Disease In Multiple Myeloma Patients Research Articles

Chromosome 1 Alterations in Multiple Myeloma: Considerations for Precision Therapy.

Multiple myeloma (MM) is an incurable blood malignancy characterized by the clonal expansion of plasma cells and the secretion of monoclonal immunoglobulins. High-risk MM, defined by specific cytogenetic abnormalities, poses significant therapeutic challenges and is associated with inferior survival outcomes compared to standard-risk disease. Although molecularly targeted therapies have shown efficacy in other hematologic malignancies, currently venetoclax is the only targeted therapy approved for MM (t(11;14)). However, chromosome 1q gains, amplifications, and 1p deletions are frequently observed in MM, and have been linked to drug resistance and poor patient prognosis. Accordingly, this review focuses on emerging MM precision therapies capable of targeting dysregulated genes within these regions. It addresses gene therapies, small molecule inhibitors and monoclonal antibodies currently under investigation to antagonize oncogenic drivers including MCL-1, BCL9, F11R, and CKS1B, all of which are implicated in cell survival, proliferation or drug resistance. In conclusion, the link between chromosome 1 abnormalities and high-risk disease in MM patients offers a compelling rationale to identify and explore therapeutic targeting of chromosome 1 gene products as a novel precision medicine approach for a poorly served patient population.

Liquid biopsy of peripheral blood using mass spectrometry detects primary extramedullary disease in multiple myeloma patients

Multiple myeloma (MM) is the second most prevalent hematological malignancy, characterized by infiltration of the bone marrow by malignant plasma cells. Extramedullary disease (EMD) represents a more aggressive condition involving the migration of a subclone of plasma cells to paraskeletal or extraskeletal sites. Liquid biopsies could improve and speed diagnosis, as they can better capture the disease heterogeneity while lowering patients’ discomfort due to minimal invasiveness. Recent studies have confirmed alterations in the proteome across various malignancies, suggesting specific changes in protein classes. In this study, we show that MALDI-TOF mass spectrometry fingerprinting of peripheral blood can differentiate between MM and primary EMD patients. We constructed a predictive model using a supervised learning method, partial least squares-discriminant analysis (PLS-DA) and evaluated its generalization performance on a test dataset. The outcome of this analysis is a method that predicts specifically primary EMD with high sensitivity (86.4%), accuracy (78.4%), and specificity (72.4%). Given the simplicity of this approach and its minimally invasive character, this method provides rapid identification of primary EMD and could prove helpful in clinical practice.

IMPACT OF CHEMOTHERAPY ON PROOXIDANT-ANTIOXIDANT STATUS IN PATIENTS WITH MULTIPLE MYELOMA AND CONCOMITANT CORONARY ARTERY DISEASE

Introduction. Multiple myeloma, an oncopathology that remains challenging to treat, is characterized by a 54% 5-year survival rate despite advancements in modern treatment strategies. Numerous studies have substantiated the correlation between high prooxidant concentrations and the progression of multiple myeloma. This progression is often associated with alterations in antioxidant defense mechanisms. Patients with coronary artery disease also experience changes in their antioxidant defense that may enhance the deleterious effects of active oxygen species and elevate the risk of chemotherapy-related organotoxic effects.
 The aim of this study is to investigate the specificities of the prooxidant-antioxidant status in patients with multiple myeloma and concomitant coronary artery disease during chemotherapy.
 Materials and methods. 42 patients with multiple myeloma were examined, 22 (52,5%) of them had concomitant coronary artery disease. According to the coronary artery disease, patients with multiple myeloma were divided into two groups. All patients obtain standard chemotherapy schemes. Assessment of the patients was performed four times: before and after the 1st and 5th chemotherapy course. TBA-reactants and catalase activity were measured in blood serum.
 Results. Both in I and II group of patients, TBA-reactants concentration in blood serum was increased in 1.85 (р1<0,0001) and in 2.35 (р1<0,0001) times respectively compare to healthy people before chemotherapy. Although in the patients with concomitant coronary artery disease TBA-reactants concentration in blood serum was increased in 1,28 (р2<0,0001) times compare to I group of patients. Simultaneously in II group of patients catalase activity was decreased in 1.28 (р4 <0,0001) times compare to healthy people and in 1,37 (р5 <0,0001) times compare to I group patients. Before the second course of chemotherapy in II group of patients, TBA-reactants level was increased in 1.03 (р<0.05) times compared to the first examination and in 1.27 (р<0.05) times compared to I group. In II group of patients, catalase activity was decreased in 1.24 (р<0.05) times compared to the first examination and in 1.38 (р<0.001) times compared to the II group. In II group of patients level of TBA-reactants was decreased in 1,13 (p<0,01) times compare to the first examination, but it was in 1,17(р<0,05) higher than TBA-reactants level in I group. Simultaneously catalase activity was decreased in 1.4 (р<0.05) times compare to the first examination.
 Conclusions. The progression of multiple myeloma is marked by the development of a prooxidant-antioxidant imbalance. In both the I and II patient groups, the level of TBA-reactants increased significantly by 1.85 (p<0.0001) and 2.35 (p<0.0001) times, respectively, compared to the reference value. The presence of concomitant coronary artery disease in multiple myeloma patients led to a 1.28-fold elevation in TBA-reactants levels (p<0.0001) compared to those without cardiovascular diseases. Simultaneously, catalase activity decreased by 1.37 times (p<0.0001) compared to patients without cardiovascular diseases.
 During chemotherapy, a progressive increase in the prooxidant-antioxidant imbalance was observed in multiple myeloma patients. This was evidenced by a 1.4-fold decrease in catalase activity (p<0.05) compared to the initial examination.

