Len-dex Treatment Research Articles

Low Frequency of CD161+CD4+ T Cells Correlate with the Occurrence of Infections in Refractory/Relapsed Multiple Myeloma Patients Receiving Lenalidomide Plus Low-Dose Dexamethasone Treatment

Background Although the combination of lenalidomide and low-dose dexamethasone (Len-dex) is known to preserve the efficacy with reduced toxicity than lenalidomide plus high-dose dexamethasone (Len-Dex) in patients with refractory/relapsed multiple myeloma (RRMM), infection is still a leading toxicity. Moreover, the patterns and risks for infection in patients with RRMM during Len-dex treatment remain unclear and there is a need to identify contributing factors associated with increased risk for infection. Considering the disease-related and treatment-related immune deficits in patients with RRMM, we explored the predictive implications of the revelation of the immune cell populations prior to Len-dex initiation for the occurrence of infection. In addition, the various clinical and laboratory parameters were analyzed. Methods Clinical and microbiology records of 90 RRMM patients during Len-dex treatment were reviewed and risk factors for infection were analyzed using the logistic regression. In addition, to develop the new immune cell biomarker, we prospectively examined immune cell populations (CD3, CD4CD161, CD8CD161, Lin-HLA-DR-CD11b+CD33+, CD14+HLA-DR-, NK and NKT cells) of the peripheral blood taken on baseline of Len-dex therapy. Results Forty-eight men and 42 women were enrolled in this study. The median age was 61 years (range, 29-84 years). During a median 11 cycles of Len-dex treatment, 52 (57.8%) patients experienced at least 1 infection episode. Of a total of 92 episodes of infection, 58 (63%) episodes were clinically defined, 29 (31.5%) episodes were microbiologically defined, and 5 (5.4%) episodes were fever of unknown focus. Severe episodes were frequently observed during early 3 cycles. In the univariate analyses, lower Hb ( + CD161 + cells in peripheral blood prior to Len-dex were associated with the occurrence of infection, especially during early 3 cycles of Len-dex therapy. Conclusions We demonstrated several clinical predictive factors for the occurrence of infection in patients with RRMM receiving Len-dex treatment. And we found that the frequency and absolute count of CD4 + CD161 + cells may provide additional information for predicting the occurrence of infection in early period of Len/dex therapy. Disclosures No relevant conflicts of interest to declare.

Low Frequency of CD161+CD4+ T Cells Correlate with the Occurrence of Infections in Refractory/Relapsed Multiple Myeloma Patients Receiving Lenalidomide Plus Low-Dose Dexamethasone Treatment

Background Although the combination of lenalidomide and low-dose dexamethasone (Len-dex) is known to preserve the efficacy with reduced toxicity than lenalidomide plus high-dose dexamethasone (Len-Dex) in patients with refractory/relapsed multiple myeloma (RRMM), infection is still a leading toxicity. Moreover, the patterns and risks for infection in patients with RRMM during Len-dex treatment remain unclear and there is a need to identify contributing factors associated with increased risk for infection. Considering the disease-related and treatment-related immune deficits in patients with RRMM, we explored the predictive implications of the revelation of the immune cell populations prior to Len-dex initiation for the occurrence of infection. In addition, the various clinical and laboratory parameters were analyzed.Methods Clinical and microbiology records of 90 RRMM patients during Len-dex treatment were reviewed and risk factors for infection were analyzed using the logistic regression. In addition, to develop the new immune cell biomarker, we prospectively examined immune cell populations (CD3, CD4CD161, CD8CD161, Lin-HLA-DR-CD11b+CD33+, CD14+HLA-DR-, NK and NKT cells) of the peripheral blood taken on baseline of Len-dex therapy.Results Forty-eight men and 42 women were enrolled in this study. The median age was 61 years (range, 29-84 years). During a median 11 cycles of Len-dex treatment, 52 (57.8%) patients experienced at least 1 infection episode. Of a total of 92 episodes of infection, 58 (63%) episodes were clinically defined, 29 (31.5%) episodes were microbiologically defined, and 5 (5.4%) episodes were fever of unknown focus. Severe episodes were frequently observed during early 3 cycles. In the univariate analyses, lower Hb (<10 g/dL) and serum albumin (<3.5 mg/dL), and higher serum creatinine (≥2 mg/dL) were associated with increased risk of infections (≥grade 3) during early 3 cycles. After adjusting for risk factors for infection on univariate analyses, multivariate analyses showed that lower Hb (<10 g/dL) was an independent factor for the occurrence of infections and lower frequency (P = 0.009) and absolute count (P = 0.072) of CD4+CD161+ cells in peripheral blood prior to Len-dex were associated with the occurrence of infection, especially during early 3 cycles of Len-dex therapy.Conclusions We demonstrated several clinical predictive factors for the occurrence of infection in patients with RRMM receiving Len-dex treatment. And we found that the frequency and absolute count of CD4+CD161+ cells may provide additional information for predicting the occurrence of infection in early period of Len/dex therapy. DisclosuresNo relevant conflicts of interest to declare.

Circulating immune cell phenotype can predict the outcome of lenalidomide plus low-dose dexamethasone treatment in patients with refractory/relapsed multiple myeloma.

