Advanced Stage Multiple Myeloma Research Articles

The levels of serum soluble CD86 are correlated with the expression of CD86 variant 3 gene and are prognostic indicators in patients with myeloma

We previously showed that cell-surface CD86 expressed on multiple myeloma (MM) cells contributed to not only tumor growth but also antitumor cytotoxic T-lymphocyte responses mediated by induction of IL-10-producing CD4+ T cells. The soluble form of CD86 (sCD86) was also detected in serum from patients with MM. Thus, to determine whether sCD86 levels are a useful prognostic factor, we investigated the association of serum sCD86 levels with disease progression and prognosis in 103 newly diagnosed patients with MM. Serum sCD86 was detected in 71% of the patients with MM but was only rarely detected in patients with monoclonal gammopathy of undetermined significance and healthy controls, and the level was significantly increased in patients with advanced-stage MM. When we examined differences in clinical characteristics according to the level of serum sCD86, those in the high (≥2.18 ng/mL, n = 38) group exhibited more aggressive clinical characteristics, with shorter overall survival times compared with those in the low (<2.18 mg/mL, n = 65) group. On the other hand, it was difficult to stratify the patients with MM into different risk groups based on the expression levels of cell-surface CD86. The levels of serum sCD86 were significantly correlated with the expression levels of the messenger RNA (mRNA) transcripts of CD86 variant 3, which lack exon 6, resulting in a truncated transmembrane region, and its variant transcripts were upregulated in the high group. Thus, our findings suggest that sCD86 can be easily measured in peripheral blood samples and is a useful prognostic marker in patients with MM.

Imaging findings in a case of leptomeningeal myelomatosis, a rare but critical central nervous system complication of multiple myeloma.

Leptomeningeal myelomatosis is a rare complication of multiple myeloma (<1% of the patients). There was an increase in the incidence of leptomeningeal myelomatosis during the last decade; the prognosis of leptomeningeal myelomatosis remains poor, (overall median survival from the time of diagnosis of 2months). We discuss a rare case of a monoclonal gammopathy evolving into multiple myeloma and finally into a rapidly progressing leptomeningeal disease. A 76year-old woman in hematologic follow-up for advanced stage multiple myeloma in sixth-line treatment had an episode of generalized tonic-clonic seizure with sphincter release followed by altered state of consciousness. The unenhanced head CT scan showed a mild enlargement of the ventricular system without intra-axial or extra-axial hemorrhages nor significant changes in brain parenchyma. The subsequent contrast-enhanced brain MRI revealed a widespread nodular leptomeningeal enhancement characterized by contrast-enhancement of the pia mater extended into the subarachnoid spaces of the sulci and cisterns, involving supra- and sub-tentorial regions and the statoacoustic nerve in the inner ear canal bilaterally. The fluid-attenuated inversion recovery MRI images demonstrated an abnormally elevated signal within the sulci in the parieto-occipital regions. The radiological diagnosis of leptomeningeal myelomatosis was made. The patient died 4 days after the examination. In patients with long-lasting multiple myeloma and onset of neurological signs or symptoms, a contrast-enhanced brain MRI should be performed to assess the actual burden of central nervous system involvement in leptomeningeal myelomatosis; CT may provide a clue to the diagnosis when progressive enlargement of the ventricles over time is noted.

Expression of NrasQ61R and MYC transgene in germinal center B cells induces a highly malignant multiple myeloma in mice

AbstractNRAS Q61 mutations are prevalent in advanced/relapsed multiple myeloma (MM) and correlate with poor patient outcomes. Thus, we generated a novel MM model by conditionally activating expression of endogenous NrasQ61R and an MYC transgene in germinal center (GC) B cells (VQ mice). VQ mice developed a highly malignant MM characterized by a high proliferation index, hyperactivation of extracellular signal-regulated kinase and AKT signaling, impaired hematopoiesis, widespread extramedullary disease, bone lesions, kidney abnormalities, preserved programmed cell death protein 1 and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain immune-checkpoint pathways, and expression of human high-risk MM gene signatures. VQ MM mice recapitulate most of the biological and clinical features of human advanced/high-risk MM. These MM phenotypes are serially transplantable in syngeneic recipients. Two MM cell lines were also derived to facilitate future genetic manipulations. Combination therapies based on MEK inhibition significantly prolonged the survival of VQ mice with advanced-stage MM. Our study provides a strong rationale to develop MEK inhibition–based therapies for treating advanced/relapsed MM.

African Americans with translocation t(11;14) have superior survival after autologous hematopoietic cell transplantation for multiple myeloma in comparison with Whites in the United States.

