Quantitative assessment of daratumumab in serum via intact light chain measurement using liquid chromatography-high resolution mass spectrometry: a method suitable for therapeutic drug monitoring.

Daratumumab, a pivotal treatment for multiple myeloma, exhibits considerable inter-patient variability in pharmacological clinical outcomes, likely attributed to serum concentration that may underscore the need for its therapeutic drug monitoring. This study aims to develop and validate a straightforward analytical method for quantifying daratumumab in serum, focusing on intact light chain determination, using liquid chromatography high-resolution mass spectrometry. The sample preparation involved immunoglobulin enrichment using Melon gel followed by a reduction step to dissociate the light from the heavy chains of immunoglobulins. The latter were then separated using a MabPac RP 2.1 × 50 mm chromatographic column and the intact light chains were detected and quantified using a Q Exactive Orbitrap mass spectrometer operating in ESI-positive ion mode at 17 500 resolution. The method demonstrated excellent linearity (R2 > 0.992) across a serum concentration range of 100 to 2000 µg mL-1 and good precision and accuracy: intra- and interday relative errors ranged from -5.1% to 6.5%, with a relative standard deviation of less than 5.8%. Clinical suitability was confirmed by analyzing 80 clinical samples from multiple myeloma patients treated with 1800 mg of daratumumab. 99% of the samples fell within the analytical range with a mean daratumumab concentration evaluated before the next administration (Ctrough) of 398 µg mL-1. These findings highlighted that intact light chain monoclonal antibody quantification could be a valid and robust alternative to either immunoassays or to LC-MS/MS targeting peptides for measuring daratumumab in clinical samples, positioning it as a suitable method for therapeutic drug monitoring applications.

Disease monitoring with quantitative serum IgA levels provides a more reliable response assessment in multiple myeloma patients.

Unlike IgG monoclonal proteins (MCPs), IgA MCP quantification is unreliable due to beta-migration of IgA MCPs on serum protein electrophoresis (SPEP). The utility of nephelometric quantitative IgA (qIgA) to monitor IgA multiple myeloma (MM) is unclear. We retrospectively studied disease response kinetics using qIgA versus MCPs by SPEP, and developed and validated novel qIgA disease assessment criteria in 491 IgA MM patients. The SPEP MCP nadir occurred a median of 41 (IQR 0-102) days before the qIgA. The median time to achieve a partial response (PR) was shorter using standard IMWG versus qIgA response criteria (32 vs 58 days, p < 0.001). Stratification by qIgA criteria, unlike IMWG criteria, led to clear separation of the progression-free survival curves of patients achieving a PR or very good PR. There was a consistent trend toward earlier detection of disease progression using qIgA versus IMWG progression criteria. In conclusion, monitoring IgA MM using MCP-based IMWG criteria may be falsely reassuring, given that MCP levels on SPEP decrease faster than qIgA levels. The qIgA response criteria more accurately stratify patients based on the progression risk and may detect disease progression earlier, which may lead to more consistent measurement of trial endpoints and improved patient outcomes.

Comparison of next-generation sequencing (NGS) and next-generation flow (NGF) for minimal residual disease (MRD) assessment in multiple myeloma.

Detecting persistent minimal residual disease (MRD) allows the identification of patients with an increased risk of relapse and death. In this study, we have evaluated MRD 3 months after transplantation in 106 myeloma patients using a commercial next-generation sequencing (NGS) strategy (LymphoTrack®), and compared the results with next-generation flow (NGF, EuroFlow). The use of different marrow pulls and the need of concentrating samples for NGS biased the applicability for MRD evaluation and favored NGF. Despite that, correlation between NGS and NGF was high (R2 = 0.905). The 3-year progression-free survival (PFS) rates by NGS and NGF were longer for undetectable vs. positive patients (NGS: 88.7% vs. 56.6%; NGF: 91.4% vs. 50%; p < 0.001 for both comparisons), which resulted in a 3-year overall survival (OS) advantage (NGS: 96.2% vs. 77.3%; NGF: 96.6% vs. 74.9%, p < 0.01 for both comparisons). In the Cox regression model, NGS and NGF negativity had similar results but favoring the latter in PFS (HR: 0.20, 95% CI: 0.09-0.45, p < 0.001) and OS (HR: 0.21, 95% CI: 0.06-0.75, p = 0.02). All these results reinforce the role of MRD detection by different strategies in patient prognosis and highlight the use of MRD as an endpoint for multiple myeloma treatment.

Pegfilgrastim improves the outcomes of mobilization and engraftment in autologous hematopoietic stem cell transplantation for the treatment of multiple myeloma.