Although the antimyeloma effect of lenalidomide is associated with activation of the immune system, the exact in vivo immunomodulatory mechanisms of lenalidomide combined with low-dose dexamethasone (Len-dex) in refractory/relapsed multiple myeloma (RRMM) patients remain unclear. In this study, we analyzed the association between immune cell populations and clinical outcomes in patients receiving Len-dex for the treatment of RRMM. Peripheral blood samples from 90 RRMM patients were taken on day 1 of cycles 1 (baseline), 2, 3, and 4 of Len-dex therapy. Peripheral blood CD3(+), CD4(+), and CD8(+) cell frequencies were significantly decreased by 3 cycles of therapy, whereas NK cell frequency was significantly increased after the 3rd cycle. For the myeloid-derived suppressor cell (MDSC) subset, the frequency of granulocytic MDSCs transiently increased after the 1st cycle, whereas there was an increase in monocytic MDSC (M-MDSC) frequency after the 1st and 3rd cycles. Among 81 evaluable patients, failure to achieve a response of VGPR or greater was associated with a decrease in CD8(+) cell frequency and increase in M-MDSC frequency after 3 cycles of Len-dex treatment. A high proportion of natural killer T (NKT)-like cells (CD3(+)/CD56(+)) prior to Len-dex treatment might predict a longer time to progression. In addition, patients with a smaller decrease in the frequency of both CD3(+) cells and CD8(+) cells by 3 cycles exhibited a longer time to the next treatment. These results demonstrated that early changes in immune cell subsets are useful immunologic indicators of the efficacy of Len-dex treatment in RRMM.

Impact of Lenalidomide Plus Low-Dose Dexamethasone Treatment on Immune Cell Populations of the Patients with Refractory/Relapsed Multiple Myeloma

Background: Lenalidomide combined with low-dose dexamethasone (Len-dex) is an effective treatment for the patients with refractory/relapsed multiple myeloma (RRMM). The anti-myeloma effect of lenalidomide is associated with activation of the immune system, but the exact immunomodulatory mechanisms in vivo and clinical impact of Len/dex in RRMM patients remains unclear. In this study, we analyzed immune cell populations in patients receiving Len-dex for the treatment of RRMM.Methods: Peripheral blood samples from 90 RRMM patients were taken on day 1 of cycles 1 (baseline), 2, 3, and 4 of Len/dex therapy. CD3+, CD4+, CD8+, CD161+ T cells, natural killer (NK) cell (CD16+/CD56+), NKT-like cell (CD3+/CD56+) and myeloid-derived suppressor cell (MDSC) including granulocytic (G-MDSC) and monocytic (M-MDSC) were analyzed by flow cytometry. In addition, response was assessed in 81 patients receiving more than 4 cycles of Len-dex and the comparison of cell populations according to an achievement of ≥very good partial response (VGPR) was performed.Results: Forty-eight men and 42 women were enrolled in this study. The median age was 61 years (range, 29-84 years). At baseline, peripheral blood CD3+ cell frequency was 51.65 ± 1.79% which was significantly decreased to 41.67 ± 2.44% (P=0.001) and 39.72 ± 2.90% (P< 0.001) after 2 and 3 cycles of therapy, respectively. Frequency of both CD4+ cell and CD8+ cells was also significantly decreased by 3 cycles of therapy, while NK cell frequency was significantly increased after Len-dex treatment (P<0.05). For the T-cell subset, the frequency of CD8+ CD161high cells was significantly decreased (1.13 ± 0.16% at baseline to 0.65 ± 0.13% at post-3 cycles, P< 0.05), while no trend was observed in CD4+ CD161+ cell frequency. No significant change was observed in frequency of G-MDSC and M-MDSC after Len-dex. Among 81 evaluable patients, 36 patients obtained ≥VGPR and 45 ≤ partial response. After adjusting for factors affecting failure of achieving a response of ≥VGPR on univariate analyses, multivariate analyses showed that decrease in CD8+ cell frequency (P=0.043) and increase in M-MDSC frequency (P=0.033) by post-3 cycles of Len-dex treatment were predictors for failure of achieving ≥VGPR. High frequency of NKT-like cell prior to Len-dex treatment could predict a longer time to progression (RR of 0.40, P=0.011). In addition, patients with less decrease in frequency of both CD3+ cell and CD8+ cells by post-3 cycles had a longer time to next treatment (RR of 0.24, P=0.024 and RR of 0.33, P=0.044, respectively).Conclusion: Our data demonstrate that Len-dex therapy in patients with RRMM is associated with decreased frequency of T cells with a trend of increased NK cell frequency. Change in CD8+ cell and M-MDSC frequency can correlate with the quality of response to Len-dex. Baseline NKT-like cell frequency and change in CD3+ and CD8+ cells early after treatment may predict continuation of anti-myeloma effect of Len-dex therapy. DisclosuresNo relevant conflicts of interest to declare.

