Elotuzumab plus pomalidomide and dexamethasone in relapsed/refractory multiple myeloma: a multicenter, retrospective, real-world experience with 200 cases outside of controlled clinical trials.

In the ELOQUENT-3 trial, the combination of elotuzumab, pomalidomide and dexamethasone (EloPd) proved to have a superior clinical benefit over pomalidomide and dexamethasone with a manageable toxicity profile, leading to its approval for the treatment of patients with relapsed/refractory multiple myeloma (RRMM) who have received at least two prior therapies, including lenalidomide and a proteasome inhibitor. We report here a real-world experience of 200 cases of RRMM treated with EloPd in 35 Italian centers outside of clinical trials. In our dataset, the median number of prior lines of therapy was two, with 51% of cases undergoing autologous stem cell transplant and 73% having been exposed to daratumumab. After a median follow-up of 9 months, 126 patients had stopped EloPd, most of them (88.9%) because of disease progression. The overall response rate was 55.4%, a finding in line with the pivotal trial results. Regarding adverse events, the toxicity profile in our cohort was similar to that in the ELOQUENT-3 trial, with no significant differences between younger (<70 years) and older patients. The median progression-free survival was 7 months, which was shorter than that observed in ELOQUENT-3, probably because of the different clinical characteristics of the two cohorts. Interestingly, International Staging System stage III disease was associated with worse progression-free survival (hazard ratio=2.55). Finally, the median overall survival of our series was shorter than that observed in the ELOQUENT-3 trial (17.5 vs. 29.8 months). In conclusion, our real-world study confirms that EloPd is a safe and possible therapeutic choice for patients with RRMM who have received at least two prior therapies, including lenalidomide and a proteasome inhibitor.

Elotuzumab plus pomalidomide and dexamethasone in relapsed/refractory multiple myeloma: Extended follow-up of a multicenter, retrospective real-world experience with 321 cases outside of controlled clinical trials.

The ELOQUENT-3 trial demonstrated the superiority of the combination of elotuzumab, pomalidomide, and dexamethasone (EloPd) in terms of efficacy and safety, compared to Pd in relapsed/refractory multiple myeloma (RRMM), who had received at least two prior therapies, including lenalidomide and a proteasome inhibitor. The present study is an 18-month follow-up update of a previously published Italian real-life RRMM cohort of patients treated with EloPd. This revised analysis entered 319 RRMM patients accrued in 41 Italian centers. After a median follow-up of 17.7 months, 213 patients (66.4%) experienced disease progression or died. Median progression-free survival (PFS) and overall survival (OS) were 7.5 and 19.2 months, respectively. The updated multivariate analysis showed a significant reduction of PFS benefit magnitude both in advanced International Staging System (ISS) (II and III) stages and previous exposure to daratumumab cases. Instead, advanced ISS (II and III) stages and more than 2 previous lines of therapy maintained an independent prognostic impact on OS. Major adverse events included grade three-fourths neutropenia (24.9%), anemia (13.4%), lymphocytopenia (15.5%), and thrombocytopenia (10.7%), while infection rates and pneumonia were 19.3% and 8.7%, respectively. A slight increase in the incidence of neutropenia and lymphocytopenia was registered with longer follow-up. In conclusion, our real-world study still confirms that EloPd is a safe and possible therapeutic choice for RRMM. Nevertheless, novel strategies are desirable for those patients exposed to daratumumab.

Dependence on glutamine uptake and glutamine addiction characterize myeloma cells: a new attractive target.

The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138(+) cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138(+) cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138(+) cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM.

The Proteasome and Myeloma-Associated Bone Disease.

