Relapsed Multiple Myeloma Patients Research Articles

Anti-CXCL12/SDF-1 Spiegelmer® Nox-A12 Alone and In Combination With Bortezomib and Dexamethasone In Patients With Relapsed Multiple Myeloma: Results From A Phase IIa Study

BackgroundNOX-A12 is a novel, potent, L-stereoisomer RNA aptamer (Spiegelmer®) that binds and neutralizes CXCL12/SDF-1, a chemokine which attracts and activates immune- and non-immune cells via interaction with its receptors, CXCR4 and CXCR7. CXCL12 plays an important role in homing and trafficking of multiple myeloma (MM) cells to the bone marrow (BM). Here, we evaluate the capacity of NOX-A12 to mobilize plasma cells into the circulation. Furthermore, we analyze a possible therapeutic impact of NOX-A12 which disrupts the interaction of MM cells with the BM stroma resulting in enhanced chemosensitivity to established therapies such as bortezomib and dexamethasone (VD). MethodsTo date, 21/28 planned patients have been enrolled into a multicenter Phase IIa study of NOX-A12 alone and in combination with VD in relapsed MM patients. Here we report interim data on PK, PD and preliminary efficacy of a pilot group consisting of 3 cohorts of 3 patients each. In the pilot phase, cohorts received single doses of 1, 2 or 4 mg/kg NOX-A12 alone, respectively, two weeks prior to 8 planned cycles of combined treatment with NOX-A12 and VD repeated every 21 days. During combination therapy, NOX-A12 was administered 1-2 hours prior to bortezomib following a dose titration design for all patients: NOX-A12 doses were increased from 1 mg/kg to 2 mg/kg and 4mg/kg at cycles 1, 2 and 3, respectively. During cycles 4-6, doses of NOX-A12 were kept at the highest individually titrated dose. Bortezomib (1.3 mg/m2) was given on days 1, 4, 8 and 11 as intravenous injection. Oral dexamethasone (20 mg) was added on the day of and the day after bortezomib administration. Response was evaluated by applying the uniform response criteria of the IMWG 2011. ResultsIn total, 10 patients were enrolled into the pilot group (one patient was replaced after withdrawal of consent). The median age was 63 years (range 56-78) with 7 women and 3 men being included. Median prior therapies were 2 (range 1-5), with 9 patients having been pretreated with dexamethasone and 3 with bortezomib prior to enrollment. At screening 1, 7 and 2 patients presented with ISS stage I, II and III, respectively. IgG, IgA and LC M-components were present in 6, 3 and 1 patients, respectively. Six patients had cytogenetic aberrations, which were high risk (t(4;14) or del17p) in 2 patients. Plasma profiles of NOX-A12 in the patient population of the pilot group (Figure 1) were similar to healthy volunteers in which a plasma half-life of approximately 38 hours was observed. After single doses of NOX-A12, a dose-linear exposure with peak plasma levels of 2.1, 3.9, and 6.7 µM was found in the corresponding cohorts. Furthermore, an increase of plasma cells in peripheral blood by approximately 200% was detected which lasted throughout the observation period of 3 days (Figure 2). NOX-A12 as single agent was safe and very well tolerated.Of nine evaluable patients who received the combination treatment of NOX-A12 and VD, 2 (22%) achieved a VGPR and 4 (44%) PR, resulting in an ORR (≥ PR) of 67%. In combination with VD, NOX-A12 was equally safe and well tolerated. [Display omitted] [Display omitted] ConclusionProof of principle was achieved as single doses of NOX-A12 reached the expected plasma exposure and translated into an effective and prolonged mobilization of plasma cells into the peripheral blood. In addition, responses (PR or better) were noted in 6/9 (67%) patients of this pilot group. Single agent NOX-A12 was not associated with any relevant toxicity and the combination of VD with NOX-A12 was well tolerated and not associated with additional toxicity on top of VD. Provided that these promising findings will be confirmed in the total sample of 28 patients, further development of this novel anti-CXCL12/SDF-1 Spiegelmer® seems warranted. Disclosures:Ludwig:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Munipharma: Honoraria, Research Funding. Weisel:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria. Engelhardt:MSD, Janssen-Cilag: Research Funding. Greil:NOXXON Pharma AG: Research Funding. Dümmler:NOXXON Pharma AG: Employment. Zöllner:NOXXON Pharma AG: Employment. Zeitler:NOXXON Pharma AG: Employment. Riecke:NOXXON Pharma AG: Employment. Kruschinski:Noxxon: Employment.

BC2059: An Orally Bioavailable β-Catenin Inhibitor Potently Induces Apoptosis As A Single Agent and In Combination With Bortezomib In Multiple Myeloma

Multiple myeloma (MM) remains an incurable malignancy of plasma cells harboured in the bone marrow. Despite the introduction of new therapeutic modalities such as lenalidomide, bortezomib and autologous stem cell transplantation, for the majority of myeloma patients treatment failure is inevitable because of the emergence of drug resistance. The canonical Wnt/β-catenin signalling pathway which plays a central role in modulating the delicate balance between stemness and differentiation in several adult stem cell niches, including the haematopoietic one in the bone marrow, has been found to be dysregulated in MM. Furthermore, it has been shown that Wnt signalling activation is associated with advanced stage MM, providing a rationale to evaluate a new orally bioavailable β-catenin inhibitor BC2059 (BetaCat Pharmaceuticals, Gaithersburg,USA) in mono- and combination therapy with proteasome inhibitors.We have shown that BC2059 induced apoptosis in 10/10 genetically heterogeneous human myeloma cell lines (HMCL) treated with dosages ranging from 50-500nM. At 72 hours the pro-apoptotic effect of BC2059 at 500nM was almost uniform with cell death (>80%) for all the HMCLs tested. BC2059 was able to inhibit the proliferation of all HMCLs in a dose and time dependent manner assessed by MTS assay and viable cell enumeration with trypan blue. Further analyses demonstrated the accumulation of cells in the G0/G1 phase suggesting G1 cell cycle arrest. The expression of the active nuclear β-catenin was confirmed by immunoblotting the nuclear fraction of the HMCLs. Furthermore we were able to demonstrate reduction of β-catenin in both cytoplasmic and nuclear compartments after 14 hours of treatment with increasing concentrations of BC2059 (for example KMS-18 treated with IC50, 2xIC50 and 4xIC50 showed a relative drop of nuclear β-catenin from 1 in the untreated cells to 0.6, 0.5 and 0.4 respectively) suggesting that the drug facilitates its degradation. MM is well known to be dependent on the bone marrow microenvironment for its survival and drug resistance. To test the efficacy of BC2059 in overcoming this protective effect we treated HMCLs cocultured with an immortalised human stromal cell line (HS5) and we were able to demonstrate that BC2059 abolished the stromal effect at dosages > IC80 (for KMS-11 with IC50=215nM at 100nM the difference in cell death between myeloma cells alone and in co-culture is statistically significant, p<0.01, whereas at 350nM the difference is not significant, p>0.05). It has been already shown that bortezomib augments the activation of canonical Wnt signaling by preventing β-catenin protein from proteosome-mediated degradation in MM cells. Nevertheless, it is not clear if this effect is pro-apoptotic or pro-survival providing us a rationale to use BC2059 with bortezomib in combination therapy. 5 HMCLs were tested with concomitant administration of BC2059 and bortezomib with 4/5 showing synergistic effect (Combination Index[CI] 0.4-1.1, CI< 1.1 is considered synergistic). As a monotherapy, BC2059 effectively killed primary MM tumour cells from relapsed and/or refractory MM patients (n=13) in an autologous bone marrow (BM) co-culture assay (46±7.5% and 59±5.6% killing at at 1µM and 5µM, respectively) and demonstrated a synergistic effect when combined with bortezomib in 3 primary MM samples (Synergy Quotient[SQ]: 2.3-8.3, SQ>1 is considered synergistic). Interestingly one sample derived from a bortezomib-resistant relapsed MM patient could be re-sensitised to bortezomib by BC2059 suggesting that up-regulation of β-catenin may be one of the mechanisms of acquired drug resistance to bortezomib. In conclusion, BC2059 induces apoptosis in HMCL and primary MM cells at therapeutically relevant concentrations and is capable of overcoming the protective influence conferred by the bone marrow microenvironment. Disclosures:Horrigan:BetaCat Pharmaceuticals: Employment.

