Multiple Myeloma Subtypes Research Articles

Macrofocal multiple myeloma in the era of novel agents in China.

Macrofocal multiple myeloma (MFMM) is characterized by clonal plasma cells comprising less than 20% of the bone marrow, multiple lytic bone lesions, and the absence of anemia, renal insufficiency, and hypercalcemia. This subtype of multiple myeloma (MM) has a relatively low incidence. Prognostic staging and cytogenetic guidance for MFMM are often insufficient due to the low tumor burden in the bone marrow. Large cohort studies on this subgroup during the era of novel agents are limited. We aim to describe the clinical characteristics and prognostic markers of MFMM patients undergoing treatment with novel agents. Consecutive cases of MM patients diagnosed at Peking University People's Hospital and Fu Xing Hospital of Capital Medical University from 2011 to 2023 were screened. A propensity score matching was conducted with a 2:1 ratio, matching classic MM patients to MFMM patients based on clinical variables of age and year of diagnosis. We identified 91 cases (4%) of MFMM and 182 matched classic MM among 2291 MM patients. The MFMM cohort had a higher proportion of male patients, those with <90% clonal plasma cells in the bone marrow by multiparameter flow cytometry, and patients with extramedullary disease, along with a lower proportion of patients with high-risk cytogenetics or advanced disease staging. MFMM patients demonstrated better overall responses compared to the control cohort (p = 0.027) in those not receiving upfront autologous stem cell transplantation (ASCT). During a median follow-up of 42.8 months for the entire cohort, the MFMM cohort exhibited significantly superior progression-free survival (PFS) and overall survival (OS) compared to the control cohort. In multivariate analysis of the entire cohort, exposure to immunomodulatory drugs and ASCT consolidation in frontline therapy were independently associated with improved PFS and OS. For the MFMM cohort, a Ki-67 index ⩾20% was associated with inferior PFS, providing valuable prognostic information in a group where staging and cytogenetic guidance are often inadequate. We concluded that treatment strategies for MFMM patients should align with those for standard MM, and a Ki-67 index ⩾20% in biopsy samples of plasmacytoma is associated with inferior PFS.

Raman spectroscopy of dried serum for the detection of rapid noninvasive multiple myeloma

Raman spectroscopy has recently emerged as an attractive focus of research interest in studies of hematological diseases. However, comprehensive Raman spectral analyses of serum samples from patients with multiple myeloma (MM) or other forms of lymphatic neoplastic disease are lacking. In this study, laser Raman spectroscopy and orthogonal partial least-squares discrimination analysis (OPLS-DA) were employed to develop a simple, noninvasive approach to MM diagnosis based on dried serum samples. To that end, systematic OPLS-DA analyses of dried serum from 35 patients with MM and 13 healthy controls were performed, revealing clear differences between these two groups in terms of the resultant serum spectral data. Specifically, significant reductions in the intensities of Raman peaks corresponding to nucleic acids (726, 781, 1579 cm−1), proteins (621, 1603, 1616 cm−1), lipids (1437, 1443, 1446 cm−1) and carotenoids (957, 1160, 1520 cm−1) were observed in MM, together with increases in the intensities of peaks corresponding to carbohydrates (920, 1123 cm−1) and collagen (1345 cm−1). Through combined analyses of serological indices associated with metabolic activity, MM patients were confirmed to exhibit elevated serum glucose levels and decreased levels of high-density lipoprotein cholesterol. These results offer a spectroscopic foundation for the relationships between MM classification and serological testing data, offering new evidence that can guide the early and efficient identification and characterization of this deadly cancer type. This exploratory study thus offers insight into the potential utility of Raman spectroscopy as a tool for the noninvasive detection of specific subtypes of MM.

Clinical Outcome of Induction Treatment in the Era of Novel Agents and the Impact of the Number of High-Risk Cytogenetic Abnormalities (HRA) on Prognosis of Patients With Newly Diagnosed Multiple Myeloma (NDMM): Insights From a Multicenter Study.

In the era of novel agents, the clinical outcomes of induction treatment and the impact of the number of high-risk cytogenetic abnormalities (HRA) in newly diagnosed multiple myeloma (NDMM) need to be explored. Through this study, we aim to analyze the effectiveness of different induction treatments and explore the survival outcomes of patients with varying numbers of HRA. A total of 734 patients from seven medical centers were included in our study. Patients in the CD38 monoclonal antibody or IMiDs plus proteasome inhibitors (PI) groups had significantly superior overall survival (OS) and progression-free survival (PFS) compared to those receiving IMiDs or PI alone. Additionally, the CD38 monoclonal antibody conferred a PFS advantage over IMiDs plus PI. Patients with ≥ 2 high-risk cytogenetic abnormalities (HRA) exhibited an extremely poor prognosis and should be considered ultra-high-risk individuals in multiple myeloma (MM). The CD38 monoclonal antibody, transplantation, and achieving minimal residual disease (MRD) negativity only partly mitigated the poor prognosis in patients with HRA. Furthermore, patients with 1q21 gain/amplification (1q21+) only had a significantly worse prognosis compared to patients without HRA, and those with 1q21+ plus del17p or t(4;14) exhibited an inferior prognosis compared to those with 1q21+ alone. Our results suggested that double-hit multiple myeloma was associated with extremely poor survival outcomes, and more effective treatments needed to be explored for this particular subtype of MM.

Physical Rehabilitation in Multiple Myeloma - A Retrospective Analysis and Future Perspectives

Multiple Myeloma is a malignancy characterized by multisystem involvement, including multiple osteolytic lesions, anemia, and renal insufficiency. The debilitating course of this disease highlights the importance of exploring the therapeutic potential of physical rehabilitation in improving patients' quality of life and providing meaningful clinical outcomes. The aim of this study is to investigate the benefits and challenges associated with the implementation of physical rehabilitation programs for patients with multiple myeloma, analyzing the evolution and characteristics of multiple myeloma cases in a medical clinic in Romania. Through this, we seek to contribute to the development of new approaches and protocols in physical rehabilitation, which may improve the therapeutic management and quality of life for patients with this complex condition. A retrospective analysis was conducted on newly diagnosed multiple myeloma patients over a 7-year period (2017-2023) at a clinic in Romania. The collected data included the time of initial diagnosis, patient age, residence (rural or urban), multiple myeloma subtype,treatments initiated, hematological parameters, presence of bone lesions, and comorbidities. We reviewed the existing literature on physical rehabilitation in multiple myeloma and assessed the associated advantages and challenges. Statistical analysis was performed to identify trends and correlations within our cohort. Out of a total of 255 patients diagnosed with multiple myeloma at a medical center in Romania, the majority were men from urban areas. It was observed that the average age at diagnosis was lower among patients from urban areas. Additionally, 69.8% of patients presented with bone lesions, while pancytopenias were rarely encountered at the time of diagnosis. Personalization of physical exercises is essential to maximize rehabilitation benefits for patients with multiple myeloma. Complications such as pancytopenias and frequently encountered bone lesions should not discourage the recommendation of rehabilitation. Decisions must be individually tailored and coordinated by a multidisciplinary team to ensure the rehabilitation program's safety and efficacy.