Lenalidomide Is Effective Treating Patients With Relapsed Multiple Myeloma and Very Severe Renal Impairment

IntroductionPatients with multiple myeloma (MM) and severe renal impairment (SRI) have shorter survival and are often excluded from clinical trials. In these patients the use of Lenalidomide (Len) is based in pharmacokinetic models and because of the risk of increased toxicity, Len dose must be adjusted to renal function. PurposeTo evaluate the outcomes of relapsed MM patients (RMM) with SRI (baseline creatinine clearance (CrCl) <30ml/min) treated with Lenalidomide-dexamethasone (Lendex), including patients under hemodyalisis. MethodsWe conducted a retrospective multicenter study in four Portuguese referral hospitals. All consecutive patients with RMM and SRI treated with Lendex between January 2007 and December 2012 were included. Lendex treatment was considered as the administration of low doses of dexamethasone and Len doses between 25mg/d and 5mg every other day. We evaluated the efficacy (response rate (RR), response duration and impact on renal function) and the relevant toxicities (myelo and neurotoxicity, thrombotic and infectious complications and second primary tumors). ResultsWe retrospectively analyzed 22 patients with SRI and RMM treated with Lendex (median CrCl at start of Lendex was 19 ml/min with 13 patients under dialysis): 55% males; median age at diagnosis 63yo; median age at start of Lendex 68yo (51-79); main subtypes were IgGk (45%) and IgGλ (18%); 3 patients with high-risk cytogenetics; 5 plasmocytomas; 1 amyloidosis; 77% received Lenalidomide ≤10mg/d x 21 days in monthly cycles; median 8 cycles (2-50). Median number of previous lines was 2 (1-6). The global RR was 72% (16/22; 12 patients with ≥PR); 6 pts had progressive disease. Five out of 6 (83%) of the patients who progressed with Lendex had prior exposure to thalidomide. Median follow-up from start of Lendex was 14 months (2 – 62). The median time until maximal response was 3 months and the median response duration was 31 months (5 – 55) and in 58% (7/12) the response was longer than 2 years. Eighteen percent had renal improvement (median CrCl improvement of 44ml/min) but all the 13 patients on hemodyalisis maintained the need for this treatment. The treatment was interrupted in 3 cases because of adverse events and all the patients were on thromboprophylaxis with aspirin. In the long-term responders a continuous Len dose adjustment was required mainly because of myelo and neurotoxicity and the dexamethasone dose was reduced according to toxicities (metabolic, fatigue and psychiatric effects). There were no critical infections or second primary tumors. ConclusionsCrCl<60 ml/min is a cut-off for Lenalidomide dose reduction. In our experience, after initial dose-adjustment of therapy, there should be a continuous process of dose-adjustment taking into account the individual tolerance, even without additional renal impairment. Lendex dosing adjusted according to CrCl does not negatively impact response or the rate of side effects. It may also have a potential to reverse RI and induce long duration of response. We concluded that Lendex is an effective and safe treatment regimen for relapsed MM patients with severe renal insufficiency. Disclosures:Joao:yassen: member of advisory board, member of advisory board Other; Celgene: member of advisory board, member of advisory board Other. Esteves:Celgene: member of advisory board, member of advisory board Other; Yassen: member of advisory board, member of advisory board Other.

Flow cytometry-based enumeration and functional characterization of cd8 t regulatory cells in patients with multiple myeloma before and after lenalidomide plus dexamethasone treatment

Multiple myeloma (MM) is a malignancy of plasma cells frequently associated with immune abnormalities. Several studies have confirmed that in MM immune deregulation can be mediated by increased numbers of CD4 T regulatory (Treg) cells, and these cells were also associated with poor outcome. In this study, we aimed to study CD8 Treg cells before and after lenalidomide plus dexamethasone (len-dex) treatment in MM patients. Using flow cytometry, we enumerated and assessed suppressive function of CD8 Treg cells in 16 MM patients before and after len-dex treatment. Numbers of CD8 Treg cells (CD8+CD25hi+FoxP3+) (P < 0.01) were significantly increased in MM patients (before treatment) compared to healthy donors. However, no significant changes were observed in CD4 and CD8 T cells. A significant increase in CD8 Treg cells was observed after len-dex treatment compared to pre-treatment but no significant difference was observed in CD4 and CD8 T cells. Proliferation assay data showed that CD8 Treg cells inhibited proliferation of CD4 T cells and IFN-γ secretion in a concentration dependent manner. Suppressive activity of CD8 Treg cells did not differ significantly between healthy donors, untreated and len-dex treated MM patients. A significant abnormal level of IL-10 was observed from proliferation assays of untreated and len-dex treated MM patients compared to healthy donors (P ≤ 0.03). Using flow cytometry, we have shown that suppressive CD8 Treg cells are increased in MM patients and len-dex treatment is unable to control these suppressive CD8 Treg cells.

Flow cytometry based enumeration and functional characterization of CD8 T regulatory cells in patients with multiple myeloma before and after lenalidomide plus dexamethasone treatment.