Measuring Minimal Residual Disease in Plasma of Multiple Myeloma Patients By Intact Mass Spectrometry

Multiple Myeloma (MM) is a hematological cancer that is currently treatable but incurable, and is characterized by the accumulation of malignant plasma cells in bone marrow. Clinical studies have shown that post-cancer treatment, the minimal residual disease (MRD) status is a prognostic factor indicative of the extent of disease remission. MRD assessment has been primarily assessed in bone marrow (BM) using flow cytometry or next generation sequencing. Not only these measurements may lead to false-negative results due to spatial heterogeneity in the BM, hemodilution and/or extramedullary disease as indicated by Derman et al. in Blood Cancer Journal ((2021), 11:19) but the procedure for BM collection is invasive and painful for the patient. These authors present a peripheral blood (PB)-based method that potentially can overcome the barriers in assessing for residual disease in MM patients. We hereby present a non-invasive blood-test for measurement of MRD using mass spectrometry (intact mass approach) for the detection of Ig product in MM patients. We will describe the assay development, analytical characterization of assay performance, and data from the analysis of plasma/serum samples from MM patients enrolled in a clinical trial. A very low volume of patient plasma/serum sample (10 μL) is required to perform this assay. The plasma/serum sample is diluted in purification buffer, applied to Melon gel resin and incubated for 5 min, followed by centrifugation and reduction with DTT. The supernatant is then analyzed by LC-MS on a Q-Exactive mass spectrometer. Deconvolution of the intact protein m/z spectra is performed to yield intact mass of the specific protein target species of interest. Overall, the method can process up to 96 samples in parallel within 3 hrs, making this procedure highly scalable for clinical applications. Data obtained from the assay characterization indicate that the assay is sensitive with an LOD <500 ng/mL and differentiates MM patient from a healthy donor as well allows for the monitoring of MM patient before and after treatment. In conclusion, although still an exploratory analysis, the blood-based mass spectrometry method can be used to screen for MRD in patients who have undetectable disease by flow cytometry or next generation sequencing.

Anti-CD38 Nanoantibody (JK36) Allows Detection of Minimal Residual Disease in Multiple Myeloma Patients Treated with Daratumumab

Objective: To evaluate the performance of the camel anti-CD38 antibody JK36 by flow cytometry in patients with Multiple Myeloma treated with Daratumumab. Materials and Methods: 7 bone marrow samples from patients with Multiple Myeloma treated with Daratumumab were evaluated. The immunophenotypic markers studied routinely were: Tube 1 CD38 FITC (T16 - Beckman Coulter), CD56 PE, CD20 PERCP-Cy5.5, CD19 PC7, Kappacy APC, Lambdacy APC-H7, CD45 V450 and CD138 V500; Tube 2 CD38 FITC (T16 - Beckman Coulter), CD28 PE, CD27 PERCP-Cy5.5, CD19 PC7, CD117 APC, CD81 APC-H7, CD45 V450 AND CD138 V500. For the detection of CD38 depletion, a Nano Tube was made with the following markers Kappacy FITC, CD56 PE, CD20 PERCP-Cy5.5, CD19 PC7, CD38 APC (nano antibody from camel JK36 - Beckman Coulter), Lambdacy APC-H7, CD45 V450 and CD138 V500. The samples were analyzed by the immunophenotyping method by flow cytometry, using the CANTO II equipment (Becton Dickinson - BD) and the analysis was performed in the Infinicyt software (Cytognos). The maximum total events acquired was 3,400,000 with merge between tubes 1 and 2. For Tube Nano, the maximum total events acquired was 1,700,000. Results: In the routinely used panel (Tube 1 and Tube 2) CD38 expression in plasma cells was negative in all samples. However, in all samples, it was possible to identify plasmocytes through expression of CD138 in conjunction with multiple gate strategies that include expressions of aberrant markers. In the Nano Tube containing the marker CD38 Nano Antibody of Camel (JK36) it was possible to visualize the expression of CD38 in the plasmocytes of all analyzed samples. Furthermore, with this marker it was possible to discriminate abnormal plasma cells from normal ones. Discussion: The expressions of CD38 together with CD138 characterize plasma cells. Anti-CD38 targeted therapy (Daratumumab) induces immune-mediated clearance of cells expressing CD38. The detection of plasma cells by flow cytometry with the use of this conventional antibody on the cell surface is impaired after the use of Daratumumab. Nano antibodies that occur naturally in camelids are capable of recognizing epitopes hidden and not blocked by targeted therapies, allowing the detection of plasma cells in patients with Multiple Myeloma after this treatment. In our study, we demonstrated that the Camel Nano Antibody CD38 antibody (JK36) was able to detect plasma cells in patients after using Daratumumab. Although it was possible to identify plasmocytes through the expression of CD138, this strategy becomes very risky and difficult to apply since the expression of this marker can often be of low intensity. The nanoantibody proved to be an additional and useful marker in the detection of MRD in patients after anti-CD38 target therapy (Dartumumab), bringing greater reliability in the analysis. Conclusion: The Camel Nano Antibody CD38 antibody (JK36) offers flow cytometry laboratories the opportunity to more accurately monitor response to anti-CD38 therapies. Nano antibody technology enhances the ability of DRM detection by flow cytometry in the era of immunotherapy.