Visibility Of Bone Lesions In Relapsed Multiple Myeloma Can Be Improved By FDG-PET Scanning

Osseous involvement is one the predominant features of patients with multiple myeloma (MM). Whole body X-ray (WBX) is the standard method for detecting skeleton abnormalities but has limited value in relapsing disease since it cannot make a distinction between old and new active skeleton lesions. Therefore changes in metabolic activity might be a more useful method to detect new skeleton lesions in MM. This can be assessed by [F-18] fluorodeoxyglucose positron emission tomography (FDG-PET) imaging. Consequently WBX and FDG-PET was studied in relapsing MM prior to treatment. In addition immunohistochemical staining on bone marrow biopsies was performed for glucose transport protein (GLUT) 3 expression, micro vessel density (MVD), and vascular endothelial growth factor (VEGF) to demonstrate whether these parameters might be predictive for the in vivo scanning results. This study included 44 patients (median age 62 yr (range 48-78)) with a median of 3.5 (range 2-12) relapses. Thus far 86% of the patients were treated with autologous stem cell transplantation in combination with bortezomib (78%) or lenalidomide (65%). New lesions on FDG-PET were more frequently demonstrated than new lesions on WBX (p=0.00001). In 78% of the patients FDG-PET demonstrated a median of 4 lesions (range 0-22) per patient with a median standard uptake value (SUV) max of 5.7 (range 0-24). In 42% of the patients new X-ray lesions (median 0 (range 0-7)) were demonstrated and these were concordant with the results of FDG-PET. In 5 % of the patients new X-ray lesions were observed without FDG-PET lesions. For the relapsing patients treatment consisted of dexamethasone in combination with bortezomib (65%) or lenalidomide (50%). CR was obtained in 27% of the patients, PR in 39% and progressive disease in 22%. Average time to next treatment (TTNT) was 11.7 months (2-43). However FDG-PET positivity was not predictive for PFS (p =0.55) and OS (p=0.40) neither if PET positivity was defined as more than 3 lesions nor if the cut off value of SUV max > 4.2 was applied. Immunohistochemical staining on bone marrow biopsies (n=20) with CD34 demonstrated a significant increased MVD of 39 ± 54 (mean ± SD, normal bone marrow MVD 3.5 ± 2.9), in particular when clusters of plasma cells were present. These enhanced MVD was associated with a significant increased expression of VEGF and GLUT-3. However MVD (p = 0.16), VEGF (p = 0.70) or GLUT-3 (p =0.70) expression were not predictive for having a positive or negative FDG-PET. In summary, our results demonstrate that FDG-PET is more informative than WBX for detecting skeleton lesions in relapsing MM patients. However, and in contrast to what was previously shown in patients with newly diagnosed MM, FDG-PET it is not predictive for treatment outcome. This might be due to heterogeneity in studied patients and treatments. Disclosures:No relevant conflicts of interest to declare.

Combination Of 2-Deoxy-D-Glucose With ABT-199 Efficiently Kills All Molecular Subgroups Of Myeloma Cells

Multiple myeloma (MM) is a heterogeneous but incurable plasma cell malignancy which still requires new therapeutic approaches. Several molecular subsets of MM have been defined based on genetic and chromosomal aberrations. Briefly, t(4;14) or t(14;16) translocations and TP53 deletion are most frequent poor-risk genetic features of MM, while t(11;14) confers a neutral prognostic value. Because cancer cells have a high glycolytic metabolism, we investigated the efficiency of 2-deoxy-d-glucose (2-DG), a competitive inhibitor of hexokinase, to kill myeloma cells in these different molecular subsets. For this purpose we used 28 human myeloma cell lines (HMCL) representative of the molecular subsets carrying a t(11;14), t(4;14) or t(14;16) translocation, which respectively deregulate CCND1, MMSET and c-MAF. Apoptosis induced by 2DG was very heterogeneous among HMCLs, ranging from 5% to 96%. Of note, HMCLs carrying t(4;14) showed a trend to be more sensitive to 2DG (p=0.06, Mann–Whitney test) while HMCLs carrying t(11;14) appeared to be more resistant (p=0.08, Mann–Whitney test). Interestingly, 2DG sensitivity was not significantly related to the constitutive glycolytic activity of HMCLs (p=0.6). In fact, 2-DG not only strongly inhibited the glycolytic activity of HMCLs but also interfered with N-glycosylation. Indeed, addition of D-mannose, an N-linked glycosylation precursor, partly reversed 2-DG-induced cell death. However, the D-mannose efficiency was also heterogeneous among HMCLs, suggesting that the inhibition of N-glycosylation was not the only mechanism of 2-DG-induced cell death. An up-regulation of GRP78, CHOP and ATF-4 expression was induced by 2-DG in both sensitive and resistant HMCLs, suggesting that 2DG-induced cell death was independent from the UPR response. Finally, 2-DG uniformly induced Mcl-1 down-regulation in HMCLs, but only those dependent on Mcl-1 for survival (as confirmed by siRNA experiments) were killed by 2-DG. While the transcription of Mcl-1 is not affected by 2-DG, preliminary results indicate that proteosomal degradation could be involved as part of a more complex mechanism. Of note, most of t(11;14) HMCLs were resistant to cell death induced by Mcl-1 down-regulation but highly sensitive to ABT-199, which targets Bcl2 and efficiently kills t(11;14) HCMLs depending on this pathway for survival. Because 2-DG uniformly down-regulated Mcl-1, we combined it with ABT-199 in ABT-199-resistant HMCLs i.e., in HMCLs expressing a Bcl-2/Mcl-1 gene expression ratio lower to the threshold required for ABT-199 response. The combination of 2-DG and suboptimal ABT-199 dosage indeed strongly synergized in both t(4;14) and t(14;16) HMCLs. The efficiency of this combination was independent from the cells' TP53 status. Finally, these results have been extended to primary myeloma cells. Finally our study highlights the fact that dual targeting of Mcl-1 by 2-DG and Bcl-2 by ABT-199, in MM cell lines or primary samples, is highly efficient to induce apoptosis whatever the molecular subtype, including those with the poorest prognostic value. ABT-199 is presently under evaluation in a phase I clinical trial in relapsed MM patients and the present study provides a biological rationale for evaluating 2-DG in combination with ABT-199 in MM patients. Disclosures:No relevant conflicts of interest to declare.

