The genomic profiling of high-risk smoldering myeloma patients treated with an intensive strategy unveils potential markers of resistance and progression.

Smoldering multiple myeloma (SMM) precedes multiple myeloma (MM). The risk of progression of SMM patients is not uniform, thus different progression-risk models have been developed, although they are mainly based on clinical parameters. Recently, genomic predictors of progression have been defined for untreated SMM. However, the usefulness of such markers in the context of clinical trials evaluating upfront treatment in high-risk SMM (HR SMM) has not been explored yet, precluding the identification of baseline genomic alterations leading to drug resistance. For this reason, we carried out next-generation sequencing and fluorescent in-situ hybridization studies on 57 HR and ultra-high risk (UHR) SMM patients treated in the phase II GEM-CESAR clinical trial (NCT02415413). DIS3, FAM46C, and FGFR3 mutations, as well as t(4;14) and 1q alterations, were enriched in HR SMM. TRAF3 mutations were specifically associated with UHR SMM but identified cases with improved outcomes. Importantly, novel potential predictors of treatment resistance were identified: NRAS mutations and the co-occurrence of t(4;14) plus FGFR3 mutations were associated with an increased risk of biological progression. In conclusion, we have carried out for the first time a molecular characterization of HR SMM patients treated with an intensive regimen, identifying genomic predictors of poor outcomes in this setting.

Natural history of relapsed myeloma, refractory to immunomodulatory drugs and proteasome inhibitors: a multicenter IMWG study.

Introduction of new myeloma therapies offers new options for patients refractory to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs). In this multicenter study, patients with relapsed multiple myeloma, who have received at least three prior lines of therapy, are refractory to both an IMiD (lenalidomide or pomalidomide) and a PI (bortezomib or carfilzomib), and have been exposed to an alkylating agent were identified. The time patients met the above criteria was defined as time zero (T0). Five hundred and forty-three patients diagnosed between 2006 and 2014 were enrolled in this study. Median age at T0 was 62 years (range 31-87); 61% were males. The median duration between diagnosis and T0 was 3.1 years. The median number of lines of therapy before T0 was 4 (range 3-13). The median overall survival (OS) from T0 for the entire cohort was 13 (95% confidence interval (CI) 11, 15) months. At least one regimen recorded after T0 in 462 (85%) patients, with a median (95% CI) progression-free survival and OS from T0 of 5 (4, 6), and 15.2 (13, 17) months, respectively. The study provides the expected outcome of relapsed multiple myeloma that is refractory to a PI and an IMiD, a benchmark for comparison of new therapies being evaluated.

Absence of spontaneous response improvement beyond day +100 after autologous stem cell transplantation in multiple myeloma.

The response evaluation after autologous stem-cell transplantation (ASCT) is usually performed at day +100 in patients with multiple myeloma (MM). A recent report suggests that improvement in the response can be observed beyond day +100. The aim of the present study has been to evaluate the rate of improved response and outcome beyond day +100 after ASCT, with and without maintenance therapy. One hundred and forty-four patients who underwent single ASCT with chemosensitive disease and achieved less than CR at day 100 post ASCT were evaluated. Seventy-four patients (51.4%) did not receive any maintenance with only one of them showing an upgrade in the response. The remaining 70 patients (48.6%) received maintenance therapy; eleven of them (15.7%) improved their response beyond day +100. The outcome of these patients was better than those who did not upgrade their response in both progression-free survival and overall survival (P=0.019 and P=0.031, respectively). In conclusion, the improvement in response beyond day +100 after ASCT in patients not receiving any therapy is exceedingly rare. A minority of patients receiving maintenance therapy after ASCT upgrades their response and this finding is associated with better outcome.

Bence Jones proteinuria in smoldering multiple myeloma as a predictor marker of progression to symptomatic multiple myeloma.

