and auto-immune cytopenias (20). Overall, we found: (i) increased (>1.0×109/l) CD8+ cells in 45/107 (42%) cases; (ii) CD4+/CD8+ cell ratios <0.7 in 56/107 (52%) cases; and, (iii) T-LGLs >20% in 66/107 (63%) cases. Within this cohort, 33 cases (group A) developed R-LON, whereas the remainder (group B, n=74) did not develop this syndrome over a comparable observation period. Importantly, no patient with autoimmune cytopenia developed R-LON. R-LON was significantly more frequent in patients with lymphoma subtypes treated with intensive chemotherapy (CLL, DLBCL, MCL), as well as patients who underwent autologous transplantation (p\q0.001 for all comparisons). No significant differences were noted between groups A or B regarding PB lymphocyte subset composition. We next evaluated the findings from the histopathological study of bone marrow biopsy samples, available in 17 Group A and 19 Group B cases, all with a diagnosis of lymphoma. The morphological and immunohistochemical examination revealed a series of features common in both groups, summarized as follows: (i) mild-to-moderate small lymphocytic infiltration by CD20-CD79a-CD3+CD45RO+CD43+ (CD3>CD45RO) cells, predominantly nodular and/or interstitial (non intrasinusoidal); (ii) pronounced hyperplasia of the erythroid and megakaryocytic series with prominent dyserythropoiesis and dysmegakaryopoiesis, respectively, including abnormal paratrabecular localization, suggestive of myelodysplasia (MDS); and (iii) remarkable shift-to-the-left of the granulocytic series, often with abnormal localization of immature progenitors (ALIP), always with \q2% CD34+ cells. A proportion of cases showed hyperplasia of the granulocytic series. However, a major difference between the two Groups concerned hypoplasia of the granulocytic series, which was noted almost exclusively in group A. We conclude that lymphoma patients treated with Rituximab often develop cytotoxic T cell expansions than can have a variable impact on hematopoiesis, with R-LON perhaps representing the end of a spectrum of T-LGL-mediated autoimmune myelopathy/myelodyplasia. The selective development of R-LON in only a proportion of cases with expanded cytotoxic T cells associated with prominent hypoplasia of the granulocytic series and MDS-like changes of the hematopoietic marrow post Rituximab raises several questions regarding the underlying (genetically determined?) immunopathogenetic mechanisms. Disclosures: No relevant conflicts of interest to declare.

Skewing of the T-Cell Receptor Repertoire in Patients Receiving Rituximab after Allogeneic Hematopoietic Cell Transplantation: What Lies Beneath?

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3962-3962
Author(s):  
Apostolia Papalexandri ◽  
Maria Karypidou ◽  
Evangelia Stalika ◽  
Michail Iskas ◽  
Anna Vardi ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Transplantation ◽  
T Cell Receptor ◽  
Hematopoietic Cell Transplantation ◽  
Late Onset ◽  
Cell Receptor ◽  
Hematopoietic Cell ◽  
Cytotoxic T Cell ◽  
Gene Repertoire ◽  
Lgl Leukemia