Leveraging diverse cellular stress patterns for predicting clinical outcomes and therapeutic responses in patients with multiple myeloma.

Tumour microenvironment harbours diverse stress factors that affect the progression of multiple myeloma (MM), and the survival of MM cells heavily relies on crucial stress pathways. However, the impact of cellular stress on clinical prognosis of MM patients remains largely unknown. This study aimed to provide a cell stress-related model for survival and treatment prediction in MM. We incorporated five cell stress patterns including heat, oxidative, hypoxic, genotoxic, and endoplasmic reticulum stresses, to develop a comprehensive cellular stress index (CSI). Then we systematically analysed the effects of CSI on survival outcomes, clinical characteristics, immune microenvironment, and treatment sensitivity in MM. Molecular subtypes were identified using consensus clustering analysis based on CSI gene profiles. Moreover, a prognostic nomogram incorporating CSI was constructed and validated to aid in personalised risk stratification. After screening from five stress models, a CSI signature containing nine genes was established by Cox regression analyses and validated in three independent datasets. High CSI was significantly correlated with cell division pathways and poor clinical prognosis. Two distinct MM subtypes were identified through unsupervised clustering, showing significant differences in prognostic outcomes. The nomogram that combined CSI with clinical features exhibited good predictive performances in both training and validation cohorts. Meanwhile, CSI was closely associated with immune cell infiltration level and immune checkpoint gene expression. Therapeutically, patients with high CSI were more sensitive to bortezomib and antimitotic agents, while their response to immunotherapy was less favourable. Furthermore, invitro experiments using cell lines and clinical samples verified the expression and function of key genes from CSI. The CSI signature could be a clinically applicable indicator of disease evaluation, demonstrating potential in predicting prognosis and guiding therapy for patients with MM.

Prognostic factors and clinical characteristics of patients with newly diagnosed non-secretory multiple myeloma in the era of new drugs in "real-world" study: Experiences of the Polish Myeloma Group.

Non-secretory multiple myeloma (NSMM) accounts for approx. 2-3% of multiple myeloma (MM) cases. Due to the rare occurrence and ineligibility of patients with NSMM to participate in clinical trials, we have limited data on treatment efficacy and the clinical course in these patients. Most of the literature consists of case reports and small retrospective studies. The study aimed to analyze patient characteristics, prognostic factors and treatment outcomes in newly diagnosed (ND) NSMM. This is a multicenter, retrospective analysis of 43 patients with NSMM diagnosed between June 2010 and September 2021, conducted in 8 Polish hematology centers. The median overall survival (OS) was 103 months (95% confidence interval (95% CI): 20-72). The most common cause of death was MM disease progression. The overall response rate (ORR) was 84.6%; complete response (CR), very good partial response (VGPR), partial response (PR), and no response (NR) rates were 20.5%, 46.2%, 17.9%, and 15.4%, respectively. In multivariable analysis, factors contributing to worse OS included International Staging System stage 3 (ISS-3) (p = 0.0277), anemia (Hb <10 g/dL or >2 below upper limit of normal value (ULN), p = 0.0270), renal insufficiency (RI, serum creatinine >2 mg/dL, p = 0.0476), and serum albumin <5.5 mg/L (0.0408). Non-secretory multiple myeloma is a rare subtype of MM. This small study demonstrates that outcomes are comparable to secretory MM. However, the inclusion of this subset of patients in clinical trials is essential to assess prognosis, treatment efficacy and clinical outcomes.

Real-world incidence and risk factors of bortezomib-related cardiovascular adverse events in patients with multiple myeloma

BackgroundAlthough most studies on the cardiovascular toxicity of proteasome inhibitors have focused on carfilzomib, the risk of cardiotoxicity associated with bortezomib remains controversial. This study aimed to evaluate the incidence and risk factors of cardiovascular adverse events (CVAEs) associated with bortezomib in patients with multiple myeloma in a real-world setting.MethodsThis cross-sectional study included patients who were treated with bortezomib at a tertiary hospital in South Korea. CVAEs, defined as hypertension, arrhythmia, heart failure, myocardial infarction, pulmonary arterial hypertension, angina, and venous thromboembolism, were detected using cardiac markers, ECG, echocardiography, medications, or documentation by clinicians. The patients were observed for at least 6 months and up to 2 years after starting bortezomib administration.ResultsAmong the 395 patients, 20.8% experienced CVAEs of any grade, and 14.7% experienced severe adverse events. The median onset time for any CVAE was 101.5 days (IQR, 42–182 days), and new-onset/worsened hypertension was the most prevalent CVAE. The risk of CVAEs increased in patients with a body mass index lower than 18.5 (adjusted HR (aHR) 3.50, 95% confidence interval (CI) 1.05-11.72), light chain (1.80, 1.04-3.13), and IgD (4.63, 1.06-20.20) as the multiple myeloma subtype, baseline stroke (4.52, 1.59-12.80), and hypertension (1.99, 1.23-3.23). However, CVAEs did not significantly affect the 2-year overall survival and progression-free survival.ConclusionApproximately 15% of the Korean patients treated with bortezomib experienced severe CVAEs. Thus, patients, especially those with identified risk factors, should be closely monitored for CVAE symptoms during bortezomib treatment.

Elucidation of molecular basis of osteolytic bone lesions in advanced multiple myeloma.