The diagnosis of smoldering multiple myeloma (SMM) includes patients with a heterogeneous risk of progression to active multiple myeloma (MM): some patients will never progress, whereas others will have a high risk of progression within the first 2 years. Therefore, it is important to improve risk assessment at diagnosis. We conducted a retrospective study in a large cohort of SMM patients, in order to investigate the role of Bence Jones (BJ) proteinuria at diagnosis in the progression to active MM. We found that SMM patients presenting with BJ proteinuria had a significantly shorter median time to progression (TTP) to MM compared with patients without BJ proteinuria (22 vs 88 months, respectively; hazard ratio=2.3, 95% confidence interval=1.4-3.9, P=0.002). We also identified risk subgroups based on the amount of BJ proteinuria: ⩾500 mg/24 h, <500 mg/24 h and without it, with a significantly different median TTP (13, 37 and 88 months, P<0.001). Thus, BJ proteinuria at diagnosis is an independent variable of progression to MM that identifies a subgroup of high-risk SMM patients (51% risk of progression at 2 years) and ⩾500 mg of BJ proteinuria may allow, if validated in another series, to reclassify these patients to MM requiring therapy before the end-organ damage development.

Phenotypic identification of subclones in multiple myeloma with different chemoresistant, cytogenetic and clonogenic potential.

Knowledge about clonal diversity and selection is critical to understand multiple myeloma (MM) pathogenesis, chemoresistance and progression. If targeted therapy becomes reality, identification and monitoring of intraclonal plasma cell (PC) heterogeneity would become increasingly demanded. Here we investigated the kinetics of intraclonal heterogeneity among 116 MM patients using 23-marker multidimensional flow cytometry (MFC) and principal component analysis, at diagnosis and during minimal residual disease (MRD) monitoring. Distinct phenotypic subclones were observed in 35/116 (30%) newly diagnosed MM patients. In 10/35 patients, persistent MRD was detected after 9 induction cycles, and longitudinal comparison of patient-paired diagnostic vs MRD samples unraveled phenotypic clonal tiding after therapy in half (5/10) of the patients. After demonstrating selection of distinct phenotypic subsets by therapeutic pressure, we investigated whether distinct fluorescence-activated cell-sorted PC subclones had different clonogenic and cytogenetic profiles. In half (5/10) of the patients analyzed, distinct phenotypic subclones showed different clonogenic potential when co-cultured with stromal cells, and in 6/11 cases distinct phenotypic subclones displayed unique cytogenetic profiles by interphase fluorescence in situ hybridization, including selective del(17p13). Collectively, we unravel potential therapeutic selection of preexisting diagnostic phenotypic subclones during MRD monitoring; because phenotypically distinct PCs may show different clonogenic and cytogenetic profiles, identification and follow-up of unique phenotypic-genetic myeloma PC subclones may become relevant for tailored therapy.

In vivo murine model of acquired resistance in myeloma reveals differential mechanisms for lenalidomide and pomalidomide in combination with dexamethasone.

The development of resistance to therapy is unavoidable in the history of multiple myeloma patients. Therefore, the study of its characteristics and mechanisms is critical in the search for novel therapeutic approaches to overcome it. This effort is hampered by the absence of appropriate preclinical models, especially those mimicking acquired resistance. Here we present an in vivo model of acquired resistance based on the continuous treatment of mice bearing subcutaneous MM1S plasmacytomas. Xenografts acquired resistance to two generations of immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide) in combination with dexamethasone, that was reversible after a wash-out period. Furthermore, lenalidomide-dexamethasone (LD) or pomalidomide-dexamethasone (PD) did not display cross-resistance, which could be due to the differential requirements of the key target Cereblon and its substrates Aiolos and Ikaros observed in cells resistant to each combination. Differential gene expression profiles of LD and PD could also explain the absence of cross-resistance. Onset of resistance to both combinations was accompanied by upregulation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway and addition of selumetinib, a small-molecule MEK inhibitor, could resensitize resistant cells. Our results provide insights into the mechanisms of acquired resistance to LD and PD combinations and offer possible therapeutic approaches to addressing IMiD resistance in the clinic.