Multiple myeloma (MM) is a malignancy of plasma cells frequently associated with immune abnormalities. Several studies have confirmed that in MM immune deregulation can be mediated by increased numbers of CD4 T regulatory (Treg) cells, and these cells were also associated with poor outcome. In the present study, we aimed to study CD8 Treg cells before and after lenalidomide plus dexamethasone (len-dex) treatment in MM patients. Using flow cytometry, we enumerated and assessed suppressive function of CD8 Treg cells in 16 MM patients before and after len-dex treatment. Numbers of CD8 Treg cells (CD8+CD25hi+FoxP3+) (P<0.01) were significantly increased in MM patients (before treatment) compared to healthy donors. However, no significant changes were observed in CD4 and CD8 T cells. A significant increase in CD8 Treg cells was observed after len-dex treatment compared to pre-treatment but no significant difference was observed in CD4 and CD8 T cells. Proliferation assay data showed that CD8 Treg cells inhibited proliferation of CD4 T cells and IFN-γ secretion in a concentration dependent manner. Suppressive activity of CD8 Treg cells did not differ significantly between healthy donors, untreated and len-dex treated MM patients. A significant abnormal level of IL-10 was observed from proliferation assays of untreated and len-dex treated MM patients compared to healthy donors (P≤0.03). Using flow cytometry, we have shown that suppressive CD8 Treg cells are increased in MM patients and len-dex treatment is unable to control these suppressive CD8 Treg cells. © 2013 Clinical Cytometry Society.

Targeted Repression of TP53 Promotes Erythropoiesis in Del(5q) MDS and Overcomes Clinical Resistance to Lenalidomide

AbstractAbstract 920 Background:Stabilization of p53 as a result of ribosomal stress is a key effector of the hypoplastic anemia in del(5q)MDS. We recently reported that lenalidomide (LEN) stabilizes MDM2 to promote p53 degradation, fostering cell cycle transition and arrest of del(5q) progenitors in G2/M (Wei S, et. al. Oncogene 2012). P53 expression in erythroid precursors (EP) decreased in responders, but was up-regulated upon emergence of resistance to LEN (Wei S, et. al. PNAS 2010). We hypothesized that targeted suppression of p53 may be an effective strategy to restore erythropoiesis in del(5q)MDS EP after LEN-treatment failure. Methods:To test this hypothesis, we first investigated the in vitro effects of cenersen (Aezea®, Eleos Inc. Plymouth, MN), a 20mer phosphorothioate antisense oligonucleotide complementary to the exon 10 coding sequence of the TP53 gene transcript, on cellular p53 expression and colony forming capacity (CFC) in EP. Cenersen, through RNase H-mediated site specific mRNA cleavage, down regulates wild type & mutant TP53 mRNA and protein expression. Using a 2-stage EP differentiation assay, CD34+ cells from MDS patient specimens (del5q, n=11; non-del5q, n=4; normal donor, n=5) were cultured in StemSpan® SFEM expansion medium (StemCell Technologies, Vancouver, Canada) supplemented with SCF, EPO, and dexamethasone for 3–10 days, then transfected with 40nM cenersen or scrambled oligodeoxynucleotides using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA). Cells were harvested after 3 days for analysis of p53 expression by fluorescence microscopy, FISH and methylcellulose cultures. CFC was assessed after 7–14 days incubation. Results:Del(5q) EP displayed greater nuclear p53 fluorescence intensity (FI) than non-del(5q)MDS or controls [del(5q) mean =82.12±11.4, non-del(5q)=41.55±13.05, controls=63.58±10.14, P=0.07; del(5q) vs. controls, P=0.33]. Treatment with cenersen significantly reduced nuclear (> 50%) and cytoplasmic p53 expression (> 59%) in EP in 9 of 11 del(5q) specimens, (mean nuclear FI, cenersen=41.75±9.02 vs. non-sense=86.84±13.12, P=0.01; mean cytoplasmic FI, cenersen= 23.38±3.14 vs. non-sense= 41.34±6.21, P=0.02). Similar but non-significant trends in nuclear p53 were observed in control (P=0.19) and non-del5qMDS (P=0.29) EP, whereas cytoplasmic p53 was significantly reduced only in controls (>52%, P=0.03; non-del5q, P=0.35). Cenersen treatment significantly increased recovery of BFU-E and CFU-E (n=15, P=0.01; n=13,P=0.0001, respectively) in MDS specimens, whereas there were no consistent changes in granulocyte-macrophage or mixed lineage colony yields. Similar but less robust improvements in CFC were observed in control samples with cenersen treatment (CFU-E, n=4, P=1.0; BFU-E, n=5, P=0.43). The magnitude of improvement in BFU-E in MDS specimens highly correlated with the extent of p53 suppression (nuclear Spearman r= -0.64, P=0.009; cytoplasmic r= -0.65, P=0.008). Changes in CFU-E showed no significant correlation with changes in nuclear (P=0.65) or cytoplasmic (P= 0.89) p53 expression. FISH analysis of cytospin preparations following oligonucleotide treatment of del(5q) EP using the EGR1 5q31/5p15.31 deletion probe (Cytocell, Cambridge UK) showed no significant differences between cenersen and non-sense treated cells (P=0.55, n=10). To determine if in vivo p53 suppression can restore transfusion independence (TI) after secondary LEN treatment failure, we treated 8 LEN-resistant, transfusion-dependent del(5q) MDS patients with dexamethasone (Dex) 20 mg weekly and continued LEN 5–10 mg/day. The glucocorticoid receptor acts as a ligand-dependent transcription factor and mutual antagonist of p53. All patients had <5% myeloblasts and Int-1 IPSS, whereas 3 patients had del(5q)+1 additional chromosome abnormality. Five of 8 patients achieved TI with LEN-Dex treatment. There was no significant reduction in the proportion of del(5q) metaphases after achievement of TI in responders. Median duration of LEN-Dex treatment was 4 months, and 9 months in responders (n=5). None of the patients experienced AML progression. Conclusions:Targeted suppression of p53 restores effective erythropoiesis in del(5q) MDS in the absence of clonal suppression. A clinical trial testing treatment with cenersen +Dex in LEN-resistant patients is currently in development. Disclosures:Smith:Smith Holdings LLC: Equity Ownership, Smith Holdings owns rights to the drug involved in this study. Patents & Royalties; Eleos, Inc: Eleos has licensed rights to the drug from Smith Holdings. Patents & Royalties. Daugherty:Eleos, Inc: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Wells:Alexion: Honoraria; Janssen: Honoraria; Celgene : Honoraria; Novartis: Honoraria, Research Funding. List:Celgene: Consultancy. Off Label Use: lenalidomide combined with Dexamethasone in lenalidomide refractory patients .

