Early relapse within 18 months is a powerful dynamic predictor for prognosis and could revise static risk distribution in multiple myeloma.

BACKGROUND: The duration of response to treatment is a major prognostic factor, and early relapse (ER) strongly predicts inferior survival in multiple myeloma (MM). However, the definitions of ER in MM vary from study to study and how to dynamically integrate risk distribution is still unsolved. METHODS: This study evaluated these ER definitions and further investigated the underlying relationship with static risk distribution in 629 newly diagnosed MM (NDMM) patients from the National Longitudinal Cohort of Hematological Diseases in China (NCT04645199). RESULTS: These data indicated that early relapse within 18 months (ER18) after initial treatment was the best time point for identifying early progression and dynamic high-risk in MM. The ER18 population (114 of 587, 19.4%) presented with more aggressive biologic features and the inferior response to treatment compared to a reference cohort (p < .001), with a significantly short median overall survival (OS) of 28.9 months. Multivariate analyses confirmed the most significant prognostic value of ER18 on OS in the context of International Staging System stage, elevated lactate dehydrogenase, thrombocytopenia, cytogenetic abnormalities, and treatment (hazard ratio, 4.467; p < .001). The authors also described the specific transitions from static risk profile to dynamic risk distribution and then constructed a mixed-risk-pattern to identify four novel populations with distinct survival (p < .001). Additionally, the authors proposed a second-state model that predicts dynamic risk changes, enabling a complementary role to the Revised International Staging System model in facilitating individualized systematic treatment. CONCLUSIONS: Collectively, this study concludes that ER18 is a simple and dynamic prognostic predictor in MM. In addition to static risk assessment, dynamic risk plays an important role in survival prediction.

Lymphoma cell-driven IL-16 is expressed in activated B-cell-like diffuse large Bcell lymphomas and regulates the pro-tumor microenvironment.

The activated B-cell-like subtype of diffuse large B-cell lymphoma (ABC-DLBCL) displays a worse outcome than the germinal center B-cell-like subtype (GCB-DLBCL). Currently, targeting tumor microenvironment (TME) is the promising approach to cure DLBCL with profound molecular heterogeneity, however, the factors affecting the tumor-promoting TME of ABCDLBCL are elusive. Here, cytokine interleukin-16 (IL-16) is expressed in tumor cells of ABCDLBCL and secreted by the cleavage of active caspase-3. The serum IL-16 levels are not only a sensitive marker of treatment response but also positively correlated with unfavorable prognosis in DLBCL patients. While IL-16 shows few direct promotional effects on tumor cell growth in vitro, its bioactive form significantly promotes tumor progression in vivo. Mechanically, IL-16 increases the infiltration of macrophages by the chemotaxis of CD4+ monocytes in the TME enhancing angiogenesis, and the expression of cytokine IL-6 and IL-10, as well as decreasing T cell infiltration to accelerate tumor progression. This study demonstrates that IL-16 exerts a novel role in coordinating the bidirectional interactions between tumor progression and the TME. IMM0306, a fusion protein of CD20 mAb with the CD47 binding domain of SIRPα, reverses the tumorpromoting effects of IL-16,which provides new insight into treatment strategy in ABC-DLBCL.

LILRB4 represents a promising target for immunotherapy by dual targeting tumor cells and myeloid-derived suppressive cells in multiple myeloma.

Multiple myeloma (MM) remains an incurable hematological malignancy. Despite tremendous advances in the treatment, about 10% of patients still have very poor outcomes with median overall survival less than 24 months. Our study aimed to underscore the critical mechanisms pertaining to the rapid disease progression and provide novel therapeutic selection for these ultra-high-risk patients. We utilized single-cell transcriptomic sequencing to dissect the characteristic bone marrow niche of patients with survival of less than two years (EM24). Notably, an enrichment of LILRB4high pre-matured plasma-cell cluster was observed in the patients in EM24 compared to patients with durable remission. This cluster exhibited aggressive proliferation and drug-resistance phenotype. High-level LILRB4 promoted MM clonogenicity and progression. Clinically, high expression of LILRB4 was correlated with poor prognosis in both newly diagnosed MM patients and relapsed/refractory MM patients. The ATAC-seq analysis identified that high chromosomal accessibility caused the elevation of LILRB4 on MM cells. CRISPR-Cas9 deletion of LILRB4 alleviated the growth of MM cells, inhibited the immunosuppressive function of MDSCs, and further rescued T cell dysfunction in MM microenvironment. The more infiltration of myeloid-derived suppressive cells (MDSCs) was observed in EM24 patients as well. Therefore, we innovatively generated a TCR-based chimeric antigen receptor (CAR) T cell, LILRB4-STAR-T. Cytotoxicity experiment demonstrated that LILRB4-STAR-T cells efficaciously eliminated tumor cells and impeded MDSCs function. In conclusion, our study elucidates that LILRB4 is an ideal biomarker and promising immunotherapy target for high-risk MM. LILRB4-STAR-T cell immunotherapy is promising against tumor cells and immunosuppressive tumor microenvironment in MM.