Multiparameter flow cytometry for the identification of the Waldenström's clone in IgM-MGUS and Waldenström's Macroglobulinemia: new criteria for differential diagnosis and risk stratification.

Although multiparameter flow cytometry (MFC) has demonstrated clinical relevance in monoclonal gammopathy of undetermined significance (MGUS)/myeloma, immunophenotypic studies on the full spectrum of Waldenström's Macroglobulinemia (WM) remain scanty. Herein, a comprehensive MFC analysis on bone marrow samples from 244 newly diagnosed patients with an immunoglobulin M (IgM) monoclonal protein was performed, including 67 IgM-MGUS, 77 smoldering and 100 symptomatic WM. Our results show a progressive increase on the number and light-chain-isotype-positive B-cells from IgM-MGUS to smoldering and symptomatic WM (P<.001), with only 1% of IgM-MGUS patients showing >10% B cells or 100% light-chain-isotype-positive B-cells (P<.001). Complete light-chain restriction of the B-cell compartment was an independent prognostic factor for time-to progression in smoldering WM (median 26 months; HR: 19.8, P=0.001) and overall survival in symptomatic WM (median 44 months; HR: 2.6, P=0.004). The progressive accumulation of light-chain-isotype-positive B-cells accompanied the emergence of a characteristic Waldenstrom's phenotype (CD22(+dim) / CD25+ /CD27+ / IgM+) that differed from other B-NHL by negative expression of CD5, CD10, CD11c or CD103. In contrast to myeloma, light-chain-isotype-positive plasma cells in IgM monoclonal gammopathies show otherwise normal antigenic expression. Our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenström's clone, as well as for the differential diagnosis, risk of progression and survival in WM.

New drugs and novel mechanisms of action in multiple myeloma in 2013: a report from the International Myeloma Working Group (IMWG).

Treatment in medical oncology is gradually shifting from the use of nonspecific chemotherapeutic agents toward an era of novel targeted therapy in which drugs and their combinations target specific aspects of the biology of tumor cells. Multiple myeloma (MM) has become one of the best examples in this regard, reflected in the identification of new pathogenic mechanisms, together with the development of novel drugs that are being explored from the preclinical setting to the early phases of clinical development. We review the biological rationale for the use of the most important new agents for treating MM and summarize their clinical activity in an increasingly busy field. First, we discuss data from already approved and active agents (including second- and third-generation proteasome inhibitors (PIs), immunomodulatory agents and alkylators). Next, we focus on agents with novel mechanisms of action, such as monoclonal antibodies (MoAbs), cell cycle-specific drugs, deacetylase inhibitors, agents acting on the unfolded protein response, signaling transduction pathway inhibitors and kinase inhibitors. Among this plethora of new agents or mechanisms, some are specially promising: anti-CD38 MoAb, such as daratumumab, are the first antibodies with clinical activity as single agents in MM. Moreover, the kinesin spindle protein inhibitor Arry-520 is effective in monotherapy as well as in combination with dexamethasone in heavily pretreated patients. Immunotherapy against MM is also being explored, and probably the most attractive example of this approach is the combination of the anti-CS1 MoAb elotuzumab with lenalidomide and dexamethasone, which has produced exciting results in the relapsed/refractory setting.

A multiparameter flow cytometry immunophenotypic algorithm for the identification of newly diagnosed symptomatic myeloma with an MGUS-like signature and long-term disease control.