Multiple myeloma (MM) with the translocation t(11;14) may have inferior outcomes in comparison with other standard-risk MM, and it has been suggested to portend a worse prognosis in African Americans in comparison with Whites. This study used the Center for International Blood and Marrow Transplant Research (CIBMTR) database to examine the impact of t(11;14) on the clinical outcomes of patients with MM of African American and White descent. This study evaluated 3538 patients who underwent autologous hematopoietic cell transplantation (autoHCT) for MM from 2008 to 2016 and were reported to the CIBMTR. Patients were analyzed in 4 groups: African Americans with t(11;14) (n = 117), African Americans without t(11;14) (n = 968), Whites with t(11;14) (n = 266), and Whites without t(11;14) (n = 2187). African Americans with t(11;14) were younger, had lower Karnofsky scores, and had more advanced stage MM with a higher Hematopoietic Cell Transplantation-Comorbidity Index (HCT-CI). Fewer African Americans with t(11;14) (21%) had a coexistent high-risk marker in comparison with Whites with t(11;14) (27%). In a multivariate analysis, race and t(11;14) had no association with progression-free survival. However, overall survival was superior among African Americans with t(11;14) in comparison with Whites with t(11;14) (hazard ratio, 0.53; 95% confidence interval, 0.30-0.93; P = .03). Survival was also associated with female sex, stage, time from diagnosis to transplant, a low HCT-CI, and receipt of maintenance. Race may have a differential impact on the survival of patients with t(11;14) MM who undergo autoHCT and needs to be further studied.

Immunotherapy for Multiple Myeloma.

Despite therapeutic advances over the past decades, multiple myeloma (MM) remains a largely incurable disease with poor prognosis in high-risk patients, and thus new treatment strategies are needed to achieve treatment breakthroughs. MM represents various forms of impaired immune surveillance characterized by not only disrupted antibody production but also immune dysfunction of T, natural killer cells, and dendritic cells, although immunotherapeutic interventions such as allogeneic stem-cell transplantation and dendritic cell-based tumor vaccines were reported to prolong survival in limited populations of MM patients. Recently, epoch-making immunotherapies, i.e., immunomodulatory drug-intensified monoclonal antibodies, such as daratumumab combined with lenalidomide and chimeric antigen receptor T-cell therapy targeting B-cell maturation antigen, have been developed, and was shown to improve prognosis even in advanced-stage MM patients. Clinical trials using other antibody-based treatments, such as antibody drug-conjugate and bispecific antigen-directed CD3 T-cell engager targeting, are ongoing. The manipulation of anergic T-cells by checkpoint inhibitors, including an anti-T-cell immunoglobulin and ITIM domains (TIGIT) antibody, also has the potential to prolong survival times. Those new treatments or their combination will improve prognosis and possibly point toward a cure for MM.

Radioimmunotherapy in Combination with Reduced-Intensity Conditioning for Allogeneic Hematopoietic Cell Transplantation in Patients with Advanced Multiple Myeloma.

Radioimmunotherapy (RIT) has the potential to reduce the incidence of relapse after allogeneic hematopoietic cell transplantation (allo-HCT) in patients with advanced-stage multiple myeloma (MM). In this study, we evaluated the efficacy of RIT in combination with chemotherapy-based reduced-intensity conditioning (RIC). RIT was based on the coupling of an anti-CD66 antibody to the beta emitter 188-rhenium (188-re) for targeted bone marrow irradiation. Between 2012 and 2018, 30 patients with MM, most of them heavily pretreated with various therapies including proteasome inhibitors, immunomodulatory drugs, anti-CD38 antibodies, and autologous hematopoietic cell transplantation (auto-HCT), were treated with a RIT-RIC combination before allo-HCT. In addition to a fludarabine plus melphalan- or treosulfan-based RIC, a median dose of 18.1 Gy (interquartile range [IQR], 14.6 to 24.1 Gy) was applied to the bone marrow. After a median duration of follow-up for surviving patients of 2.1 years (IQR, 1.3 to 3.0 years), the 2-year progression-free survival and overall survival rates were 43% (95% confidence interval [CI], 26% to 73%) and 55% (95% CI, 38% to 79%), respectively. The 2-year nonrelapse mortality and cumulative incidence of progression were 17% (95% CI, 3% to 30%) and 46% (95% CI, 25% to 67%), respectively. Renal toxicity and mucositis were the most frequent extramedullary side effects. In conclusion, the addition of RIT to RIC was safe and feasible and resulted in promising outcomes compared with those previously reported for RIC-based allo-HCT without the addition of RIT in patients with relapsed/refractory MM. Nevertheless, despite the addition of RIT, relapse after allo-HCT remained a major determinant of therapeutic failure. Therefore, the development of novel RIT strategies (eg, dual targeting strategies or combinations with adapter chimeric antigen receptor T cell-based therapies) is needed.

