ALBERT

Description: Dendritic cells (DCs) are the main antigen presenting cells and play a pivotal role in the stimulation of T-cell immune responses. DCs cultured in the presence of a single tumor antigen can elicit an immune response against tumor cells expressing that antigen. However, simultaneous use of several tumor antigens may be advantageous since polyclonal activation of T cells against different tumor antigens may be a better approach to eradicate tumor cells. In this sense, fusions of dendritic and tumor cells (FCs) show a broad spectrum of tumor antigens, both known and unidentified, to be presented by class I and II MHC. Although prophylactic vaccines were successful in murine models, the results in the therapeutic setting have been unsatisfactory. We hypothesised that enhancing costimulation of FCs would help to break tumor tolerance once the tumor is established. To this purpose, we transduced FCs with a recombinant adenovirus encoding CD40L (AdvCD40L or AdvGFP as control) and we studied the therapeutic antitumoral effect of the administration of FC-CD40L in a murine model of myeloma. DCs obtained from day 7-bone marrow cultures of Balb/c mice were fused with tumor cells, a syngeneic murine myeloma cell line (4TOO). FCs hybrids were generated with PEG and selected after culturing in HAT medium plus GM-CSF for 7 days. FC were quantified by determining the percentage of cells that coexpress specific DC (CD11c) and tumor markers (CD138). Mean fusion efficiency was 30% (20–40%) and FCs expressed moderate levels of CD80, CD83, CD86, CD54, CD40 and MHC II and did not express CD40L. FC-CD40L showed a significant increase of expression of costimulatory molecules (CD80, CD86, CD54, and MHC II) compared to FC-GFP (p=0.011). Moreover, in a syngeneic mixed lymphocyte reaction, FC-CD40L induced a two-fold higher T-cell proliferation than FC-GFP or FC alone. In addition, FC-CD40L had improved migration to lymphoid tissues, preferentially to spleen, in comparison with FC-GFP (2.8% versus 1.6%). The antitumor effect of FC-CD40L was analyzed in vivo. Mice (n=10 per group) were injected i.v. with 2.5×105 tumor cells and treated with irradiated FC, FC-GFP or FC-CD40L (1×106 cells each) on days 2, 6 and 10 after tumor challenge. 40% of mice treated with FC-CD40L had long-term survival (〉120 days). In contrast, all of mice treated with FC or FC-GFP died between days 25 and 35 (p=0.012). In parallel, treatment with mixed cells (not fused DC+ tumor cells), mix transduced with AdvGFP, or mix transduced with AdvCD40L did not provide any significant antitumor effect. We conclude that FCs transduced with AdvCD40L better stimulate in vitro and in vivo immune responses than FC alone and may provide a new strategy for treating patients with multiple myeloma or lymphoma.