Osteolytic bone lesion is a major cause of lower quality of life and poor prognosis in patients with multiple myeloma (MM), but molecular pathogenesis of the osteolytic process in MM remains elusive. Fms-like tyrosine kinase 3 ligand (FLT3L) was reported to be elevated in bone marrow (BM) and blood of patients with advanced MM who often show osteolysis. Here, we investigated a functional link of FLT3L to osteolytic process in MM. We recruited 86, 306, and 52 patients with MM, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL), respectively. FLT3L levels of patients with hematologic malignancies were measured in BM-derived plasma and found to be significantly higher in MM than in AML or ALL, which rarely show osteolysis. FLT3L levels were further elevated in MM patients with bone lesion compared with patients without bone lesion. In vitro cell-based assays showed that the administration of FLT3L to HEK293T, HeLa, and U2OS cells led to an increase in the DKK1 transcript level through STAT3 phosphorylation at tyrosine 705. WNT reporter assay showed that FLT3L treatment reduced WNT signaling and nuclear translocation of β-catenin. These results collectively show that the FLT3L-STAT3-DKK1 pathway inhibits WNT signaling-mediated bone formation in MM, which can cause osteolytic bone lesion. Finally, transcriptomic profiles revealed that FLT3L and DKK1 were predominantly elevated in the hyperdiploidy subtype of MM. Taken together, FLT3L can serve as a promising biomarker for predicting osteolytic bone lesion and also a potential therapeutic target to prohibit the progression of the osteolytic process in MM with hyperdiploidy.

Prevalence of Exclusion Criteria in Multiple Myeloma Randomized Controlled Trials: A Systematic Review

Introduction: Patients with the most aggressive, hardest-to-treat, and rarer forms of multiple myeloma (MM), such as patients with severe renal impairment, central nervous system (CNS) disease, extramedullary disease, and plasma cell leukemia, have the greatest need of novel therapeutic approaches. Strict eligibility criteria for randomized controlled trials (RCTs) both deny access to clinical trials to these patients in greatest need, and also limit generalizability of trial results. We thus systematically assessed newly diagnosed MM RCT's from 2006 to 2023 to ascertain the prevalence of restrictive eligibility criteria, and their trends over time. Methods: We searched MEDLINE/PubMed, Embase, and the Cochrane Registry of Controlled Trials to measure the prevalence of restrictive inclusion criteria MM RCTs. We included all published newly-diagnosed MM RCTs from 2006-2023. Restrictive eligibility criteria evaluated included 1) age exclusions, 2) projected life expectancy requirements, 3) performance status 4) requiring thresholds of “secretory disease”, 5) CNS disease, 6) plasma cell leukemia, 7) hepatic and/or 8) renal function requirements, 9) hematological thresholds, 10) prior malignancy, 11) history of neuropathy, and 12) exclusion of extramedullary disease. We searched manuscripts, clinicaltrials.gov listings, and individual protocols whenever publicly available to ascertain these criteria. We ascertained what proportion of trials had each of these twelve restrictive criteria and assessed trends over time. A logistical regression was used to compare these variables over time. Results: 85 RCTs were included (Table 1). Exclusion criteria included: age in 43 (51%); projected life expectancy in 20 (24%); performance status in 74 (87%); thresholds of “secretory or measurable” disease in 47 (55%), , hepatic function in 64 (79%), renal function in 63 (74%), hematological thresholds in 50 (59%), prior malignancy (various time windows) in 68 (80%), neuropathy in 50 (59%) (various grades), plasma cell leukemia in 16 (18.8%), extramedullary disease in 4 (4.7%) and CNS disease in 9 (10.6%). Trends in exclusion criteria were assessed over time (Figure 1) showing all criteria (other than performance status) have failed to improve from 2006 to 2023, and there has been an increase in exclusionary criteria pertaining to plasma cell leukemia, extramedullary disease, CNS involvement and pre-existing neuropathy. Specific definitions of secretory disease varied with trials that required secretory disease (n=47), with 24 (51%) of these trials requiring a serum monoclonal protein of 1 g/dL or 0.5 if IgA/IgD/IgE/IgM MM, 2 (4.3%) requiring serum monoclonal protein of 2 g/dL regardless of MM subtype, 3 (6.4%) requiring serum monoclonal protein of 0.5 regardless of MM subtype, 11 (23.4%) requiring a serum free light chain of 10 mg/dL, and one (2.1%) requiring a serum free light chain of 5 mg/dL. Conclusion: The majority of MM RCTs have restrictive eligibility criteria that limit enrollment of the sickest MM patients and subsequent external validity of these trials, which perpetuates the status quo where limited data are available to guide the treatment of these challenging MM subtypes. It is very likely our methodology under-estimates the true prevalence of exclusionary criteria, as protocols were not universally available, and a trial was considered as having an exclusionary criteria only if explicitly listed. The presence of these restrictive eligibility criteria has failed to decrease over time.

Artificial Intelligence of the 2-D and 3-D Bone Marrow Microenvironment to Identify Cytogenetic Subtypes of Multiple Myeloma

Multiple myeloma is the second most common hematologic malignancy with an annual incidence of 35,000 new cases every year in the U.S. Due to improved access to effective therapies, overall survival has improved from 1-3 years to over 10-20 years. However, there is not yet any available curative therapy for multiple myeloma and most patients will suffer from recurrent relapses, with an unmet clinical need for novel therapies. The interplay between the bone marrow microenvironment and malignant plasma cells is critically important to better understand pathogenesis of the disease, to predict response to therapy (in particular, immunotherapy), and to identify novel drug targets. Leveraging the power of computer vision, we are developing novel machine learning algorithms to label and characterize individual cells from digitalized H&amp;E and CD138 stained plasma cells in bone marrow core biopsies. A total of 188 patients were included in this analysis: 157 with multiple myeloma, 6 with monoclonal gammopathy of undetermined significance, 17 with smoldering multiple myeloma, and 6 with primary amyloidosis. The deep learning framework to build our artificial intelligence (AI) models is implemented by Pytorch. Using the surface creation tool from the Imaris 10.0.1 software, we are able to measure pixel intensity, area, sphericity, and other cellular features that are then correlated with myeloma clinical outcomes as well as defined cytogenetic subtypes of multiple myeloma in 2-D. As a preliminary step in this project we analyzed the IMARIS software's ability to accurately determine the number of plasma cells compared with clinical standards through a linear regression plot with strong correlation (R 2=0.9). In addition, we rendered the 3-D microenvironment by combining more than 60 sequential formalin-fixed paraffin embedded (FFPE) tissue block bone marrow biopsy samples sliced at a thickness of 4 microns. Through utilizing the surface creation tool on the 2-D images within the 3-D model, we can show distance measurements between plasma cells and other features of the microenvironment such as adipocytes. This insight allows us to better understand and control the immune microenvironment through observing the various 2-D tissue slices within the 3-D model. Furthermore, we have assessed whether multiple myeloma digital images are able to detect translocation [t(11;14)] multiple myeloma subtype, which is present in 15% to 24% of patients, shown to predict treatment response to Bcl-2 inhibitor therapy (such as venetoclax). A small cohort (n=25) of patients with t(11;14) have been compared to non-t(11;14) cases using the machine learning model and preliminary data indicate that there is a correlation between digital images and multiple myeloma subtype. In the future, we anticipate our AI model will improve the accuracy of diagnostic methods while reducing costs and offering deeper translational insights that will predict drug response in vivo.