Abstract The development of CD3+/CD8+/CD57+ cytotoxic cell expansions after allogeneic hematopoietic cell transplantation (allo-HCT) driven by antigenic stimulation, viral or associated with chronic graft-versus-host disease (cGvHD), has been suggested as related with favorable outcome. Rituximab, an anti-CD20 humanized monoclonal antibody, has been linked to the development of oligo- or even monoclonal expansions of CD3+/CD8+/CD57+ T-large granular lymphocytes (T-LGLs) that can manifest with neutropenia of delayed origin in relation to Rituximab administration. We have recently reported remarkable skewing of the T-cell receptor (TR) gene repertoire in two allo-HCT transplanted patients with delayed neutropenia associated with T-LGL expansions developing in a context of GvHD and Rituximab administration for EBV reactivation. Prompted by these preliminary findings, we here extend our immunogenetic studies of the TR repertoire in patients receiving Rituximab post allo-HCT. The study group was comprised of 9 patients (including the two previously reported) aged 14-50 years (median 41) who were subjected to myeloablative allo-HCT (4 from matched related, 3 from matched unrelated donors), haplo-identical transplantation (1) or Reduced Intensity Conditioning-allo-HCT from sibling donor (1), all for hematological malignancies. All patients received Rituximab consecutively between 2010-2013 either as pre-emptive treatment for EBV reactivation or against refractory cGvHD. In all patients TR gene repertoire analysis was performed at least one year after the transplantation (range 12-72 months), when immune reconstitution normally would have been achieved, and 5-24 months after the first treatment with Rituximab. Each patient received a mean of 7 cycles of Rituximab (range, 1-14). TRBV-TRBD-TRDJ gene rearrangements were PCR-amplified on genomic DNA isolated from bone marrow samples using the BIOMED2 protocol and subjected to classic subcloning/Sanger sequencing. Sequence data was interpreted using the IMGT/V-QUEST tool. A total of 164 sequences were analyzed (9-25/case, median=18) revealing 106 productive TRBV-TRBD-TRBJ rearrangements. Among the 29 TRBV functional genes identified only three accounted for 48% of cases: (i) TRBV27*01 (25%), (ii) TRBV6-5*01 (13%), (iii) TRBV6-2*01 (10%). Of note, TRBV27*01 has been reported as the most frequent TRBV gene in Rituximab-related late-onset neutropenia in CLL. All cases were found to carry clusters of identical (>=2) rearrangements corresponding to clonotypes. In the majority of cases (5/9), 2-4 (median 3) immunodominant clonotypes accounted for over 30% of the analyzed sequences (frequency of immunodominant clonotype/case 13-40%). Lymphocyte subpopulation analysis by flow cytometry in 6 patients revealed T-LGL expansion. Samples from additional time points (spanning a period of 10 years), pre- and post- Rituximab, were studied in one patient. Analysis of 71 sequences demonstrated progressive expansion of a certain clonotype overtime, associated with the emergence of steroid-refractory autoimmune hemolytic anemia in a context of CD3+CD8+CD57+ lymphoproliferation. This particular clonotype dominated the repertoire by far, thus establishing a diagnosis of T-LGL leukemia. which, remarkably, proved to be of donor origin (97% and 30% donor chimerism in T lymphocytes and total hematopoeisis, respectively). No association of oligoclonality to stronger GvL effect could be found among the rest of the patients. However, a strong correlation with cGvHD (100% vs 25% among polyclonal cases) was identified. Late-onset neutropenia was documented in 4/9 patients, regardless of the composition of the repertoire i.e whether it was polyclonal or oligo(mono)clonal. In conclusion, we report frequent development of oligoclonal cytotoxic T-cell populations after Rituximab treatment post allo-HCT likely of multifactorial evidence. Direct evidence of the anti-leukemic effect of this phenomenon could not be provided, however, the observed association of oligoclonality with GvHD and the development of a possible “T-LGL leukemia vs leukemia” effect in one patient is noteworthy and merits further investigation. Finally, the observed skewing of the TR gene repertoire strongly implicates antigen selection in the development of cytotoxic T-cell expansions after allo-HCT. Disclosures No relevant conflicts of interest to declare.

Skewing of the T Cell Receptor Gene Repertoire and Public Clonotypes in Cytotoxic T Cells of Patients with Chronic Idiopathic Neutropenia: A Role for Antigen Selection in Disease Development

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 831-831
Author(s):  
Evangelia Stalika ◽  
Michalis Spanoudakis ◽  
Nikos Darzentas ◽  
Maria Karypidou ◽  
Achilles Anagnostopoulos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Sequence Data ◽  
Cytotoxic T Cell ◽  
Gene Rearrangements ◽  
Gene Repertoire ◽  
Autoimmune Reaction ◽  
Chronic Idiopathic Neutropenia ◽  
Antigen Selection