Description: Introduction: Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous clonal plasma cell disorder. A number of prognostic factors to identify patients at a higher risk of progression have been described, such as the size of the M protein, proportion of abnormal bone marrow plasma cells (BMPCs), immunoparesis and serum free light chain (FLC) k/l ratio. More recently, isotype-specific uninvolved heavy and light chain (HLC) pair suppression measured with the Hevylite assay was also associated with an increased risk of progression. Recent studies have evaluated the key prognostic impact of an increase in M-protein levels during follow-up ("evolving" pattern). However, an important limitation could be the evaluation of M-protein level variations based on serum protein electrophoresis (SPE) in patients with a small size M-spike. The aim of this study was to prospectively analyze the changes in M-protein according to SPE and HLC measurements, as well as other risk factors for progression, in patients with SMM. Methods: Thirty patients newly diagnosed with SMM at a single institution from January 2014 through September 2017 were prospectively included in the study. For each patient, baseline levels of known prognostic factors (serum M-protein, serum and urine immunofixation, clonal BMPCs percentage, total immunoglobulins, involved/uninvolved FLC and involved/uninvolved HLC pairs) were recorded. During the follow up, M-protein level, FLC and isotype specific HLC pairs were also analyzed. Evolving change in M-protein level according to SPE was defined as ³ 10% increase within the first 6 months of diagnosis (if M-protein was ³ 30 g/L) and/or ³ 25% increase within the first 12 months (for any level of M-protein); evolving change according to HLC was defined as a ³ 10% increase in the involved pair. A sequential increase in each of three or more consecutive measurements from diagnosis was considered an evolving change regardless of its magnitude. Results: The clinical characteristics of the total of patients, as well as of the patients with evolving changes in M-protein according to HLC are summarized in Table 1. During the study period, 5/30 (17%) of patients demonstrated an evolving behavior of the M-protein according to SPE. Four of these patients (4/5) also showed a progressive increase in the M-protein in the HLC measurements. One patient showed stable HLC levels even though both the M-protein and the involved FLC progressively increased. This patient was of intermediate and low risk according to Mayo Clinic and PETHEMA scores, respectively. On follow up, no progressive suppression of the isotype-specific uninvolved HLC pair or increase in the FLC ratio was noted, and there have been no signs of progression after a follow up of 3 years. According to involved HLC-pair levels, 12/30 (40%) of patients demonstrated an evolving behavior. Five out of 7 patients that were not classified as evolving by SPE, were IgA isotype. Eight out of 12 patients showed severe isotype-specific suppression of the uninvolved HLC-pair (〉 50% below lower level of normal) as well as a highly abnormal FLC ratio (8). Three out of the 4 remaining patients showed either severe isotype-specific HLC pair suppression or highly abnormal FLC ratio in follow up measurements. Compared to patients with no "HLC-evolving pattern", evolving patients were more likely to have highly abnormal FLC ratios (90 vs. 33%, p=0.009), severe suppression of the other isotypes (64 vs. 19%, p=0,024), highly abnormal isotype-specific HLC ratios (67 vs. 33%, p=NS), severe isotype-specific HLC-pair suppression (75 vs. 50%, p=NS), and immunoparesis (67 vs. 39%p=NS). Five patients progressed to symptomatic multiple myeloma during follow up; 4 of them showed a progressive increase in the involved HLC pair from diagnosis. The remaining patient demonstrated a progressive increase in the involved HLC pair that started 19 months prior to progression, followed 4 months later with an increase in M-protein as measured by SPE. Conclusions: In our series, the Hevylite assay allowed us to identify patients with a progressive increase in M-protein (clonal heavy/light chain pair) that was not evident with SPE measurements. This "HLC evolving pattern" was associated with other risk factors for progression to symptomatic disease and with worsening of other prognostic parameters during follow up. Disclosures Rosinol: Janssen, Celgene, Amgen, Takeda: Honoraria. Bladé:Janssen: Honoraria.

Description: Introduction Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism in the clinical activity of rituximab for the treatment of B-cell malignancies. The NK cells, through the activating receptor FcγRIIIa, play a major role in the rituximab-mediated ADCC. We have studied the effect of NK stimulators such IL-15 and CpG oligodeoxynucleotides (ODN) in the enhancement of rituximab-mediated ADCC against B-cell lymphoma. Methods ADCC was performed by standard 51Cr release assays using peripheral blood mononuclear cells (PBMC) purified from 4 normal volunteers by Ficoll-Hypaque gradient centrifugation. Cells were cultured for 18 hours in the presence of human recombinant interleukin 15 (rhIL-15; 10 ng/ml) or CpG ODN (2,5 μg/ml). The B-lymphoma cell line Raji was used as target in the presence of rituximab (10 μg/ml) or human IgG1 as control (10 μg/ml) for 30 min at room temperature. Target cells and effector cells were incubated at different ratios ranging from 1:1 to 30:1, for 4 hours at 37°C. The polymorphism at amino acid 158 of the activating FcγRIIIa receptor (FcγRIIIa V→F) was analyzed in effector cells from normal donors with a polymerase chain reaction (PCR)-based allele-specific restriction analysis assay. Results Lysis of B lymphoma cells treated with rituximab alone was 40% ± 5% (mean ± SD) at the highest ratio, as compared to 12% ± 7% (p