More accurate prediction of treatment response than mean apparent diffusion coefficient value in multiple myeloma using whole body MRI histogram analysis.

Aim: To assess baseline histogram parameters from apparent diffusion coefficient (ADC) images in predicting early treatment response in newly diagnosed multiple myeloma (NDMM) patients. Methods: The histogram parameters of lesions in 68 NDMM patients were obtained with the Firevoxel software. The presence of deep response after two cycles of induction was recorded. Results: Some parameters were significantly different between the two groups, for example, ADC 75% in lumbar spine (p = 0.026). No significant difference in mean ADC for any anatomic site was found (all p > 0.05). The combination of ADC 75, ADC 90 and ADC 95% in lumbar spine; ADC skewness and ADC kurtosis in rib achieved a sensitivity of 100% in predicting deep response. Conclusion: Histogram analysis of ADC images can describe NDMM heterogeneity and accurately predict treatment response.

Single-cell profiles reveal tumor cell heterogeneity and immunosuppressive microenvironment in Waldenström macroglobulinemia.

BACKGROUND: Waldenström macroglobulinemia (WM) is a rare and incurable indolent B-cell malignancy. The molecular pathogenesis and the role of immunosuppressive microenvironment in WM development are still incompletely understood. METHODS: The multicellular ecosystem in bone marrow (BM) of WM were delineated by single-cell RNA-sequencing (scRNA-seq) and investigated the underlying molecular characteristics. RESULTS: Our data uncovered the heterogeneity of malignant cells in WM, and investigated the kinetic co-evolution of WM and immune cells, which played pivotal roles in disease development and progression. Two novel subpopulations of malignant cells, CD19+CD3+ and CD138+CD3+, co-expressing T-cell marker genes were identified at single-cell resolution. Pseudotime-ordered analysis elucidated that CD19+CD3+ malignant cells presented at an early stage of WM-B cell differentiation. Colony formation assay further identified that CD19+CD3+ malignant cells acted as potential WM precursors. Based on the findings of T cell marker aberrant expressed on WM tumor cells, we speculate the long-time activation of tumor antigen-induced immunosuppressive microenvironment that is involved in the pathogenesis of WM. Therefore, our study further investigated the possible molecular mechanism of immune cell dysfunction. A precursor exhausted CD8-T cells and functional deletion of NK cells were identified in WM, and CD47 would be a potential therapeutic target to reverse the dysfunction of immune cells. CONCLUSIONS: Our study facilitates further understanding of the biological heterogeneity of tumor cells and immunosuppressive microenvironment in WM. These data may have implications for the development of novel immunotherapies, such as targeting pre-exhausted CD8-T cells in WM.

Prediction of Early Treatment Response in Multiple Myeloma Using MY-RADS Total Burden Score, ADC, and Fat Fraction From Whole-Body MRI: Impact of Anemia on Predictive Performance.