A Synthetic Lethality Druggable Genome RNAi Screen Identifies Genes Mediating Sensitivity To Lenalidomide In Multiple Myeloma Including RSK2

Immunomodulatory drugs (IMiDs) are widely used in the treatment of patients with Multiple Myeloma (MM) however, only 30% of relapsed MM patients respond to single agent therapy and most patients eventually develop drug resistance. The molecular target of IMiDs in MM is cereblon but other parallel pathways or downstream events which enhance or preclude drug responsiveness are unknown. We therefore conducted a genome scale small interfering RNA (siRNA) lethality study in MM in the presence of increasing concentrations of lenalidomide. Primary screening was performed in a single-siRNA-per-well format with the human druggable genome siRNA set V4 comprising four siRNAs targeting each of 6,992 genes (total 27968 siRNAs). Lenalidomide was added 24 hours post transfection and cell viability was measured by ATP-dependent luminescence at 144 hours after transfection. Primary screen data was rigorously evaluated for multiple quality control metrics and found to exceed all expected performance parameters with >98% global transfection efficiency, <0.25 CV values, and minimal plate-to-plate and set-to-set variations observed. Hit selection was performed by analysis of IC50 value shift in the presence of each testing siRNA compared with three different control siRNA oligos. 160 candidate genes that enhance lenalidomide sensitivity upon silencing (sensitizers) were selected and re-screened with four siRNA oligos targeting each gene. 50 genes were identified as reproducible lenalidomide sensitizers including three Peroxisome (PEX) family proteins (PEX1, PEX10 and PEX7) and seven RAB family proteins (RAB17, RAB1A, RAB26, RAB30, RAB36, RAB4A and RAB8A). Four kinase genes were also identified in sensitizer hits and two of these, I-Kappa-B Kinase-Alpha (IKK1 or CHUK) and ribosomal protein S6 kinase (RPS6KA3 or RSK2), encode proteins that associate with significance together with a phosphorylation dependent transcription factor (CREB1) in Toll signaling pathways (p-value 0.0068). RSK2 is a serine/threonine-protein kinase that acts downstream of oncogenic FGFR3 mediated signaling and is phosphorylated by ERK (MAPK1/ERK2 and MAPK3/ERK1 signaling) during hematopoietic transformation. Phosphorylated RSK2 was previously reported to be frequently expressed in myeloma cell lines and primary myeloma cells. Using lentiviral shRNA expression, we demonstrated that knockdown of RSK2 in three genetically variable MM cell lines induced cyctocytoxiticy and consistently sensitized to lenalidomide. Two selective small molecular inhibitors of RSK2 (SL 0101-1 and BI-D1870) were then demonstrated to synergize with lenalidomide to induce myeloma cell cytotoxicixity. To further understand the mechanism underlying sensitization, immunoblotting analysis was performed to look at downstream changes after either RSK2 knockdown or RSK2 inhibition by BI-D1870. We found that both RSK2 knockdown and BI-D1870 treatment, mimicking lenalidomide treatment or cereblon inhibition, induced downregulation of both IRF4 and MYC in MM cells. The combination of lenalidomide and BI-D1870 not only produced a substantial synergistic effect inducing MM cytotoxicity, but also demonstrated a significant enhancement of downregulation of IRF4 and MYC. Forced overexpression of RSK2 attenuated the synergistic effects of lenalidomide and BI-D1870. In summary, our high throughput screen identified multiple gene targets that associate with increasing sensitivity to IMiDs in MM cells, of which, RSK2 was further validated by both shRNA silencing and specific inhibitors as an effective target to cooperate with IMiDs to induce myeloma cytotoxicity. Clinical studies of RSK2 inhibition in concert with IMiD (cereblon inhibitor) therapy would be appropriate. Disclosures:Stewart:Onyx: Consultancy, Research Funding; Millennium: Honoraria, Research Funding; Celgene: Honoraria; BMS: Honoraria.

Elevated Nuclease Activity Correlates With Clinical Spectrum Of Plasma Cell Dyscrasias