Efficacy and safety of lenalidomide and dexamethasone in overtreated multiple myeloma patients.

e18566 Background: Lenalidomide is a therapeutic option for pretreated patients with multiple myeloma, especially in association with steroids. Methods: Oraldaily lenalidomide (10-25 mg) for 21 days every month (median courses 8.6) and weekly dexamethasone (Dex) were administered until progression in 33 pretreated patients (median age 68.1 yrs) observed between June 2008 and January 2012 at Hematology Department of Sant’Andrea Hospital in Rome. Previous treatment included alkylants, anthracyclines, IFN-α, thalidomide, bortezomib and single or double autograft. Fifteen patients were evaluated after 6 courses of therapy, administered as second to fifth line, and prospectively followed-up; two patients weren’t considered because of early death. Treatment response was assessed according to IMWG uniform response criteria. Results: The overall response rate was 53.3% (8/15 pts) with a median duration of response of 12 months: 8 patients reached a partial response (PR), 6 showed a stable disease (SD) and 1 progressive disease (PD). The median progression free survival was 14 months. Among patients in PR, 5 are alive in continous treatment, 2 died due to PD after 17 courses administered as fifth line therapy and 1 died for unrelated surgical complicancies. The whole responses were achieved indifferently if lenalidomide was administered as second, third or fifth line of therapy (2, 3 and 3 patients respectively). Out of patients in SD, all experienced progressive disease after a median of 12 courses: 3 are still alive in salvage therapy, 3 died during treatment. The only patient in PD died 9 months after the end of Len-Dex treatment during salvage chemotherapy. The reported side effects included pneumonia, neutropenia, diarrhea, peripheral sensitive polyneuropathy, transient liver failure, lower urinary tract infection, skin rash, transient aphasia, conjunctival hemorrhage and deep vein thrombosis, all resolved with brief discontinuance and specific support therapy without hospitalization. Conclusions: Len-Dex association is a completely oral effective salvage therapy with good response rate and manageable side effects also for overtreated relapsed and refractory multiple myeloma patients.

Influence of Lenalidomide Treatment on Immune Effector Cells From High-Risk Smoldering Multiple Myeloma (SMM) Patients,