BACKGROUND. The recently released Myeloma Response Assessment and Diagnosis System (MY-RADS) for multiple myeloma (MM) evaluation using whole-body MRI (WB-MRI) describes the total burden score. However, assessment is confounded by red bone marrow hyperplasia in anemia. OBJECTIVE. The purpose of this study is to assess the utility of the MY-RADS total burden score, ADC, and fat fraction (FF) from WB-MRI in predicting early treatment response in patients with newly diagnosed MM and to compare the utility of these measures between patients with and without anemia. METHODS. This retrospective study included 56 patients (40 men, 16 women; mean age, 57.4 ± 9.6 [SD] years) with newly diagnosed MM who underwent baseline WB-MRI including DWI and modified Dixon sequences. Two radiologists recorded total burden score using MY-RADS and measured the ADC and FF of diffuse and focal disease sites. Mean values across sites were derived. Interobserver agreement was evaluated, and the mean assessments of the readers were used for further analyses. Presence of deep response after four cycles of induction chemotherapy was recorded. Patients were classified as having anemia if their hemoglobin level was less than 100 g/L. The utility of WBMRI parameters in predicting deep response was assessed. RESULTS. A total of 24 of 56 patients showed deep response, and 25 of 56 patients had anemia. Interobserver agreement, which was expressed using intraclass correlation coefficients, ranged from 0.95 to 0.99. Among patients without anemia, those with deep response compared with those without deep response had a lower total burden score (9.0 vs 18.0), a lower ADC (0.79 × 10-3 mm2/s vs 1.08 × 10-3 mm2/s), and a higher FF (0.21 vs 0.10) (all p < .001). The combination of these three parameters (optimal cutoffs: ≤ 15 for total burden score, ≤ 0.84 × 10-3 mm2/s for ADC, and > 0.16 for FF) achieved sensitivity of 93.8%, specificity of 93.3%, and accuracy of 93.5% for predicting deep response. In patients with anemia, none of the three parameters were significantly different between patients with and without deep response (all p > .05), and the combination of parameters achieved sensitivity of 56.3%, specificity of 100.0%, and accuracy of 72.0%. CONCLUSION. Low total burden score, low ADC, and high FF from WB-MRI may predict deep response in patients with MM, although only among those without anemia. CLINICAL IMPACT. WB-MRI findings may help guide determination of prognosis and initial treatment selection in MM.

IgH translocation with undefined partners is associated with superior outcome in multiple myeloma patients.

BACKGROUND: In multiple myeloma (MM), impact of specific chromosomal translocations involving IgH (14q21 locus, including t(4;14), t(11;14), and t(14;16)) has been explored extensively. However, over 15% MM patients harboring IgH translocation with undefined partners have long been ignored. METHODS: A prospective non-randomized cohort study with a total of 715 newly-diagnosed MM cases was conducted, 13.6% of whom were t(14;undefined) positive. The whole cohort was divided into four groups: no IgH split (47.7%); t(14;undefined) (13.6%); t(11;14) (17.6%); and t(4;14) or t(14;16) group (21.1%). RESULTS: Median OS for the four groups was 84.2, not reached (NR), 58.7, and 44.2 months, respectively, with P values for t(14;undefined) vs no IgH split, t(11;14), and t(4;14)/t(14;16) groups of 0.197, 0.022, and 0.001, respectively. In bortezomib-based group, the survival advantage gained by t(14;undefined) group was much more significant compared to t(11;14) and t(4;14)/t(14;16) groups. Importantly, t(14;undefined) turned out to be an independent predictive factor for longer OS of MM patients in multivariate analysis, especially in the context of bortezomib treatment. Similar results were also observed in the PUMCH external validation cohort. CONCLUSION: Collectively, our data confirmed and externally validated the favorable prognosis of the t(14;undefined) groups, especially in the era of novel agents.

[Treatment and Prognosis of Adult T Cell Acute Lymphoblastic Leukemia].

To analyze the treatment and prognosis of T cell acute lymphoblastic leukemia(T-ALL)in adults. Method The clinicobiogical and survival data of 68 adult patients with newly diagnosis T-ALL were retrospectively analzyed. Results The median age of these 68 patients was 23 years(14-60 years).T-ALL was more common in men(81%).After the first cycle of treatment,complete remission was achieved in 50 patients(73%).The highest complete remission(CR) rate was in patients with cortex T-ALL(100%),followed by other T-ALL(73%)and early T-cell precursor lymphoblastic leukemia(54%),(χ 2=5.712,P=0.058).The CR rate for adults aged >35 years was significantly lower than that of patients aged ≤ 35 years(40% vs. 79%,χ 2=6.364,P=0.012).The overall CR rate after the second treatment course was 93%.For patients treated with chemotherapy,autograft hematopoietic stem cell transplantation(auto-SCT),and allogeneic SCT,the median relapse free survival was 10 months,24 months,and not reached,respectively(P=0.002).The 5-year overall survival rate was 25% for all patients;for patients treated with chemotherapy,auto-SCT and allogeneic SCT,the median overall survival was 24 months,34 months,and 30 months,respectively(P=0.007),and the 5-year overall survival rate was 9%,33%,and 38%(P=0.037).Multivariate analysis showed leukocyte count ≥100×10 9/L was a risk factor for decreased relapse free survival(risk ratio 2.540,95%CI=1.058-6.099,P=0.037). Conclusion Adult T-ALL patients have poor prognosis,which may be improved by SCT.