Infectious Complications in the Outpatient and Inpatient Autologous Stem Cell Transplantation Setting for Patients with Multiple Myeloma. Princess Margaret Cancer Center Experience

Background: Autologous stem cell transplant (ASCT) for multiple myeloma (MM) can be feasible and safe in the outpatient setting and has become standard of care at many centers. Limited literature, however, exists on the types of infectious complications, including specific pathogenic isolates and resistance patterns in the outpatient (OP) setting and whether these differ from the inpatient (IP) setting. This creates uncertainty and variation among local treatment guidelines on antibiotic prophylaxis and treatment of infections in the outpatient population.Methods:In this retrospective study, at total of 240 patients who underwent ASCT at our institution from 2015 to 2016 were reviewed. Study objectives included: 1) to describe the types of infections and pathogenic isolates in MM patients during the acute transplant period and 2) compare isolate results between patients undergoing OP vs IP ASCT. We also aimed to assess 30-day post-ASCT all-cause mortality, readmission rates, and incidence of multidrug resistant bacterial infections in each cohort. As per standard transplant protocol at our institution, all patients received ciprofloxacin and fluconazole prophylaxis and standardized initial therapy for febrile neutropenia with piperacillin-tazocin. Daily GCSF support was routinely used starting day +7 for IPs, and on demand only for OPs.Results:Patient, disease and transplant characteristics:Baseline demographics of both OP and IP cohorts are shown in Table 1 (total n=240; 109 OP, 131 IP). As expected, IPs had poorer performance status (KPS<90), more comorbidities (HCT-CI ≥3), more advanced stage MM, had worse pre-transplant renal function, and were more likely to receive Melphalan dose reductions. Median time to engraftment was similar between groups - 12 days for IP and 13 days for OP (range 9-25), with duration of neutropenia <0.5x10^9/L exceeding 7 days in 46% of the cohort. Median length of stay in hospital was 17 days (12-55) for the inpatients. In the OP cohort, 63.9% of patients required readmission to hospital, with median 3-day hospital stay. In total, only 4 (1.7%) patients required ICU admission.Infections in total cohort (OP and IP):Overall, 121 of 240 patients (50.4%) developed febrile neutropenia, with most common causes including: blood stream infection (BSI) (n=35, 29%), pneumonia (n=18, 14.8%) and respiratory virus infections (n=16, 13.2%). Opportunistic infections were uncommon (n=6, 2.5%) with 1 case of invasive aspergillosis and 5 cases of CMV infection.Of the 43 cases of BSI, 33 (76%) were identified as central line associated blood stream infections (CLABSI), 20 (46.5%) due to gram positive and 20 (46.5%) due to gram negative organisms. Multi-drug resistant gram negative organism isolates were observed in 22 patients (51%), including 5 cases of extended spectrum beta lactamase producing bacteria (ESBL; 11.6%), 9 cases of gram negative bacteria carrying inducible AmpC gene (SPICE; 20.1%), and 10 (23%) cases of quinolone resistant gram negative BSI.Comparison of IP vs OP infections:Although the OP cohort were less frail with fewer comorbidities than the IP cohort, they had more episodes of febrile neutropenia (64% vs 51%; p=0.042), worse mucositis (22.0% vs 10.8%, p= 0.021), and more BSI (24.7% vs 12.9% p=0.019). In the multivariable analysis, only OP status (OR 2.4, p=0.03) and baseline ANC<1.5 pre ASCT (OR 6.3, p=0.003) predicted for BSI occurrence.At 18 months, OS was 98.5% and PFS was 68% for all patients, with no difference between IP and OP (p=0.772). PFS was worse in patients with BSI (p=0.0031, HR 8.7), with gram positive infections in particular (p=0.0264, HR 4.9)Conclusions:In our autotransplant experience, blood stream and lung infections are common, with multi-drug resistance a concern. We were surprised to report that patients undergoing outpatient-based transplant had more BSI, presumably central line-related, and that this may impact disease outcomes (PFS). We hypothesize that outpatients may undergo additional line manipulation or inferior line hygiene, suggesting that enhanced patient/staff education on infection control measures should be a focus. In general, though, outpatient ASCT patients had excellent outcomes, with similar engraftment rates, short readmission rates, low ICU usage, as the entire cohort. DisclosuresTiedemann:Novartis: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Merck: Consultancy; Roche: Consultancy. Chen:Amgen: Honoraria.

Immunopathogenesis and immunotherapy of multiple myeloma.

Despite the advent of novel therapies and improvements in survival, multiple myeloma (MM) remains an incurable disease. Thus, new treatment strategies including immunotherapies are needed for MM patients with stable disease after induction chemotherapy as well as for disease control in patients with advanced disease. However, profound immune dysregulation not only of B cells, but also of other immune cells such as natural killer cells, T cells, and dendritic cells and increase in the number of immunosuppressive cells, i.e., regulatory T and B cells and myeloid-derived suppressor cells, have been demonstrated in advanced MM patients, which may be involved in disease progression. Because of immune dysfunction, immunotherapies have not shown clinical efficacy in MM patients. It is therefore crucial to resolve immunosuppressive mechanisms and improve immune responses, especially in advanced MM patients. Recently, excellent clinical efficacy of new immunotherapeutic strategies such as immunomodulatory drug-intensified monoclonal antibody treatment, immune checkpoint inhibitors, and chimeric antigen receptor T-cell therapy targeting B cell maturation antigen has been reported in advanced-stage MM patients. Those new treatments or their combination will improve prognosis and possibly point toward a cure for myeloma.