Achieving complete remission (CR) in multiple myeloma (MM) translates into extended survival, but two subgroups of patients fall outside this paradigm: cases with unsustained CR, and patients that do not achieve CR but return into a monoclonal gammopathy of undetermined significance (MGUS)-like status with long-term survival. Here, we describe a novel automated flow cytometric classification focused on the analysis of the plasma-cell compartment to identify among newly diagnosed symptomatic MM patients (N=698) cases with a baseline MGUS-like profile, by comparing them to MGUS (N=497) patients and validating the classification model in 114 smoldering MM patients. Overall, 59 symptomatic MM patients (8%) showed an MGUS-like profile. Despite achieving similar CR rates after high-dose therapy/autologous stem cell transplantation vs other MM patients, MGUS-like cases had unprecedented longer time-to-progression (TTP) and overall survival (OS; ~60% at 10 years; P<0.001). Importantly, MGUS-like MM patients failing to achieve CR showed similar TTP (P=0.81) and OS (P=0.24) vs cases attaining CR. This automated classification also identified MGUS patients with shorter TTP (P=0.001, hazard ratio: 5.53) and ultra-high-risk smoldering MM (median TTP, 15 months). In summary, we have developed a biomarker that identifies a subset of symptomatic MM patients with an occult MGUS-like signature and an excellent outcome, independently of the depth of response.

MYD88 L265P is a marker highly characteristic of, but not restricted to, Waldenström's macroglobulinemia.

We evaluated the MYD88 L265P mutation in Waldenström's macroglobulinemia (WM) and B-cell lymphoproliferative disorders by specific polymerase chain reaction (PCR) (sensitivity ∼10(-3)). No mutation was seen in normal donors, while it was present in 101/117 (86%) WM patients, 27/31 (87%) IgM monoclonal gammapathies of uncertain significance (MGUS), 3/14 (21%) splenic marginal zone lymphomas and 9/48 (19%) non-germinal center (GC) diffuse large B-cell lymphomas (DLBCLs). The mutation was absent in all 28 GC-DLBCLs, 13 DLBCLs not subclassified, 35 hairy cell leukemias, 39 chronic lymphocytic leukemias (16 with M-component), 25 IgA or IgG-MGUS, 24 multiple myeloma (3 with an IgM isotype), 6 amyloidosis, 9 lymphoplasmacytic lymphomas and 1 IgM-related neuropathy. Among WM and IgM-MGUS, MYD88 L265P mutation was associated with some differences in clinical and biological characteristics, although usually minor; wild-type MYD88 cases had smaller M-component (1.77 vs 2.72 g/dl, P=0.022), more lymphocytosis (24 vs 5%, P=0.006), higher lactate dehydrogenase level (371 vs 265 UI/L, P=0.002), atypical immunophenotype (CD23-CD27+ +FMC7+ +), less Immunoglobulin Heavy Chain Variable gene (IGHV) somatic hypermutation (57 vs 97%, P=0.012) and less IGHV3-23 gene selection (9 vs 27%, P=0.014). These small differences did not lead to different time to first therapy, response to treatment or progression-free or overall survival.

The epoxyketone-based proteasome inhibitors carfilzomib and orally bioavailable oprozomib have anti-resorptive and bone-anabolic activity in addition to anti-myeloma effects.

Proteasome inhibitors (PIs), namely bortezomib, have become a cornerstone therapy for multiple myeloma (MM), potently reducing tumor burden and inhibiting pathologic bone destruction. In clinical trials, carfilzomib, a next generation epoxyketone-based irreversible PI, has exhibited potent anti-myeloma efficacy and decreased side effects compared with bortezomib. Carfilzomib and its orally bioavailable analog oprozomib, effectively decreased MM cell viability following continual or transient treatment mimicking in vivo pharmacokinetics. Interactions between myeloma cells and the bone marrow (BM) microenvironment augment the number and activity of bone-resorbing osteoclasts (OCs) while inhibiting bone-forming osteoblasts (OBs), resulting in increased tumor growth and osteolytic lesions. At clinically relevant concentrations, carfilzomib and oprozomib directly inhibited OC formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Accordingly, carfilzomib and oprozomib increased trabecular bone volume, decreased bone resorption and enhanced bone formation in non-tumor bearing mice. Finally, in mouse models of disseminated MM, the epoxyketone-based PIs decreased murine 5TGM1 and human RPMI-8226 tumor burden and prevented bone loss. These data demonstrate that, in addition to anti-myeloma properties, carfilzomib and oprozomib effectively shift the bone microenvironment from a catabolic to an anabolic state and, similar to bortezomib, may decrease skeletal complications of MM.