Low serum miR-19a expression as a novel poor prognostic indicator in multiple myeloma.

Multiple myeloma (MM) is the second most common hematologic malignancy characterized by the clonal expansion of plasma cells. Despite continuing advances, novel biomarkers are needed for diagnosis and prognosis of MM. In our study, we characterized the diagnostic and prognostic potential of circulating microRNAs (miRNAs) in MM. Serum miRNA levels were analyzed in 108 newly diagnosed symptomatic MM patients and 56 healthy donors (HDs). Our analysis identified 95 dysregulated miRNAs in newly diagnosed MM patients. Of the 95 dysregulated miRNAs, dysregulation of miR-19a, miR-92a, miR-214-3p, miR-135b-5p, miR-4254, miR-3658 and miR-33b was confirmed by quantitative reverse transcription PCR (RT-qPCR). Receiver operating characteristic analysis revealed that a combination of miR-19a and miR-4254 can distinguish MM from HD with a sensitivity of 91.7% and specificity of 90.5%. Decreased expression of miR-19a was positively correlated with international staging system advancement, del(13q14) and 1q21 amplification. Furthermore, downregulation of miR-19a resulted in significantly decreased progression-free survival (PFS) and overall survival (OS). Our analysis indicated that the poor prognostic correlation of miR-19a expression was independent of genetic abnormalities in MM. Multivariate analysis revealed that miR-19a was a significant predictor of shortened PFS and OS. Interestingly, although miR-19a levels portend a poor prognosis, patients with low miR-19a levels had an improved response to bortezomib compared to those with high miR-19a profile. Patients with downregulated miR-19a experienced a significantly extended survival upon bortezomib-based therapy. These data demonstrate that the expression patterns of serum microRNAs are altered in MM, and miR-19a levels are a valuable prognostic marker to identify high-risk MM.

Multiple myeloma patients with low proportion of circulating plasma cells had similar survival with primary plasma cell leukemia patients.

The common features shared by primary plasma cell leukemia (pPCL) and multiple myeloma (MM) with circulating plasma cells (CPCs) are peripheral blood invasion and expansion of plasma cells independent of the protective bone marrow (BM) microenvironment niche. However, few studies have addressed the relationship between pPCL and MM with CPCs. Here, we quantitated the number CPCs by conventional morphology in 767 patients with newly diagnosed MM; their clinic features were compared with those of 33 pPCL cases. When the presence of CPCs was defined as more than 2 % plasma cells per 100 nucleated cells on Wright-Giemsa stained peripheral blood smears, the incidence of MM with CPCs was 14.1 % in newly diagnosed MM. Patients with CPCs shared many clinical features with pPCL, especially clinical parameters related to tumor burden. However, no commonalities were found in immunophenotyping and cytogenetics. The prognosis of pPCL was poor, with a median progression free survival (PFS) of 12 months and an overall survival (OS) of 15 months. MM patients with CPCs had a clearly inferior PFS and OS as compared with the control cohort. Most interestingly, although the CPCs were not high enough to meet the diagnostic criteria for pPCL, the survival of MM patients with CPCs was comparable with that of pPCL, with a median PFS of 17 months and an OS of 25 months.

Critical role of AKT protein in myeloma-induced osteoclast formation and osteolysis.