CXCR4-directed endoradiotherapy induces high response rates in extramedullary relapsed Multiple Myeloma.

C-X-C-motif chemokine receptor 4 (CXCR4) is a key factor for tumor growth and metastasis in several types of human cancer. We have recently reported promising first-in-man experience with CXCR4-directed endoradiotherapy (ERT) in multiple myeloma (MM).Eight heavily pretreated MM patients underwent a total of 10 ERT cycles (7 patients with 1 cycle and a single patient with 3 cycles). ERT was administered in combination with chemotherapy and autologous stem cell support. End points were occurrence and timing of adverse events, progression-free and overall survival.ERT was overall well tolerated without any unexpected acute adverse events or changes in vital signs. With absorbed tumor doses >30-70 Gy in intra- or extramedullary lesions, significant anti-myeloma activity was observed with 1 patient achieving complete remission and 5/8 partial remission. Directly after ERT major infectious complications were seen in one patient who died from sepsis 22 days after ERT, another patient with high tumor burden experienced lethal tumor lysis syndrome. Median progression-free survival was 54 days (range, 13-175), median overall survival was 223 days (range, 13-313). During follow-up (6 patients available), one patient died from infectious complications, 2/8 from disease progression, the remaining 3/8 patients are still alive.CXCR4-directed ERT was well-tolerated and exerted anti-myeloma activity even at very advanced stage MM with presence of extramedullary disease. Further assessment of this novel treatment option is highly warranted.

Palliative Care of Multiple Myeloma Patients - a Nigerian Retrospective Outcome

Background: According to World Health Organization, Palliative care is an approach that improves the quality of life of patients and their families facing the problems associated with life-threatening illness, through the prevention and relief of suffering by means of early identification, assessment and treatment of pain, and other problems, physical, psychosocial and spiritual. Multiple Myeloma (MM) is one of the hematological malignancies that requires palliative care. This is because of the diagnostic dilemma it poses in resource-limited settings, its life-threatening nature and the suffering it inflicts on people living with it, especially in sub-Saharan Africa. Late presentation coupled with the complications of the disease worsen the prognosis of MM in this region, hence the need for palliative intervention. This study gives insight to the complications presented by people living with MM in the Niger-delta region of Nigeria and the outcome of various palliative interventions recruited to improve their quality of life.Methodology: A-10-year multi-centered retrospective analysis of 26 patients diagnosed and managed in three major centers from January, 2003 to December, 2013. Information on the clinical, laboratory, radiological data as well as palliative treatment (supportive and definitive) was obtained at presentation and at 3 monthly intervals until patients were lost to follow-up.Result: The median age of patients was 60.6 years with M:F ratio of 2.3:1. The mean duration before presentation was 11.8 months (11-48 months) with 61.5% (16), 30.8% (8),and 7.7% (2) presenting in Durie-salmon (DS) stage III,II and I diseases respectively. About 65.4% of the patients had a Performance status (PS) of III-IV (based on Eastern Co-operative Oncology Group (ECOG) classification)) while 34.6% had PS of I-II. The complications presented at diagnosis were anaemia (61.5%), pathological fracture (42.0%), nephropathy (23.1%), and hemiplegia (35%). The mean Hemoglobin concentration, Erythrocyte sedimentation rate (ESR), Bone marrow plasma cells (BMPC), serum creatinine, serum calcium and serum albumin were 7.8±5.1g/dl, 126.9±59.0 mm/hour, 38.5±33.5%, 256±192.5µmol/L, 2.51±0.8mmol/L and 36±9.3g/dl respectively. 25% (1/4) and 75% (3/4) were IgA- and IgG-types myeloma respectively. 70% (14/20) had osteopenic bone lesions. All (100%) the patients received analgesics (mainly NSAID regimens-non could access oral morphine) and hematinics (Iron supplements) as supportive interventions while 56.7%, 50.0%, and 19% had surgery, blood transfusion and renal hemodialysis respectively. Radiotherapy, Bisphosphonates, Erythropoietin and G-CSF (Neupogen) were received by 3.8%, 38%, 38%, and 11.4% of the patients respectively. 57.6% were on melphalan-prednisone (MP) double regimen while 19% and 8% were on MP-Thalidomide and MP-Bortezomib triple regimens respectively.8% were on Cyclophosphamide plus Prednisolone (CP). 3.8% at DS stage III-B disease had an Autologous Stem Cell Transplantation (ASCT). The mean survival interval was 13.12 months (95% CI, 6.65-19.58). The patients on MPV had longest duration of 72 months while the patients on CP had least duration of 2 months.Conclusion: The PC of people living with advanced stage MM in Niger-Delta Nigeria is grossly inadequate. This could account significantly for the poor prognostic outcome of MM in the region. There is need to scale up palliative care of people living with MM via proper diagnosis, good supportive and definitive interventions. Oral morphine should be made availabe to alleviate the pains and sufferings due to bone lesions in this condition. DisclosuresNo relevant conflicts of interest to declare.

