Isatuximab plus pomalidomide and dexamethasone in patients with relapsed/refractory multiple myeloma in real-world: The retrospective IMAGE study.

BACKGROUND: IMAGE is a retrospective cohort study of patients enrolled in early access programs (EAPs) in France with relapsed/refractory multiple myeloma (RRMM) receiving isatuximab with pomalidomide and dexamethasone (Isa-Pd). METHODS: Patients aged ≥18 years with RRMM who received ≥1 dose of Isa under the EAPs between July 29, 2019 and August 30, 2020 were included. Effectiveness endpoints included progression-free survival (PFS) and response rates. Verbatim terms for adverse events (AEs) were coded using the Medical Dictionary for Regulatory Activities and not graded for severity. RESULTS: A total of 294 and 299 patients were included in the effectiveness and safety populations, respectively. IMAGE included patients who received one prior line of treatment (10.2%) and were daratumumab-refractory (19.1%). At median follow-up of 14.2 months, median PFS in the effectiveness population was 12.4 months (95% CI 9.0-15.0). Overall response and very good partial response rates were 46.3% and 27.9%, respectively. Subgroup analyses reflected similar results. In the safety population, 26.4% of patients reported at least one AE; the most common any-grade AE was neutropenia (9.4%). CONCLUSION: IMAGE demonstrated Isa-Pd had meaningful effectiveness in median PFS and depth of response and no new safety signals in a real-world context, consistent with clinical trial results.

RAS/RAFlandscape in monoclonal plasma cell conditions.

Multiple Myeloma (MM) is characterized by a huge heterogeneity at the molecular level. The RAS/RAF pathway is the most frequently mutated, in about 50% of the patients. However, these mutations are frequently subclonal, suggesting a secondary event. Since these genes are part of our routine next-generation sequencing (NGS) panel, we analyzed >10,000 patients with different plasma cell disorders in order to describe the RAS/RAF landscape. In this large cohort of patients, almost 61% of the patients presented a RAS/RAF mutation at diagnosis or relapse, but much lower frequencies in pre-symptomatic cases. Of note, the mutations were different from that observed in solid tumors (higher proportions of Q61 mutations). In 29 patients with two different mutations, we were able to perform single cell sequencing, showing that in most cases, mutations occurred in different subclones, suggesting an ongoing mutational process. These findings suggest that RAS/RAF pathway is not an attractive target, both on therapeutic and residual disease assessment points of vue.

Daratumumab Carfilzomib Lenalidomide and Dexamethasone with tandem transplant in high-risk newly diagnosed myeloma.

High-risk (HR) cytogenetics are associated with poor outcomes in newly diagnosed multiple myeloma (NDMM) and dedicated studies should address this difficult-to-treat population. The phase 2 study 2018-04 from the Intergroupe Francophone du Myelome evaluated feasibility of an intensive strategy with quadruplet induction and consolidation plus tandem transplant in HR transplant eligible (TE) NDMM (NCT03606577). HR cytogenetics were defined by the presence of del(17p), t(4;14) and/or t(14;16). Treatment consisted in daratumumab-carfilzomib-lenalidomide-dexamethasone (D-KRd) induction (6 cycles), autologous stem cell transplantation (ASCT), D-KRd consolidation (4 cycles), second ASCT, and daratumumab-lenalidomide maintenance for 2 years. The primary endpoint was feasibility. Fifty patients with previously untreated NDMM were included. Median age was 57. Del(17p), t(4;14) and t(14;16) were found in 40%, 52% and 20% of patients respectively. At data cut-off, the study met the primary endpoint with 36 (72%) patients completing second transplant. Twenty patients (40%) discontinued the study due to stem-cell collection failure (n=8), disease progression (n=7), adverse event (n=4), consent withdrawal (n=1). Grade 3-4 Dara-KRd induction/consolidation related adverse events (>5% of patients) were neutropenia (39%), anemia (12%), thrombocytopenia (7%) and infection (6%). The overall response rate was 100% for patients completing second transplant (n=36), including 81% complete response. Pre-maintenance Minimal Residual Disease (MRD) negativity rate (NGS, 10-6) was 94%. After a median follow up of 33 months, the 30-month progression-free (PFS) and overall survival were 80% and 91%, respectively. In conclusion, D-KRd with tandem transplant is feasible in high-risk TE NDMM patients and resulted in high rates of MRD negativity and PFS.