Abnormal osteoclast formation and osteolysis are the hallmarks of multiple myeloma (MM) bone disease, yet the underlying molecular mechanisms are incompletely understood. Here, we show that the AKT pathway was up-regulated in primary bone marrow monocytes (BMM) from patients with MM, which resulted in sustained high expression of the receptor activator of NF-κB (RANK) in osteoclast precursors. The up-regulation of RANK expression and osteoclast formation in the MM BMM cultures was blocked by AKT inhibition. Conditioned media from MM cell cultures activated AKT and increased RANK expression and osteoclast formation in BMM cultures. Inhibiting AKT in cultured MM cells decreased their growth and ability to promote osteoclast formation. Of clinical significance, systemic administration of the AKT inhibitor LY294002 blocked the formation of tumor tissues in the bone marrow cavity and essentially abolished the MM-induced osteoclast formation and osteolysis in SCID mice. The level of activating transcription factor 4 (ATF4) protein was up-regulated in the BMM cultures from multiple myeloma patients. Adenoviral overexpression of ATF4 activated RANK expression in osteoclast precursors. These results demonstrate a new role of AKT in the MM promotion of osteoclast formation and bone osteolysis through, at least in part, the ATF4-dependent up-regulation of RANK expression in osteoclast precursors.

Novel Insights into the Initiation, Evolution, and Progression of Multiple Myeloma by Multi-Omics Investigation.

The evolutionary history of multiple myeloma (MM) includes malignant transformation, followed by progression to pre-malignant stages and overt malignancy, ultimately leading to more aggressive and resistant forms. Over the past decade, large effort has been made to identify the potential therapeutic targets in MM. However, MM remains largely incurable. Most patients experience multiple relapses and inevitably become refractory to treatment. Tumor-initiating cell populations are the postulated population, leading to the recurrent relapses in many hematological malignancies. Clonal evolution of tumor cells in MM has been identified along with the disease progression. As a consequence of different responses to the treatment of heterogeneous MM cell clones, the more aggressive populations survive and evolve. In addition, the tumor microenvironment is a complex ecosystem which plays multifaceted roles in supporting tumor cell evolution. Emerging multi-omics research at single-cell resolution permits an integrative and comprehensive profiling of the tumor cells and microenvironment, deepening the understanding of biological features of MM. In this review, we intend to discuss the novel insights into tumor cell initiation, clonal evolution, drug resistance, and tumor microenvironment in MM, as revealed by emerging multi-omics investigations. These data suggest a promising strategy to unravel the pivotal mechanisms of MM progression and enable the improvement in treatment, both holistically and precisely.

CRIP1 involves the pathogenesis of multiple myeloma via dual-regulation of proteasome and autophagy.

BACKGROUND: Multiple myeloma (MM) is an incurable hematological malignancy of the plasma cells. The maintenance of protein homeostasis is critical for MM cell survival. Elevated levels of paraproteins in MM cells are cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as a backbone agent and successfully improved the outcome of patients; however, the increasing activity of autophagy suppresses the sensitivity to PIs treatment. METHODS: The transcription levels of CRIP1 were explored in plasma cells obtained from healthy donors, patients with newly diagnosed multiple myeloma (NDMM), and relapsed/refractory multiple myeloma (RRMM) using Gene expression omnibus datasets. Doxycycline-inducible CRIP1-shRNA and CRIP1 overexpressed MM cell lines were constructed to explore the role of CRIP1 in MM pathogenesis. Proliferation, invasion, migration, proteasome activity and autophagy were examined in MM cells with different CRIP1 levels. Co-immunoprecipitation (Co-IP) with Tandem affinity purification/Mass spectrum (TAP/MS) was performed to identify the binding proteins of CRIP1. The mouse xenograft model was used to determine the role of CRIP1 in the proliferation and drug-resistance of MM cells. FINDINGS: High CRIP1 expression was associated with unfavorable clinical outcomes in patients with MM and served as a biomarker for RRMM with shorter overall survival. In vitro and in vivo studies showed that CRIP1 plays a critical role in protein homeostasis via the dual regulation of the activities of proteasome and autophagy in MM cells. A combined analysis of RNA-seq, Co-IP and TAP/MS demonstrated that CRIP1 promotes proteasome inhibitors resistance in MM cells by simultaneously binding to de-ubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promoted proteasome activity and autophagosome maturation by facilitating the dequbiquitination and stabilization of PA200. INTERPRETATION: Our findings clarified the pivotal roles of the CRIP1/USP7/PA200 complex in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the pathogenesis of MM. FUNDING: A full list of funding sources can be found in the acknowledgements section.