Noncanonical SQSTM1/p62-Nrf2 pathway activation mediates proteasome inhibitor resistance in multiple myeloma cells via redox, metabolic and translational reprogramming.

Multiple Myeloma (MM) is a B-cell malignancy characterized by the accumulation of clonal plasma cells in the bone marrow, with drug resistance being a major cause of therapeutic failure. We established a carfilzomib-resistant derivative of the LP-1 MM cell line (LP-1/Cfz) and found that the transcription factor NF-E2 p45-related factor 2 (Nrf2; gene symbol NFE2L2) contributes to carfilzomib resistance. The mechanism of Nrf2 activation involved enhanced translation of Nrf2 as well as its positive regulator, the autophagy receptor sequestosome 1 (SQSTM1)/p62. The eukaryotic translation initiation factor gene EIF4E3 was among the Nrf2 target genes upregulated in LP-1/Cfz cells, suggesting existence of a positive feedback loop. In line with this, we found that siRNA knockdown of eIF4E3 decreased Nrf2 protein levels. On the other hand, elevated SQSTM1/p62 levels were due at least in part to activation of the PERK-eIF2α pathway. LP-1/Cfz cells had decreased levels of reactive oxygen species as well as elevated levels of fatty acid oxidation and prosurvival autophagy. Genetic and pharmacologic inhibition of the Nrf2-EIF4E3 axis or the PERK-eIF2α pathway, disruption of redox homeostasis or inhibition of fatty acid oxidation or autophagy conferred sensitivity to carfilzomib. Our findings were supported by clinical data where increased EIF4E3 expression was predictive of Nrf2 target gene upregulation in a subgroup of patients with chemoresistant minimal residual disease and relapsed/refractory MM. Thus, our data offer a preclinical rationale for including inhibitors of the SQSTM1/p62-Nrf2 pathway to the treatment regimens for certain advanced stage MM patients.

In Vitro and In Vivo Efficacy of a Novel Orally Bioavailable Beta-Catenin Inhibitor-BC2059- As Monotherapy or in Combination with Proteasome Inhibitors in Multiple Myeloma

Background: The currently available treatment options are unlikely to be curative for the majority of Multiple Myeloma (MM) patients, emphasizing a continuing role for the introduction of investigational agents that overcome drug resistance. The canonical Wnt/β-catenin signalling pathway has been found to be dysregulated in MM, and its activation is associated with advanced stage MM, providing a rationale to evaluate the novel β-catenin inhibitor BC2059 in mono- and combination therapy with proteasome inhibitors in vitro and in vivo.Methods and Results: We evaluated the activation status of the canonical Wnt pathway in 12 genetically heterogeneous Human Myeloma Cell Lines (HMCL) by assessing the expression of β-catenin protein in the nuclear compartment (active form). This showed that nuclear β-catenin was present in all HMCL tested and absent in plasma cells derived from a healthy donor. Moreover, additional stimulation of the canonical pathway with rhWnt3a was shown to be pro-proliferative, in contrast, no proliferation was seen with activation of the non canonical pathway following treatment with rhWnt5a. BC2059 (50nM to 500nM) induced apoptosis of all 12 HMCL and was able to inhibit the proliferation of all HMCL tested in a dose and time dependent manner assessed by MTS assay and viable enumeration with trypan blue (IC50: 53nM to 247nM). Mimicking the bone marrow (BM) microenvironment by co-culturing HMCL with the immortalised human stromal cell line HS-5, BC2059 was able to overcome the protective effect of HS-5 (for example KMS18 at IC90=220nM had no stromal pro-survival effect). Similarly, BC2059 was able to abolish the pro-proliferative effect of rh-Wnt3a or conditioned media derived from MM patients' BM when used at doses >100nM or 50nM, respectively. BC2059 facilitated the degradation of β-catenin protein in the nuclear cellular compartment ( >50% decrease of nuclear β-catenin in KMS18 treated with 1.5xIC50 when compared with untreated cells), furthermore, using a reporter assay we showed that BC2059 inhibited TCF/LEF transcriptional activity in a dose-dependent manner and decreased the transcription of axin2, a down-stream target gene of β-catenin - 78% reduction in KMS18 cells treated with 1.5x IC50 when compared to untreated controls. BC2059-induced HMCL cell death was associated with activation of both the intrinsic and extrinsic caspase-dependent apoptotic pathways, as shown by the accumulation of the activated forms of caspases 8, 9 and 3 following BC2059 treatment. However, inhibition of the caspase-pathway by the addition of caspase inhibitors (pan-caspase inhibitor Z-VAD, and caspase-3 inhibitor Z-DEVD) could not abolish the pro-necrotic effect of BC2059 or BC2059 plus bortezomib, suggesting a possible role for autophagy-induced cell death. As β-catenin undergoes proteasome-mediated destruction and has been found to increase following bortezomib treatment, we evaluated the effect of combining BC2059 with Bortezomib. The combination was synergistic for 6/8 HMCL tested (e.g. for LP1 CI:0.64-0.55, where CI<1.1=synergism). We also evaluated the effect of the combination of BC2059 with next generation proteasome inhibitors (carfilzomib and marizomib) where it was shown to have synergistic and/or additive effects (e.g. for carfilzomib LP1 CI:0.33-0.99). Single agent BC2059 effectively killed primary MM tumour cells from relapsed/refractory MM patients (n=13) and the combination with bortezomib was synergistic (n=2) with no effect on healthy peripheral blood mononuclear cells (n=4). Finally, BC2059 (10mg/kg) prolonged survival of xenografted NSG mice compared to untreated controls with no major side effects in Wnt/β-catenin dependent tissues (GI track and haematopoiesis).Conclusion: We have demonstrated that BC2059 at nano-molar concentrations has a strong anti-MM effect both in vitro and in vivo and synergises with proteasome inhibitors. These data strongly support the clinical evaluation of BC2059 for the treatment of MM. DisclosuresHorrigan:BetaCat Pharmaceuticals: Employment.