Therapy Related Multiple Myeloma: A Distinct Entity

Background The development of therapy-related multiple myeloma (t-MM) is a poorly described entity. Multiple myeloma (MM) is the second most common hematologic malignancy. Despite significant strides in MM treatment over recent decades, identifying modifiable risk factors, optimizing screening methodologies, and identifying potential treatment strategies or targets to address t-MM remains of utmost importance, as this disease continues to pose a significant therapeutic challenge and remains incurable. This retrospective review seeks to explore the incidence and characteristics of t-MM at a medium-sized, academic center. Methods We performed a single institution retrospective chart review of patients diagnosed with multiple myeloma between 2008-2023 and evaluated for prior diagnosis of solid malignancy, excluding all skin cancers, lymphomas or myeloproliferative neoplasm (MPN). We collected data for the following: prior treatment for solid malignancy, lymphoma or MPN, lapsed time between prior therapy and development of MM, cytogenetics, prominent subtype present, renal failure and hypercalcemia if present at time of diagnosis, management of t-MM including transplant data, disease progression including relapse status, and survival data. Results In our study, we identified a subgroup of 17 individuals who had a previous history of solid malignancy, 1 patient with unknown prior solid tumor, 1 patient with history of Hodgkin's lymphoma, and 1 additional patient with history of MPN who received chemotherapy and/or radiation therapy, with 3 patients having MGUS prior to development of MM. Notably, among this population, 70% were male. All patients developed MM following the treatment for a solid malignancy, lymphoma or MPN. The average amount of time from diagnosis of solid tumor, lymphoma or MPN to development of MM was 9.53 years (1-25 years). 50% of the individuals were White Americans, while the remaining 50% were African Americans. A significant majority (85%) of the population had a single primary solid tumor. Among the primary solid tumors, 45% were prostate cancer, 15% were breast cancer, and 10% were renal cell carcinoma (Figure 1). 5 patients received chemotherapy alone without documented prior radiation exposure while 13 had prior radiation exposure for prior solid malignancy. At time of diagnosis, 45% of patients had renal failure with 10% of patients having both renal failure and hypercalcemia. Within the t-MM subtypes, we discovered that 50% had IgA type, with 70% of these individuals exhibiting the IgA Kappa subtype. 45% of the individuals who developed t-MM had the IgG type, with 78% of them with IgG Kappa subtype. 15 (75%) of the patients received transplant and only 2 patients had confirmed relapse with 2 patients unknown and 2 additional patients who passed away prior to starting maintenance therapy following transplant. 4 patients are very early in their disease process and as of yet have not undergo transplant. 55% of our patients are presumed living. The OS for patients with t-MM was 1190 days compared to the average OS of 2326 days in our institutional MM database without t-MM. Conclusion Our analysis focused on patients diagnosed with MM who had a prior history of solid primary tumors, lymphoma or MPN treated with chemotherapy and/or radiotherapy in hopes of providing valuable insights into the characteristics and outcomes of individuals who developed MM following treatment for solid tumors, lymphoma or MPN. Our findings highlighted specific risk factors associated with t-MM in this population. Being male and having a history of prostate cancer was associated with higher incidence of development of t-MM. This is likely related to the radiation therapy associated with prostate cancer, as radiation exposure has been a known risk factor for the development of MM. We observed that the predominant subtype of MM among these individuals was IgA kappa, which diverges from the prevalent subtype, IgG kappa, observed in the general population. IgA is known to typically have more cytogenetic abnormalities and overall poorer prognosis, likely contributing to the difference in OS compared to patients with t-MM and MM not associated with prior malignancy. These insights shed light on the unique characteristics and potential influences of treatment-related MM in patients with prior malignancies or MPN.

Transcriptomic Features Influencing Anti-Myeloma Drug Resistance in Human Multiple Myeloma Cell Lines