Abstract Abstract 831 Chronic idiopathic neutropenia (CIN) is a disorder of neutrophil production usually characterized by benign and asymptomatic course and female predominance. The defective hematopoiesis in CIN can be mainly attributed to accelerated Fas-mediated death of the CD34+/CD33+ granulocytic progenitor cells, secondary to an inflammatory bone marrow (BM) microenvironment. Crucial to CIN pathogenesis are the increased numbers of activated T cells identified in both peripheral blood (PB) and BM of CIN patients. Recently, using flow cytometry and CDR3 spectratyping, we obtained for the first time evidence for expanded T cell populations alluding to repertore skewing in both PB and BM of patients with CIN, but more prominent in the BM CD8+ subset. Prompted by these fndings, here we significantly extended our studies of the cytotoxic T cell responses in CIN in order to obtain a comprehensive view of the role of antigen selection in CIN pathogenesis. The study included 18 patients with CIN, 17 females and 1 male, diagnosed according to the established criterial for CIN. TRBV-TRBD-TRBJ gene rearrangements were amplified on either genomic DNA or cDNA isolated from CD8+ cells of PB (n=6) or BM (n=12) samples. PCR products were subcloned by transformation into E.Coli/TOP10F' competent bacteria and individual colonies were chosen randomly and subjected to Sanger sequencing. Sequence data were analyzed using the International imMunoGenetics information system and more particularly the IMGT/V-QUEST tool. Overall, 507 TRBV-TRBD-TRBJ gene rearrangements were analyzed (19-30/case) of which 466 were productive since they used functional TRBV genes and also carried in-frame CDR3s. A polyclonal profile was seen in only 3/18 cases (16.7%). The remaining cases were found to carry clusters of identical rearrangements corresponding to distinct immunodominant clonotypes. The frequency of each clonotype was determined by dividing the number of identical sequences by the total number of subcloned sequences analyzed. In 10/18 cases (55.5%), the dominant clonotype accounted for 12.8–22.6% of the total, whereas in 5/18 cases (27.8%) it accounted for 34.2–68.4% of the total. The TRBV28 gene was used by the dominant clonotype of three different CIN cases; the TRBV10-2, TRBV10-3, TRB19, and TRBV7-8 genes were identified in the major clonotypes of two cases each. Importantly, cluster analysis of the CDR3 sequences of all CIN cases of the present study identified 4 different rearrangements that were shared by different patients (public clonotypes). In conclusion, the present study strongly suggests that CIN may result from an autoimmune reaction directed against granulocytic progenitors triggered by a restricted range of, as yet, unidentified antigen(s). The finding of public clonotypes may indicate that public antigenic stimuli and/or shared immune processes underlie CIN development, at least for a proportion of cases. Overall, our results further support the concept that CIN may share common pathophysiologic mechanisms with other disorders characterized by T-cell and cytokine mediated suppression of hematopoiesis, perhaps representing a milder extreme within the spectrum of these acquired immune-mediated BM failure syndromes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Use of Intravenous Immunoglobulin (IVIG) during Severe Neurotoxicity Among the Recipients of Chimeric Antigen Receptor T-Cell (CAR-T) Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5627-5627 ◽  
Author(s):  
Sepideh Mokhtari ◽  
Justin M Asquith ◽  
Christina A Bachmeier ◽  
Michael D. Jain ◽  
Peter Forsyth ◽  
...  
Keyword(s):  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
T Cell Therapy ◽  
Significant Difference ◽  
Car T ◽  
Group A ◽  
Grade 3 ◽  
Group B