An overview of pim kinase as a target in multiple myeloma.

Multiple myeloma (MM) is the second common hematologic malignancy manifesting as a clonal proliferation of plasma cells in the bone marrow. In recent years, high expression and activity of pim kinase have been found to be associated with both the progression and prognosis of a significant proportion of malignant diseases. Therefore, pim kinase has become a potential therapeutic target in the treatment of MM and some pim kinase inhibitors have demonstrated good efficacy in clinical trials. Based on nearly the entire literature searched from PubMed in the field of pim kinase in MM, the paper concluded how pim kinase got involved in the proliferation of myeloma cells, the progression of bone disease infiltration, and even in the regulation of the immune microenvironment. Next as a very promising drug, the effectiveness of pim kinase inhibitors as single agents or in combination with other drugs in the treatment of MM was also summarized. Our analysis will guide the clinical use of pim kinase inhibitors for managing tumor load and bone disease in MM patients.

Use of Oral Antivirals Ritonavir-Nirmatrelvir and Molnupiravir in Patients with Multiple Myeloma Is Associated with Low Rates of Severe COVID-19: A Single-Center, Prospective Study

In patients with multiple myeloma (MM), SARS-CoV-2 infection has been associated with a severe clinical course and high mortality rates due to the concomitant disease- and treatment-related immunosuppression. Specific antiviral treatment involves viral replication control with monoclonal antibodies and antivirals, including molnupiravir and the ritonavir-boosted nirmatrelvir. This prospective study investigated the effect of these two agents on SARS-CoV-2 infection severity and mortality in patients with MM. Patients received either ritonavir-nirmatrelvir or molnupiravir. Baseline demographic and clinical characteristics, as well as levels of neutralizing antibodies (NAbs), were compared. A total of 139 patients was treated with ritonavir-nirmatrelvir while the remaining 30 patients were treated with molnupiravir. In total, 149 patients (88.2%) had a mild infection, 15 (8.9%) had a moderate infection, and five (3%) had severe COVID-19. No differences in the severity of COVID-19-related outcomes were observed between the two antivirals. Patients with severe disease had lower neutralizing antibody levels before the COVID-19 infection compared to patients with mild disease (p = 0.04). Regarding treatment, it was observed that patients receiving belantamab mafodotin had a higher risk of severe COVID-19 (p < 0.001) in the univariate analysis. In conclusion, ritonavir-nirmatrelvir and molnupiravirmay prevent severe disease in MM patients with SARS-CoV-2 infection. This prospective study indicated the comparable effects of the two treatment options, providing an insight for further research in preventing severe COVID-19 in patients with hematologic malignancies.

Microfluidic isolation of aptamers with affinity towards multiple myeloma monoclonal immunoglobulins.

Multiple myeloma (MM) is a bone marrow cancer of resident plasma cells that affects 125,000 patients in the U.S. with about 30,000 new cases per year. Its signature is the clonal proliferation of a single plasma cell that secretes a patient specific monoclonal immunoglobulin (M-Ig). Targeting the M-Ig in patient serum could allow sensitive and noninvasive identification of minimal residual disease in multiple myeloma. Aptamers, which are single-stranded oligonucleotides with affinity and specificity to a target molecule, have recently been introduced as affinity reagents for recognition of MM M-Igs. Here we exploit microfluidic SELEX technology to enable rapid and efficient generation of aptamers against M-Ig proteins from MM patients. We first characterize the technology by isolating aptamers with affinity towards the monoclonal antibody rituximab as a model M-Ig and then apply the technology to isolating aptamers specifically targeting M-Igs obtained from serum samples of MM patients. We demonstrate that high-affinity DNA aptamers (KD < 50 nM) for M-Ig proteins from a patient sample could be isolated via microfluidic SELEX within approximately 12h and using less than 100 micrograms of patient M-Ig. Such aptamers can potentially be used in personalized monitoring of minimal residual disease in MM patients.