Despite increasing number of new drugs, multiple myeloma (MM) remains a significant challenge in hematology. This problem primarily arises from the emergence of drug resistance, which could either result from pre-existing drug-resistant clones, or from the development of secondary genomic and phenotypic drug resistance features upon drug exposure. The use of primary MM patient-derived samples, while considered the gold standard in preclinical MM research, presents challenges due to notoriously low plasma cell yields and short ex vivo lifespan. Moreover, these cells often fail to survive cryopreservation, making them difficult to use in reproducible drug sensitivity screens. To address this issue, the MM research community has established numerous culture-adapted MM cell lines (HMCLs), representative of all cytogenetic MM subtypes and characterized by DNA and RNA sequencing. However, a comprehensive evaluation of their drug sensitivity has been lacking. In this study, we sought to bridge this gap by profiling the sensitivity of over 30 HMCLs to both established and novel drugs relevant to MM treatment. We explored the transcriptomic and genomic features that correlate with drug sensitivity of these cell lines, and investigated whether these features were associated with the outcomes of patients enrolled in the MMRF CoMMpass study. Our initial results suggest potential pathways universally implicated in MM drug resistance, which could be targeted therapeutically. We performed drug sensitivity screening in 96-well plates, testing established drugs such as proteasome inhibitors, immunomodulatory drugs, dexamethasone, and melphalan, alongside novel treatment modalities including the BCL2 inhibitor venetoclax, XPO1 inhibitor selinexor, and histone deacetylase inhibitor panobinostat. Optimal cell density and drug dilutions were ensured to capture the full spectrum of drug response. After 4 days of incubation, cell proliferation was measured using a metabolic MTS assay, and EC50 values were computed using four-parameter logistic regression. Genomic and transcriptomic data were obtained from HMCL66 dataset from the Keats laboratory. Drug sensitivity was analyzed using a log-transformed EC50 matrix, which was used for unsupervised hierarchical clustering of cell lines, revealing two primary clusters: multi-drug resistant (MDR) and multi-drug sensitive (MDS) cell lines. Gene set enrichment analysis revealed that MDR cell lines showed positive enrichment for KEAP1/NFE2L2 (Figure 1A), UPR/PERK pathways, and type I interferon signaling. Negative enrichment in MDR cell lines was observed for TP53-regulated transcription of cell death genes, with the top leading edge genes being key apoptosis inducers such as BAX, BID, APAF1, and FAS. We used the obtained leading edge gene lists to assign respective pathway activation scores to the CoMMpass patients, based on their transcriptomic profile, using the ssGSEA singscore package. High activation score of KEAP1/NFE2L2 was associated with shorter overall survival (HR = 1.57, p = 0.016), irrespective of type of first-line therapy (Figure 1A). Our study represents a significant milestone in characterizing the HMCL drug sensitivity landscape, suggesting that MDR cell lines inherently exhibit transcriptional features associated with high activity of the KEAP1/NFE2L2 and PERK pathways. These pathways have been previously associated with PI and IMiD resistance, but interestingly, our findings also link these signatures with the sensitivity to a variety of other anti-MM drugs. Conversely, MDS cell lines displayed enhanced basal activity of TP53-regulated pro-apoptotic genes, which might increase their apoptotic priming and consequently increase their susceptibility to drug-induced apoptosis. With the rapid evolution of protein-protein interaction inhibitors, future exploration of KEAP1-NRF2 or MDM2-TP53 interaction inhibitors could potentially enhance myeloma cell sensitivity. In the future, our team plans to expand the drug panel used in the screen and provide this enriched dataset as a public resource, thereby facilitating wider access and collaborative advancements in the field. Study supported by National Science Centre Grants PRELUDIUM 2018/31/N/NZ5/03214, ETIUDA 2020/36/T/NZ5/00610 and Polish Stem Cell Bank ExCELLent grant.

Biological and Clinical Significance of Undetectable Circulating Tumor Cells (CTCs) in Patients (Pts) with Multiple Myeloma (MM)

Background: There is evidence suggesting that CTCs are responsible for the spreading of clonal plasma cells (PC) inside and outside the bone marrow (BM). However, some pts unexpectedly have no CTCs at diagnosis. Here, we hypothesize that the absence of CTC defines a specific MM subtype with unique biological and clinical features. To our knowledge, this has never been investigated. Aim: Define the frequency and the characteristics of MM pts with undetectable CTCs. Methods: The presence of CTCs was assessed by NGF in 1,687 blood samples from pts with MGUS (n=467), smoldering (SMM, n=368), newly diagnosed (NDMM, n=652), and relapsed refractory MM (RRMM, n=200). All pts were enrolled in prospective PETHEMA/GEM multicenter studies. BM clonal PC from pts with (n=239) and without (n=17) detectable CTCs were isolated by FACS prior exome and RNA sequencing. Results: The frequency of pts with undetectable CTCs progressively decreased ( p &amp;lt;.001) from MGUS (54%) to SMM (21%) and NDMM (8%) and RRMM (11%). In those with detectable CTCs, the median percentage was 0.0003, 0.002, 0.01 and 0.008, respectively ( p &amp;lt;.001). The 1-log increment observed in the progression from MGUS to SMM and NDMM underpins the link between CTC levels and malignant transformation. The similarity between NDMM and RRMM is probably related with the fact that the latter were analyzed in between different lines of therapy. When compared to other SMM pts, those with undetectable CTCs were more frequently staged with low risk according to the 20/2/20 IMWG model (32% vs 49%; p &amp;lt;.001). Absence of CTCs (HR: 0.38; p =.042) and the 20/2/20 IMWG model (HR: 0.46; p =.011) had independent prognostic value in a multivariate analysis. SMM pts with undetectable vs detectable CTCs showed lower risk of transformation (progression rates at 2 years of 7% vs 24%, p =.004). When compared to other NDMM pts, those with undetectable CTCs had significantly less anemia and BM infiltration. There were no differences in the incidence of lytic lesions, renal insufficiency and hypercalcemia. Only one pt without CTCs was staged with R-ISS 3. These findings, together with a higher frequency of plasmacytomas, suggest that the MM subtype with undetectable CTCs may include pts with macrofocal disease. In transplant eligible NDMM, pts with undetectable vs detectable CTCs had PFS rates at 7 years of 90% and 44% (HR: 0.14; p &amp;lt;.001), and OS rates of 97% and 72% (HR: 0.10; p =.02). In transplant ineligible NDMM, pts with undetectable vs detectable CTCs had PFS rates at 2 years of 83% and 54% (HR: 0.31; p =.02), and OS rates of 94% and 76% (HR: 0.33; p =.13). In a multivariate analysis including the R-ISS and transplant eligibility, the absence of CTCs showed independent prognostic value for PFS (HR: 0.25; p &amp;lt;.001) and OS (HR: 0.24; p =.014). Among pts with undetectable CTCs, there were no differences in PFS and OS according to the presence of standard vs high risk cytogenetic abnormalities. In contrast with pts having detectable CTCs, the PFS and OS of those without CTCs was not significantly reduced if CR or MRD negativity were not achieved after treatment. The cytogenetic profile of BM clonal PC from pts with undetectable (n=112) vs detectable (n=846) CTCs was characterized by a lower frequency of +1q21 (16% vs 45%, p &amp;lt;.001) and del(1p32) (2% vs 8%, p =.02). Other alterations were not significant. There was no correlation between the number of mutations and the percentage of CTCs. There were 66 differentially expressed genes (DEG) between BM clonal PC from pts with detectable vs undetectable CTCs. Of note, 9 of the 66 DEG were in Chr1, with up- and downregulation of genes in 1p and 1q, respectively. There was a significant correlation between CTC levels and the expression of genes associated with MM dissemination ( FLNA , LGALS1and CXCR4). These findings, together with the modest correlation between the percentage of CTCs and BM clonal PC determined by morphology (r=.45, p &amp;lt;.001) and NGF (r=.49, p &amp;lt;.001), suggest that the selective egress of tumor cells from BM into the PB might depend on specific genetic alterations and transcriptional priming more than tumor burden. Conclusions:This study uncovered that ~8% of MM pts may progress due to mechanisms different from CTC dissemination. This subgroup showed unique features and achieved unprecedented outcomes, particularly if eligible to intensive therapy. Accordingly, our results endorse the recognition of pts with undetectable CTCs as a new biological and clinical subtype of MM.