Immunomodulation of NK, NKT and B/T cell subtypes in relapsed/refractory multiple myeloma patients treated with pomalidomide along with velcade and dexamethasone and its association with improved progression-free survival.

Background: Multiple Myeloma (MM) patients exhibit dysregulated immune system, which is further weakened by chemotherapeutic agents. While cereblon-modulating agents, such as pomalidomide and lenalidomide, have been found to improve the immune profile, the efficacy of their impact in combination with other treatments is yet unknown. Methods: We conducted an immune-profiling of a longitudinal cohort of 366 peripheral blood samples from the CC4047-MM-007 (OPTIMISMM, NCT01734928) study. This study followed relapsed/refractory Multiple Myeloma (RRMM) patients who were treated with Velcade + dexamethasone (Vd), or Vd with pomalidomide (PVd). 366 blood samples from 186 patients were evaluated using multi-color flow cytometry at 3 timepoints: screening, day 8 of cycle 1, and cycle 3. Results: Among NK and NKT cell populations, adding pomalidomide showed no inhibition in the frequency of NK cells. When expression of double positivity for activation markers like, p46/NKG2D, on NK cells was higher than the median, PVd treated patients showed significantly better (p=0.05) progression-free survival (PFS) (additional 15 months) than patients with lower than the median expression of p46/NKG2D on NK cells. PVd treated patients who expressed CD158a/b below the median at cycle 1 demonstrated a significantly better PFS (more than 18months). Among B cell subtypes, PVd treatment significantly increased the abundance of B1b cells (p<0.05) and decreased Bregs (p<0.05) at day 8 of both cycle 1 and cycle 3 when compared to screening samples. Of all the B cell-markers evaluated among paired samples, a higher expression of MZB cells at day 8 of cycle 1 has resulted in enhanced PFS in PVd treated patients. Within T cells, pomalidomide treatment did not decrease the frequency of CD8 T cells when compared with screening samples. The higher the surface expression of OX-40 on CD8 T cells and the lower the expression of PD-1 and CD25 on CD4 T cells by PVd treatment resulted in improved PFS. Conclusion: The prognostic significance for the number of immune markers is only seen in the PVd arm and none of these immune markers exhibit prognostic values in the Vd arm. This study demonstrates the importance of the immunomodulatory effects and the therapeutic benefit of adding pomalidomide to Vd treatment.

Deneddylation of ribosomal proteins promotes synergy between MLN4924 and chemotherapy to elicit complete therapeutic responses.

The neddylation inhibitor MLN4924/Pevonedistat is in clinical trials for multiple cancers. Efficacy is generally attributed to cullin RING ligase (CRL) inhibition, but the contribution of non-CRL targets is unknown. Here, CRISPR screens map MLN4924-monotherapy sensitivity in retinoblastoma to a classic DNA damage-induced p53/E2F3/BAX-dependent death effector network, which synergizes with Nutlin3a or Navitoclax. In monotherapy-resistant cells, MLN4924 plus standard-of-care topotecan overcomes resistance, but reduces DNA damage, instead harnessing ribosomal protein nucleolar-expulsion to engage an RPL11/p21/MYCN/E2F3/p53/BAX synergy network that exhibits extensive cross-regulation. Strikingly, unneddylatable RPL11 substitutes for MLN4924 to perturb nucleolar function and enhance topotecan efficacy. Orthotopic tumors exhibit complete responses while preserving visual function. Moreover, MLN4924 plus melphalan deploy this DNA damage-independent strategy to synergistically kill multiple myeloma cells. Thus, MLN4924 synergizes with standard-of-care drugs to unlock a nucleolar death effector network across cancer types implying broad therapeutic relevance.

Multiomic Mapping of Acquired Chromosome 1 Copy-Number and Structural Variants to Identify Therapeutic Vulnerabilities in Multiple Myeloma.

