Immunogenicity of COVID-19 vaccines in patients with follicular lymphoma receiving frontline chemoimmunotherapy.

Immune responses to primary COVID-19 vaccination were investigated in 58 patients with follicular lymphoma (FL) as part of the PETReA trial of frontline therapy (EudraCT 2016-004010-10). COVID-19 vaccines (BNT162b2 or ChAdOx1) were administered before, during or after cytoreductive treatment comprising rituximab (depletes B cells) and either bendamustine (depletes CD4+ T cells) or cyclophosphamide-based chemotherapy. Blood samples obtained after vaccine doses 1 and 2 (V1, V2) were analysed for antibodies and T cells reactive to the SARS-CoV-2 spike protein using the Abbott Architect and interferon-gamma ELISpot assays respectively. Compared to 149 healthy controls, patients with FL exhibited lower antibody but preserved T-cell responses. Within the FL cohort, multivariable analysis identified low pre-treatment serum IgA levels and V2 administration during induction or maintenance treatment as independent determinants of lower antibody and higher T-cell responses, and bendamustine and high/intermediate FLIPI-2 score as additional determinants of a lower antibody response. Several clinical scenarios were identified where dichotomous immune responses were estimated with >95% confidence based on combinations of predictive variables. In conclusion, the immunogenicity of COVID-19 vaccines in FL patients is influenced by multiple disease- and treatment-related factors, among which B-cell depletion showed differential effects on antibody and T-cell responses.

Changes associated with the development of resistance to imatinib (STI571) in two leukemia cell lines expressing p210 Bcr/Abl protein.

BACKGROUND: Although various mechanisms have been recognized as being associated with the development of resistance to imatinib mesylate in vitro and in clinical situations, their relative significance and contributions remain poorly understood, as is the sequence of events leading to the selection of the resistant phenotype. Experimental in vitro systems involving well defined cell lines and conditions can be used to some advantage to answer specific questions and to develop in vitro models of imatinib resistance that would reflect its potential heterogeneity. METHODS: Two cell lines, KBM5 and KBM7, which expressed p210 Bcr/Abl and which differed in their inherent sensitivity to imatinib, the number of copies of the BCR/ABL fusion gene, and the activation of apoptotic pathways, were grown in vitro in the presence of increasing concentrations of imatinib. The resistant cells were analyzed for cell cycle progression, apoptotic response after exposure to imatinib, expression of Bcr/Abl, tyrosine kinase activity, and the presence of mutations within the adenosine triphosphate (ATP) coding domain of BCR/ABL. At various levels of resistance, the cells were transferred into drug-free media, and the stability of the resistant phenotype was determined in the absence of the drug. RESULTS: In KBM7 cells, the development of resistance was characterized by loss of apoptotic response to the drug, amplification of BCR/ABL, increased levels of expression of p210 Bcr/Abl, and decreased inhibition of Bcr/Abl tyrosine kinase (TK) activity by imatinib. No mutations within the ATP-binding domain of Bcr/Abl were identified, and resistance remained stable in the absence of the drug. In KBM5 cells, which previously were found to be characterized by the acquisition of a single C-T mutation at ABL nucleotide 944 (T315I) at high levels of resistance, this same mutation was detected at an intermediate level, but not at a low level, of resistance. The response of KBM5 cells to imatinib was characterized by a low level of apoptotic response, a marginal increase in BCR/ABL copy number, a modest increase in p210 expression, and a highly imatinib-resistant Bcr/Abl TK. Partial reversal of resistance was observed in highly resistant KBM5-STI571(R1.0) cells, which continued to display the C-T mutation. In KBM5 cells with an intermediate level of resistance, the T315I mutation was no longer detectable upon their reversal to the sensitive phenotype. CONCLUSIONS: BCR/ABL amplification with subsequent overexpression of Bcr/Abl protein, loss of apoptotic response, or point mutation of the ATP-binding site of BCR/ABL was associated alternatively with the acquisition of the resistant phenotype, supporting the notion that multiple mechanisms are involved in the induction of resistance to imatinib. The initial number of BCR/ABL copies itself was not related directly to the degree of resistance. The reversibility of the resistance may be complete, partial, or irreversible, depending on the mechanism(s) involved and the degree of resistance. Both cell lines serve as models for further elucidation of various aspects of imatinib-resistance mechanisms.