Assessment of Bone Disease in Multiple Myeloma Patients After Autologous Stem Cell Transplant

Background : Detection of lytic bone lesions is crucial in the workup for multiple myeloma (MM) and very often dictates the decision to start treatment. Modern imaging techniques such as MRI, PET, and CT offer superior detection of myeloma bone disease and extramedullary manifestations of plasma cell dyscrasias. Autologous stem cell transplant (ASCT) is regarded as frontline therapy in candidate patients. Recent advances in understanding of myeloma bone disease showed that the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) system plays a key role in this regard. Objectives : to assess the response of bone disease in patients with multiple myeloma using whole body low dose CT (WBLD CT) scan after different modalities of treatment including autologous stem cell transplant. Patients and method : This is a prospective cohort study in which patients with multiple myeloma had baseline WBLD CT scan with basic evaluation. They were then received induction chemotherapy with and then grouped into two groups according to the modality of consolidation (ASCT vs no ASCT). Both groups had second WBLD CT and were compared for the osteolytic lesions concerning size and site. Results : 20 patients were included in the study (12 males, 8 females), 8 treated with chemotherapeutic protocols (mainly bortezomib based) (group A) while 12 patients underwent ASCT during their course of treatment (group B). The improved score was found only in two patients, one in each group. Conclusion : myeloma bone disease needs to be assessed initially and promptly prevented and treated since it increases morbidity and mortality. Using newer imaging modalities like WBLD CT, MRI, PET scan is encouraged over conventional X ray skeletal survey.

Assessment of Minimal Residual Disease in Multiple Myeloma patients undergoing Autologous Stem Cell Transplant using Multiparameter Flow Cytometry

Abstract BACKGROUND Multiple Myeloma (MM) is still considered an incurable relapsing disease, despite major advances in multidrug combinations, incorporation of autologous transplant and achieving higher rates of CR. This suggests persistent presence of residual disease, not detected by current techniques. Minimal residual disease (MRD) monitoring by multiparameter flow cytometry (MFC) is a powerful tool to quantitatively measure residual disease and allow tailoring of the management plan on individual basis. AIM To assess the value of MRD by MFC in determining the efficacy of treatment, monitoring depth of remission and predicting impending relapse. PATIENTS AND METHODS We included 33MM patients, who were candidates for ASCT in this prospective study. MFC (with a 0.01% limit of detection) on a BM aspirate obtained before transplant and at day 100 post-transplant, was used to measure MRD for all patients. Patients were then later assessed for progression 1-year post-transplant. RESULTS MRD status at 100 days post-transplant was strongly related to the risk of 1-year post-transplant progression (p-value 0.001), and by using a ROC curve, we found that MRD (%) at 100 days post-transplant can predict progression after 1 year with a best cutoff value ≥ 0.04 achieving a 75% sensitivity and 81.2% specificity. On dividing the patients according to their MRD status before and after transplant, group 3 who were MRD positive pre- and post-transplant, had a high risk of progression 1year post-transplant (p-value 0.003). The use of bortezomib-based regimens resulted in a deeper response and a more negative MRD pre-transplant (p-value 0.01), that was maintained post-transplant. Also, ASCT resulted in the development of deeper remission (p-value 0.004) and more MRD negativity (p-value 0.02). CONCLUSION we recommend the use of MRD by MFC as a powerful prognostic tool that should be incorporated in routine myeloma workup.

MM-299: Fibroblast Growth Factor 23 in Multiple Myeloma Patients with Renal Impairment

Context Intact fibroblast growth factor (iFGF23) is a 32 kDa factor containing 251 amino acids and belongs to the FGF family. iFGF23 is thought to increase the renal excretion of phosphates and decrease vitamin D levels. It is a novel factor introduced to the medical field, and the increased expression of iFGF23 has been reported in renal impairment and multiple myeloma. Objective To measure the level of serum iFGF23 in multiple myeloma patients with renal impairment and to investigate its possible predictive role in multiple myeloma patients with renal impairment. Design Cross-sectional comparative study. Setting Clinical Hematology and Bone Marrow Transplantation Unit, Ain Shams University Hospital, Cairo, Egypt. Patients and Method 80 subjects were classified into 4 groups. Group 1: 20 multiple myeloma patients with renal impairment (on dialysis and not on dialysis). Group 2: 20 multiple myeloma patients without renal impairment. Group 3: 30 healthy controls who were group-matched with patients for age and sex. Group 4: 10 non-multiple myeloma patients with renal impairment. Results Serum iFGF23 was higher in patients with multiple myeloma than in healthy controls (p Conclusion iFGF23 was observed to be associated with renal disease in multiple myeloma patients, so it may have predictive value as a biomarker, especially in multiple myeloma patients with renal impairment. Moreover, it could be used as a targetable marker for therapeutic intervention.