PURPOSE: Chromosome 1 (chr1) copy-number abnormalities (CNA) and structural variants (SV) are frequent in newly diagnosed multiple myeloma (NDMM) and are associated with a heterogeneous impact on outcomes, the drivers of which are largely unknown. EXPERIMENTAL DESIGN: A multiomic approach comprising CRISPR, gene mapping of CNAs and SVs, methylation, expression, and mutational analysis was used to document the extent of chr1 molecular variants and their impact on pathway utilization. RESULTS: We identified two distinct groups of gain(1q): focal gains associated with limited gene-expression changes and a neutral prognosis, and whole-arm gains, which are associated with substantial gene-expression changes, complex genetics, and an adverse prognosis. CRISPR identified a number of dependencies on chr1 but only limited variants associated with acquired CNAs. We identified seven regions of deletion, nine of gain, three of chromothripsis (CT), and two of templated insertion (TI), which contain a number of potential drivers. An additional mechanism involving hypomethylation of genes at 1q may contribute to the aberrant gene expression of a number of genes. Expression changes associated with whole-arm gains were substantial and gene set enrichment analysis identified metabolic processes, apoptotic resistance, signaling via the MAPK pathway, and upregulation of transcription factors as being key drivers of the adverse prognosis associated with these variants. CONCLUSIONS: Multiple layers of genetic complexity impact the phenotype associated with CNAs on chr1 to generate its associated clinical phenotype. Whole-arm gains of 1q are the critically important prognostic group that deregulate multiple pathways, which may offer therapeutic vulnerabilities.

High-dose melphalan treatment significantly increases mutational burden at relapse in multiple myeloma.

High-dose melphalan (HDM) improves progression-free survival in multiple myeloma (MM), yet melphalan is a DNA-damaging alkylating agent; therefore, we assessed its mutational effect on surviving myeloma cells by analyzing paired MM samples collected at diagnosis and relapse in the IFM 2009 study. We performed deep whole-genome sequencing on samples from 68 patients, 43 of whom were treated with RVD (lenalidomide, bortezomib, and dexamethasone) and 25 with RVD + HDM. Although the number of mutations was similar at diagnosis in both groups (7137 vs 7230; P = .67), the HDM group had significantly more mutations at relapse (9242 vs 13 383, P = .005). No change in the frequency of copy number alterations or structural variants was observed. The newly acquired mutations were typically associated with DNA damage and double-stranded breaks and were predominantly on the transcribed strand. A machine learning model, using this unique pattern, predicted patients who would receive HDM with high sensitivity, specificity, and positive prediction value. Clonal evolution analysis showed that all patients treated with HDM had clonal selection, whereas a static progression was observed with RVD. A significantly higher percentage of mutations were subclonal in the HDM cohort. Intriguingly, patients treated with HDM who achieved complete remission (CR) had significantly more mutations at relapse yet had similar survival rates as those treated with RVD who achieved CR. This similarity could have been due to HDM relapse samples having significantly more neoantigens. Overall, our study identifies increased genomic changes associated with HDM and provides rationale to further understand clonal complexity.

In Multiple Myeloma, High-Risk Secondary Genetic Events Observed at Relapse Are Present From Diagnosis in Tiny, Undetectable Subclonal Populations.

PURPOSE: Multiple myeloma (MM) is characterized by copy number abnormalities (CNAs), some of which influence patient outcomes and are sometimes observed only at relapse(s), suggesting their acquisition during tumor evolution. However, the presence of micro-subclones may be missed in bulk analyses. Here, we use single-cell genomics to determine how often these high-risk events are missed at diagnosis and selected at relapse. MATERIALS AND METHODS: We analyzed 81 patients with plasma cell dyscrasias using single-cell CNA sequencing. Sixty-six patients were selected at diagnosis, nine at first relapse, and six in presymptomatic stages. A total of 956 newly diagnosed patients with MM and patients with first relapse MM have been identified retrospectively with required cytogenetic data to evaluate enrichment of CNA risk events and survival impact. RESULTS: A total of 52,176 MM cells were analyzed. Seventy-four patients (91%) had 2-16 subclones. Among these patients, 28.7% had a subclone with high-risk features (del(17p), del(1p32), and 1q gain) at diagnosis. In a patient with a subclonal 1q gain at diagnosis, we analyzed the diagnosis, postinduction, and first relapse samples, which showed a rise of the high-risk 1q gain subclone (16%, 70%, and 92%, respectively). In our clinical database, we found that the 1q gain frequency increased from 30.2% at diagnosis to 43.6% at relapse (odds ratio, 1.78; 95% CI, 1.58 to 2.00). We subsequently performed survival analyses, which showed that the progression-free and overall survival curves were superimposable between patients who had the 1q gain from diagnosis and those who seemingly acquired it at relapse. This strongly suggests that many patients had 1q gains at diagnosis in microclones that were missed by bulk analyses. CONCLUSION: These data suggest that identifying these scarce aggressive cells may necessitate more aggressive treatment as early as diagnosis to prevent them from becoming the dominant clone.