DNp73 enhances tumor progression and immune evasion in multiple myeloma by targeting the MYC and MYCN pathways.

Introduction: Multiple myeloma (MM) is an incurable hematological malignancy with high chromosome instability and heavy dependence on the immunosuppressive bone marrow microenvironment. P53 mutations are adverse prognostic factors in MM; however, clinically, some patients without P53 mutations also exhibit aggressive disease progression. DNp73, an inhibitor of TP53 tumor suppressor family members, drives drug resistance and cancer progression in several solid malignancies. Nevertheless, the biological functions of DNp73 and the molecular mechanisms in myelomagenesis remain unclear. Methods: The effects of DNp73 on proliferation and drug sensitivity were assessed using flow cytometry and xenograft models. To investigate the mechanisms of drug resistance, RNA-seq and ChIP-seq analyses were performed in MM cell lines, with validation by Western blot and RT-qPCR. Immunofluorescence and transwell assays were used to assess DNA damage and cell invasion in MM cells. Additionally, in vitro phagocytosis assays were conducted to confirm the role of DNp73 in immune evasion. Results: Our study found that activation of NF-κB-p65 in multiple myeloma cells with different p53 mutation statuses upregulates DNp73 expression at the transcriptional level. Forced expression of DNp73 promoted aggressive proliferation and multidrug resistance in MM cells. Bulk RNA-seq analysis was conducted to assess the levels of MYCN, MYC, and CDK7. A ChIP-qPCR assay was used to reveal that DNp73 acts as a transcription factor regulating MYCN gene expression. Bulk RNA-seq analysis demonstrated increased levels of MYCN, MYC, and CDK7 with forced DNp73 expression in MM cells. A ChIP-qPCR assay revealed that DNp73 upregulates MYCN gene expression as a transcription factor. Additionally, DNp73 promoted immune evasion of MM cells by upregulating MYC target genes CD47 and PD-L1. Blockade of the CD47/SIRPα and PD-1/PD-L1 signaling pathways by the SIRPα-Fc fusion protein IMM01 and monoclonal antibody atezolizumab significantly restored the anti-MM activity of macrophages and T cells in the microenvironment, respectively. Discussion: In summary, our study demonstrated for the first time that the p53 family member DNp73 remarkably induces proliferation, drug resistance, and immune escape of myeloma cells by directly targeting MYCN and regulating the MYC pathway. The oncogenic function of DNp73 is independent of p53 status in MM cells. These data contribute to a better understanding of the function of TP53 and its family members in tumorigenesis. Moreover, our study clarified that DNp73 overexpression not only promotes aggressive growth of tumor cells but, more importantly, promotes immune escape of MM cells through upregulation of immune checkpoints. DNp73 could serve as a biomarker for immunotherapy targeting PD-L1 and CD47 blockade in MM patients.

Fluorescence in situ hybridization reveals the evolutionary biology of minor clone of gain/amp(1q) in multiple myeloma.

Growing evidence suggests that gain or amplification [gain/amp(1q)] accumulates during disease progression of multiple myeloma (MM). Previous investigations have indicated that small gain/amp(1q) subclones present at the time of diagnosis may evolve into dominant clones upon MM relapse. However, the influence of a minor clone of gain/amp(1q) on MM survival, as well as the correlation between different clonal sizes of gain/amp(1q) and the chromosomal instability (CIN) of MM, remains poorly understood. In this study, we analyzed fluorescence in situ hybridization (FISH) results of 998 newly diagnosed MM (NDMM) patients. 513 patients were detected with gain/amp(1q) at diagnosis. Among these 513 patients, 55 had a minor clone (≤20%) of gain/amp(1q). Patients with a minor clone of gain/amp(1q) displayed similar survival outcomes compared to those without gain/amp(1q). Further analysis demonstrated patients with a minor clone of gain/amp(1q) exhibited a clonal architecture similar to those without gain/amp(1q). Lastly, our results showed a significant increase in the clonal size of the minor clone of gain/amp(1q), frequently observed in MM. These findings suggested that a minor clone of gain/amp(1q) might represent an earlier stage in the pathogenesis of gain/amp(1q) and propose a "two-step" process in the clonal size changes of gain/amp(1q) in MM.