Salvage Transplant Versus CAR-T Cell Therapy for Relapsed Multiple Myeloma

Background: Ide-cel and cilta-cel received regulatory approval for the treatment of patients with relapsed or refractory multiple myeloma (MM) after ≥4 prior lines of therapy, and almost all eligible patients in the US and Europe have previously received at least one autologous hematopoietic cell transplant (autoHCT). Even for those who received upfront autoHCT, salvage transplant (either second autoHCT or allogeneic HCT) for patients with relapsed MM was shown to be effective and feasible. Therefore, comparing CAR-T cell therapy with salvage transplant could facilitate future selection of patients with relapsed MM, as eligibility for transplant (especially allogeneic HCT) differs significantly from that of CAR-T. Methods: We leveraged a large European dataset to identify relapsed MM patients who received either salvage autoHCT, allogeneic HCT, or CAR-T cell therapy. To minimize selection bias, we only included patients who received salvage treatment from 2018 onwards and who had documented time of relapse. Furthermore, to control for differences in patient characteristics, we applied propensity score (PS) matching for the comparison: CAR-T versus auto HCT and versus allogeneic HCT. We performed individual matching with the “nearest neighbour” method, using a caliper=0.2. The model for PS estimation included patient sex, MM classification at diagnosis, previous transplant history (single or tandem auto), interval between upfront transplant and relapse and between relapse and CAR-T/salvage transplant, age, performance status and disease status at CAR-T/salvage transplant. Endpoints of matched groups were compared by Log-Rank test or Gray test stratified on the pair. Primary endpoint was progression-free survival (PFS) at 1 year with 95% confidence interval (CI). Secondary endpoints constituted non-relapse mortality, incidence of relapse, and overall survival (OS). Results: We included 4292 patients, of whom 3858 received salvage autoHCT, 371 allogeneic HCT, and 63 CAR-T cell therapy (60% ide-cel, 33% cilta-cel, 7% other investigational). Median age at time of salvage treatment was 62 years for autoHCT, 54 years for allogeneic HCT, and 61 years for CAR-T cell therapy (P&amp;lt;0.001). More than 60% of patients were male in all groups, and IgG subtype of MM was present in 52% of autoHCT, 48% of allogeneic HCT, and 44% of CAR-T cell therapy. More patients receiving CAR-T cell therapy had a Karnofsky performance status &amp;lt;80% (41%) versus 32% of autoHCT and 29% of allogeneic HCT. In terms of response, complete response to treatment was observed in 40% for autoHCT, 63% for allogeneic HCT, and 44% for CAR-T cell therapy. Partial remission or very good partial remission was achieved 11%, 9%, and 35%, respectively (P&amp;lt;0.001). Median follow-up was 16.7 months for the entire cohort. The PS method could successfully match almost all patients for the comparison of CAR-T cell therapy versus salvage autoHCT (59 patient, respectively). The 1-year PFS for CAR-T cell therapy versus autoHCT was 68% (95% CI, 56-80%) versus 44% (95% CI, 28-59%; P=0.048; Figure 1). The 1-year non-relapse mortality was 9% (95% CI, 2-17%) versus 0% (P=0.31). Cumulative incidence of relapse at 1 year was 23% versus 57% (P=0.028), and 1-year overall survival was 81% versus 68% (P=0.059). The PS method matched 33 patients for the comparison of CAR-T cell therapy versus salvage allogeneic HCT. The 1-year PFS was 72% (95% CI, 56-88%) versus 39% (95% CI, 18-60%; P=0.346). Non-relapse mortality at 1-year was 13% (95% CI, 1-25%) versus 23% (95% CI, 7-43%; P=0.257). The 1-year cumulative incidence of relapse was 15% versus 36% (P=0.763), and the 1-year overall survival was 84% (95% CI, 71-97%) versus 60% (95% CI, 39-81%; P=0.083). Conclusion: This multicenter retrospective study compared CAR-T cell therapy with salvage transplantation strategies for relapsed MM. CAR- T cell therapy appeared to improve outcomes, especially in comparison with salvage autoHCT. The higher absolute rates of non-relapse mortality observed with CAR-T cell therapy may not counterbalance overall beneficial results, and rather highlights the need for careful selection of patients. Longer follow-up will be needed for definitive results.

Discovery and Development of CD70 As a Cellular Therapy Target in High Risk Multiple Myeloma