Abstract 3944Lenalidomide is an immunomodulatory agent that enhances T and NK cell activation, being this consideration as a major player in its anti-myeloma effect. However, in MM lenalidomide is usually combined with the immunosuppressant dexamethasone, which has raised questions regarding a potential abrogation of this immunomodulatory effect. In fact, this may be a dilemma upon treating early stage MM patients with lenalidomide +/− dexamethasone. Moreover, our current knowledge of the immune system in SMM is limited.Herein we evaluated by multiparameter flow cytometry (MFC) immunophenotyping peripheral blood (PB) T and NK cells from high-risk SMM patients (N=33), treated according to the QUIREDEX trial (NCT 00480363): an induction phase of 9 four-week cycles of LenDex followed by maintenance with lenalidomide until disease progression. To evaluate the immune status of T and NK cells of SMM patients, we compared them at baseline vs healthy adults (HA) aged over 60 years (N=10). To assess the effect of LenDex on T and NK cells of SMM patients, we compared baseline samples vs those studied after 3 and 9 cycles of LenDex. To address the question whether dexamethasone antagonizes the immunomodulatory properties of lenalidomide, we compared in 11 of the 33 patients, the PB T and NK cells at the end of induction (9th cycle of LenDex) vs during maintenance (lenalidomide alone and at least 3 months after dexamethasone discontinuation).The percentage of PB T cells in high-risk SMM patients at baseline was increased when compared to HA (23% vs 17%; P=.02), mainly due to expansion of CD8 T cells (P=.03). Of note, γδ T cells were also increased in SMM (0.8% vs 0.3%; P=.003). In turn, no differences (P>.05) were noted for both the CD56dim and CD56bright NK cell compartments. However, when a more detailed immunophenotypic characterization was carried out, CD4 and/or CD8 T cells from SMM patients showed decreased expression of activation markers (CD25, P≤.04; CD54, P<.001 and CD154, P=.002), as well as decreased production of the Th1 related cytokines (IFNγ, P=.03; TNFα, P≤.003; and IL-2, P=.02).We then investigated the effect of LenDex treatment. After 3 and 9 cycles of LenDex both CD4 and/or CD8 T cells showed up-regulation of Th1related chemokines (CCR5; p<.001) and cytokine production (IFNγ, P=.03; TNFα, P=.03 and IL-2, P=.02), as well as increased expression of activation markers (CD69, P≤.005; CD25, P<.001; CD28, P≤.04; CD54, P<.001 and HLA-DR, P<.001). Similarly, CD56dim and CD56bright NK cells showed up-regulation of HLA-DR (P<.001), the antibody-dependent cell-mediated cytotoxicity associated receptor CD16 (p≤.005), and the adhesion molecules CD11a (p≤.001) and CD11b (p≤.005). Concerning cell cycle analysis, the percentage of cells in S-phase was significantly increased from baseline vs. 3 vs. 9 cycles of LenDex for T CD4 (0.04% vs 0.13% vs 0.13%; p<.001), CD8 (0.05% vs 0.13% vs 0.18%; p<.001) and NK cells (0.07% vs 0.16% vs 0.15%; p<.001). Interestingly, an unsupervised cluster analysis of the overall immunophenotypic expression profile obtained after 9 cycles of LenDex was able to discriminate two groups of patients with different activation profiles particularly on T CD8 cells, with differences (P<.05) in both their percentage in PB and expression of activation, Th1 and maturation markers. Patients displaying a higher activation profile showed a trend towards increased depth of response after 9 cycles of LenDex (sCR+CR: 31% vs 15%; p=.229), as well as time-to progression (TTP) to symptomatic MM (TTP at 2-years: 100% vs 79%; P=.177).Finally, we explored whether the immunomodulatory properties of lenalidomide could be increased when dexamethasone was removed for the maintenance phase. Regarding T and NK cell distribution, only an increase in the percentage of CD4 T cells was found (9% vs. 12%, P=.04), whereas no differences (P>.05) were noted regarding the immunophenotypic expression profile of T and NK cells studied.In summary, we show that in high-risk SMM patients at baseline CD8 and γδ T cells are increased but overall T cells show an impaired activation profile. Treatment with LenDex induces an activation and proliferation of T and NK cells which may contribute to disease control. Finally, our results do not show an inhibition of the immunomodulatory effects of lenalidomide by the concomitant use of dexamethasone. Disclosures:Paiva:Celgene: Honoraria; Janssen: Honoraria. Off Label Use: lenalidomide is not approved for smoldering myeloma. Mateos:Janssen: Honoraria; Celgene: Honoraria. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Lahuerta:Janssen: Honoraria; Celgene: Honoraria. Blade:Janssen: Honoraria; Celgene: Honoraria. San Miguel:Janssen-Cilag: Honoraria; Celgene: Honoraria.

Multiparameter Flow Cytometry Analysis of Peripheral Blood T, NK and Dendritic Cells From High-Risk Smoldering Multiple Myeloma Patients Treated with Lenalidomide and Dexamethasone