First-in-Human Experience of CXCR4-Directed Endoradiotherapy with 177Lu- and 90Y-Labeled Pentixather in Advanced-Stage Multiple Myeloma with Extensive Intra- and Extramedullary Disease.

Chemokine receptor 4 (CXCR4) is a key factor for tumor growth and metastasis in several types of human cancer. Based on promising experiences with a radiolabeled CXCR4 ligand ((68)Ga-pentixafor) for diagnostic receptor targeting, (177)Lu- and (90)Y-pentixather were recently developed as endoradiotherapeutic vectors. Here, we summarize the first-in-human experience in 3 heavily pretreated patients with intramedullary and extensive extramedullary manifestations of multiple myeloma undergoing CXCR4-directed endoradiotherapy. CXCR4 target expression was demonstrated by baseline (68)Ga-pentixafor PET. Each treatment was approved by the clinical ethics committee. Pretherapeutic (177)Lu-pentixather dosimetry was performed before (177)Lu-pentixather or (90)Y-pentixather treatment. Subsequently, patients underwent additional chemotherapy and autologous stem cell transplantation for bone marrow rescue. A remarkable therapeutic effect was visualized in 2 patients, who showed a significant reduction in (18)F-FDG uptake. CXCR4-targeted radiotherapy with pentixather appears to be a promising novel treatment option in combination with cytotoxic chemotherapy and autologous stem cell transplantation, especially for patients with advanced multiple myeloma.

Long‐term survival after allogeneic stem cell transplantation for advanced stage multiple myeloma

Despite the use of novel drugs and autologous stem cell transplantation (SCT), multiple myeloma (MM) is still considered to be an incurable disease. Both autologous and allogeneic SCT (allo-SCT) improve long-term survival. Allo-SCT can result in long-term disease control and remains a potentially curative therapeutic modality, related in part to the immune anti-myeloma effect of the allograft, but it is hampered by high transplant-related toxicities. Reduced intensity conditioning (RIC) allo-SCT has been developed in order to reduce transplant-related mortality while maintaining a graftversus-myeloma (GVM) effect. Few studies have reported long-term outcomes in MM after allo-SCT, with a median follow-up typically ranging from 2 to 5y ears. Recently, Sahebiet al (2013) reported an overall survival (OS) of 60%, progression-free survival (PFS) of 31% and relapse incidence of 59% at 7 years in 60 MM patients undergoing allo-SCT after RIC, with a median follow-up of 98 years. Very late relapses occurred in 10% of patients, even as late as 115 years after transplantation (Sahebi et al, 2013). We report on the long-term follow-up (median

Bone Marrow Stromal Mediated Pro-Oncogenic Wnt/β-Catenin Antagonize sDKK1 and Promote Osteoclast Activity In Multiple Myeloma