Bone disease is the hallmark of multiple myeloma (MM), a hematological malignancy characterized by osteolytic lesions due to a severe uncoupled and unbalanced bone remodeling with pronounced osteoblast suppression. Bone metastasis is also a frequent complication of solid tumors including advanced breast or prostate cancer. In the past years, the ubiquitin-proteasome pathway has been proved critical in regulating the balance between bone formation and bone resorption. Proteasome inhibitors (PIs) are a new class of drugs, currently used in the treatment of MM, that affect both tumor cells and bone microenvironment. Particularly, PIs stimulate osteoblast differentiation by human mesenchymal stromal cells and increase bone regeneration in mice. Interestingly, in vitro data indicate that PIs block MM-induced osteoblast and osteocyte cell death by targeting both apoptosis and autophagy. The preclinical data are supported by the following effects observed in MM patients treated with PIs: increase of bone alkaline phosphatase levels, normalization of the markers of bone turnover, and reduction of the skeletal-related events. Moreover, the histomorphometric data indicate that the treatment with bortezomib stimulates osteoblast formation and maintains osteocyte viability in MM patients. This review updates the evidence on the effects of PIs on bone remodeling and on cancer-induced bone disease while focusing on MM bone disease.

IL21R expressing CD14+CD16+ monocytes expand in multiple myeloma patients leading to increased osteoclasts.

Bone marrow monocytes are primarily committed to osteoclast formation. It is, however, unknown whether potential primary alterations are specifically present in bone marrow monocytes from patients with multiple myeloma, smoldering myeloma or monoclonal gammopathy of undetermined significance. We analyzed the immunophenotypic and transcriptional profiles of bone marrow CD14+ monocytes in a cohort of patients with different types of monoclonal gammopathies to identify alterations involved in myeloma-enhanced osteoclastogenesis. The number of bone marrow CD14+CD16+ cells was higher in patients with active myeloma than in those with smoldering myeloma or monoclonal gammopathy of undetermined significance. Interestingly, sorted bone marrow CD14+CD16+ cells from myeloma patients were more pro-osteoclastogenic than CD14+CD16-cells in cultures ex vivo Moreover, transcriptional analysis demonstrated that bone marrow CD14+ cells from patients with multiple myeloma (but neither monoclonal gammopathy of undetermined significance nor smoldering myeloma) significantly upregulated genes involved in osteoclast formation, including IL21RIL21R mRNA over-expression by bone marrow CD14+ cells was independent of the presence of interleukin-21. Consistently, interleukin-21 production by T cells as well as levels of interleukin-21 in the bone marrow were not significantly different among monoclonal gammopathies. Thereafter, we showed that IL21R over-expression in CD14+ cells increased osteoclast formation. Consistently, interleukin-21 receptor signaling inhibition by Janex 1 suppressed osteoclast differentiation from bone marrow CD14+ cells of myeloma patients. Our results indicate that bone marrow monocytes from multiple myeloma patients show distinct features compared to those from patients with indolent monoclonal gammopathies, supporting the role of IL21R over-expression by bone marrow CD14+ cells in enhanced osteoclast formation.

Multiple Myeloma in 2023 Ways: From Trials to Real Life.

Multiple myeloma is a chronic hematologic malignancy that obstinately tends to relapse. Basic research has made giant strides in better characterizing the molecular mechanisms of the disease. The results have led to the manufacturing of new, revolutionary drugs which have been widely tested in clinical trials. These drugs have been approved and are now part of the therapeutic armamentarium. As a consequence, it is essential to combine what we know from clinical trials with real-world data in order to improve therapeutic strategies. Starting with this premise, our review aims to describe the currently employed regimens in multiple myeloma and compare clinical trials with real-life experiences. We also intend to put a spotlight on promising therapies such as T-cell engagers and chimeric antigen receptor T-cells (CAR-T) which are proving to be effective in changing the course of advanced-stage disease.

Targeting LAG3/GAL-3 to overcome immunosuppression and enhance anti-tumor immune responses in multiple myeloma.

Immune profiling in patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and multiple myeloma (MM) provides the framework for developing novel immunotherapeutic strategies. Here, we demonstrate decreased CD4+ Th cells, increased Treg and G-type MDSC, and upregulation of immune checkpoints on effector/regulatory and CD138+ cells in MM patients, compared MGUS/SMM patients or healthy individuals. Among the checkpoints profiled, LAG3 was most highly expressed on proliferating CD4+ Th and CD8+ Tc cells in MM patients BMMC and PBMC. Treatment with antibody targeting LAG3 significantly enhanced T cells proliferation and activities against MM. XBP1/CD138/CS1-specific CTL generated in vitro displayed anti-MM activity, which was further enhanced following anti-LAG3 treatment, within the antigen-specific memory T cells. Treg and G-type MDSC weakly express LAG3 and were minimally impacted by anti-LAG3. CD138+ MM cells express GAL-3, a ligand for LAG3, and anti-GAL-3 treatment increased MM-specific responses, as observed for anti-LAG3. Finally, we demonstrate checkpoint inhibitor treatment evokes non-targeted checkpoints as a cause of resistance and propose combination therapeutic strategies to overcome this resistance. These studies identify and validate blockade of LAG3/GAL-3, alone or in combination with immune strategies including XBP1/CD138/CS1 multipeptide vaccination, to enhance anti-tumor responses and improve patient outcome in MM.