The Significance of CD319 and CD269 in the Detection of Immunophenotyping and Minimal Residual Disease in Multiple Myeloma Patients

To investigate the expression of CD319 and CD269 in plasma cells of patients with multiple myeloma (MM) and the feasibility of using CD319 instead of CD38 as a gating antigen in immunophenotyping and minimal residual disease (MRD) monitoring. The expression levels of CD319 and CD269 antigens in clonal bone marrow plasma cells of 387 patients were detected by CD38/CD138 gating strategy with 8-color flow cytometry, and the stability of antigens was also analyzed, and the sensitivity and correlation of two different gating strategies employing CD319/CD138 and CD38/CD138 were compared as well. The control group consisted of 53 cases with non-malignant blood disease matched by age and sex. Monoclonal plasma cells were detected in 303 of 387 MM patients, among which 277 cases (91.42%) were positive for CD269, and all cases were positive for CD319 (100%). In newly diagnosed MM (NDMM) and recurrent refractory MM (RRMM) patients, the expression levels of CD269 were 97.53% (0-99.92%) and 94.96% (0.22%-99.99%), respectively, while levels of CD319 were 99.90% (87.77%-100%) and 99.78% (63.12%-100%), respectively. The expression levels of CD269 and CD319 in the control group were 97.00% (77.00%-100%) and 100% (89.00%-100%), respectively. There were no significant differences in the expression levels of CD269 and CD319 among NDMM, RRMM and the control group. Patients acquiring therapeutic effects were divided into complete remission (CR) group, very good partial response (VGPR) group and partial response (PR) group. Gating with CD38/CD138, median MRD values were 0.76% (0-1.88%), 0.77% (0-4.96%) and 1.75% (0.09%-10.90%) in the three groups, respectively, while gating with CD319/CD138, median MRD values were 0.57% (0.18%-1.96%), 1.07% (0.12%-4.85%) and 1.77% (0.08%-8.22%), respectively. There was no significant difference in MRD level by the two gating strategies, but a good correlation between the two (r=0.808, P<0.05). In addition, in 4 patients treated by CD38 monoclonal antibody (DARA), the expression level of CD38 was observed to be down-regulated or even negative after treatment. When the MRD level was very low, CD38/CD138 gating resulted in false MRD-, while CD319/CD138 gating resulted in MRD+. CD319 and CD269 express stably and continuously in plasma cells of MM patients at different disease stages. CD319 can be used as an alternative of CD38 for immunophenotyping and MRD detection, especially for MRD detection after DARA treatment, while CD269 is suitable for detection before BCMA-CAR-T treatment.

Is whole body low dose CT still necessary in the era of 18F-FDG PET/CT for the assessment of bone disease in multiple myeloma patients?

Whole body low dose computed tomography (WBLDCT) is the first-choice imaging modality to identify bone involvement in multiple myeloma (MM). Because the unenhanced LDCT co-registered to positron emission tomography (PET) (LDCT/PET) has similar technical characteristics to WBLDCT, we aimed to assess its reliability in the detection of bone disease, for employing fluorine-18-fluorodeoxyglucose (18F-FDG) PET/CT as unique multimodality imaging method in MM patients. Thirty three consecutive MM patients were prospectively enrolled and evaluated with WBLDCT to assess bone involvement. In addition, patients underwent 18F-FDG PET/CT using a disease-tailored optimized LDCT protocol. To compare both methods, skeletal anatomical regions were identified and a per-region and per-patient analysis were performed using Cohen's k test. Low dose computed tomography/PET sensitivity, specificity and accuracy were also calculated. The two imaging modalities resulted highly concordant considering both patient-based (k=0.841) and region-based analysis; some discrepancies were observed in dorsal spine (k=0.809) and thorax (k=0.756). Low dose computed tomography/PET sensitivity, specificity and accuracy were 89.4%, 98.3% and 93.5%, respectively. Low dose computed tomography co-registered PET has comparable performance to WBLDCT. If confirmed on a lager sample, these encouraging results suggest the possibility to use this multimodal hybrid imaging as the only method for MM evaluation, rather than both exams, providing both morphologic and metabolic information in one session with impact on patient compliance, health care spending and especially radiation exposure.

Role of Imaging in the Evaluation of Minimal Residual Disease in Multiple Myeloma Patients.