AbstractAbstract 1906Lenalidomide is an immunomodulatory agent that interacts with different components of the immune system by altering cytokine production, regulating T cells and increasing NK cell cytotoxicity. In multiple myeloma (MM), lenalidomide is approved for use in combination with dexamethasone in patients who have received at least one prior therapy. Recent observations have shown that dexamethasone enhances the anti-myeloma effect of lenalidomide; however, dexamethasone may also antagonize the immunomodulatory properties of lenalidomide.In the present study we evaluated by multiparameter flow cytometry (MFC) peripheral blood (PB) T, NK and dendritic (plasmacytoid, myeloid and monocytic) cells (DC) from high-risk smoldering MM (SMM) patients, defined by the presence of at least 2 of the 3 following criteria at diagnosis: bone marrow plasma cell (BMPC) infiltration ≥10%; and/or high M-component (IgG≥30g/L or IgA≥20g/L or B-J Protein>10g/L); and/or ≥95% myelomatous-PC/BMPC and immune paresis. SMM patients were treated according to the QuiReDex trial (NCT 00480363): an induction phase of nine four-week cycles of lenalidomide plus dexamethasone (LenDex) followed by maintenance with lenalidomide until disease progression. In this ongoing study, immunophenotypic data is available in 53 patients at diagnosis (baseline), 30 after 3 cycles of LenDex and 22 at the end of induction therapy (9th cycle). Here we will focus on the 22 cases with information at the 3 time points. For MFC analysis, PB samples were stained using a four-color direct immunofluorescence technique that allowed the quantification and characterization of T, NK and DC cells, including cell cycle analysis.The percentage of PB T cells in total PB cellularity was stable from baseline vs 3 vs 9 cycles of LenDex (22% vs 21% vs 21%; respectively, NS), with similar results also obtained for T CD4 (12% vs 11% vs 9%; respectively, NS) and T CD8 (8% vs 6% vs 8%; respectively, NS) cells. NK cells were slightly increased after 9 cycles of LenDex for both the CD56dim (4.1%, 3.4% and 6%; respectively; NS) and CD56bright (0.05%, 0.04% and 0.15%; respectively; NS) NK cell compartments. Similarly, the percentage of DC slightly increased along treatment, especially for plasmacytoid DC (0.2% at baseline vs 0.4% after 9 cycles; p=0.09). However, when a more detailed immunophenotypic characterization of T and NK cells was carried out significant differences emerged following LenDex treatment (Figure 1A). Accordingly, after 3 and 9 cycles of LenDex both T CD4 and CD8 cells showed increased expression of activation markers such as CD69 (p=.03), CD25 (p=.02 and NS, respectively), CD54 (p<.001), CD28 (p≤.03) and CD120b (p≤.01), together with increased production of IFNγ (p=.03) and IL-2 (p=.1 and p=.008, respectively). Interestingly, after induction therapy an up-regulation of chemokine receptors related to the Th1 (CCR5; p<.001) but also Th2 (CCR4; p≤.002) immune response was detectable in CD4 and CD8 T cells. T CD4 cells displayed a clear maturation into a central memory phenotype following LenDex treatment (38% at baseline vs 50% and 66% at 3 and 9 cycles, respectively; p<.001) while T CD8 cells displayed an increased effector memory phenotype (44% vs 59% vs 62%; p=.004). Further analysis showed increased expression of HLA-DR (p≤.008), the antibody-dependent cell-mediated cytotoxicity associated receptor CD16 (p≤.03), and the adhesion molecules CD11a (p'.006) and CD11b (p≤.004) both on NK (CD56dim and CD56bright) and T cells. No consistent changes were observed in other NK cell receptors, such as CD94 and the immunoglobulin like receptors CD158a, CD161, NKB1 (3DL1) and NKAT2 (2DL3). Concerning cell cycle analysis, the percentage of cells in S-phase was significantly increased from baseline vs 3 vs 9 cycles of LenDex in T CD4 (0.05% vs 0.15% vs 0.16%; p<.001), CD8 (0.05% vs 0.11% vs 0.23%; p<.001) and NK cells (0.09% vs 0.17% vs 0.20%; p=.001). Finally, an unsupervised cluster analysis of the overall immunophenotypic profile obtained after 9 cycles of LenDex (Figure 1B) was able to discriminate two groups of patients (A and B). Interestingly, within the group with higher activation profile (A) 50% of patients achieved ≥VGPR vs 23% in group B (p=.2).In summary, these preliminary results show that in high risk SMM patients the combination of lenalidomide and dexamethasone modulates PB T and NK cells, with increased activation status that may contribute to disease control. [Display omitted] Disclosures:Off Label Use: Lenalidomide is not approved for the treatment of smoldering multiple myeloma. De La Rubia:Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosiñol:Celgene: Honoraria. Oriol:Celgene: Consultancy; Janssen-Cilag: Consultancy; Novartis: Consultancy. Hernández:Celgene: Honoraria. de Arriba:Janssen-Cilag: Honoraria; Celgene: Honoraria. Mateos:Celgene: Honoraria. San Miguel:Janssen-Cilag: Honoraria; Celgene: Honoraria, Speakers Bureau.

In Vivo Antagonistic Effects of Dexamethasone On Lenalidomide-Induced NK Cell Activation.