The synergy of panobinostat plus doxorubicin in acute myeloid leukemia suggests a role for HDAC inhibitors in the control of DNA repair.

Acute myeloid leukemia (AML) is a clonal disorder characterized by the accumulation of myeloid blasts in the bone marrow. Here, we report the effects of the novel histone deacetylase inhibitor panobinostat (LBH589) in combination with doxorubicin on AML cells. Panobinostat exhibited potent anti-AML activity in all AML cell lines tested and in primary AML cells from patients (IC(50)<20 nM). In addition, panobinostat potentiated the action of several standard-of-care anti-AML compounds, particularly, doxorubicin. The molecular effects induced by panobinostat and doxorubicin treatment were investigated by analyzing gene expression, cell cycle, apoptosis and signaling pathways. Analyses of gene expression profiles identified 588 genes whose expression was exclusively affected by the combination of panobinostat and doxorubicin. The combination induced AML cell death by an increase in the mitochondrial outer membrane permeability and release of cytochrome c from the mitochondria, resulting in caspase-dependent apoptosis and accompanied by the upregulation of Bax, Bak and, particularly, Bad. The drug combination provoked a strong activation of a DNA damage response, indicating that this combination may trigger cell death by a mechanism that induced DNA double-strand breaks. These data indicate that the combination of panobinostat and doxorubicin may be an effective therapy for the treatment of AML.

Pemetrexed acts as an antimyeloma agent by provoking cell cycle blockade and apoptosis.

Multiple myeloma (MM) is a malignancy of terminally differentiated B lymphocytes, characterized by accumulation of a monotypic plasma cell population in the bone marrow, monoclonal immunoglobulin in serum and/or urine and osteolytic lesions. Despite recent advances in the treatment, MM remains an incurable disease. This calls for an effort to develop novel therapeutics in order to eradicate the disease. Here we have evaluated the potential antimyeloma action of Pemetrexed, an antifolate drug that has shown promising results in other neoplastic diseases. Pemetrexed had a potent antimyeloma effect on cell lines sensitive and resistant to conventional therapeutic agents, and was also efficient on fresh cells from patients and in a murine MM model. Furthermore, Pemetrexed abrogated the protective action on MM cell death of several growth factors produced by the bone marrow microenvironment. Mechanistic studies indicated that Pemetrexed provoked this action by a combined effect that included cell cycle blockade, probably by p21 upregulation, and induction of apoptosis through caspase-dependent and -independent mechanisms. These data, together with the fact that Pemetrexed is already licensed for the therapy of other neoplastic diseases, opens the possibility for the inclusion of Pemetrexed in the therapeutic armamentarium to battle MM.

Novel etodolac analog SDX-308 (CEP-18082) induces cytotoxicity in multiple myeloma cells associated with inhibition of beta-catenin/TCF pathway.

We have reported previously that R-enantiomer of etodolac (R-etodolac), which is under investigation in phase 2 clinical trials in chronic lymphocytic leukemia, induces potent cytotoxicity at clinically relevant concentrations in multiple myeloma (MM) cells. In this study, we demonstrated that SDX-308 (CEP-18082), a novel analog of etodolac, has more potent cytotoxicity than R-etodolac against both MM cell lines and patient MM cells, including tumor cells resistant to conventional (dexamethasone, doxorubicine, melphalan) and novel (bortezomib) therapies. SDX-308-induced cytotoxicity is triggered by caspase-8/9/3 activation and poly (ADP-ribose) polymerase cleavage, followed by apoptosis. SDX-308 significantly inhibits beta-catenin/T-cell factor pathway by inhibiting nuclear translocation of beta-catenin, thereby downregulating transcription and expression of downstream target proteins including myc and survivin. Neither interleukin-6 nor insulin-like growth factor-1 protect against growth inhibition triggered by SDX-308. Importantly, growth of MM cells adherent to bone marrow (BM) stromal cells is also significantly inhibited by SDX-308. Our data therefore indicate that the novel etodolac analog SDX-308 can target MM cells in the BM milieu.