INTRODUCTION: Severe neurotoxicity occurs in ~30% of Diffuse Large B Cell Lymphoma (DLBCL) patients treated with CAR-T cell therapy. The current treatment for severe neurotoxicity is glucocorticoids +/- tocilizumab (an IL-6 antagonist) depending on concurrent cytokine release syndrome. Even with these treatments, neurotoxicity can have a complicated course. It is therefore essential to find the optimal treatment to reverse neurotoxicity timely. METHOD:This is a retrospective cohort study of neurologic and oncologic outcomes among patients with grade ≥ 3 neurotoxicity treated with glucocorticoids and IVIG compared to glucocorticoids only. Severe neurotoxicity was defined as grade 3 and graded by CRES/CARTOX score. Time to resolution of severe neurotoxicity (TTR) was defined as improvement of severe neurotoxicity to grade ≤ 2. RESULTS: We identified a total of 20 patients who received CAR-T therapy and developed severe neurotoxicity. Ten patients received glucocorticoids and IVIG (group A) and ten patients received glucocorticoids alone (group B). The median age was 62 (range: 52-74) for group A vs 64 years (range: 48-75) for group B. Both groups had similar ECOG performance status (p=0.17), IPI scores (p=0.34), and onset of severe neurotoxicity (median=6 days in both groups). Median TTR was 3 days (range, 1-7) for group A and 4.5 days (range, 2-22) for group B. There was no significant difference in TTR of severe neurotoxicity among both groups (Log-rank p=0.18, Figure). The median time to administration of IVIG after initiation of glucocorticoids was 2 days (range, 0.5-8). The median TTR following initiation of IVIG was 0.5 day (0.5-4). The objective response rate at 30 days was 80% in both groups. None of the patients who received IVIG developed thromboembolism, renal failure, autoimmune hemolytic anemia or acute lung injury. CONCLUSIONS: Although the use of IVIG during severe neurotoxicity after CAR-T therapy appeared to be safe, this pilot retrospective analysis demonstrated no significant difference in resolution of severe neurotoxicity with addition of IVIG to glucocorticoids. Further controlled studies limiting selection bias inherent in this retrospective analysis will help to determine the efficacy of IVIG in severe neurotoxicity in the context of CAR-T cell therapy. Disclosures Mokhtari: KITE PHARMA: Other: Clinical Advisor; NOVARTIS: Other: Clinical Advisor. Bachmeier:Kite/Gilead: Speakers Bureau. Jain:Kite/Gilead: Consultancy. Forsyth:Department of Defense: Research Funding; Pfizer: Research Funding; State of Florida Bankhead Coley: Research Funding; Moffitt Center of Excellence Celgene Project: Research Funding; Florida Academic Cancer Center Alliance: Research Funding; NIH/NCI 1R21 Grant: Research Funding; NIT DT Study Section Grant Review: Membership on an entity's Board of Directors or advisory committees; AbbVie Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ziopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tocagen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BTG: Consultancy, Membership on an entity's Board of Directors or advisory committees; Inovio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novocure: Consultancy, Membership on an entity's Board of Directors or advisory committees. Locke:Novartis: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy; Kite: Other: Scientific Advisor. Lazaryan:Kadmon: Consultancy.

Ofatumumab Combined with Fludarabine and Cyclophosphamide (O-FC) Shows High Activity in Patients with Previously Untreated Chronic Lymphocytic Leukemia (CLL): Results From a Randomized, Multicenter, International, Two-Dose, Parallel Group, Phase II Trial.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 207-207 ◽  
Author(s):  
William G Wierda ◽  
Thomas J Kipps ◽  
Jan Dürig ◽  
Laimonas Griskevicius ◽  
Stephan Stilgenbauer ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Phase Ii Trial ◽  
Lymphocytic Leukemia ◽  
Parallel Group ◽  
Group A ◽  
Grade 3 ◽  
Group B