Monitoring Minimal Residual Disease in Patients with Multiple Myeloma by Targeted Tracking Serum M-Protein Using Mass Spectrometry (EasyM).

PURPOSE: We investigated both the clinical utilities and the prognostic impacts of the clonotypic peptide mass spectrometry (MS)-EasyM, a blood-based minimal residual disease (MRD) monitoring protocol in multiple myeloma. EXPERIMENTAL DESIGN: A total of 447 sequential serum samples from 56 patients with multiple myeloma were analyzed using EasyM. Patient-specific M-protein peptides were sequenced from diagnostic samples; sequential samples were quantified by EasyM to monitor the M-protein. The performance of EasyM was compared with serum immunofixation electrophoresis (IFE), bone marrow multiparameter flow cytometry (MFC), and next-generation flow cytometry (NGF) detection. The optimal balance of EasyM sensitivity/specificity versus NGF (10-5 sensitivity) was determined and the prognostic impact of MS-MRD status was investigated. RESULTS: Of the 447 serum samples detected and measured by EasyM, 397, 126, and 92 had time-matching results for comparison with serum IFE, MFC-MRD, and NGF-MRD, respectively. Using a dotp >0.9 as the MS-MRD positive, sensitivity was 99.6% versus IFE and 100.0% versus MFC and NGF. Using an MS negative cutoff informed by ROC analysis (<1.86% of that at diagnosis), EasyM sensitivity remained high versus IFE (88.3%), MFC (85.1%), and NGF (93.2%), whereas specificity increased to 90.4%, 55.8%, and 93.2%, respectively. In the multivariate analysis, older diagnostic age was an independent predictor for progression-free survival [PFS; high risk (HR), 3.15; 1.26-7.86], the best MS-MRD status (MS-MRD negative) was independent predictor for both PFS (HR, 0.25; 0.12-0.52) and overall survival (HR, 0.16; 0.06-0.40). CONCLUSIONS: EasyM is a highly sensitive and minimal invasive method of MRD monitoring in multiple myeloma; MS-MRD had significant predictive ability for survival outcomes.

Identification of Therapy-Induced Clonal Evolution and Resistance Pathways in Minimal Residual Clones in Multiple Myeloma through Single-Cell Sequencing.

PURPOSE: In multiple myeloma (MM), therapy-induced clonal evolution is associated with treatment resistance and is one of the most important hindrances toward a cure for MM. To further understand the molecular mechanisms controlling the clonal evolution of MM, we applied single-cell RNA sequencing (scRNA-seq) to paired diagnostic and posttreatment bone marrow (BM) samples. EXPERIMENTAL DESIGN: scRNA-seq was performed on 38 BM samples from patients with monoclonal gammopathy of undetermined significance (n = 1), MM patients at diagnosis (n = 19), MM posttreatment (n = 17), and one healthy donor (HD). The single-cell transcriptome data of malignant plasma cells (PC) and the surrounding immune microenvironment were analyzed. RESULTS: Profiling by scRNA-seq data revealed three primary trajectories of transcriptional evolution after treatment: clonal elimination in patients with undetectable minimal residual disease (MRD-) and clonal stabilization and clonal selection in detectable MRD (MRD+) patients. We noted a metabolic shift toward fatty acid oxidation in cycling-resistant PCs, whereas selective PCs favored the NF-κB pathway. Intriguingly, when comparing the genetic and transcriptional dynamics, we found a significant correlation between genetic and nongenetic factors in driving the clonal evolution. Furthermore, we identified variations in cellular interactions between malignant PCs and the tumor microenvironment. Selective PCs showed the most robust cellular interactions with the tumor microenvironment. CONCLUSIONS: These data suggest that MM cells could rapidly adapt to induction treatment through transcriptional adaptation, metabolic adaptation, and specialized immune evasion. Targeting therapy-induced resistance mechanisms may help to avert refractory disease in MM.

Clinical implications of residual normal plasma cells within bone marrow across various disease stages in multiple myeloma.