Monoclonal and Bispecific Anti-BCMA Antibodies in Multiple Myeloma.

B-cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor superfamily, is universally expressed by normal and neoplastic plasma cells and plays a critical role in the proliferation, survival and tumor progression in multiple myeloma (MM). B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) have been recognized as proliferation ligands for BCMA in the bone marrow microenvironment. Soluble BCMA levels in the serum correlates with disease phase and tumor burden and is a predictor of progression-free survival (PFS) and overall survival (OS). Recently, the introduction of new monoclonal antibodies against CD38 (Daratumumab and Isatuximab) and SLAM7 (Elotuzumab) has changed the therapeutic approach to MM, improving the response rate and the time to progression, both in newly diagnosed and refractory/relapsed patients. Among the surface antigens on MM cells, BCMA is a suitable target for the design of new antibody-based strategies. Experimental approaches targeting BCMA are currently being investigated and include antibody-drug conjugates (ADCs), bispecific antibodies (bsAbs) and genetically engineered T-cells with chimeric antigen receptors (CAR). In this review we summarize the more recent findings about BCMA biologic rationale as a therapeutic target and report the updated results of preclinical and clinical studies focused on ADCs and bsAbs targeting BCMA.

Novel targets for the treatment of relapsing multiple myeloma.

Introduction: Multiple myeloma (MM) is characterized by the high tendency to relapse and develop drug resistance. Areas covered: This review focused on the main novel targets identified to design drugs for the treatment of relapsing MM patients. CD38 and SLAMF7 are the main surface molecules leading to the development of monoclonal antibodies (mAbs) recently approved for the treatment of relapsing MM patients. B cell maturation antigen (BCMA) is a suitable target for antibody-drug conjugates, bispecific T cell engager mAbs and Chimeric Antigen Receptor (CAR)-T cells. Moreover, the programmed cell death protein 1 (PD)-1/PD-Ligand (PD-L1) expression profile by MM cells and their microenvironment and the use of immune checkpoints inhibitors in MM patients are reported. Finally, the role of histone deacetylase (HDAC), B cell lymphoma (BCL)-2 family proteins and the nuclear transport protein exportin 1 (XPO1) as novel targets are also underlined. The clinical results of the new inhibitors in relapsing MM patients are discussed. Expert opinion: CD38, SLAMF7, and BCMA are the main targets for different immunotherapeutic approaches. Selective inhibitors of HDAC6, BCL-2, and XPO1 are new promising compounds under clinical investigation in relapsing MM patients.

Bone Marrow CX3CL1/Fractalkine is a New Player of the Pro-Angiogenic Microenvironment in Multiple Myeloma Patients.

C-X3-C motif chemokine ligand 1 (CX3CL1)/fractalkine is a chemokine released after cleavage by two metalloproteases, ADAM metallopeptidase domain 10 (ADAM10) and ADAM metallopeptidase domain 17 (ADAM17), involved in inflammation and angiogenesis in the cancer microenvironment. The role of the CX3CL1/ C-X3-C motif chemokine receptor 1(CX3CR1) axis in the multiple myeloma (MM) microenvironment is still unknown. Firstly, we analyzed bone marrow (BM) plasma levels of CX3CL1 in 111 patients with plasma cell disorders including 70 with active MM, 25 with smoldering myeloma (SMM), and 16 with monoclonal gammopathy of undetermined significance (MGUS). We found that BM CX3CL1 levels were significantly increased in MM patients compared to SMM and MGUS and correlated with BM microvessel density. Secondly, we explored the source of CX3CL1 in MM and BM microenvironment cells. Primary CD138⁺ cells did not express CXC3L1 but up-regulated its production by endothelial cells (ECs) through the involvement of tumor necrosis factor alpha (TNFα). Lastly, we demonstrated the presence of CX3CR1 on BM CD14⁺CD16⁺ monocytes of MM patients and on ECs, but not on MM cells. The role of CX3CL1 in MM-induced angiogenesis was finally demonstrated in both in vivo chick embryo chorioallantoic membrane and in vitro angiogenesis assays. Our data indicate that CX3CL1, present at a high level in the BM of MM patients, is a new player of the MM microenvironment involved in MM-induced angiogenesis.

