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Abstract: Tisagenlecleucel is approved for adults with relapsed/refractory (r/r) follicular lymphoma (FL) in the third- or later-line setting. The primary analysis (median follow-up, 17 months) of the phase 2 ELARA trial reported high response rates and excellent safety profile in patients with extensively pretreated r/r FL. Here, we report longer-term efficacy, safety, pharmacokinetic, and exploratory biomarker analyses after median follow-up of 29 months (interquartile range, 22.2-37.7). As of 29 March 2022, 97 patients with r/r FL (grades 1-3A) received tisagenlecleucel infusion (0.6 × 108-6 × 108 chimeric antigen receptor-positive viable T cells). Bridging chemotherapy was allowed. Baseline clinical factors, tumor microenvironment, blood soluble factors, and circulating blood cells were correlated with clinical response. Cellular kinetics were assessed by quantitative polymerase chain reaction. Median progression-free survival (PFS), duration of response (DOR), and overall survival (OS) were not reached. Estimated 24-month PFS, DOR, and OS rates in all patients were 57.4% (95% confidence interval [CI], 46.2-67), 66.4% (95% CI, 54.3-76), and 87.7% (95% CI, 78.3-93.2), respectively. Complete response rate and overall response rate were 68.1% (95% CI, 57.7-77.3) and 86.2% (95% CI, 77.5-92.4), respectively. No new safety signals or treatment-related deaths were reported. Low levels of tumor-infiltrating LAG3+CD3+ exhausted T cells and higher baseline levels of naïve CD8+ T cells were associated with improved outcomes. Tisagenlecleucel continued to demonstrate highly durable efficacy and a favorable safety profile in this extended follow-up of 29 months in patients with r/r FL enrolled in ELARA. This trial was registered at www.clinicaltrials.gov as #NCT03568461., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The transfer of membrane proteins between cells during contact, known as trogocytosis, can create novel cells with a unique phenotype and altered function. We demonstrate that trogocytosis is more common in multiple myeloma (MM) than chronic lymphocytic leukemia and Waldenstrom macroglobulinaemia; that T cells are more probable to be recipients than B or natural killer cells; that trogocytosis occurs independently of either the T-cell receptor or HLA compatibility; and that after trogocytosis, T cells with acquired antigens can become novel regulators of T-cell proliferation. We screened 168 patients with MM and found that CD86 and human leukocyte antigen G (HLA-G) were antigens commonly acquired by T cells from malignant plasma cells. CD3+ CD86acq+ and CD3+ HLA-Gacq+ cells were more prevalent in bone marrow than peripheral blood samples. The presence of either CD86 or HLA-G on malignant plasma cells was associated with a poor prognosis. CD38++ side population cells expressed HLA-G, suggesting that these putative myeloma stem cells could generate immune tolerance. HLA-G+ T cells had a regulatory potency similar to natural Tregs, thus providing another novel mechanism for MM to avoid effective immune surveillance.

T cells contribute to host-tumor interactions in patients with monoclonal gammopathies. Expansions of CD8(+)CD57(+) T-cell receptor Vbeta-positive (TCRVbeta(+))-restricted cytotoxic T-cell (CTL) clones are found in 48% of patients with multiple myeloma and confer a favorable prognosis. We now report that CTL clones with varying TCRVbeta repertoire are present in 70% of patients with Waldenström macroglobulinemia (WM; n = 20). Previous nucleoside analog (NA) therapy, associated with increased incidence of transformation to aggressive lymphoma, significantly influenced the presence of TCRVbeta expansions (chi(2) = 11.6; P < .001), as 83% of patients without (n = 6) and only 7% with (n = 14) TCRVbeta expansions had received NA. Clonality of CD3(+)CD8(+)CD57(+)TCRVbeta(+)-restricted CTLs was confirmed by TCRVbeta CDR3 size analysis and direct sequencing. The differential expression of CD3(+)CD8(+)CD57(+)TCRVbeta(+) cells was profiled using DNA microarrays and validated at mRNA and protein level. By gene set enrichment analysis, CTL clones expressed not only genes from cytotoxic pathways (GZMB, PRF1, FGFBP2) but also genes that suppress apoptosis, inhibit proliferation, arrest cell-cycle G1/S transition, and activate T cells (RAS, CSK, and TOB pathways). Proliferation tracking after stimulation confirmed their anergic state. Our studies demonstrate the incidence, NA sensitivity, and nature of clonal CTLs in WM and highlight mechanisms that cause anergy in these cells.

Limited response to idiotype vaccination in patients with myeloma suggests that there is a need to develop better immunotherapy strategies. It has been determined that the number of high-potency CMRF44+CD14-CD19- dendritic cells (DCs) in the blood of patients with myeloma (range, 0.03%-0.8% of mononuclear cells [MNCs]; n = 26) was not significantly different from that in controls (range, 0.05%-0.8% of MNCs; n = 13). Expression of the costimulatory molecules CD80 and CD86 on DCs from these patients (mean, 29%+/-17% of MNCs and 85%+/-10% of MNCs, respectively) was also normal (mean, 29%+/-17% and 86%+/-16% of MNCs, respectively). Up-regulation of CD80 expression in response to stimulation by human huCD40LT + interleukin (IL)-2 was significantly reduced on the DCs of patients with myeloma during stable disease (n = 9) and was absent during progressive stages (n = 7) of disease. Similar effects were seen on B cells but not on monocytes of the same group of patients. CD86 expression on DCs was high before (86%) and after (89%) stimulation. Inhibition of CD80 up-regulation was neutralized by either anti-transforming growth factor (TGF)-beta1 or anti-IL-10. Up-regulation of CD80 on DCs of controls was inhibited by rTGF-beta1 in a dose-dependent manner. Serum TGF-beta1 and IL-10 levels were normal in most patients studied. Cytoplasmic TGF-beta1 was increased in plasma cells during progressive disease. Thus patients with myeloma have normal numbers of DCs, but CD80 expression may fail to be up-regulated in the presence of huCD40LT because of tumor-derived TGF-beta1 or IL-10. Autologous high-potency DCs may have to be tested for CD80 up-regulation and biologically modified ex vivo before idiotype priming for immunotherapy.