Abstract Abstract 207 Introduction: Chemoimmunotherapy regimens have become the treatment standard for patients with CLL. Ofatumumab is a human monoclonal antibody that targets a unique small-loop epitope on CD20 and elicits rapid and efficient in vitro complement-dependent cytotoxicity, as well as antibody-dependent cellular cytotoxicity. Recent studies demonstrated single-agent ofatumumab activity, with high overall response rates (ORR) in patients with refractory CLL. We conducted an international, randomized, parallel group, Phase II trial with two doses of ofatumumab combined with fludarabine and cyclophosphamide (FC) in previously untreated patients with CLL to evaluate the efficacy and tolerability of this chemoimmunotherapy regimen. Methods: Previously untreated patients (N=61) with active CLL (by NCI-WG guidelines) were randomized to receive ofatumumab 500 mg (Group A) or 1000 mg (Group B) on day 1, combined with fludarabine (25 mg/m2 IV daily; days 1–3) and cyclophosphamide (250 mg/m2 IV daily; days 1–3) every 4 weeks for a total of 6 courses. In both Groups, the first dose of ofatumumab was 300 mg. Dose reduction of FC, but not ofatumumab, was allowed. Premedication for ofatumumab was paracetamol and antihistamine prior to each infusion, and glucocorticoid prior to infusions 1 and 2. Neutrophil growth factor and anti-infective prophylaxis were not mandated. The primary endpoint was complete response (CR) rate (1996 NCI-WG criteria) assessed by an Independent Review Committee (IRC), measured from the start of treatment until 3 months after the last infusion. Safety evaluations included investigator-reported adverse events (AEs) and deaths. Follow-up continues for collection of time-to-event endpoints. Results: Data from all 61 patients were available for response assessment (primary endpoint). Pretreatment characteristics are shown in the Table. 71% and 57% of patients in Groups A and B, respectively, completed all 6 courses of O-FC treatment. The CR rate (95% CI) by IRC evaluation was 32% (17, 51%) for Group A and 50% (31, 69%) for Group B; the ORR (95% CI) was 77% (59, 90%) and 73% (54, 88%), respectively (Table). The median progression-free survival has not been reached with the short median follow up of 8 months. No CTC grade 3–4 infusion-related reactions on the day of ofatumumab infusion were reported. During treatment and up to 30 days following the last dose, the most common (>10% of patients) grade 3–4 AEs reported by investigators were infections in 11 patients (Group A, n=4; Group B, n=7) including febrile neutropenia in 3 patients in each Group, and hematologic AEs including neutropenia in 29 patients (Group A, n=11; Group B, n=18), anemia in 8 patients (Group A, n=2; Group B, n=6) and thrombocytopenia in 9 patients (Group A, n=2; Group B, n=7); grade 3–4 hemolytic anemia occurred in 2 patients in Group A and 1 in Group B; one patient in Group B died (19 days from last dose) with dyspnea (etiology unknown). Beyond the AE reporting period mentioned above, one patient in Group A died (50 days from last dose) due to febrile neutropenia during the follow up period. Results from additional analysis of data will be presented at the meeting. Conclusions: The O-FC regimen is highly active in previously untreated patients with CLL at both ofatumumab doses investigated and may offer a new chemoimmunotherapy treatment option in these patients. AEs with the O-FC regimen were manageable with no unexpected toxicities. The 1000 mg dose of ofatumumab is currently being evaluated in combination with chemotherapy in other studies for patients with CLL. Disclosures: Wierda: Genmab, GlaxoSmithKline: Honoraria, Research Funding. Off Label Use: Ofatumumab is an investigational anti-CD20 monoclonal antibody, currently under development for the treatment of B-cell malignancies (chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Waldenstroms macroglobulinemia and follicular lymphoma) as well as autoimmune diseases (rheumatoid arthritis and multiple sclerosis). Kipps:Physicians' Education Resource, Educational Concepts: Speakers Bureau; Genmab, Abbott Industries, Celgene, Biogen Idec, Cephalon, sanofi-aventis, Medimmune, Memgem, Genentech: Research Funding. Dürig:GlaxoSmithKline: Honoraria. Griskevicius:GlaxoSmithKline, Genmab: Research Funding. Stilgenbauer:GlaxoSmithKline, Genmab: Consultancy, Honoraria, Research Funding. Mayer:GlaxoSmtihKline: Consultancy. Smolej:Bayer Schering: Honoraria. Padmanabhan:GlaxoSmithKline: Consultancy, Honoraria; Celgene, Genentech: Consultancy. Gorczyca:GlaxoSmithKline: Employment. Chan:GlaxoSmithKline: Employment. Gupta:GlaxoSmithKline: Employment. Andersen:H. Lundbeck A/S: Shares ownership; Novo Nordisk A/S, H. Lundbeck A/S and Genmab A/S: Consultancy. Strange:Genmab: Employment. Nielsen:Genmab: Employment. Russell:Genmab: Employment, Equity Ownership.