Over the last decade, there has been increasing interest in mechanisms that underlie genomic instability which is implicated in tumor progression, development of drug resistance and possibly even oncogenesis. We have observed that dysregulated homologous recombination (HR) is an important contributor to genomic instability. In this study, we aim to broaden our understanding of HR by exploring alternate pathways that may affect genomic instability such as nuclease activity. MethodsWe have developed an assay to measure total cellular nuclease activity and compare activity levels across a spectrum of plasma cell dyscrasias. Total nuclease activity in cell lines and patient samples was assessed using a novel plasmid degradation assay. Briefly, multiple myeloma (MM) cell lines and primary MM patient cells were lysed at equal cell concentration. The lysates were mixed with plasmid DNA in triplicate/quadruplicate and incubated at 37°C for fixed time points ranging from 0 to 30 minutes. Following forced cessation of enzymatic activity, samples were fractionated on agarose gel and resultant image analyzed for band intensity by Kodak 1D Image analysis software. The ratio of degraded plasmid over time was analyzed via nonlinear regression to calculate a half-life and k-constant. ResultsNon-linear regression analysis of data derived from assays involving 6 MM cell lines (RPMI, OPM1, H292, U266, INA6 & MM1S) with three pellets each of equal size taken from non-confluent cell culture with > 85% viability showed statistically significant reproducibility of best fit curve with an average R-squared of 0.93. Similar regression analysis could not be performed on data derived from normal PBMC pellets because they showed minimal degradation with linear regression slope of (-0.62). Lastly, non-linear regression analysis on 22 patient samples with a plasma cell dyscrasia diagnosis, revealed a broader range of activity by half-life. Further differentiation of plasma cell dyscrasia patients into 2 groups revealed a significantly higher half-life in MGUS & smouldering (9499.8 minutes) versus newly diagnosed and refractory or relapsed multiple myeloma patients (9.4,19.3 minutes respectively). ConclusionsIn conclusion, we show that nuclease activity, implicated in elevated recombination and genomic instability, can be measured in a statistically reproducible fashion by our in vitro assay and may be used to differentiate plasma cell dyscrasias across a clinical spectrum. Further analysis of nuclease activity in clinically annotated samples for correlation with clinical features, genomic data and survival outcomes is ongoing. Disclosures:Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

CD57+ NK CELLS ARE Increased In Patients With Multiple Myeloma and ARE Primed Effectors For ADCC, But NOT Natural Cytotoxicty

IntroductionCD57+ NK cells are a subset of CD56lo/CD16+ NK cells which have been alternatively described as terminally differentiated or memory NK cells; however their physiological role remains unclear. In this study, we explored this unique NK subset and define their role in the context of MM. ResultsCD57+ NK cell frequency was initially assessed by FACS. Thus, newly diagnosed untreated (NEW) Multiple Myeloma (MM) patients had a significantly increased frequency of CD57+ NK cells in the PB (p=0.0029) and BM (p=0.0095) compared with age matched normal donor controls. Refractory relapsed (RR) MM patients also had significantly increased CD57+ cells in the BM (p=0.008) (Figure 1). There was no change in CD57+ NK cell numbers (in either patient cohort) following three cycles of lenalidomide+dexamethasone (len+dex) therapy. Normal donor CD57+ NK cells had significantly less natural cytotoxicity function (compared with CD57- NK cells) against K562 (p=0.04) or U266 (p=0.0265) (Figure 2A). The same trend was observed in MM patient CD57+ NK cells; however this did not reach statistical significance (Figure 2B). In contrast, the ADCC activity for CD57+ and CD57- NK cells was the same whether the effector cells were from normal donors (Figure 2A) or MM patients (Figure 2B). To explain the reason for this difference in natural cytotoxicity function between the two NK cell subsets, the balance of NK activating (KAR) and inhibitory receptors (KIR) was examined (Figure 3). CD57+NK cells expressed significantly lower levels of the NK activating receptor Nkp46 than CD57-NK cells, in the PB of normal donors (p=0.001), NEW MM patients at presentation (p=0.008) or NEW MM patients after three cycles of len-dex therapy (p=0.0237) (Figure 3). In contrast, both CD57+ and CD57- NK cell subsets in RR MM patients had the same levels of Nkp46, this did not change with len-dex therapy. To further explore reasons for the CD57+ NK cell functional state, expression of NK effector molecules (perforin and granzyme) and NK effector transcription factors (TF) T-bet and Eomesdermin (Eomes) was assessed. Surprisingly, PB CD57+ NK cells had significantly higher perforin expression than CD57- NK cells in the PB of normal donors (p=0.0089) and NEW (p=0.0815) MM patients, and the BM of NEW MM patients (p=0.0487). Similarly, granzyme B was significantly elevated in CD57+ compared to CD57- NK cells in normal donors (p=0.013), NEW MM patients in the PB (p=0.0024) and BM (p=0.0007). There was no statistical difference in the expression of Eomes or T-bet in either NK subset, whether the cells were from normal donors or MM patients. Nonetheless, a trend in higher Eomes expression was observed in CD57+ NK cells from MM patients compared to age matched controls. Lastly, CD57+ (compared to CD57-) NK cells have a significantly lower proliferation rate in a NK homeostatic proliferation assay. [Display omitted] [Display omitted] [Display omitted] Discussion and ConclusionsStudies to date indicate CD57+ NK cells are terminally differentiated NK cells; however to date their role remains undefined. We observed an increased frequency of CD57+ NK in the PB and BM of newly diagnosed untreated MM patients. These CD57+ NK cells have the same functional and phenotypic features in normal donors and MM patients, whether at diagnosis or in RR patients. The strikingly reduced natural cytotoxicity in CD57+ NK cells, coupled with reduced Nkp46 expression, indicates these cells are not important for surveillance of viral infected or tumour cells. Rather CD57+ NK express elevated effector proteins and the effector TF Eomes and are ‘primed' for killing of target cells via ADCC. Unlike CD57- NK cells, CD57+ NK cells do not undergo homeostatic proliferation; whilst this is consistent with terminally differentiated cells, it is not clear how these cells persist in the periphery of MM patients. Nonetheless, CD57+ NK cells are elevated in MM patients at presentation, whilst this likely contributes to the overall decreased natural cytotoxicity observed in MM patients, it also provides an opportunity to activate NK cells via CD16 (FcγRIII) to induce ADCC against MM. Disclosures:Harrison:Celgene Corp: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; Virolytics: Consultancy.

Phase 2 Study Of Bendamustine, Bortezomib (Velcade) and Prednisone (BVP) For Newly Diagnosed Multiple Myeloma (MM)