Background: Even in the BCMA CAR-T era, multiple myeloma (MM) patients with high-risk genotypes have the poorest outcomes. We thus leveraged MMRF CoMMpass data to identify potential novel surface immunotherapeutic targets in high-risk MM. As described below, we focused on CD70, an immunotherapeutic target known to be upregulated on many hematologic and solid tumors but minimally expressed on normal tissue. While CD70 was previously explored in MM (McEarchern et al, Clin Cancer Res (2008)), it was not further investigated due to heterogeneous expression in newly diagnosed samples. Here we revisit this target to suggest cellular therapies against CD70 could be highly beneficial in high-risk MM patients, currently the greatest unmet need in the field. Methods: Primary patient samples were obtained under an IRB-approved protocol. CAR-T lentiviral transduction was performed on paired CD4/CD8 human T cells from at least 3 healthy donors. Effector function was assessed by cytotoxicity against MM cell lines and CAR-T profiling for memory and exhaustion markers. In vivo efficiency was assessed using bioluminescence. ATAC-seq peaks were called using Promo and HOMER (data from Jin et al. Blood (2018)). Machine learning was performed using XGBoost approach applied to CoMMpass RNA-seq data with 80% of samples for training and 20% as test set. Results: Analyzing CoMMpass mRNA data (release IA19) we first evaluated surface protein encoding-genes upregulated in tumors at relapse compared to diagnosis. Among &amp;gt;100 significantly upregulated genes, we focused on CD70 given known promise as an immunotherapy target. Further analysis across genomic subtypes revealed a specific, marked upregulation of CD70 in t(4;14) and gain 1q ( Fig. 1), as well as R-ISS 3 at diagnosis. Flow cytometry profiling of 8 cell line models, including t(4;14) models KHM11, KMS26, and LP1, revealed consistent CD70 protein expression with antigen density similar to BCMA. Profiling of relapsed primary MM samples confirmed that 19 of 28 samples had CD138+ plasma cells positive for CD70 expression. Of 5 patients in whom both BCMA and CD70 were measured, antigen densities were also similar. To target CD70, we then designed 10 unique anti-CD70 CAR-T constructs. 9 of these were scFv-based binders derived from therapeutic antibody fragments currently in preclinical or clinical development. By a structure-guided design based on the CD70:CD27 co-crystal structure (PDB: 7KX0), we also derived a unique truncated fragment of CD27 as an alternative CD70 binding element. All CD70 CARs were efficacious in in vitro cytotoxicity assays targeting MM cell lines AMO1, MM1.S, LP1, and RPMI8226. We then selected the top 3 performing scFv based-CARs as well as our natural ligand (CD27) CAR-T for in vivo studies. All 4 CD70 CAR-Ts prolonged survival of MM.1S-bearing mice compared to empty CAR ( n=5 mice/arm, p&amp;lt;0.005 by log-rank), similar to BCMA CAR-T controls. Remarkably, the CD27-based CAR showed superior expansion (50-85x, p&amp;lt; 0.0001 by ANOVA) and persistence compared to scFv-based ( Fig. 2). These results underscore that targeting CD70 using a natural ligand-based CAR-T represents a promising preclinical candidate for treatment of relapsed and high risk MM. We further investigated why CD70 may be upregulated in high risk MM. Analysis of ChIP-seq in ENCODE and primary MM tumor ATAC-seq was used to nominate a set of transcription factors that bind the CD70 locus. Extending from CoMMpass data, we built a predictive machine learning model for CD70 expression as a function of transcription factor gene expression, nominating key transcription factors TFAP2A, LEF1, MYB, and PAX5 as CD70 regulators; validation is now underway. Additionally, we showed that knockout of NSD2 in t(4;14) models KMS34 and KMS11 led to significantly decreased CD70 surface expression, implicating this central epigenetic driver in CD70 overexpression in this high-risk MM subtype. Finally, we also demonstrated that treatment with the hypomethylating agent azacytidine can drive increased CD70 expression, similar to that shown in AML models. Conclusion: Taken together, targeting CD70 appears to be a highly promising cellular therapeutic strategy for MM, with particularly high utility in high-risk patients after BCMA CAR-T relapse. We are continuing preclinical and future clinical development of this CAR-T strategy, using our natural ligand CD27 design with extended in vivo persistence.

Somatic Hypermutation in Enhancer Regions Shapes Non-Coding Myeloma Genome, Generating DNA Breaks and Driving Etiology through Mutation and Structural Variation

INTRODUCTION: Little is known about the pattern and function of mutations within the 98% of the genome which is non-coding (nc). Whole-genome sequencing (WGS) can identify the full range of single nucleotide variants (SNVs), insertions/deletions (InDels), copy-number variants (CNVs), and structural variants (SVs), which are critical to disease progression. Here, we characterize the non-coding genome to gain significant insight into the role of mutations in gene regulatory elements in the etiology of multiple myeloma (MM) and to models of how it develops. METHODS: We studied 302 of MM precursor and newly diagnosed MM (NDMM) patients with high-coverage WGS, where each SNV/InDel was confirmed by two or more algorithms. Results were validated on an independent cohort of 256 NDMM with 80X WGS data. A pipeline employing a consensus mechanism for determining the final set of somatic events was used, including Mutect2, Strelka2, and VarScan2 for SNVs; Mutect2, Strelka2, VarScan2, and SvABA for InDels; Battenberg and FACETS for CNVs; Manta, SvABA, DELLY2, and IgCaller for SVs (https://github.com/pblaney/mgp1000). The R package fishHook was used toidentify statistically significant enrichment of mutations. To identify nc-variants we partitioned the genome into 10 kb tiles that were iteratively shifted by 500 bp and tested each tile against a regression model built into fishHook. The model includes a series of covariates that inform replication timing, sequencing context, and chromatin states. RESULTS: We identified 2,039,841 SNVs and 492,746 InDels in total. The tumor mutational burden (TMB) varies between molecular subgroups with the t(4;14) being significantly higher at 3.23 (somatic mutations per Mb) in comparison to the t(11;14) at 2.57 (FDR adj. P=0.035), which was closer to patients without a subtype translocation at 2.78. For ncSNVs and ncInDels, we identified 4,374 and 272 tiles respectively with significant mutation enrichment genome-wide (FDR adj. P&amp;lt;0.05). As tiles may overlap, we collapsed contiguous segments into consensus regions assigning the nearest coding gene as an identifier and termed these “mutation-enriched regions” or MERs. We identified 282 MERs associated with 203 genes for ncSNVs and 26 MERs associated with 25 genes for ncInDels. The two types of regions overlap at six loci ( TENT5C, OR2T2, FOXD4L1, BCL6, BLOC1SS- TXNDC5, PLD5P1). Thus, we identified 302 MERs associated with 221 genes, with some of the most highly mutated MERs included BCL6 (76.2% of patients), BLOC1S5- TXNDC5 (28.1%), ZFP36L1 (22.2%), BTG2 (21.2%), IRF8 (16.2%), TENT5C (13.6%), and CCND1 (12.3%). In total 19,743 of the 2,532,587 mutations fall into MERs with 1.3-65.6% of patients having one of these mutations. We evaluated the MERs for functional relevance by intersecting the regions with a list of 8,357 genome-wide enhancer (E) and super-enhancer (SE) elements derived from germinal-center B cells (GCB), DLBCL (Bal et al. Nature 2022) and MM (Lovén et al. Cell 2013). In total, 17.9% (54/302) of the MERs were identified, involving 45 genes. These MERs intersected with 28 Es and 20 SEs, with a non-random distribution of mutations within them. Of the total MER mutations, 21.9% (4,317/19,743) fell into some form of enhancer element. Breaking these down further 41.6% (1,798/4,317) are in Es, and 58.4% (2,519/4,317) are in SEs. All the E mutations were MM specific; of the SE mutations, 6.0% (18/302) of patients had a mutation in an ABC-DLBCL specific SE, 16.6% (50/302) in a GCB specific SE and 64.2% (194/302) in a MM specific SE. We examined the distribution of mutations within the SE regions and found they are non-random suggesting a selective mechanism. We intersected the SE regions with SV and found an excess at TENT5C, BTG2, BLOC1S5-TXNDC5 and ZFP36L1. A focused analysis of chr1p, chr1q, chr6q and chr14 revealed the importance of mutationally induced breaks within the SE and its translocation to a receptor site often 8q the site of MYC. CONCLUSIONS: We provide evidence for an important contribution of mutations within E and SE regions to the etiology of MM. This may involve either direct selection of mutations within the GC or by the re-entry of a memory B-cell carrying a pattern of mutations it acquired in a pre-MM phase, which then acquires a MM-specific driver. FIGURE: Distribution of mutations across MM genomes. A) Tumor mutational burden across MM subtypes. B) Q-Q plots of fishHook model for SNVs