IntroductionBone marrow stromal cells (BMSC) provide a suitable microenvironment for multiple myeloma (MM) cell survival, resistance and related osteolytic bone disease. Wnt signaling enhances osteoblast proliferation and mineralization, whereas it blocks osteoblast apoptosis and osteoclast differentiation by increasing the OPG/RANKL ratio. The Wnt inhibitor Dickkopf 1 (Dkk1) negatively regulates canonical Wnt signaling by binding to and antagonizing the Wnt co-receptors Lrp5/6 (He et al., 2004). Serum levels of DKK1 have been reported to be elevated in patients with MM, while osteoprotegrin levels are decreased (Tian et al 2003). Crosstalk between Wnt/β-catenin and other signaling pathways within the bone marrow microenvironment that regulate the osteogenic differentiation of mesenchymal stem cells (MSCs) has been reported previously (Cawthorne et al. 2012 and Krishnan et al. 2006). However the question of whether the pro-oncogenic effect of Wnt-β-catenin signal from the stroma antagonizes the elevated serum DKK1 and thus actually enhance the osteolytic actvities in MM relapse remains to be answered. MethodsBone marrow-derived stromal fibroblasts from myeloma patients were maintained in culture for 21 days. Adherent stromal fibroblast (confirmed with staining for Vimentin) and non-adherent cells were examined for DKK1 by ELISA and for Wnt β-catenin by performing (a) immunofuoresence detection, (b) luciferase reporter assays and (c) Western blot analysis. ResultsAdherent stromal fibroblasts showed β-catenin expression after 21 days in culture (Fig 1), the signal for β-catenin expression was 68% compared to that in non-adherent BM cells (27%). Consistently, luciferase reporter assays of adherent stromal fibroblasts transfected with Wnt/β-cat responsive reporter plasmids (STF16) showed >4 fold increase in the reporter activity compared to that in the MM cells. As expected, the level of soluble DKK1 was significantly lower in patients with early MM (ranges from 5167 ± 379 pg/ml) vs. advanced stage MM (53967±1323 pg/ml) among patients who are relapsed (n=16), and the difference in the DKK1 levels between these two groups of samples (early vs. advanced stage MM, n=8 each) are statistically significant (p<0.05). The increase in serum DKK1 is also directly correlated with increase in the serum VEGF and vascular tube formation suggesting the stromal influence of Wnt/β-catenin in mediating angiogenesis in MM relapse patients. Transfection of stromal fibroblasts with siRNA for NF-κBp65 down regulated both β-catenin and DKK1 at the mRNA and protein levels, suggesting a close interaction between Wnt/β catenin and inflammatory signaling within the BM stromal microenvironment.To further determine the effect of potential drugs on the regulation of β-catenin and DKK1, we measured soluble DKK1 activity in the cell culture medium of growing BMSC cells stimulated with human M-CSF and RANKL to generate osteoclasts and osteoblasts, followed by treatment with different concentrations of Bortezomib, or Dipyridamole (an agent which increases exracellular adenosine levels) at a concentration of 2.5, 5.0 and 10ug/ml respectively) for 24h. As previously reported, dipyridamole increased obsteoblast differentiation, surprisingly, diminished sDKK1 levels, as well as bortezomib (11667±1528 pg/ml) at 10 ug/ml. SummaryOur preliminary findings suggest that Wnt/β-catenin and sDKK1are co-produced in BM stromal cells and are both diminished by drugs that modulate both Wnt and RANKL pathways. Another interesting message from this study is that the stromal derived β-catenin may influence bone metastasis of myeloma, as seen in other cancer types, via transcriptionally regulating DKK1 within the BM niche. Since the bone marrow stromal cells from MM relapse patients still express an elevated level of β-catenin, in spite of the higher serum DKK1 level in MM relapse patients, we believe that stromal contribution of β-catenin is critical in patients with multiple myeloma.▪ Disclosures:No relevant conflicts of interest to declare.

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.

MiR-15a and miR-16 affect the angiogenesis of multiple myeloma by targeting VEGF

Deregulated microRNAs (miRNAs) and their roles in cancer development have attracted much attention. Two miRNAs, miR-15a and miR-16, which act as putative tumor suppressor by targeting the oncogene BCL2, have been implicated in cell cycle, apoptosis and proliferation. In this study, we investigated the possible role of miR-15a/16 in the angiogenesis of multiple myeloma (MM). Using a stem-loop quantitative reverse transcription-PCR, we analyzed miR-15a/16 expressions in bone marrow samples from newly diagnosed MM patients and a panel of MM cell lines. miRNA transfection, western blotting analysis and assay of luciferase activity were used to examine whether vascular endothelial growth factor (VEGF) is the target of miR-15a/16. The functional roles of miR-15a/16 on tumorigenesis and angiogenesis were examined by in vitro angiogenesis models and in vivo tumor xenograft model. We showed that miR-15a and miR-16 were significantly underexpressed in primary MM cells as well as in MM cell lines. The aberrant expression of miR-15a/16 was detected especially in advanced stage MM. In human MM cell lines and normal plasma cells, expression of miR-15a/16 inversely correlated with the expression of VEGF-A. Western blotting combined with the luciferase reporter assay demonstrated that VEGF-A was a direct target of miR-15a/16. Ectopic overexpression of miR-15a/16 led to decreased pro-angiogenic activity of MM cells. Finally, infection of lentivirus-miR-15a or lentivirus-miR-16 resulted in significant inhibition of tumor growth and angiogenesis in nude mice. This study suggest that miR-15a/16 could play a role in the tumorigenesis of MM at least in part by modulation of angiogenesis through targeting VEGF-A.