Neurofibromatosis type I and multiple myeloma coexistence: A possible link?

The association between Neurofibromatosis type I (NF1) and multiple myeloma (MM), a plasma cell, dyscrasia is very rare. Here we put to the attention of the scientific community two new cases. The first one is a patient with active MM whereas the second with smoldering MM. Both patients present typical features of NF1 but skeletal alterations were present only in the second case including dysplasia, marked scoliosis and osteoporosis. MM osteolytic lesions were absent in both patients. In addition to the clinical diagnosis of NF1, a molecular testing for NF1 gene mutations has been performed finding that patient one was heterozygous for the c.6855C>A (Tyr2285Ter) mutation, while patient two was heterozygous for the c.7838dupC (Lys2614GlufsTer20) mutation. The two mutations were diagnosed both in genomic DNA from peripheral blood and from MM cells. The potential link between NF1 mutation and the increased risk of MM is discussed in the report.

A Rare Case of Systemic AL Amyloidosis with Muscle Involvement: A Misleading Diagnosis.

Muscle involvement in AL amyloidosis is a rare condition, and the diagnosis of amyloid myopathy is often delayed and underdiagnosed. Amyloid myopathy may be the initial manifestation and may precede the diagnosis of systemic AL amyloidosis. Here, we report the case of a 73-year-old man who was referred to our center for a monoclonal gammopathy of undetermined significance (MGUS) diagnosed since 1999. He reported a progressive weakness of proximal muscles of the legs with onset six months previously. Muscle biopsy showed mild histopathology featuring alterations of nonspecific type with a mixed myopathic and neurogenic involvement, and the diagnostic turning point was the demonstration of characteristic green birefringence under cross-polarized light following Congo red staining of perimysial vessels. Transmission electron microscopy (TEM) confirmed amyloid fibrils around perimysial vessels associated with collagen fibrils. A stepwise approach to diagnosis and staging of this disorder is critical and involves confirmation of amyloid deposition, identification of the fibril type, assessment of underlying amyloidogenic disorder, and evaluation of the extent and severity of amyloidotic organ involvement.

Lenalidomide increases human dendritic cell maturation in multiple myeloma patients targeting monocyte differentiation and modulating mesenchymal stromal cell inhibitory properties.

The use of Lenalidomide (LEN), to reverse tumor-mediated immune suppression and amplify multiple myeloma-specific immunity is currently being explored. Particularly, LEN effects on dendritic cells (DCs) are still unclear. In this study, we investigated the potential effect of LEN on DC differentiation and activity. DCs were differentiated either from CD14+ cells obtained from patients with multiple myeloma or from a human monocytic cell line. LEN, at the concentration range reached in vivo, significantly increased the median intensity expression of HLA-DR, CD86 and CD209 by DCs derived from both bone marrow and peripheral myeloma monocytes and enhanced the production of Interleukin-8, C-C motif chemokine ligand (CCL) 2, CCL5 and tumor necrosis factor-α. Consistently, LEN pre-treated DCs showed an increased ability to stimulate autologous CD3+ cell proliferation. LEN effect on dendritic differentiation was associated with the degradation of the Cereblon-related factors Ikaros and Aiolos. Moreover, we showed that LEN also blunted mesenchymal stromal cell inhibitory effect on dendritic differentiation, inhibiting Casein Kinase-1α levels. Finally, in vitro data were confirmed in ex vivo cultures obtained from relapsed myeloma patients treated with LEN, showing a significant increase of DC differentiation from peripheral blood monocytes. In conclusion, LEN increased the expression of mature dendritic markers both directly and indirectly and enhanced DC ability to stimulate T cell proliferation and to release chemokines. This suggests a new possible mechanism by which LEN could exert its anti-myeloma activity.