Biallelic deletion of 1p32 defines ultra-high-risk myeloma, but monoallelic del(1p32) remains a strong prognostic factor.

Cytogenetic abnormalities (CAs) are known to be the preponderant prognostic factor in multiple myeloma. Our team has recently developed a prognostic score based on 6 CAs, with which del(1p32) appears to be the second worst abnormality after del(17p). This study aimed to confirm the adverse effect of 1p32 deletion in patients with newly diagnosed multiple myeloma (NDMM). Among 2551 patients with newly diagnosed multiple myeloma, 11% were harboring del(1p32). Their overall survival (OS) was significantly inferior compared with patients without del(1p32) (median OS: 49 months vs 124 months). Likewise, progression-free survival was significantly shorter. More importantly, biallelic del(1p32) conferred a dramatically poorer prognosis than a monoallelic del(1p32) (median OS: 25 months vs 60 months). As expected, the OS of patients with del(1p32) significantly decreased when this abnormality was associated with other high-risk CAs [del(17p), t(4;14), or gain(1q)]. In the multivariate analysis, del(1p32) appeared as a negative prognostic factor; after adjustment for age and treatment, the risk of progression was 1.3 times higher among patients harboring del(1p32), and the risk of death was 1.9 times higher. At the dawn of risk-adapted treatment strategies, we have confirmed the adverse effect of del(1p32) in multiple myeloma and the relevance of its assessment at diagnosis.

Heterogeneity in long-term outcomes for patients with Revised International Staging System stage II, newly diagnosed multiple myeloma.

In the era of personalized treatment in multiple myeloma, high-risk patients must be accurately identified. The International Myeloma Working Group recommends using the Revised International Staging System (R-ISS) to pick out high-risk patients. The main purpose of our work was to explore the heterogeneity of outcome among R-ISS stage II patients assessing the impact of International Staging System (ISS) stage, chromosomal abnormalities and lactate dehydrogenase level in this subgroup. Data were collected from 1,343 patients up to 65 years old with newly diagnosed myeloma, enrolled in three clinical trials implemented by the Intergroupe Francophone du Myélome. All patients were eligible for intensive treatment. Patients in R-ISS stage II but ISS stage I had 1.6 times higher risk of death than patients in R-ISS stage I (adjusted hazard ratio=1.6; 95% confidence interval: 1.1-2.2; P=0.01) and patients in R-ISS stage II but with ISS stage III had a better overall survival than patients in R-ISS stage III (adjusted hazard ratio=0.7; 95% confidence interval: 0.4-0.9, P=0.02). However, among patients classified in R-ISS II, ISS stage and chromosomal abnormalities (del[17p] and t[4;14]) were still relevant prognostic factors for death. Dividing R-ISS stage II into three subgroups: ISS I with standard-risk chromosomal abnormalities, ISS II or III with standard-risk chromosomal abnormalities and patients with high-risk chromosomal abnormalities, median overall survival times were, respectively, not reached, 112 months and 71 months (P<0.001). In conclusion, stratification of patients in the R-ISS stage II group can be improved by taking into account chromosomal abnormalities and ISS. However, this does not improve predictive performance of survival models.

The location of the t(4;14) translocation breakpoint within the NSD2 gene identifies a subset of patients with high-risk NDMM.

Although translocation events between chromosome 4 (NSD2 gene) and chromosome 14 (immunoglobulin heavy chain [IgH] locus) (t(4;14)) is considered high risk in newly diagnosed multiple myeloma (NDMM), only ∼30% to 40% of t(4;14) patients are clinically high risk. We generated and compared a large whole genome sequencing (WGS) and transcriptome (RNA sequencing) from 258 t(4;14) (n = 153 discovery, n = 105 replication) and 183 non-t(4;14) NDMM patients with associated clinical data. A landmark survival analysis indicated only ∼25% of t(4;14) patients had an overall survival (OS) <24 months, and a comparative analysis of the patient subgroups identified biomarkers associated with this poor outcome, including translocation breakpoints located in the NSD2 gene and expression of IgH-NSD2 fusion transcripts. Three breakpoints were identified and are designated as: "no-disruption" (upstream of NSD2), "early-disruption" (in the 5' UTR), and "late-disruption" (within the NSD2 gene). Our results show a significant difference in OS based on the location of DNA breakpoints (median OS 28.6 "late-disruption" vs 59.2 "early disruption" vs 75.1 months "no disruption"). These findings have been replicated in an independent replication dataset. Also, univariate and multivariate analysis suggest high-risk markers such as del17p, 1p independently contribute to poor outcome in t(4;14) MM patients.