Autologous stem cell transplantation (ASCT) is the only curable therapy for multiple myeloma (MM), while its success primarily relies on mobilization to obtain sufficient hematopoietic stem/progenitor cells (HPC). Although the role of Pegfilgrastim (PEG), a novel PEGylated form of the recombinant G-CSF filgrastim (FIL), in mobilization has been demonstrated, it remains unclear whether this approach is cost-effective in MM treatment. Here, we performed a real-world analysis to evaluate the efficacy and cost of PEG for mobilization in a cohort of MM patients, of which 53% carried high-risk cytogenetic abnormalities. A total of 91 patients who received either a single dose of PEG (6 or 12 mg, n = 42) or multiple dosing of 10 µg/kg/day FIL (n = 49) after chemotherapy for HPC mobilization were included. The yield of MNCs and CD34+ cells per milliliter of blood collected via apheresis was significantly greater in the PEG group than that in the FIL group (P = 0.014 and P = 0.038). Mobilization with PEG yielded significantly higher median number of collected CD34+ cells than FIL (5.56 vs. 4.82 × 106/kg; P = 0.038). Moreover, the average time-to-recovery of leukocytes and platelets after transplantation was markedly shorter in the PEG group than that in the FIL group (leukocyte, 11.59 ± 1.98 vs 12.93 ± 2.83 days, P = 0.019; platelet, 12.86 ± 2.62 vs 14.80 ± 5.47, P = 0.085). However, the total cost of mobilization and apheresis using PEG or FIL was comparable (P = 0.486). Of note, mobilization with 12 mg PEG further shortened time-to-recovery of leukocytes (10.64 ± 0.51 vs. 12.04 ± 2.26 days, P = 0.05) and platelets (10.60 ± 2.89 vs. 13.33 ± 2.35 days, P = 0.031) compared with 6 mg PEG. Our results support a notion that PEG (especially 12 mg) combined with chemotherapy is a cost-effective and convenient regimen of mobilization, which might improve the outcome of ASCT in MM.

Incidence and Management of Therapeutic Monoclonal Antibody Interference in Monoclonal Gammopathy Monitoring.

BACKGROUND: The treatment of multiple myeloma (MM) has been revolutionized by the introduction of therapeutic monoclonal antibodies (tmAbs). Daratumumab, a human IgG1/κ tmAb against CD38 on plasma cells, has improved overall survival in refractory MM and was recently approved as a frontline therapy for MM. Work on tmAb interference with serum protein electrophoresis (SPE) during MM monitoring has failed to provide information for laboratories on incidence of interference and effective methods of managing the interference at a practicable level. We aimed to evaluate daratumumab and elotuzumab interference in a large academic hospital setting and implement immediate solutions. METHODS: We identified and chart reviewed all cases of possible daratumumab interference by electrophoretic pattern (120 of 1317 total cases over 3 months). We retrospectively reviewed SPE cases in our laboratory to assess clinical implications of tmAb interference before the laboratory was aware of tmAb treatment. We supplemented samples with daratumumab and elotuzumab to determine the limits of detection and run free light chain analysis. RESULTS: Approximately 9% (120 of 1317) of tested cases have an SPE and/or immunofixation electrophoresis (IFE) pattern consistent with daratumumab, but only approximately 47% (56) of these cases were associated with daratumumab therapy. Presence of daratumumab led to physician misinterpretation of SPE/IFE results. Limits of daratumumab detection varied with total serum gammaglobulin concentrations, but serum free light chain analysis was unaffected. CONCLUSIONS: Clinical laboratories currently rely on interference identification by electrophoretic pattern, which may be insufficient and is inefficient. Critical tools in preventing misinterpretation efficiently include physician education, pharmacy notifications, separate order codes, and interpretive comments.

Mass Spectrometry-Based Method Targeting Ig Variable Regions for Assessment of Minimal Residual Disease in Multiple Myeloma.

Multiple myeloma is a systemic malignancy of monoclonal plasma cells that accounts for 10% of hematologic cancers. With development of highly effective therapies for multiple myeloma, minimal residual disease (MRD) assessment has emerged as an important end point for management decisions. Currently, serologic assays lack the sensitivity for MRD assessment, and invasive bone marrow sampling with flow cytometry or molecular methods has emerged as the gold standard. We report a sensitive and robust targeted mass spectrometry proteomics method to detect MRD in serum, without the need of invasive, sequential bone marrow aspirates. The method detects Ig-derived clonotypic tryptic peptides predicted by sequencing the clonal plasma cell Ig genes. A heavy isotope-labeled Ig internal standard is added to patient serum at a known concentration, the Ig is enriched in a light chain type specific manner, and proteins are digested and analyzed by targeted mass spectrometry. Peptides from the constant regions of the λ or κ light chains, Ig heavy chains, and clonotypic peptides unique to the patient monoclonal Igs are targeted. This technique is highly sensitive and specific for the patient-specific monoclonal Igs, even in samples negative by multiparametric flow cytometry. Our method can accurately and precisely detect monoclonal protein in serum of patients treated for myeloma and has broad implications for management of hematologic patients.

Assessment of early treatment response on MRI in multiple myeloma: Comparative study of whole-body diffusion-weighted and lumbar spinal MRI.