The International Myeloma Working Group (IMWG) recently introduced the evaluation of minimal residual disease (MRD) within the multiple myeloma (MM) response criteria, and MRD negativity assessed inside and outside the bone marrow is currently considered the most powerful predictor of favorable long-term outcomes. However, MRD evaluation has thus far relied on flow-cytometry or molecular-based methods, despite the limitations associated with the patchy infiltration of bone marrow (BM) plasma cells and the presence of extra-medullary (EMD). On the contrary, imaging-based sensitive response assessment through the use of functional rather than morphological whole-body (WB) imaging techniques, such as positron emission tomography with computed tomography (PET/CT) and magnetic resonance imaging (MRI), likely is a promising strategy to overcome these limitations in evaluating response to therapy and in the assessment of the MRD status in MM patients. However, despite the significant advances in the development and availability of novel functional imaging techniques for MRD evaluation, a worldwide standardization of imaging criteria for acquisition, interpretation, and reporting is yet to be determined and will be object of future investigations.

The Glasgow Prognostic Score at Diagnosis Is a Predictor of Clinical Outcome in Patients with Multiple Myeloma Undergoing Autologous Haematopoietic Stem Cell Transplantation.

Background: Immunity and inflammatory response affect the tumour microenvironment and the progression of malignancies. Metabolic and inflammatory parameters and ratios of the peripheral blood correlate with outcome in cancer patients. There exist several established and validated inflammation-based scores of prognostic significances including the Glasgow Prognostic Score (GPS). Methods: In this retrospective, multicentre study, we investigated the prognostic capabilities of baseline GPS in patients with multiple myeloma (MM) undergoing autologous stem cell transplantation as a complementary resource for risk stratification. For GPS calculation, a C-reactive-protein (CRP) value of >10 mg/dL counts as one point and an albumin value of <35 g/L connotes another point, resulting in three different subgroups (group I: 0 points; group II: 1 point; and group III: 2 points). Patients with MM admitted to the participating institutions between January 2010 and July 2018 were screened, and established prognostic scores and ratios were assessed. Characteristics significantly associated with overall survival (OS) or progression-free survival (PFS), upon univariate analysis, were included in a Cox proportional hazards model. Results: Following initial assessment, we identified 224 fully evaluable patients who underwent autologous haematopoietic stem cell transplantation for multiple myeloma. A centralised review of pathology and cytogenetic reports was conducted, and a central hematopathology assessment was performed in 175 of 224 cases (78.1%). Proceeding to high-dose chemotherapy and subsequent autologous stem cell transplantation was the main inclusion criterion for all transplant-eligible patients in the study. The median age at diagnosis was 59 years (range: 35–76 years) with a median follow-up of 76 months. Multivariate analysis revealed neutrophil–platelet score (NPS) (HR = 0.528, 95% CI = 0.284–0.984) and B symptoms at primary diagnosis (HR = 1.838, 95% CI = 1.232–2.740) to be independent predictors of PFS while high-risk cytogenetic changes (HR = 2.358, 95% CI = 1.413–3.934, p = 0.001) could be identified as an independent predictor of OS, and GPS to be the only independent predictor of both OS and PFS (OS: HR = 2.127, 95% CI = 1.431–3.162, p < 0.0001 and PFS: HR = 1.405; 95% CI = 1.058–1.867, p = 0.019). Conclusions: Our data show that baseline GPS correlates with rates of relapse and refractory disease in MM patients undergoing autologous transplantation. In a multivariate analysis, these effects were proven to hold prognostic capabilities beyond and independent from established prognosticators. These results require further validation in a prospective setting.

Assessment of TP53 lesions for p53 system functionality and drug resistance in multiple myeloma using an isogenic cell line model

Recent advances in molecular diagnostics have shown that lesions affecting both copies of the gene for tumor suppressor protein 53 (TP53) count among the most powerful predictors for high-risk disease in multiple myeloma (MM). However, the functional relevance and potential therapeutic implications of single hits to TP53 remain less well understood. Here, we have for the first time approximated the different constellations of mono- and bi-allelic TP53 lesions observed in MM patients within the frame of a single MM cell line model and assessed their potential to disrupt p53 system functionality and to impart drug resistance. Both types of common first hit: point mutation with expression of mutant p53 protein or complete loss of contribution from one of two wildtype alleles strongly impaired p53 system functionality and increased resistance to melphalan. Second hits abolished remaining p53 activity and increased resistance to genotoxic drugs even further. These results fit well with the clinical drive to TP53 single- and double-hit disease in MM patients, provide a rationale for the most commonly observed double-hit constellation (del17p+ TP53 point mutation), and underscore the potential increases in MM cell malignancy associated with any type of initial TP53 lesion.