Description: BACKGROUND Smoldering multiple myeloma (SMM) is an asymptomatic and biologically heterogeneous clonal plasma cell disorder. Prognostic factors to identify patients at higher risk of progression have been described, including the suppression of uninvolved polyclonal immunoglobulins (Ig) or immunoparesis (IP). Novel serum assays allow the quantification of each isotype-specific heavy and light chain pair (HLC), enabling the study of a new type of IP: the suppression of the uninvolved light chain counter part of the same heavy-chain Ig isotype (isotype matched immunoparesis). AIM: To prospectively characterize the prevalence and severity of immunoparesis as determined by HLC measurements, its association with other risk factors for progression and its prognostic significance, in patients with SMM diagnosed at a single institution. METHODS: Fifty-four patients newly diagnosed with SMM from January 2014 through March 2019 were prospectively included. Serum samples obtained at baseline and during follow-up were tested for M-protein level, free light chain (FLC) and HLC concentrations (all three isotype pairs at baseline, involved isotype pair at follow up visits). Isotype matched immunoparesis (IMI) was defined by the HLC assay, based on the levels of the Ig with the same heavy chain of the monoclonal isotype but of the alternative light chain (e.g. IgG-kappa suppression in a patient with IgG-lambda SMM). Aditionally, HLC immunoparesis (IP) was defined by suppression of any Ig with a heavy chain different to the M-protein (e.g. IgA kappa/lambda pair suppression in a patient with IgG-lambda SMM). Severe immunoparesis was defined by Ig values suppressed by 50% or greater below the lower limit of normal (LLN). RESULTS: Median age at diagnosis was 73 years, with the following heavy chain isotype distribution: 31 IgG, 20 IgA, 2 biclonal IgG-IgA and 1 light-chain only SMM. Median follow up for the series was 2.5 years. Isotype specificity of immunoparesis was reflected in the greater prevalence of IMI vs. noninvolved isotype IP (Figure 1, A). HLC measurements identified IMI in 42 out of the 53 patients with IgG, IgA or biclonal SMM (82%), with severe IMI observed in 27 patients (53%). Suppression of at least one HLC pair of an uninvolved isotype was present in 37 patients (72%), with severe suppression observed in 20 patients (38%). Eleven patients presented severe IMI without severe IP of uninvolved isotypes. On the other hand, only one out of the 37 patients with IP of uninvolved isotypes presented without IMI. In the case of the 12 patients that showed IP of any IgM HLC pair, they all had concomitant severe IMI and IP of the other uninvolved heavy chain isotype. Analysis of other risk factors for progression between groups of patients with i) no severe immunoparesis (n=22), ii) severe IMI without severe IP of uninvolved isotypes (n=11) and iii) severe IMI and severe IP of one or more HLC pairs of uninvolved isotypes (n=18), showed significantly different FLC ratios and lower % of normal plasma cells in bone marrow (Figure 1, B). There was a trend, although statistically not significant, towards higher M-protein levels. Progression to malignant symptomatic gammopathy was observed in 12 patients. The suppression of any IgM HLC pair was associated with a shorter time to progression (p=0.007; HR=4.2; 95% CI, 0.84-21) (Figure 1, C). Severe IMI alone or severe IMI plus severe IP of a different isotype were associated only with a statistically not significant trend towards a shorter time to progression. Eight patients demonstrated an evolving behavior (≥ 10% increase in the involved HLC pair in 3 or more consecutive determinations from diagnosis), 7 of which presented with severe IMI at diagnosis. Of the five evolving patients that progressed, four had severe IMI and the remaining one developed severe IMI during follow up. Patients with severe IMI at diagnosis maintained the same level of HLC suppression throughout the follow up period. CONCLUSION: The significantly greater incidence of IMI over IP of uninvolved isotypes pairs supports an isotype specificity of early Ig suppression mechanisms in the case of IgG and IgA SMM. That would be of special interest at the time of initial evaluation of patients with SMM using HLC pairs. Both IMI and IP of uninvolved isotypes are associated with other recognized risk factors for progression, but the later appears to develop with more advanced disease and associate with a higher risk of progression. Disclosures Cibeira: Amgen: Honoraria, Other: Educational lectures; Celgene: Honoraria, Other: Educational lectures; Akcea Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational lectures. Bladé:Oncopeptides: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Takeda: Honoraria.

Description: An amendment to this paper has been published and can be accessed via a link at the top of the paper.