Expression of CD38 in myeloma bone niche: A rational basis for the use of anti-CD38 immunotherapy to inhibit osteoclast formation.

It is known that multiple myeloma (MM) cells express CD38 and that a recently developed human anti-CD38 monoclonal antibody Daratumumab mediates myeloma killing. However, the expression of CD38 and other functionally related ectoenzymes within the MM bone niche and the potential effects of Daratumumab on bone cells are still unknown. This study firstly defines by flow cytometry and immunohistochemistry the expression of CD38 by bone marrow cells in a cohort of patients with MM and indolent monoclonal gammopathies. Results indicate that only plasma cells expressed CD38 at high level within the bone niche. In addition, the flow cytometry analysis shows that CD38 was also expressed by monocytes and early osteoclast progenitors but not by osteoblasts and mature osteoclasts. Indeed, CD38 was lost during in vitro osteoclastogenesis. Consistently, we found that Daratumumab reacted with CD38 expressed on monocytes and its binding inhibited in vitro osteoclastogenesis and bone resorption activity from bone marrow total mononuclear cells of MM patients, targeting early osteoclast progenitors. The inhibitory effect was not observed from purified CD14+ cells, suggesting an indirect inhibitory effect of Daratumumab. Interestingly, all-trans retinoic acid treatment increased the inhibitory effect of Daratumumab on osteoclast formation. These observations provide a rationale for the use of an anti-CD38 antibody-based approach as treatment for multiple myeloma-induced osteoclastogenesis.

Cutaneous localization in multiple myeloma in the context of bortezomib-based treatment: how do myeloma cells escape from the bone marrow to the skin?

The skin is a possible site of extramedullary localization in multiple myeloma (MM) patients; however, the mechanisms involved in this process are poorly understood. We describe the case of a refractory MM patient who developed a cutaneous localization under bortezomib treatment and we further expanded observations in other eight MM patients. We focused on the expression of genes involved in plasma cell skin homing, including CCR10, which was highly expressed. Moreover, we observed a lack of CXCR4 surface expression and the down-regulation of ICAM1/CD54 throughout the progression of the disease, suggesting a possible mechanism driving the escape of MM cells from the bone marrow into the skin.

The Proteasome Inhibitor Bortezomib Maintains Osteocyte Viability in Multiple Myeloma Patients by Reducing Both Apoptosis and Autophagy: A New Function for Proteasome Inhibitors.

Multiple myeloma (MM) is characterized by severely imbalanced bone remodeling. In this study, we investigated the potential effect of proteasome inhibitors (PIs), a class of drugs known to stimulate bone formation, on the mechanisms involved in osteocyte death induced by MM cells. First, we performed a histological analysis of osteocyte viability on bone biopsies on a cohort of 37 MM patients with symptomatic disease. A significantly higher number of viable osteocytes was detected in patients treated with a bortezomib (BOR)-based regimen compared with those treated without BOR. Interestingly, both osteocyte autophagy and apoptosis were affected in vivo by BOR treatment. Thereafter, we checked the in vitro effect of BOR to understand the mechanisms whereby BOR maintains osteocyte viability in bone from MM patients. We found that osteocyte and preosteocyte autophagic death was triggered during coculturing with MM cells. Our evaluation was conducted by analyzing either autophagy markers microtubule-associated protein light chain 3 beta (LC3B) and SQSTM1/sequestome 1 (p62) levels, or the cell ultrastructure by transmission electron microscopy. PIs were found to increase the basal levels of LC3 expression in the osteocytes while blunting the myeloma-induced osteocyte death. PIs also reduced the autophagic death of osteocytes induced by high-dose dexamethasone (DEX) and potentiated the anabolic effect of PTH(1-34). Our data identify osteocyte autophagy as a new potential target in MM bone disease and support the use of PIs to maintain osteocyte viability and improve bone integrity in MM patients.