P671Impacts of echocardiography-defined pulmonary hypertension on clinical outcome in patients with multiple myeloma

Abstract Background Pulmonary hypertension (PH) is a rarely reported complication of multiple myeloma (MM). PH of MM is usually mild to moderate and can be secondary to a variety of conditions, including left ventricular dysfunction, diastolic dysfunction, chronic heart failure, treatment-related toxicity, thrombophilic condition and precapillary involvement. However, only few reports regarding PH in MM incidence and prognosis exist up to now. Purpose The purpose of this study was to investigate the risk factors of transthoracic echocardiography-defined PH and its impact on clinical outcome in patients with MM. Methods A total of 277 MM patients was included and divided into 2 groups–those non-pulmonary hypertension (PH) or those with PH, based on the results of the transthoracic echocardiography (TTE); PH group (n=143, 60.9±9.2 years, 68 males) versus non-PH group (n=134, 55.9±11.5 years, 72 males). We analyzed propensity score matching and multiple imputation method were used to deal with the missing data in echocardiographic characteristics. Results During the follow-up period (median 618 days), all-cause death occurred in 79 (28.5%) patients and 41 patients (14.8%) died from cardiovascular causes (including acute decompensated heart failure, fatal MI, sudden cardiac arrest). In the Kaplan-Meier survival analysis of crude population and propensity-matched population, cumulative overall survival and cardiovascular death (CVD)-free survival were significantly lower in the PH group than in the non-PH group (p&lt;0.001). In propensity-matched population, estimated pulmonary artery pressure &gt;35mmHg in TTE, congestive heart failure, and DM were significant independent predictors of all-cause death. KM curves in MM stratified by PH Conclusion This study demonstrates that the presence of PH, congestive heart failure, and DM is an independent prognostic factor for all-cause death in MM patients with MM. These results highlight the risk associated cardiovascular disease in MM patients and emphasize that management strategies that prevent deterioration of cardiac function are essential.

Epigenetic silencing of miR-335 induces migration by targeting insulin-like growth factor-1 receptor in multiple myeloma

Multiple myeloma (MM) is a common hematological malignancy and remains incurable. MiRNA-335 is a classic tumor suppressor, yet its expression pattern and biological role in MM is unclear. The aim of the present study was to determine the expression pattern, biological role, and mechanism of miR-335 in MM. In this study, we found that miR-335 expression was decreased in MM. The promoter of miR-335 was also hypermethylated in MM. It was found that over-expression of miR-335 or 5-azacytidine treatment suppressed migration of MM cells and down-regulated the expression of IGF-1R. MiR-335 thus acts as a metastatic suppressor by targeting IGF-1R in MM. Moreover, aberrant promoter hyper-methylation is critical for miR-335 silencing in MM. We also found that miR-335 assisted in predicting both the prognosis and progression of disease in MM patients. Observations might offer a new complementary diagnostic and therapeutic target in MM.

Comparison of minimal residual disease in multiple myeloma patients detected by 8-color panels and next generation flow cytometry

目的比较8色组合和二代流式细胞术（NGF）检测多发性骨髓瘤（MM）患者治疗后微小残留病（MRD）的敏感性。方法分别建立一组检测胞膜抗原CD45、CD38、CD138、CD19、CD56、CD117、CD81和CD27的8C-Mem组，及一组检测胞膜及胞质抗原CD45、CD38、CD138、CD19、CD56、CD27、cκ和cλ的8C-Cyto组，将8C-Mem组和8C-Cyto组合成的8C-2tubes组，3组采用Diva软件分析浆细胞胞膜抗原表达与胞质轻链表达，统称8色组合；NGF是采用Infinicyt软件将8C-2tubes组两管数据融合，进一步分析浆细胞各抗原的表达。以20名正常对照和76例治疗后达完全缓解的MM患者骨髓样本进行检测与比较分析。结果NGF组合识别异常浆细胞（aPC）和克隆性浆细胞（cPC）的阳性率均为88.2%（67/76），一致率为94.7%（72/76），cPC的中位值为0.3530%，均为胞质轻链限制性表达，NGF检测的敏感度为0.0003%。8色组合中，8C-Mem组、8C-Cyto组和8C-2tubes组MRD阳性率分别为84.2%、85.5%和86.8%，均低于NGF的88.2%（P<0.001）；8C-Cyto组和8C-Mem组MRD阳性率低于8C-2tubes组（P<0.001），8C-Mem组MRD阳性率低于8C-Cyto组（P<0.001）。NGF敏感性和特异性优于8色组合，8色组合中8C-2tubes组敏感性和阴性预测值、特异性和阳性预测值及准确性最高，缺点是获取数据太大，采用Diva软件分析耗时长，效率低；其次是8C-Cyto组，8C-Mem最低。结论反应浆细胞克隆性的胞质轻链较胞膜抗原更特异，胞膜抗原异常表达不能完全代表cPC，建议胞膜抗原和胞质轻链同时检测，有条件者优先选择NGF，8色组合中建议选择8C-Cyto组。