Alloreactive T Cell Clonotypes Identified By In Vitro Mixed Lymphoid Reaction and High-Throughput Sequencing Exhibit Increased Frequency In Peripheral Blood Samples From Patients Following Allogeneic Hematopoietic Cell Transplantation For Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 149-149
Author(s):  
Aaron C. Logan ◽  
Mark R. Krampf ◽  
Mark Klinger ◽  
Martin Moorhead ◽  
Jianbiao Zheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
High Throughput Sequencing ◽  
Hematopoietic Cell Transplantation ◽  
Lymphocytic Leukemia ◽  
Cell Populations ◽  
Blood Samples ◽  
Donor T Cells

Abstract Introduction Allogeneic hematopoietic cell transplantation (allo-HCT) provides long-term immunologic disease control for a substantial portion of patients with hematologic malignancies. Chronic lymphocytic leukemia (CLL) is sensitive to graft-versus-leukemia (GVL) effects as evidenced by responses to reduced-intensity conditioning (RIC) allo-HCT, and to donor lymphocyte infusions (DLI) for post-HCT relapse. To identify potential alloreactive (AR)/GVL T cells, we performed in vitro mixed lymphocyte reactions (MLRs) between recipient CLL cells and donor T cells derived from blood apheresis products acquired for DLI. Responder and non-responder T cell populations from MLRs and recipient post-HCT blood samples underwent T cell receptor beta (TCRB) high-throughput sequencing (HTS). The prevalence of candidate AR/GVL TCRB clonotypes at various times following HCT was quantified and correlated with CLL disease burden and graft-versus-host disease (GVHD). Methods CLL cells isolated from cryopreserved PBMC aliquots of 7 patients who experienced post-HCT relapse were pre-stimulated in vitro for 72 hours with CpG oligodeoxynucleotides in X-VIVO 15 supplemented with IL-4, IL-7, BAFF, and GM-CSF. Upregulation of CD80, CD86, CD40L, MHCI, and/or MHCII was confirmed by flow cytometry. Donor T cells were isolated from cryopreserved DLI using pan-T cell isolation beads (Miltenyi), labeled with CFSE, and incubated with CpG-stimulated CLL cells for 7 days. Upon the conclusion of the MLR incubation, T cell populations were sorted into rapid responders (RR; CFSEdim), slow responders (SR; CFSEbrightCD69pos) and non-responders (NR; CFSEbrightCD69neg) and RNA was isolated from each cell population. RNA was then amplified and TCRB sequenced using the LymphoSIGHT platform (Sequenta). PBMC samples collected and cryopreserved pre-HCT and regularly following HCT and DLI were also subjected to TCRB-HTS. Results RR cells comprised 11.5 +/- 9.2% of MLR T cells, whereas SR were 4.2 +/- 3.5% and NR were 84.3 +/- 10.1% (Fig 1A). RR, SR, and NR populations demonstrated clonotypic exclusion with a mean 4.4% +/- 5.5% coincidence between populations (Fig 1B). TCRB diversity in the RR population was more restricted compared with diversity in the SR and NR populations, with the mean number of clonotypes comprising the top 50th percentile of total TCRB reads being 11.8 +/- 6.5%, 17.7 +/- 8.5%, and 20.2 +/- 1.8% of unique reads, respectively (p<0.01). Candidate AR/GVL TCRB clonotypes, specified as those enriched in MLR-responder populations compared to pre-stimulation samples, were validated by comparison with pre-stimulation frequency-matched clonotypes. The mean frequency of AR/GVL TCRB clonotypes in blood samples post-HCT were 10- to 100-fold greater than control clonotypes, depending on the patient (Fig 1C). AR/GVL T cells were present within the post-HCT PBMC TCRB repertoires at mean frequencies of 5x10-4 at day +90 rising to 2x10-3 by day +360 post-HCT in 3/4 patients experiencing at least 2 year remissions post-HCT. In patients experiencing early relapse (within 1yr post-HCT), no candidate AR/GVL clonotypes were identified in 1/3 patients and 2/3 exhibited AR/GVL clonotypes at roughly one log lower frequencies (mean 8x10-5 at day +90 and 4x10-4 at day 360) than those with longer remissions (Fig 1D). One patient who experienced fatal post-DLI steroid-refractory GVHD exhibited striking changes in AR/GVL clonotype prevalence following DLI (Fig 1D). Conclusions In vitro MLR between donor T cells and CpG-stimulated CLL cells selects clonotypically distinct T cell populations with an oligoclonal RR population. Persistence of adoptively transferred candidate AR/GVL clones identified by MLR appears to correlate with likelihood of maintaining clinical remission beyond 2 years in CLL patients undergoing RIC allo-HCT. Failure to adoptively transfer AR/GVL clonotypes may be associated with early treatment failure. Disclosures: Klinger: Sequenta, Inc.: Employment, Research Funding. Moorhead:Sequenta, Inc.: Employment, Research Funding. Zheng:Sequenta, Inc.: Employment, Research Funding. Faham:Sequenta Inc.: Employment, Stockholder Other.