Young and Distinct: Clinical Characteristics and Outcomes of Patients 50 or Younger with Multiple Myeloma

Introduction: Multiple Myeloma (MM) presents at a median age of 69. Patients age 50 or younger represent only 10% of diagnoses and are less well-characterized. Some studies have suggested that younger MM patients are more likely to present with lytic lesions, be IgD subtype, have high-risk cytogenetics, and have relatively improved survival. We aimed to characterize this understudied population in the modern era. Methods: We conducted a retrospective chart review of patients age &amp;lt;50 meeting IMWG criteria for symptomatic MM treated across our institution's cancer center network between 2010 and 2023. Overall survival was defined as time in months from diagnosis to last follow-up or death. Time to relapse after transplant was captured. Results: We identified 97 younger MM patients, with a median age of 44 (range 26-50). Males and females represented 50.5% and 49.5% of cases respectively. Complete demographics are listed in the table. The most common MM subtype was IgG Kappa (40%), and 23% had light chain-only disease. Incidentally found cases, defined as abnormalities on imaging or labs ordered for another reason, represented 11% of cases. 61% of patients presented with a skeletal issue such as bone pain and/or pathologic fracture. Presenting features are described in the table. At time of diagnosis, 77% of patients were found to have lytic lesions. Few patients had previously identified MGUS (3%) or smoldering MM (5%). Regarding cytogenetics, 18% had one or more high-risk features. The most common frontline treatment was VRD. As shown in the table, 66% and 29% received 3 and 4 drug induction regimens respectively. Of note, 91% were referred for autologous stem cell transplant (ASCT), and 72% proceeded. 4 patients were deceased at the time of analysis. Survival for the entire population was 95.9% at a median follow up time of 65.5 months. In the patients who underwent ASCT, 41.2% relapsed with median progression-free survival of 57 (44-71) months. Conclusions: MM patients age &amp;lt;50 were found to be distinct from the known characteristics of the general population in several notable ways. There was equipoise among demographic features, with a nearly equivalent prevalence in males and females, and similar median ages across racial groups. We found a greater enrichment of light chain disease than expected, but less renal dysfunction at presentation. IgD subtype does not appear increased in this population. Younger patients do not appear more likely to have high risk cytogenetics. Lastly, survival for younger patients was higher compared to the reported 5-year survival of MM, which may be explained by the finding that these patients were treated primarily with triplet or quadruplet anti-myeloma regimens and often proceeded to ASCT.

Effect of interleukin-2 (IL-2) polymorphisms on multiple myeloma: IL-2RA rs2104286, IL-2 rs2069762 and rs2069763 polymorphisms

Interleukin-2 (IL-2) is a cytokine secreted from T helper type 1 cells and released after induction of T helper cells with major histocompatibility complexes or antigens presented by antigen presenting cells. IL-2 activity and gene polymorphisms have been studied in both solid and hematological malignancies. In the present study, it was aimed to examine the effects of IL-2RA rs2104286, IL-2 rs2069762 and rs2069763 polymorphisms on multiple myeloma (MM) susceptibility, progression-free survival (PFS) and overall survival (OS). A total of 300 patients diagnosed with MM in our clinic between January 2010 and January 2021, and 170 healthy individuals were included. In addition to the demographic data of the patients, MM subtypes, initial stages, prognostic index scores, laboratory results, treatment preferences, and survival data were recorded. The genotypes of the IL-2RA rs2104286, IL-2 rs2069762 and rs2069763 polymorphisms were statistically compared between patients and healthy controls to reveal their effects on MM susceptibility and survival. In the statistical analysis performed to examine the effect of IL-2RA rs2104286, IL-2 rs2069762 and rs2069763 polymorphisms on disease susceptibility, no significant difference was found between the patient and healthy control groups. Patients with the TG genotype of IL-2 rs2069762 had a significantly shorter median PFS and OS compared to others. Patients with the GG genotype of IL-2 rs2069763 had a significantly shorter median PFS compared to others. Having the TG genotype of IL-2 rs2069762 has been shown to be protective for short PFS and OS. Our study results will be guiding in terms of IL-2 based therapies, the future for MM and MM epigenetics.

Integrated analysis of next generation sequencing minimal residual disease (MRD) and PET scan in transplant eligible myeloma patients

Minimal residual disease (MRD) assays allow response assessment in patients with multiple myeloma (MM), and negativity is associated with improved survival outcomes. The role of highly sensitive next generation sequencing (NGS) MRD in combination with functional imaging remains to be validated. We performed a retrospective analysis on MM patients who underwent frontline autologous stem cell transplant (ASCT). Patients were evaluated at day 100 post-ASCT with NGS-MRD and positron emission tomography (PET-CT). Patients with ≥ 2 MRD measurements were included in a secondary analysis for sequential measurements. 186 patients were included. At day 100, 45 (24.2%) patients achieved MRD negativity at a sensitivity threshold of 10−6. MRD negativity was the most predictive factor for longer time to next treatment (TTNT). Negativity rates did not differ according to MM subtype, R-ISS Stage nor cytogenetic risk. PET-CT and MRD had poor agreement, with high rates of PET-CT negativity in MRD-positive patients. Patients with sustained MRD negativity had longer TTNT, regardless of baseline risk characteristics. Our results show that the ability to measure deeper and sustainable responses distinguishes patients with better outcomes. Achieving MRD negativity was the strongest prognostic marker and could help guide therapy-related decisions and serve as a response marker for clinical trials.

Is t(11;14)(q13;q32) good or bad for newly diagnosed multiple myeloma?

Is t(11;14)(q13;q32) good or bad for newly diagnosed multiple myeloma?