BackgroundBortezomib-based combinations, including alkylating agents (VMP or CyBorD) or immunomodulatory drugs (VTD or RVD) have been established as regimens widely used in newly diagnosed MM patients. Bendamustine is a bifunctional alkylating agent effective in relapsed and/or refractory MM patients, and approved in Europe in combination with prednisone for elderly newly diagnosed MM. Since bendamustine may be more efficient than other alkylators, an attractive possibility would be to explore it in combination with bortezomib and prednisone (BVP) in newly diagnosed MM patients both transplant and non transplant candidates. Patients and Methods60 newly diagnosed MM patients were included in the trial. The first cycle consisted on bendamustine at 90 mg/m2 given IV on days 1 and 4, in combination with bortezomib at 1,3 mg/m2 given IV on days 1, 4, 8, 11, 22, 25, 29 and 32 and prednisone at 60 mg/m2 given PO on days 1 to 4. In the following cycles, bendamustine was given on days 1 and 8, and bortezomib on days 1, 8, 15 and 22 (weekly schedule), and prednisone as it was previously described. Patients younger than 65 years proceeded to peripheral blood stem cell collection (PBSC) using growth factors alone after 4 cycles; HDT-ASCT was performed after 6 cycles. Patients older than 65 years received up to nine 28-day cycles. ResultsBetween May 2011 and July 2012 enrollment was completed (60 pts). Median age was 61 years (range 38-82; 18 pts ≥65), 67% had ISS stage II/III, and 67% had unfavorable cytogenetics: t(4;14), t(14;16), del 17p or 1q gains by FISH.After a median of 6 cycles (2-9), 75% of patients achieved at least PR, including 16% of sCR, 9% CR and 28% of VGPR. Although the differences were not statistically significant, there was a trend to higher CR rate in the group of patients <65 years (31%) compared with elderly patients (11%). No differences were observed in overall response rates and CR rates in patients with standard and high risk cytogenetic abnormalities.Forty patients proceeded to stem cell collection after a median of 4 cycles of BVP. Upon using G-CSF alone, 14 pts (35%) failed to collect a minimun of 2 x 106 CD34+ cells/Kg. An ammendment was done and plerixafor was recomended for poor mobilizers (peripheral CD34 cell count inferior to 10/μL on day 4); all patients but 2 achieved, with G-CSF plus plerixafor, the minimum of CD34+ cells required to proceed to ASCT. These 2 patients successfully collected CD34+ cells using chemotherapy plus G-CSF and plerixafor. Of the 31 patients who received HDT-ASCT, sCR and CR rate before transplant was 18% and 13%, respectively, upgrading up to 39% of sCR and 13% CR after transplant. 7 pts (22%) achieved immunophenotypic CR.After a median f/u of 12 months (5-25), 8 pts have progressed, resulting in a 15-m TTP of 85%. Concerning OS, 89% of patients remained alive at 15 months. None of patients achieving sCR and CR have progressed and all of them are alive at 15 months. Regarding cytogenetic abnormalities, although there were not significant differences, one patient progressed in standard risk group and five in the high risk subgroup resulting in a 15 m-TTP of 93% vs 85%. No significant differences have been observed in terms of 15 m-OS between standard and high risk cytogenetic subgroup (100% vs 92%, respectively).As far as toxicity is concerned, hematologic toxicities included: G3/4 anemia (11%), neutropenia (23%), and thrombocytopenia (14%). The most common G3/4 non-hematologic toxicities were: asthenia (10%), infections (9%), and peripheral neuropathy (4%). ConclusionsIn patients candidates to HDT-ASCT, response rates obtained before and after transplant are comparable to other three drug bortezomib-based combinations, such as VTD or CyBorD. However, growth factors alone for stem cell collection after four BVP cycles as induction resulted in a 35% of poor mobilizers who were rescued with plerixafor. In the elderly population, although the number of patients included was small, BVP seems not superior to VMP in response rates. Disclosures:Mateos:Janssen, Mundipharma: Honoraria. Off Label Use: bendamustine plus bortezomib and prednisone is not an approved combination for first line of therapy. Oriol:Celgene: Consultancy. Ocio:Onyx: Consultancy, Research Funding; Novartis: Consultancy; Array Biopharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding. Alegre:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Bladé:Janssen, Mundipharma: Honoraria. San Miguel:Janssen, Mundipharma: Membership on an entity's Board of Directors or advisory committees.

High-Risk Multiple Myeloma: Different Definitions, Different Outcomes?

Multiple myeloma (MM) is a clonal plasma cell malignancy. Although MM is still not completely curable, it can be maintained at the level of a long-term chronic condition. Irrespective of the treatment strategy, relapse is still a major problem for most patients. Approximately 10% to 15% of all MM patients relapse early and have poor prognosis and outcome. Currently, there are many ways of identifying these high-risk patients using cytogenetics or molecular biology. Despite these various approaches to definition of high risk patients, a clear definition of high-risk MM has not been widely accepted. In this review, we discuss and compare various approaches, and their strengths and weaknesses in early identification of high-risk MM patients.

Attenuation of dexamethasone-induced cell death in multiple myeloma is mediated by miR-125b expression

Dexamethasone is a key front-line chemotherapeutic for B-cell malignant multiple myeloma (MM). Dexamethasone modulates MM cell survival signaling but fails to induce marked cytotoxicity when used as a monotherapy. We demonstrate here the mechanism behind this insufficient responsiveness of MM cells toward dexamethasone, revealing in MM a dramatic anti-apoptotic role for microRNA (miRNA)-125b in the insensitivity toward dexamethasone-induced apoptosis. MM cells responding to dexamethasone exhibited enhanced expression of oncogenic miR-125b. Dexamethasone also induced expression of miR-34a, which acts to suppress SIRT1 deacetylase, and thus allows maintained acetylation and inactivation of p53. p53 mRNA is also suppressed by miR-125b targeting. Reporter assays showed that both these dexamethasone-induced miRNAs act downstream of their target genes to prevent p53 tumor suppressor actions and, ultimately, resist cytotoxic responses in MM. Use of antisense miR-125b transcripts enhanced expression of pro-apoptotic p53, repressed expression of anti-apoptotic SIRT1 and, importantly, significantly enhanced dexamethasone-induced cell death responses in MM. Pharmacological manipulations showed that the key regulation enabling complete dexamethasone sensitivity in MM cells lies with miR-125b. In summary, dexamethasone-induced miR-125b induces cell death resistance mechanisms in MM cells via the p53/miR-34a/SIRT1 signaling network and provides these cells with an enhanced level of resistance to cytotoxic chemotherapeutics. Clearly, such anti-apoptotic mechanisms will need to be overcome to more effectively treat nascent, refractory and relapsed MM patients. These mechanisms provide insight into the role of miRNA regulation of apoptosis and their promotion of MM cell proliferative mechanisms.

Association of Th1 and Th2 cytokines with transient inflammatory reaction during lenalidomide plus dexamethasone therapy in multiple myeloma

Transient inflammatory reactions have been reported in a subpopulation of patients with multiple myeloma (MM) during lenalidomide (Len) plus dexamethasone (DEX) therapy. Here, we examined serum levels of Th1 (IL-2 and IFN-γ) and Th2 cytokines (IL-6 and TNF-α) in nine refractory or relapsed MM patients treated with Len plus low-dose DEX. Six patients showed elevation of C-reactive protein (CRP) after the initiation of therapy. In these patients, IFN-γ and IL-6 were also elevated in two and three patients, respectively. The remaining three patients showed no appreciable changes in CRP or these cytokines. Furthermore, Len enhanced the production of both Th1 and Th2 cytokines in normal peripheral blood mononuclear cells and in patient bone marrow mononuclear cells containing primary myeloma cells and lymphocytes. These results suggest that the modulation of the Th1 and Th2 cytokine production by Len may contribute to transient inflammatory reaction in MM patients.