scholarly journals Self-sparing of long-term in vitro-cloned or uncloned cytotoxic T lymphocytes.

1986 ◽  
Vol 164 (3) ◽  
pp. 962-967 ◽  
Author(s):  
M F Luciani ◽  
J F Brunet ◽  
M Suzan ◽  
F Denizot ◽  
P Golstein
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Cytotoxic T Cell ◽  
Cell Populations ◽  
Con A ◽  
T Cell Clones ◽  
Cell Clones

At least some long-term in vitro-cultured cytotoxic T cell clones and uncloned cell populations are able, in the presence of Con A, to lyse other cells, to be lysed by other cells, but not to lyse themselves. This as-yet-unexplained result may have implications as to the mechanism of T cell-mediated cytotoxicity.

Immunity to Diphtheria, Pertussis, Tetanus, and Poliomyelitis in Children with Acute Lymphocytic Leukemia After Cessation of Chemotherapy

PEDIATRICS ◽  
1981 ◽  
Vol 67 (2) ◽  
pp. 222-229 ◽  
Author(s):  
A. van der Does-van den Berg ◽  
J. Hermans ◽  
J. Nagel ◽  
G. van Steenis
Keyword(s):  
Acute Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
First Year ◽  
Healthy Children ◽  
Healthy Control ◽  
Antibody Titers ◽  
Group A ◽  
One Year ◽  
Group B ◽  
Antibody Levels

Antibody titers to diphtheria, pertussis, tetanus, and poliomyelitis (types I to III) were measured in previously vaccinated children with acute lymphocytic leukemia in remission after cessation of therapy. The response to revaccination one year after therapy was stopped was also studied. The patients' antibody titers were compared with those of healthy children, matched for age and sex. Two groups of patients were studied: one group (group A, N = 30) was given two drugs (6-mercaptopurine, methotrexate); the other group (group B, N= 19) was given three drugs (6-mercaptopurine, methotrexate, and cyclophosphamide) for maintenance treatment. In general, the patients' antibody titers were lower than those of healthy children, but in most patients they were still at levels considered to be protective. No significant differences in antibody levels between the two patient groups were found. A spontaneous rise in antibody titers in the first year after termination of therapy was not observed. After revaccination the rise in antibody titers was correlated with preexisting antibody titers in the same way in patients as in healthy children, and the antibody titers in patients and in healthy control subjects were on roughly the same level.