The proteasome inhibitor PS-341 inhibits growth and induces apoptosis in Bcr/Abl-positive cell lines sensitive and resistant to imatinib mesylate.

BACKGROUND AND OBJECTIVES: Imatinib mesylate (IM) is the choice treatment for Bcr/Abl-positive malignancies. Emergence of resistance to IM warrants the exploration of novel well-tolerated anticancer agents. We intended to evaluate the effect of PS-341 on proliferation, survival, and cellular events in Bcr/Abl-positive cells sensitive and resistant to IM, and to investigate the effect of PS-341 and IM in conjunction. DESIGN AND METHODS: Bcr/Abl-positive cell lines sensitive (p210Bcr/Abl KBM5, p210Bcr/Abl KBM7, and p190Bcr/Abl Z-119) or resistant (KBM5-R) to IM were treated with PS-341 alone or in combination with IM. The effect on cell growth was determined using the MTT assay. Cell-cycle analysis was performed by propidium iodide staining. Apoptosis was evaluated by measurement of sub-G1 DNA content, annexin V binding, and caspase 3 activity assays. Levels of apoptotic proteins, P-IkBalpha, Bcr/Abl, and phosphorylated Bcr/Abl were determined by western blotting. NF-kappaB activity was evaluated by electromobility gel shift assays. RESULTS: PS-341 exerted growth inhibition effects in IM-sensitive and -resistant cells. This phenomenon correlated with accumulation of cells in the G2/M phase of cell cycle; transient downregulation of NFkappaB DNA binding activity; downregulation of Bcl-xL; activation of caspase 3, induction of apoptosis; inhibition of expression and phosphorylation of Bcr/Abl. Sequential combination of PS-341 followed by IM demonstrated a synergistic pro-apoptotic effect in IM-sensitive cells; concomitant exposure was antagonistic. INTERPRETATION AND CONCLUSIONS: PS-341 suppresses growth and induces apoptosis in Bcr/Abl-positive cells sensitive and resistant to IM. The use of PS-341 should be explored in patients with chronic myelogenous leukemia resistant to IM. Trials of combinations of PS-341 and IM require cautious design.

Contribution of beta-2 microglobulin levels to the prognostic stratification of survival in patients with myelodysplastic syndrome (MDS).

Prospective analysis of the importance of the plasma levels of beta-2 microglobulin (B2M) in 553 patients with myelodysplastic syndrome (MDS) found that B2M is an independent prognostic variable for survival with weighted significance second only to the karyotype. The incorporation of the B2M covariate into risk assessment of MDS patients added significantly to the power of the IPSS to stratify MDS patients into risk categories. Our results further document that the 2 objectively measured covariates that display the highest power to predict survival, that is, karyotype and B2M, can alone be used for risk stratification. While the results must be verified in an independent and comparable population, our data strongly recommend routine measurement of B2M in patients with MDS.

Mutation in the ATP-binding pocket of the ABL kinase domain in an STI571-resistant BCR/ABL-positive cell line.

The major mechanism of action of STI571 is a competitive interference with the ATP-binding site of the Bcr/Abl tyrosine kinase. In the BCR/ABL-positive cell line KBM5, we studied cellular events associated with the in vitro acquisition of resistance to STI571. The emergence of the STI571-resistant phenotype was accompanied by only a marginal increase in the number of copies of the BCR/ABL gene and its level of expression. The activity of the Bcr/Abl kinase (level of autophosphorylation) in resistant cells was, however, incompletely inhibited by STI571, and the acquisition of the high degree of resistance was associated with a single-point mutation leading to a substitution of a threonine-to-isoleucine at position 315 of Abl. In the resistant KBM5-STI571(R1.0) cells, 20% of the BCR/ABL transcripts and 10% of BCR/ABL gene copies on the DNA level were mutated. The mutation was present in all 10 STI571-resistant clones derived from low density clonogenic assay, confirming its presence in all colony-forming cells but only in a fraction of the BCR/ABL gene copies in each cell. The contribution of this mutation to STI571-resistant phenotype remains unknown. Preliminary data showing partial reversibility of resistance in these cells suggest that resistance may be multifactorial. No other mutations were identified in the kinase domain of the BCR/ABL gene.