The myeloma plasma cell is a postgerminal center, isotype-switched B cell. Chromosomal translocations into immunoglobulin heavy chain (IgH) switch regions, recombination sites in isotype switching, were initially demonstrated in myeloma cell lines but only a limited number of primary tumors. Molecular cytogenetics have since been applied to a series of primary tumors, in which IgH translocations accounted for many recurrent aberrations, among numerous nonrecurrent changes of unknown significance. This study, therefore, examined primary myeloma for IgH switch translocations using an established Southern blot assay that detected illegitimate switch recombinations. Sensitivity of the method was established by confining the analysis to 21 samples (4 stable, 17 progressive disease) with demonstrable legitimate isotype switches, of a total of 60 samples. Illegitimate recombinations were found in 12 or 57% (1 stable, 11 progressive) of 21 samples, comparable with estimates by molecular cytogenetics. The presence of switch translocations was supported by demonstrating up-regulated expression in myeloma marrow of cyclin D1 and fibroblast growth factor receptor 3 (FGFR3), candidate oncogenes on chromosomes 11q13 and 4p16, respectively. Illegitimate switches were detected most frequently in Smu, with more than one region involved in 6 cases. Although these results confirmed the presence of switch translocations in primary myeloma, their absence in 43% of cases may imply heterogeneity of pathogenesis. In progressive disease, there was no significant difference between patients with and without illegitimate switches in survival, nor the prognostic indicators of beta(2) microglobulin (beta(2)m) and serum thymidine kinase (STK). Hence IgH switch translocations as a single entity are unlikely to be a feature of disease progression or have prognostic significance.

The human beta-globin gene complex spans a region of 70 kb and contains numerous sequence variants. These variant sites form a 5' cluster (5' beta-haplotype) and a 3' cluster (3' beta-haplotype) with strong linkage disequilibrium among the sites within each cluster, but not between the two clusters. The 9-kb region between the 5' and 3' clusters has been estimated to have rates of recombination that are 3 to 30 times normal, and the region has therefore been proposed as a 'hotspot' of recombination. We describe three families with evidence of meiotic recombination within this 'hotspot' of the beta-globin gene cluster and in which the cross-over breakpoints have been defined at the sequence level. In one family, the recombination has occurred in the maternal chromosome within a region of 361 bp between positions -911 and -550 5' to the beta-globin gene. In the other two families, the recombination has occurred in the paternal chromosome within a region of approximately 1,100 bp between positions -542 and +568 relative to the beta-globin gene cap site. Both regions occur within the 2-kb region of replication initiation (IR) in the beta-globin gene domain with no overlap. The IR region contains a consensus sequence for a protein (Pur), which binds preferentially to single-stranded DNA, a role implicated in recombination events.

A common beta-thalassemia mutation in Asian populations is the C --> T substitution at position 654 of intron 2, which leads to the activation of two cryptic splicing sites and the incorporation of 73 extra nucleotides into the mutant mRNA. Like most beta-thalassemia mutations, it normally exhibits recessive inheritance. We investigated the unusually severe phenotype in two heterozygotes for this mutation, father and son, who had thalassemia intermedia and an apparent dominant mode of inheritance. An increased level of aberrantly spliced transcript in the reticulocytes of the probands compared with asymptomatic beta654 heterozygotes led us to investigate the production and processing of beta654 RNA. We showed that large amounts of the aberrant beta654 transcript were detectable in erythroblasts from one of the asymptomatic cases. The translation product of this mRNA was not detectable in vivo, and we were unable to demonstrate the translation of the mutant mRNA in a cell-free translation system. Although the reticulocyte alpha:beta mRNA ratios in the two probands were within the range observed in the asymptomatic heterozygotes, globin chain biosynthesis studies showed that the probands had considerably greater alpha:beta chain imbalance. These results imply that the more severe phenotype may be due to a second defect, possibly unlinked to the beta-globin cluster, that acts at the translational or posttranslational level., (Copyright 1998 by The American Society of Hematology)

While the precipitation of unstable variant beta-globin chains has been implicated as a major pathogenic mechanism in dominantly inherited beta thalassemia, their instability and presence in intra-erythroblastic inclusions have not been conclusively shown. We report the investigation of two cases of dominantly inherited beta thalassemia due to heterozygosity for the beta-codon 121 G-T mutation. In one case, we were able to demonstrate the presence of an abnormal beta-globin chain in both peripheral blood reticulocytes and bone marrow erythroblasts, and to assess its stability in relation to the substantial amounts of mutant beta mRNA transcript. The serum transferrin receptor (TfR) level was markedly increased, an indication of increased erythropoietic activity. In both cases, we could show by immunoelectron microscopy that the intra-erythroblastic inclusion bodies, a prominent feature of diseases in this category, contained not only precipitated alpha-globin chains, but also beta chains. The data confirm previous suggestions that the cellular pathology underlying this group of beta thalassemias is related to the synthesis of highly unstable beta-globin chain variants, which fail to form functional tetramers and precipitate intracellularly with the concomitant excess alpha chains, leading to increased ineffective erythropoiesis.