Genetic relationships among Brazilian strains of Aspergillus ochraceus based on RAPD and ITS sequences

2004 ◽  
Vol 50 (11) ◽  
pp. 985-988 ◽  
Author(s):  
Maria Helena Pelegrinelli Fungaro ◽  
Marciane Magnani ◽  
Laurival Antônio Vilas-Boas ◽  
Patrícia Cristina Vissotto ◽  
Márcia Cristina Furlaneto ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Sequence Data ◽  
Genetic Relationships ◽  
Its Region ◽  
Aspergillus Ochraceus ◽  
Coffee Bean ◽  
Coffee Beans ◽  
Major Species ◽  
Group A ◽  
Group B

Ochratoxin A (OA) is a mycotoxin that has been found in coffee beans and coffee beverages. Its toxicological profile includes carcinogenicity, nephrotoxicity, and immunotoxicity. Aspergillus ochraceus is the major species responsible for OA production in Brazilian coffee beans. The genetic relationships among 25 A. ochraceus strains collected from Brazilian coffee-bean samples were determined based on RAPD and internal transcribed spacer (ITS) sequence data. The isolates were resolved into 2 distinct groups, one with 4 strains (group A) and the other with 21 strains (group B). Specific nucleotide variations characterizing group A and B were found for both ITS1 and ITS2 regions. Group B is a new group proposed here to accommodate the majority of the Brazilian isolates. Each group was found to contain both toxigenic and nontoxigenic strains, indicating that there is no association between molecular genotypes and the ability to produce OA.Key words: Aspergillus ochraceus, ochratoxin A, ITS region (ITS1–5.8S–ITS2), RAPD.

scholarly journals Friedreich's Ataxia Frequency in a Large Cohort of Genetically Undetermined Ataxia Patients

2021 ◽  
Vol 12 ◽  
Author(s):  
Alexander F. Brown ◽  
Michael H. Parkinson ◽  
Hector Garcia-Moreno ◽  
Ese Mudanohwo ◽  
Robyn Labrum ◽  
...  
Keyword(s):  
Late Onset ◽  
Friedreich’S Ataxia ◽  
Friedreich's Ataxia ◽  
Diagnostic Process ◽  
Spinocerebellar Ataxias ◽  
Screening Process ◽  
Group A ◽  
Improve Patient Management ◽  
Group B ◽  
Testing Group

Background: Patients with suspected genetic ataxia are often tested for Friedreich's ataxia (FRDA) and/or a variety of spinocerebellar ataxias (SCAs). FRDA can present with atypical, late-onset forms and so may be missed in the diagnostic process. We aimed to determine FRDA-positive subjects among two cohorts of patients referred to a specialist ataxia centre either for FRDA or SCA testing to determine the proportion of FRDA cases missed in the diagnostic screening process.Methods: 2000 SCA-negative ataxia patients, not previously referred for FRDA testing (group A), were tested for FRDA expansions and mutations. This group was compared with 1768 ataxia patients who had been previously referred for FRDA testing (group B) and were therefore more likely to have a typical presentation. The phenotypes of positive cases were assessed through review of the clinical case notes.Results: Three patients (0.2%) in group A had the FRDA expansion on both alleles, compared with 207 patients (11.7%) in group B. The heterozygous carrier rate across both cohorts was of 41 out of 3,768 cases (1.1%). The size of the expansions in the three FRDA-positive cases in group A was small, and their presentation atypical with late-onset.Conclusions: This study demonstrates that FRDA is very rare among patients who were referred purely for SCA testing without the clinical suspicion of FRDA. Such cases should be referred to specialist ataxia centres for more extensive testing to improve patient management and outcomes.

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