In-depth analysis of alternative splicing landscape in multiple myeloma and potential role of dysregulated splicing factors.

Splicing changes are common in cancer and are associated with dysregulated splicing factors. Here, we analyzed RNA-seq data from 323 newly diagnosed multiple myeloma (MM) patients and described the alternative splicing (AS) landscape. We observed a large number of splicing pattern changes in MM cells compared to normal plasma cells (NPC). The most common events were alterations of mutually exclusive exons and exon skipping. Most of these events were observed in the absence of overall changes in gene expression and often impacted the coding potential of the alternatively spliced genes. To understand the molecular mechanisms driving frequent aberrant AS, we investigated 115 splicing factors (SFs) and associated them with the AS events in MM. We observed that ~40% of SFs were dysregulated in MM cells compared to NPC and found a significant enrichment of SRSF1, SRSF9, and PCB1 binding motifs around AS events. Importantly, SRSF1 overexpression was linked with shorter survival in two independent MM datasets and was correlated with the number of AS events, impacting tumor cell proliferation. Together with the observation that MM cells are vulnerable to splicing inhibition, our results may lay the foundation for developing new therapeutic strategies for MM. We have developed a web portal that allows custom alternative splicing event queries by using gene symbols and visualizes AS events in MM and subgroups. Our portals can be accessed at http://rconnect.dfci.harvard.edu/mmsplicing/ and https://rconnect.dfci.harvard.edu/mmleafcutter/ .

Circulating Tumor and Immune Cells for Minimally Invasive Risk Stratification of Smoldering Multiple Myeloma.

PURPOSE: Early intervention in smoldering multiple myeloma (SMM) requires optimal risk stratification to avoid under- and overtreatment. We hypothesized that replacing bone marrow (BM) plasma cells (PC) for circulating tumor cells (CTC), and adding immune biomarkers in peripheral blood (PB) for the identification of patients at risk of progression due to lost immune surveillance, could improve the International Myeloma Working Group 20/2/20 model. EXPERIMENTAL DESIGN: We report the outcomes of 150 patients with SMM enrolled in the iMMunocell study, in which serial assessment of tumor and immune cells in PB was performed every 6 months for a period of 3 years since enrollment. RESULTS: Patients with >0.015% versus ≤0.015% CTCs at baseline had a median time-to-progression of 17 months versus not reached (HR, 4.9; P < 0.001). Presence of >20% BM PCs had no prognostic value in a multivariate analysis that included serum free light-chain ratio >20, >2 g/dL M-protein, and >0.015% CTCs. The 20/2/20 and 20/2/0.015 models yielded similar risk stratification (C-index of 0.76 and 0.78). The combination of the 20/2/0.015 model with an immune risk score based on the percentages of SLAN+ and SLAN- nonclassical monocytes, CD69+HLADR+ cytotoxic NK cells, and CD4+CXCR3+ stem central memory T cells, allowed patient' stratification into low, intermediate-low, intermediate-high, and high-risk disease with 0%, 20%, 39%, and 73% rates of progression at 2 years. CONCLUSIONS: This study showed that CTCs outperform BM PCs for assessing tumor burden. Additional analysis in larger series are needed to define a consensus cutoff of CTCs for minimally invasive stratification of SMM.

Perspectives on the Risk-Stratified Treatment of Multiple Myeloma.

The multiple myeloma treatment landscape has changed dramatically. This change, paralleled by an increase in scientific knowledge, has resulted in significant improvement in survival. However, heterogeneity remains in clinical outcomes, with a proportion of patients not benefiting from current approaches and continuing to have a poor prognosis. A significant proportion of the variability in outcome can be predicted on the basis of clinical and biochemical parameters and tumor-acquired genetic variants, allowing for risk stratification and a more personalized approach to therapy. This article discusses the principles that can enable the rational and effective development of therapeutic approaches for high-risk multiple myeloma.