Safety and Efficacy From Intravenous Busulfan with PK-Directed Dosed Adjustment and Bortezomib Conditioning Regimen in Relapsed Multiple Myeloma Patients Undergoing a Second Autologous Hematopoietic Stem Cell Transplantation

s / Biol Blood Marrow Transplant 19 (2013) S178eS193 S186 may be warranted to discern if DM pts may benefit from different methods of mobilization or if long term transplant outcomes are impacted.

A prospective, international phase 2 study of bortezomib retreatment in patients with relapsed multiple myeloma

Multiple myeloma (MM) typically follows a relapsing course with many patients requiring multiple therapies. This single-arm phase 2 study prospectively evaluated the efficacy and safety of bortezomib retreatment in MM patients who had relapsed after achieving at least a partial response (≥ PR) to prior bortezomib-based therapy. Patients aged ≥ 18 years, with measurable, secretory MM, who relapsed ≥ 6 months after prior bortezomib treatment were eligible. Patients received up to eight cycles of bortezomib (± dexamethasone). The primary endpoint was best confirmed response at retreatment; secondary endpoints included duration of response (DOR), time to progression (TTP), and safety. Adverse events (AEs) were graded by National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0. A total of 130 patients (median of two prior lines of therapy) were enrolled and received retreatment. At retreatment, 28% and 72% of patients received bortezomib and bortezomib-dexamethasone, respectively. Overall response rate was 40%. In patients who achieved ≥ PR, median DOR and TTP were 6.5 and 8.4 months, respectively. Thrombocytopenia was the most common grade ≥ 3 AE (35%). Forty percent of patients experienced neuropathy events, which improved and resolved in a median of 1.5 and 8.9 months, respectively. In conclusion, bortezomib retreatment was effective and tolerable in relapsed MM patients, with no evidence of cumulative toxicities.

Good clinical activity and favorable toxicity profile of once weekly bortezomib, fotemustine, and dexamethasone (B‐MuD) for the treatment of relapsed multiple myeloma

Since multiple myeloma (MM) is still not-curable, the management of relapse remains challenging. Given the known efficacy of alkylating agents in MM, we conducted a phase I/II study to test a new three drug combination in which Fotemustine (Muphoran), an alkylating agent of nitrosurea family, was added to bortezomib + dexamethasone backbone (B-MuD) for the treatment of MM relapsed patients. Fotemustine was administered at two dose levels (80–100 mg/m2 i.v.) on day 1. The original 21-day schedule was early amended for extra-hematological toxicity and a 35-day schedule was adopted (Bortezomib 1.3 mg/m2 i.v. on days 1, 8, 15, and 22, Dexamethasone 20 mg i.v. on days 1, 8, 15, and 22) for a total of six courses. Twenty-four patients were enrolled. The maximum tolerated dose of Fotemustine was 100 mg/m2. The overall response rate was of 62% (CR 8%, VGPR 33%, and PR 21%). The median OS was 28.5 months, the median progression-free survival (PFS) was 19.1 months. B-MuD resulted effective in patients previous exposed to bortezomib without difference of response (P = 0.25) and PFS (P = 0.87) when compared to bortezomib-naive patients. Thrombocytopenia was the most common AE overall. In conclusion, B-MuD is an effective and well tolerated combination in relapsed MM patients even in advanced disease phase. © Am. J. Hematol., 88:102–106, 2013. © 2012 Wiley Periodicals, Inc.

Relapse Pattern After Bortezomib Based Salvage Therapy in Patients with Multiple Myeloma

AbstractAbstract 1869 Backgrounds & Aims:More intensive and novel therapy options in multiple myeloma (MM) improve the treatment outcome. However, disease evolution, induced with long disease duration and extensive pretreatment, has resulted in changes in the biological behavior of MM and unusual relapse emergence. Therefore, we studied the relapse pattern after bortezomib based salvage treatment in patients with MM and also we analyzed the prognostic significance according to relapse pattern. Methods:We have retrospectively analyzed the relapse pattern. Eligibility criteria included primary refractory or relapsed MM patients who must have received previous chemotherapy and they also received at least 2 cycles of bortezomib based salvage treatment. Immunoglobulin M type of MM, primary amyloidosis and plasma cell leukemia were excluded in this study. For evaluation of disease response, International Myeloma Working Group (IMWG) uniform response criteria were used. Results:Between November 2004 and August 2011, 132 patients received bortezomib based salvage chemotherapy. In 132 patients, 91 (68.9%) patients showed the disease relapse. The pattern of relapse after bortezomib salvage treatment was heterogeneous, the 14/91 (15.4%) patients relapsed as a novel manifestation comparison with the initiation of bortezomib (plasmacytoma: 7 patients, light chain escape pattern: 4 patients and, plasma cell leukemia: 2 patients). There was no statistical difference in the duration from diagnosis to bortezomib treatment according to novel (group A) vs isoform (group B) relapse. In the group B, the median overall survival after relapse was 16.1 months (95% CI: 11.4–20.8) and the group A was 2.5 months (95% CI: 0.0–5.7)(p=0.000). The 27 out of 132 patients received the retreatment of bortezomib and 23 out of 27 patients showed the disease progression. The 4/24 (16.7%) patients relapsed as a novel manifestation (plasmacytoma: 2 patients, light chain escape pattern: 1 patients and intact immunoglobulin secretion from plasmacytoma type: 1 patient). In patients received retreatment of bortezomib, novel relapsed group showed the trend of poor survival without statistical significance (2.2 months vs 10.0 months) (p=0.30). Conclusion:Our report suggests that bortezomib treatment contribute as a selective pressure and it has culminated in novel relapse manifestation. The relapsed patients as novel pattern after bortezomib treatment show the extreme poor prognosis. These findings suggest that these patients may be worth consideration for intensive treatment or early clinical trials. Disclosures:No relevant conflicts of interest to declare.

Preclinical Evaluation of AT219, a Small Molecule Inhibitor of MDM2 As an Anti-Myeloma Agent.