Transcriptional Silencing of the Wnt-Antagonist DKK1 by Promoter Methylation Is Associated with Enhanced Wnt Signaling in Advanced Multiple Myeloma

The Wnt/β-catenin pathway plays a crucial role in the pathogenesis of various human cancers. In multiple myeloma (MM), aberrant auto-and/or paracrine activation of canonical Wnt signaling promotes proliferation and dissemination, while overexpression of the Wnt inhibitor Dickkopf1 (DKK1) by MM cells contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since DKK1 itself is a target of TCF/β-catenin mediated transcription, these findings suggest that DKK1 is part of a negative feedback loop in MM and may act as a tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is low or undetectable in a subset of patients with advanced MM as well as in MM cell lines. This absence of DKK1 is correlated with enhanced Wnt pathway activation, evidenced by nuclear accumulation of β-catenin, which in turn can be antagonized by restoring DKK1 expression. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling.

Autologous Stem Cell Transplant in Patients with Multiple Myeloma Previously Treated with Lenalidomide: A Single-Institution Experience Using An Intermediate-Dose Cyclophosphamide-Based Regimen

Abstract 4391 Background:High dose chemotherapy followed by autologous transplantation of peripheral blood stem cells plays an important role in the management of intermediate- and advanced-stage multiple myeloma. In order for successful engraftment to occur, adequate numbers of high quality peripheral stem cells must be harvested prior to transplantation. Mobilization of PBSC in patients treated with lenalidomide has been associated with poor apheresis collections after G-CSF mobilization. The purpose of this study is to present our institution's experience utilizing an intermediate-dose cyclophosphamide-based mobilization regimen. Methods:We retrospectively analyzed data for patients with multiple myeloma who underwent autologous stem cell transplant at UCLA between 2006 and 2010. Data were obtained from the database of the UCLA Heme Malignancy/Stem Cell Transplant Unit, the electronic health record system and stem cell processing laboratory. All patients underwent mobilization with a regimen of cyclophosphamide 2.5g/m2 IVPB, G-CSF 10 mcg/kg/day for 4 days SQ, and prednisone 2mg/kg/day for 4 days po. The number of CD 34+ cells was used as a marker for the number of peripheral stem cells collected. Minimum dose collected to ensure adequate engraftment was 2×106/kg CD34+ cells. Patients were conditioned with melphalan 100mg/m2/d x2 (unless there was evidence of renal failure, in which case the dose was reduced to 100mg/m2) with subsequent infusion of stem cells. Neutrophil engraftment was defined as the first day of absolute neutrophil count greater than 500×106/L ≥ 7 days after transplant. Results:Autologous stem cell transplant was performed in 103 patients with multiple myeloma at UCLA between 2006 and 2010. Median number of apheresis procedures was 1 (1–12) with a median of 4.4×106/kg (1.4–33.5) CD34+ cells collected. Median time to engraftment was 10 (8–18) days. Thirty-five patients received lenalidomide at some point in their pretransplant treatment. Median number of apheresis procedures was 1 in both lenalidomide and non lenalidomide treated groups. In the lenalidomide treated group 54% required only one collection versus 75% in the non lenalidomide treated group (p=0.033). In the lenalidomide treated group 31% required 3 more or more collections versus 10% in non lenalidomide treated group (p=0.0075). Three patients in the lenalidomide group had subsequent mobilization with plerixafor with one of these requiring bone marrow harvesting. Median CD34+ cells collected was 3.5×106/kg and 4.9×106/kg (p=0.246) in the lenalidomide and non-lenalidomide groups respectively. Both groups had a median time to neutrophil engraftment of 10 days with a similar range. Conclusion:Pre-transplant use of lenalidomide adversely affected the number of stem cell apheresis procedures required to procure adequate stem cell dose as evidenced by a greater percentage requiring 3 or more collections. However, despite prior lenalidomide exposure, the use of our mobilization regimen permitted adequate collection, with the majority of patients requiring only one apheresis procedure, and led to an equivalent time to neutrophil recovery. Disclosures:Off Label Use: Cyclophosphamide and G-CSF for mobilization of stem cells.

Bendamustine, thalidomide and dexamethasone is an effective salvage regimen for advanced stage multiple myeloma - response to Grey-Davies et al

Bendamustine, thalidomide and dexamethasone is an effective salvage regimen for advanced stage multiple myeloma - response to Grey-Davies et al