6q deletion in Waldenström macroglobulinemia is associated with features of adverse prognosis.

Fluorescence in situ hybridisation (FISH) is an effective technique for the cytogenetic analysis of Waldenström macroglobulinemia (WM), but the potential impact of molecular cytogenetics on disease evolution and as a prognostic marker is still unknown. Deletion of the long arm of chromosome 6 (6q-) is the most frequent cytogenetic abnormality in WM. This study analysed the prevalence of this aberration in 102 WM patients, and correlated it with disease characteristics. The incidence of 6q21 deletion was 7% by conventional cytogenetics and 34% when analysed by FISH (54% when cytoplasmic immunoglobulin M-FISH was used). Patients with deletion of 6q displayed features of adverse prognosis, such as higher levels of beta2-microglobulin and monoclonal paraprotein and a greater tendency to display anaemia and hypoalbuminemia. Interestingly, there was a correlation between the presence of 6q deletion and the International Staging System prognostic index (incidence of 6q- among patients stratified in stages 1, 2 and 3 was 24%, 42% and 67% respectively). Those patients diagnosed with smouldering WM who displayed the abnormality showed a trend to an earlier requirement of treatment. Finally, the survival analysis did not show differences between the two groups of patients, probably due to the short follow up of our series.

Gene expression profiling of B lymphocytes and plasma cells from Waldenström's macroglobulinemia: comparison with expression patterns of the same cell counterparts from chronic lymphocytic leukemia, multiple myeloma and normal individuals.

The tumoral clone of Waldenström's macroglobulinemia (WM) shows a wide morphological heterogeneity, which ranges from B lymphocytes (BL) to plasma cells (PC). By means of genome-wide expression profiling we have been able to identify genes exclusively deregulated in BL and PC from WM, but with a similar expression pattern in their corresponding cell counterparts from chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), as well as normal individuals. The differentially expressed genes have important functions in B-cell differentiation and oncogenesis. Thus, two of the genes downregulated in WM-BL were IL4R, which plays a relevant role in CLL B-cell survival, and BACH2, which participates in the development of class-switched PC. Interestingly, one of the upregulated genes in WM-BL was IL6. A set of four genes was able to discriminate clonal BL from WM and CLL: LEF1 (WNT/beta-catenin pathway), MARCKS, ATXN1 and FMOD. We also found deregulation of genes involved in plasma cell differentiation such as PAX5, which was overexpressed in WM-PC, and IRF4 and BLIMP1, which were underexpressed. In addition, three of the target genes activated by PAX5 - CD79, BLNK and SYK - were upregulated in WM-PC. In summary, these results indicate that both PC and BL from WM are genetically different from the MM and CLL cell counterpart.

CD34 + cell dose and outcome of patients undergoing reduced-intensity-conditioning allogeneic peripheral blood stem cell transplantation.

While in bone marrow allogeneic transplantation the infusion of high doses of progenitor stem cells has a favourable impact on outcome, due to a faster hematopoietic and immune recovery, in the peripheral blood allo-setting the infusion of a high number of CD34 cells increases the risk of extensive chronic graft vs. host disease (cGVHD). This higher incidence of extensive cGVHD has an adverse impact on outcome due to a higher transplant related mortality, specially among patients receiving T-cell depleted allogeneic transplantation with myeloablative conditioning. By contrast, patients undergoing reduced intensity conditioning regimen may benefit from increasing higher CD34 + cell doses, especially those categorized as high risk according to disease status at transplant. Thus, the source of progenitors cells, type of conditioning and GVHD prophylaxis, among other factors, may influence the effect of the progenitor cell dose on outcome after allogeneic transplant.