AbstractAbstract 2948 Background:Deletions and mutations in the tumor suppressor protein p53 are an uncommon observation in new multiple myeloma (MM) patients and are observed more commonly in patients with advanced disease. p53 deletion has been observed to correlate with poor overall and progression free survival in MM patients. Wild type p53 modulates the expression levels of a broad array of proteins involved in cell cycle progression, apoptosis ultimately leading to cell cycle arrest and apoptosis. p53 is negatively regulated by MDM2. MDM2 binds to and ubiquitinates p53 marking it for proteasomal degradation. In addition, MDM2 is a direct downstream regulator of p53. Targeting the p53-MDM2 interaction by developing agents that bind to the p53 binding motif of MDM2 and reactivating p53 has therefore been an active area of research. Here, we present results from our pre-clinical studies using AT219, a small molecule inhibitor that binds to MDM2 preventing its interaction with p53. Methods:AT219 was obtained from Ascenta Therapeutics. Stock solutions were made using DMSO and working stock solutions were made using RPMI 1640 media containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Akt1/2 kinase inhibitor (Akti) was purchased from Sigma. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In addition, apo-2.7 was also used to measure apoptosis in patient cells. Mitocapture and cytochrome-c assays were also performed to confirm the induction of apoptosis in MM cell lines. In order to study the mechanism of action of the drug, immunoblotting studies were performed on lysates made from cell lines incubated with the drug for various time points. Results:AT219 induced potent cytotoxicity in MM cell lines MM1S, MM1R and H929, all three expressing wild type p53 with IC50 values of 2.5–5μM. Similar effects were observed when the above mentioned cell lines were treated with AT219 and the inhibitory effect of proliferation of these cells were examined. When MM1S or H929 cells were cultured with bone marrow stromal cells (BMSCs) derived from MM patients or with one of the three tumor promoting cytokines implicated in MM (IL6, IGF or VEGF) and treated with AT219, the drug was able to inhibit the proliferation of both cell lines to similar extents as observed when cultured independently without BMSCs or the cytokines. The increase in cytotoxicity was found to be due to cells undergoing apoptosis as observed when MM1S or H929 cells were cultured with AT219 and % apoptotic cells were measured as measured by annexin/PI, mitocapture and cytochrome c assays. AT219 was also observed to induce more potent apoptosis in primary cells obtained from new MM patients with wild type p53 than in cells obtained from relapsed MM patients with wild type p53. AT219 clearly upregulated p53 as observed by performing immunoblots after treatment with the drug in MM1S and H929 cells. In addition, MDM2 and p21 were also found to be significantly upregulated and Bax was slightly upregulated post drug treatment. Bcl2, Mcl1 and Xiap levels were down regulated. In MM1S cells AT219 treatment resulted in a slight down regulation of pAkt (Ser 473). However, in H929 cells we observed a transient upregulation of pAkt following AT219 treatment. This prompted us to test AT219 in combination with Akti on MM cell lines. Our results on both MM1S and H929 cells using AT219 in combination with Akti demonstrated synergy. We are currently testing this combination in primary cells drawn from MM patients with both wild type p53 and those with p53 deletions and mutations. Conclusions:Our studies validate the anti-MM activity of AT219 in MM patients with wild type p53. In addition to using AT219 in combination with Akti, we are testing AT219 in combination with existing anti- MM chemotherapeutic agents. Interesting results from our studies will form the basis for clinical evaluation of AT219 as a single agent or in combination with an Akt inhibitor or other agents in MM patients. Disclosures:Kumar:Celgene: Consultancy, Research Funding; Merck: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; Novartis: Research Funding; Genzyme: Research Funding; Cephalon: Research Funding.

BT062, an Antibody-Drug Conjugate Directed Against CD138, Given Weekly for 3 Weeks in Each 4 Week Cycle: Safety and Further Evidence of Clinical Activity

AbstractAbstract 4042 Background:CD138 (Syndecan-1) is highly overexpressed in various solid tumors and hematological malignancies, and represents one of the most specific target antigens for identification of multiple myeloma (MM) cells. BT062 (Biotest AG Dreieich, Germany) is an antibody-drug conjugate, comprised of the anti-CD138 chimerized MAb (nBT062) and the cytotoxic agent DM4. Once bound to CD138 on a target cell, the conjugate is internalized and releases DM4, leading to target cell death. Data from the first in human study (969) of BT062 demonstrated an acceptable toxicity profile and evidence of clinical activity in heavily pretreated relapsed and/or refractory MM patients(1). Based on these data, a Phase I/IIa study in MM (975) was initiated to further evaluate the safety and efficacy of BT062 when given more frequently. Objectives:To determine the maximum tolerated dose (MTD), the dose-limiting toxicities (DLTs), pharmacokinetics (PK), and anti-MM activity of increasing doses of BT062, when administered on a repeated, multiple dose schedule (Days 1, 8, and 15, every 4 weeks) in patients with relapsed and/or refractory MM. Methods:This is a prospective, open label, dose-escalation, multicenter Phase I/IIa study. The Phase I part includes dose escalation, and the Phase IIa the expansion of the MTD or Recommended Phase II Dose (RPTD) cohort. Patients aged ≥18 years with relapsed or relapsed/refractory MM who have failed previous treatment, including an immunomodulatory agent and a proteasome inhibitor, were eligible to participate. Patients with clinical response (or no evidence of progressive disease) and without unacceptable toxicities were eligible for further treatment cycles. Patients were enrolled in cohorts of at least 3 at each dose level; DLT in the first cycle triggered cohort expansion. Toxicities were assessed by CTCAE v4 and clinical response was assessed according to the international myeloma working group criteria. Results:A total of 29 patients have been treated with BT062, receiving 1 of the first 8 dose levels ranging from 40 mg/m2 to 160 mg/m2. One out of 6 patients treated at 140 mg/m2 experienced a palmar-plantar erythrodysesthesia syndrome that was assessed as DLT (SAE, CTC grade 3). Dose was escalated to 160 mg/m2, and 1 of the 2 patients treated experienced an elevation of liver enzymes that was reported as DLT (non-serious, CTC grade 3). Maximum administered dose has not been reached and recruitment into the 160 mg/m2 cohort is ongoing. About 90% of the reported Adverse Events (AE) are grades 1–2. The most frequently reported AEs are anemia, diarrhea, and fatigue. Among the 23 patients evaluable for efficacy, 1 patient achieved a partial response (PR) and has been on study treatment for more than 1 year. Stable disease (SD) for at least 3 months was noted in an additional 11 patients, with median progression free survival of 112 (90–245) days. Thus disease control (PR + SD) was noted in more than 50% (12/23) of patients. The most common reason for discontinuation was disease progression. Preliminary pharmacokinetic results indicate rapid early clearance of BT062 from plasma consistent with rapid delivery and binding of BT062 to the MM cells. No significant accumulation of BT062 was noted, even after the end of the 2nd and 3rd infusions within a cycle. Conclusion:Preliminary data from this ongoing study indicate that BT062 is well tolerated even in this multiple dose schedule and provide further evidence of its clinical activity. Dose escalation is ongoing (now at 160 mg/m2) to define the MTD/RPTD, with cohort expansion then planned to further evaluate the safety and efficacy of BT062 at this dose. Based on the favorable safety profile, a Phase I/IIa study (983) has been initiated to evaluate the safety and efficacy of BT062 in combination with lenalidomide and dexamethasone. Disclosures:Heffner:Millennium: Research Funding. Jagannath:Millennium: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Zimmerman:Novartis: Consultancy; Millennium: Honoraria; Celgene: Honoraria. Lonial:Onyx: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Merck and Co: Consultancy. Lutz:ImmunoGen, Inc.: Employment. Czeloth:Biotest AG: Employment. Osterroth:Biotest AG: Employment. Ruehle:Biotest AG: Employment. Beelitz:Biotest Pharmaceuticals Corporation: Employment. Wartenberg-Demand:Biotest AG: Employment. Haeder:Biotest AG: Employment. Anderson:Celgene: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Acetylon: Membership on an entity’s Board of Directors or advisory committees; Oncopep: Membership on an entity’s Board of Directors or advisory committees; Millennium Pharmaceuticals: Consultancy. Munshi:Oncopep: Patents & Royalties; Merck: Consultancy; Onyx: Membership on an entity’s Board of Directors or advisory committees; Millennium: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees.