Relationship between Bone Marrow PD-1 and PD-L1 Expression and the Presence of Osteolytic Bone Disease in Multiple Myeloma Patients

Alterations of the bone marrow (BM) immune-microenvironment characterize the progression of monoclonal gammopathies and the development of osteolytic bone disease in multiple myeloma (MM). MM patients exhibit immune dysfunctions as impaired dendritic, NK and T cells, whereas the onset of MM osteolytic lesions is associated to an increased prevalence of Th17 cells. Recently, the pathophysiological role of the programmed cell death protein 1 (PD-1)/PD-1 ligand (PD-L1) pathway together with an increase of myeloid derived suppressor cells (MDSCs) in the induction of tumor tolerance and immune evasion has been underlined with a therapeutic relevance. However, unclear data on the expression profile of PD-1/PD-L1 axis in MM patients have been reported and it is not known if this axis could be related with the presence of osteolytic bone disease.In order to elucidate these aspects, we analyzed a total cohort of 80 consecutives patients with monoclonal gammopathies, including 15 monoclonal gammopathy of undetermined significance (MGUS), 23 smoldering MM (SMM), 21 newly diagnosed MM (MMD) and 21 relapsed/refractory MM (MMR) patients. The presence of bone disease was checked by low-dose computerized tomography (CT) with or without positron emission tomography (PET) scan and by X-rays skeletal survey in 11 MM patients. 87% of MM patients displayed osteolytic lesions. High bone disease (HBD) was defined by the presence of 3 or more osteolytic lesions (62% of our cohort). Patients without bone lesions or with minus of 3 lesions were defined as low bone disease (LBD). BM mononuclear cells were analyzed by flow cytometry, evaluating plasma cells (PCs) (CD138+), monocytes (CD14+) and T cells (total CD3+, CD3+CD4+ and CD3+CD8+). PD-L1 (CD274) expression was evaluated on CD138+ and CD14+ cells, and PD-1 (CD279) on CD3+, CD4+ and CD8+ cells. Lastly, in a subgroup of patients we analysed MDSC populations, including both granulocytic (gMDSCs) (CD11b+HLA-DR-CD14-CD15+) and monocytic MDSCs (mMDSCs), (CD11b+HLA-DR-/lowCD14+CD15-).The percentage of BM CD3+PD-1+ cells increased from MGUS to MMR patients with a significant trend (p=0.004). Indeed, patients with active MM (MMD and MMR) showed both higher % of CD3+PD-1+ cells (median value: 48.5% vs 40.6%, p=0.001) and PD-1 median fluorescence intensity (MFI) on CD3+ (median value: 18.9 vs 16.7 MFI, p=0.024) as compared to patients with SMM and MGUS. CD4+PD-1+, but not CD8+PD-1+ cells are increased in active MM compared to SMM and MGUS patients (p=0.023). On the other hand, any significant difference was not observed in the PD-L1 expression on both BM CD138+ and CD14+ cells across patient groups. The percentage of BM MDSC populations did not significantly change across the different monoclonal gammopathies (p=0.14); moreover, comparing MM with SMM and MGUS patients, the % of gMDSCs was significantly reduced (median %: 12.5% vs 17%, p=0.04) and the % of mMDSCs was increased (median %: 0.36% vs. 0.24%) without reaching a statistical significance.Focusing on MM bone disease, we found that osteolytic MM patients displayed higher CD4+/CD8+ ratio compared to non-osteolytic ones (p=0.043). Regarding the PD-1 expression, the % of CD3+PD-1+ cells did not change (p=0.192), whereas the % of CD8+PD-1+ cells was significantly reduced in osteolytic patients compared to non-osteolytic ones (p=0.016). Consistently, among the BM CD8+ cells, a significant decrease of %PD-1+ cells was found in HBD vs LBD MM patients (median value: 46.9% vs 57.4%, p=0.045). Interestingly, when compared to LBD MM patients, HBD MM patients displayed a significant reduction of PD-L1 expression on both BM CD138+ PCs (median MFI value: 13.3 vs 21.6 MFI, p=0.007) and CD14+ cells (median MFI value: 15.4 vs 23.8 MFI, p=0.007). In a multivariate analysis, PD-1+ expression on CD8+ cells and PD-L1 MFI on CD138+ were significant independent factors related to the presence of HBD.In conclusion, our study indicates that the frequency of PD-1+ T cells increases across the progression of the monoclonal gammopathies. On the other hand, for the first time, we show in MM patients a significant relationship between the presence of extensive osteolytic bone disease and a reduced expression profile of BM PD-1/PD-L1 axis on CD8+ and CD138+ cells. We hypothesize that a less immune-suppressive profile could be related to the development of osteolysis consistent with the negative cross talk existing between PD-1/PD-L1 axis and Th17 cells. DisclosuresAversa:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding; Celgene Italy: Other: Avisory Board, Research Funding.