Abstract 1639Poster Board I-665Dexamethasone (dex) and lenalidomide (len) is a potent treatment for multiple myeloma (MM). In vitro, len directly inhibits MM tumor cell proliferation via cell cyle arrest, and can also costimulate T cells and augment natural killer (NK) cell activity, leading to enhanced anti-tumour immunity. Conversely, dex also directly inhibits MM cell proliferation but is profoundly immuno-suppressive and may therefore subvert the full capacity of len to act via immune mechanisms against MM. We previously reported that MM patients responding to len-dex combination show an increase in Treg numbers, and little evidence in recovery of their B and T cell numbers (Quach et al. Blood 2008; 112: abstract 1696). We have since undertaken a prospective and systematic analysis of NK cell number and function in MM patients treated with len-dex, and evaluated the mechanisms by which dex downregulates len-induced NK activation in in vitro assays using patients' and normal donors' blood samples. 25 relapsed MM patients (aged 58-77 years) were treated with low dose len (15mg Days 1-21 of each 28-day cycle) and dex (20mg/day, Days 1-4,9-12,17-20). After a median of 9 (2-19) cycles, 19 patients responded (24% CR/VGPR, 52% PR). At baseline, NK cell numbers and function [assessed by % lysis of 51Cr labelled K562 target cells at 50 (effector):1 (target) ratio] in MM patients were similar to age matched controls (0.2 vs. 0.3× 105/ml in controls, p=0.09 and 49% K562 cell lysis vs. 58% in controls, p=0.44 respectively) (fig.1A). Whilst NK cell numbers slightly increased in vivo after len-dex treatment [2.0 (baseline) vs. 3.9×105/l (cycle 6), p=0.04, paired t test] (fig.1A), mean NK cell function progressively decreased compared to baseline after 6 and 9 len-dex cycles [mean 49% K562 cell lysis at baseline vs. 28% after 6 cycles (p=0.007) and 31% after 9 cycles (p=0.02)] (fig.1B). Following 72 hours of in vitro treatment with len (10mM), there was increased NK function in healthy donor peripheral blood mononuclear cells (PBMC) [mean 54% K562 cell lysis from len-treated PBMC vs. 38% lysis in untreated PBMC, p=0.04] (fig. 2). In PBMCs from MM patients at baseline, ex vivo treatment with len (10mM) did not significantly increase NK cell function [mean 47% K562 cell lysis (untreated) vs. 52% (len treated), p=0.17], nor did it increase NK cell function after 6 len-dex treatment cycles [mean 32% K562 cell lysis (untreated) vs. 30% (treated), p=0.4].Conversely, dex (0.1mM) decreased NK cell function in healthy donors' PBMC [mean 7.6% K562 cell lysis (dex treated) vs. 38% (untreated) p=0.01], even in the presence of len [mean 7% K562 cell lysis (len+dex) vs. 38% (untreated), p=0.002] (fig. 2). Dex-induced in vitro NK inhibition was dose dependent and could be rescued by the addition of IL-2 to normal donors [mean 7.6 % K562 cell lysis (dex) vs. 28% lysis (Dex +IL2),p=0.03] as well as PBMC from MM patients at baseline [mean lysis 16% (dex) vs. 59% (Dex+IL2) p=0.0002]. However, IL-2 was less able to rescue dex-induced NK dysfunction in PBMC from patients post 6 treatment cycles compared to patients at baseline [mean 59% K562 cell lysis (baseline) vs. 28% (C6), p=0.03]. Dex induced NK dysfunction was reversible as NK cell function recovered after a 3 days dex washout. In summary, NK function in MM patients, whilst similar to healthy controls at baseline, progressively decreases after prolonged len-dex treatment despite a clinical response. The observed decrease in NK function in vivo and in vitro is directly due to the effects of dex, which could not be reversed by the NK activating effects of len. Our results suggest that the efficacy of len and dex co-therapy is not due to augmentation of NK cytolytic activity, due to the immunosuppressive effects of dex against NK cells. This suggests that alternative dosing schedules of dex, after initial induction with len and dex co-therapy, may optimise len-induced immunostimulation of NK cells and subsequent sustained disease control via anti-MM immunity. [Display omitted] [Display omitted] DisclosuresLynch:Celgene Corporation: Employment. Prince:Celgene Corporation: Research Funding.

Regulatory T Cells (Treg) Are Depressed in Patients with Relapsed/Refractory Multiple Myeloma (MM) and Increases towards Normal Range in Responding Patients Treated with Lenalidomide (LEN).

CD4+CD25+Foxp3+ regulatory T cells (Tregs) are a group of immune-suppressive T cells responsible for the maintenance of self-tolerance and autoimmunity. Increased Tregs have been implicated in suppression of tumour-immunity, and are associated with poor prognosis in gastric, oesophageal and ovarian cancers. Conversely, reduced Tregs are associated with aggressive disease and poor prognosis in lymphoproliferative disorders such as follicular lymphoma and Hodgkin lymphoma. Meanwhile, conflicting literature exists with regards to the role of Tregs in the pathogenesis of multiple myeloma (MM). Lenalidomide (LEN) is an immunomodulatory drug (ImiD™) that increases T cell numbers and function in vitro but the impact on Tregs in vivo is unknown. As part of an ongoing clinical trial of low-dose lenalidomide, peripheral blood lymphocyte subsets (LSS) and Treg numbers were examined in patients [n=18, median age 67 years (49–77)] with relapsed/refractory MM at baseline and at various time points during treatment with combination LEN (15mg/o/d d1–21q28)-dexamethasone (dex) (20mg/o/d d1–4,9–12,17–20 q28), in order to measure changes induced by LEN-dex treatment. Results were compared to 10 age matched healthy donors. LSS and Tregs were assessed by flow cytometry, with Tregs strictly defined as CD4+CD25hiCD127dimFOXP3+. At baseline, MM patients had depressed B cells (median 0.76 vs. 3.2 ×105/ml; p=0.02) and CD3+ T cells (median 7.2 vs. 12.1 ×105/ml; p<0.001) compared to healthy donors, with an inverse CD4:CD8 T cell ratio (0.7:1 vs. 2.9:1, p=0.008). Tregs in MM patients were markedly reduced at baseline compared to healthy donors [median 0.40 (range: <0.01–1.9) vs. 4.8 (range 2.4–7.1) ×104/ml; p=0.0005]. In 11 patients completing at least 4 treatment cycles, no recovery of B cells (7.6 vs. 2.3 ×105/ml, p=0.053), NK cells (2.1 vs. 2.7×105/ml, p=0.1) or T cells (5.6 at baseline vs. 7.5 × 105/ml, p=0.45) were seen despite all achieving at least a PR (one with IF negative CR). In contrast, median Treg numbers progressively increased towards normal range, from a baseline of 0.3 to 1.3 × 104/ml (p=0.005) and 2.0 × 104/ml (p=0.009) after 4 (n=11) and 6 (n=7) cycles of LEN-dex respectively (figure 1). Patients with relapsed/refractory MM have profoundly disturbed LSS including marked decreased in Treg numbers. Normalisation of Treg numbers in responding patients treated with LENdex suggest that, as in other tumours, Tregs are actively involved in the pathophysiology of MM and their modulation may be an important component of the immunomodulatory mechanisms of LEN. [Display omitted]