Fas apoptosis inhibitory molecule is upregulated by IGF-1 signaling and modulates Akt activation and IRF4 expression in multiple myeloma

Multiple myeloma (MM) is an incurable malignancy of terminally differentiated B-lymphoid cells. Here, we investigate the role of Fas apoptosis inhibitory molecule (FAIM) in MM. We demonstrate that insulin-like growth factor 1 (IGF-1) treatment upregulated FAIM expression in MM cells in a dose-dependent manner. Silencing of FAIM expression attenuates Akt signaling downstream of IGF-1 and compromises the viability of MM cells. We further showed that IGF-1 stimulation of MM cells leads to enhanced expression of IRF4, a known 'addictive' factor for MM. This upregulation of IRF4 expression by IGF-1 treatment of MM cells is abrogated when FAIM expression is silenced or Akt activation is inhibited. Thus, FAIM modulates IGF-1-induced Akt activation and IRF4 expression and has a role in MM cell survival. Consistent with these findings, FAIM expression is shown to be higher in plasma cells of symptomatic MM patients compared with normal individuals or patients with premalignant conditions. Moreover, a higher level of FAIM expression is shown to correlate with poorer survival outcomes of newly diagnosed MM patients treated with stem cell transplantation or relapsed MM patients treated in clinical trials with Bortezomib. Thus taken together, our study reveals a novel, as well as clinically relevant role for FAIM in MM.

Increased T Regulatory Cells Are Associated with Adverse Clinical Features and Predict Progression in Multiple Myeloma

BackgroundRegulatory T (Treg) cells play an important role in the maintenance of immune system homeostasis. Multiple myeloma (MM) is a plasma cell disorder frequently associated with impaired immune cell numbers and functions.MethodsWe analyzed Treg cells in peripheral blood (n = 207) and bone marrow (n = 202) of pre-malignant and malignant MM patients using flow cytometry. Treg cells and their subsets from MM patients and healthy volunteers were functionally evaluated for their suppressive property. A cohort of 25 patients was analyzed for lymphocytes, CD4 T cells and Treg cells before and after treatment with cyclophosphamide, thalidomide plus dexamethasone (CTD).ResultsWe found elevated frequencies of Treg cells in newly diagnosed (P<0.01) and relapsed MM patients (P<0.0001) compared to healthy volunteers. Also, Treg subsets including naïve (P = 0.015) and activated (P = 0.036) Treg cells were significantly increased in MM patients compared to healthy volunteers. Functional studies showed that Treg cells and their subsets from both MM and healthy volunteers were similar in their inhibitory function. Significantly increased frequencies of Treg cells were found in MM patients with adverse clinical features such as hypercalcemia (>10 mg/dL), decreased normal plasma cell (≤5%) count and IgA myeloma subtype. We also showed that MM patients with ≥5% of Treg cells had inferior time to progression (TTP) (13 months vs. median not reached; P = 0.013). Furthermore, we demonstrated the prognostic value of Treg cells in prediction of TTP by Cox regression analysis (P = 0.045). CTD treatment significantly reduced frequencies of CD4 T cells (P = 0.001) and Treg cells (P = 0.018) but not Treg cells/CD4 T cells ratio compared to pre-treatment.ConclusionsOur study showed immune deregulation in MM patients which is evidenced by elevated level of functionally active Treg cells and patients with increased Treg cells have higher risk of progression.

Phase I Trial of Vorinostat Combined With Bortezomib for the Treatment of Relapsing and/or Refractory Multiple Myeloma

Preclinical studies have shown that targeted combination therapy consisting of vorinostat and bortezomib has antitumor activity in multiple myeloma (MM). We examined this drug combination in advanced relapsing and/or refractory MM patients (n = 34). Although the maximum tolerated dose was not reached, the study found this combination regimen generally well tolerated and clinically active in relapsed and/or refractory MM patients. Development of targeted therapies for MM has improved response rates and increased patient survival, but ultimately the disease becomes refractory and progresses. Vorinostat combined with bortezomib has demonstrated synergistic antiproliferative and proapoptotic activity in preclinical models of MM. The objectives of this study were to determine the maximum tolerated dose for vorinostat with bortezomib in patients with advanced MM and to evaluate the clinical benefit of this new drug combination. Patients ≥ 18 years old with relapsed and/or refractory MM were enrolled into escalating dose cohorts of vorinostat and bortezomib combination therapy. Thirty-four patients were enrolled and were evaluable for safety and efficacy analyses. All patients reported adverse events, 89% of which were mild to moderate in severity. Thirteen patients experienced 29 serious adverse events, 12 (41%) of which were considered drug-related. The maximum tolerated dose was not reached. Partial responses were observed in 9 (27%) patients. Minimal responses were observed in 2 additional patients (6%), and another 20 patients (59%) experienced disease stabilization. Vorinostat with bortezomib is generally well-tolerated and has clinical activity in patients with relapsed and/or refractory MM. Response rates were similar in patients previously exposed to bortezomib and patients who were naive to bortezomib therapy.