Residual normal plasma cells (NPCs), which compete with tumor plasma cells, play an important role in multiple myeloma. However, large-scale cohort studies investigating residual NPCs, especially at the minimal residual disease (MRD) phase, are currently lacking. In this study, we conducted a comprehensive investigation into the clinical significance of residual NPCs throughout the entire disease course in 1363 myeloma patients from the NICHE cohort (NCT04645199). Our results revealed that myeloma patients with high baseline NPCs ratio (≥5%) exhibited distinct indolent features, characterized by lower tumor burden, reduced frequencies of cytopenia, immunoparesis, and high-risk cytogenetics. Importantly, high residual NPCs ratio at diagnosis or relapse was independently associated with favorable survival. High absolute percentages of NPCs at undetectable MRD were related with superior clinical benefit and immune reconstitution. At MRD-positive phases, grouping based on NPCs ratio (<50%, 50-90%, ≥90%) demonstrated better risk stratification compared to residual tumor log levels. Based on the time-dependent NPCs ratio trend, we developed a dynamic MRD model that classifies patients into three groups with diverse longitudinal trends, leading to distinct prognoses. Collectively, residual NPCs serves not only as a valuable complementary biomarker for risk stratification but also provides valuable insights on reclassifications and kinetics of MRD.

Determination of MYD88 and CXCR4 mutation for clinical detection and their significance in Waldenström macroglobulinemia.

PURPOSE: This study aims to explore the incidence and clinical features of MYD88 and CXCR4 mutations in patients with Waldenström macroglobulinemia (WM) and determine the optimal method for routine clinical practice. Additionally, we seek to evaluate the prognostic significance of these features across various therapeutic backgrounds [cytotoxic group, the Rituximab/Bortezomib-based group, and the Bruton's tyrosine kinase inhibitor (BTKi) group]. EXPERIMENTAL DESIGN: 385 symptomatic WM patients were analyzed for MYD88 and CXCR4 mutations using Sanger sequencing, next-generation sequencing (NGS), allele-specific quantitative polymerase chain reaction (AS-PCR), and/or droplet digital PCR (ddPCR). RESULTS: The overall MYD88 mutation rate was 87.8%, relatively lower than that in Western cohort. Both AS-PCR and ddPCR demonstrated high sensitivity in unsorted samples, detecting 98.5% and 97.7% of mutations, respectively, including those with low tumor burdens. The total CXCR4 mutation rate was 30.9%, with NGS exhibiting the highest sensitivity of 78.0%. CXCR4 mutation was significantly linked to shorter OS only within the BTKi treatment group. The multivariate analysis indicated that MYD88 and CXCR4 mutations were not independent prognostic factors in the non-BTKi group when considering IPSSWM clinical staging. However, in the BTKi treatment group, these mutations emerged as independent adverse prognostic factors, overshadowing the prognostic significance of IPSSWM classification (MYD88: HR=0.229, P=0.030; CXCR4: HR=3.349, P=0.012). CONCLUSIONS: Testing for MYD88 mutations using AS-PCR or ddPCR in unsorted samples is viable for routine clinical practice. Under BTKi treatment, MYD88 and CXCR4 mutations hold greater prognostic importance than IPSSWM staging in WM.

[Establishment of Patient-Derived Xenograft (PDX) Zebrafish Model of Multiple Myeloma and Its Application in Drug Screening].

OBJECTIVE: To establish a MM patient-derived tumor xenograft model (MM-PDX) in zebrafish, and to evaluate the anti-myeloma activity of indirubin-3'-monoxime(I3MO) using this model. METHODS: Zebrafish embryos 2 days after fertilization were transplanted with fluorescence labeled myeloma primary tumor cells, the survival of primary tumor cells in zebrafish was observed at 0,16 and 24 hours after cell injection. The zebrafish embryos after tumor cell transplantation were randomly divided into control group, BTZ treatment and I3MO treatment group. Before and 24 hours after treatment with BTZ and I3MO, the positive area with calcein or Dil in zebrafish were observed under fluorescence microscope to reflect the survival of tumor cells, and it was verified. RESULTS: MM patient derived tumor cells survived in zebrafish. The construction of MM-PDX was successful. Compared with control group, the fluo- rescence area of the BTZ and I3MO treatment groups in zebrafish were significantly decreased(P<0.05), and BTZ and I3MO significantly inhibited the survival of MM cells in zebrafish. CONCLUSION: MM-PDX model was successfully established. Zebrafish model derived from tumor cells of MM patients can be used as a tool for drug screening of MM.