OBJECTIVES: To compare remission status at completion of chemotherapy for multiple myeloma (MM) with changes in total diffusion volume (tDV) calculated from whole-body diffusion-weighted imaging (WB-DWI) and fat fraction (FF) of lumbar bone marrow (BM) by modified Dixon Quant (mDixon Quant) soon after induction of chemotherapy, and to assess the predictive value of MRI. METHODS: Fifty patients (mean age, 66.9 ± 10.5 years) with symptomatic myeloma were examined before and after two cycles of chemotherapy. From WB-DWI data, tDV was obtained with the threshold for positive BM involvement. Mean FF was calculated from lumbar BM using the mDixon Quant sequence. At the completion of chemotherapy, patients were categorized into a CR/very good PR (VGPR) group (n = 15; mean age, 67.6 ± 10.3 years) and a PR, SD or PD group (n = 35; mean age, 69.1 ± 8.6 years). ROC curves were plotted to assess performance in predicting achievement of CR/VGPR. RESULTS: At second examination, serum M protein, ß2-microglobulin, and tDV were significantly decreased and hemoglobin, mean ADC, and FF were significantly increased in the CR/VGPR group and serum M protein was significantly increased in the PR/SD/PD group. The general linear model demonstrated that percentage changes in FF and M protein contributed significantly to achieving CR/VGPR (P = 0.02, P = 0.04, respectively). AUCs of ROC curves were 0.964 for FF and 0.847 for M protein. CONCLUSIONS: Early change in FF of lumbar BM and serum M protein soon after induction of chemotherapy contributed significantly to prediction of CR/VGPR.

Challenges and opportunities in the assessment of measurable residual disease in multiple myeloma.

Treatment response assessment in multiple myeloma (MM) relies on the detection of paraprotein in serum and/or urine, bone marrow morphology and immunohistochemistry. With remarkable advances in therapy, particularly in the newly diagnosed setting, achievement of complete remission became frequent, creating the need to identify smaller amounts of residual disease and understand their prognostic and therapeutic implications. Measurable residual disease (MRD) can be assessed primarily by flow cytometry and next generation sequencing and state-of-the-art assays have sensitivity approaching 1 in 106 cells. This review discusses the existing challenges in utilizing MRD to inform management of MM and highlights open research questions and opportunities as MRD is more routinely incorporated into clinical practice for patients with MM.

Monitoring tumour burden and therapeutic response through analysis of circulating tumour DNA and extracellular RNA in multiple myeloma patients.

Monitoring tumour burden and therapeutic response through analyses of circulating cell-free tumour DNA (ctDNA) and extracellular RNA (exRNA) in multiple myeloma (MM) patients were performed in a Phase Ib trial of 24 relapsed/refractory patients receiving oral azacitidine in combination with lenalidomide and dexamethasone. Mutational characterisation of paired BM and PL samples at study entry identified that patients with a higher number of mutations or a higher mutational fractional abundance in PL had significantly shorter overall survival (OS) (p = 0.005 and p = 0.018, respectively). A decrease in ctDNA levels at day 5 of cycle 1 of treatment (C1D5) correlated with superior progression-free survival (PFS) (p = 0.017). Evaluation of exRNA transcripts of candidate biomarkers indicated that high CRBN levels coupled with low levels of SPARC at baseline were associated with shorter OS (p = 0.000003). IKZF1 fold-change <0.05 at C1D5 was associated with shorter PFS (p = 0.0051) and OS (p = 0.0001). Furthermore, patients with high baseline CRBN coupled with low fold-change at C1D5 were at the highest risk of progression (p = 0.0001). In conclusion, this exploratory analysis has provided the first demonstration in MM of ctDNA for predicting disease outcome and of the utility of exRNA as a biomarker of therapeutic response.

Assessment of Red Blood Cell Distribution Width and Multiple Myeloma in a Guangxi Population: a Retrospective Study.

BACKGROUND: Red blood cell distribution width (RDW) has been reported as a marker for inflammation and tumors. The present study aims to investigate the use of RDW in patients with multiple myeloma (MM). METHODS: Seventy-three patients with newly diagnosed symptomatic multiple myeloma (SMM), 39 patients with relapsed multiple myeloma (RMM), and 91 healthy individuals were recruited into this study. The demographic and laboratory parameters were reviewed retrospectively, and the correlation between RDW and other parameters among groups were evaluated by Spearman's correlation analysis. The sensitivity and specificity of RDW were determined by receiver operating characteristic (ROC) curve analysis. RESULTS: The RDW values in both SMM and RMM were significantly higher than in the healthy individuals (p < 0.001). In SMM patients with International Staging System (ISS) Stages II and III, the level of RDW was higher than in the patients with ISS Stage I; however, there was no significant difference between each ISS stage in RMM patients. The RDW strongly correlated with platelet distribution width (PDW), cystatin C, serum beta2-microglobulin (Sß2M), hemoglobin (HGB), hematocrit (HCT), albumin (Alb), and calcium (p < 0.05) in SMM patients, and RDW in RMM patients had a positive or negative correlation with PDW, Sß2M, globulin, HGB, absolute neutrophil count, platelet count, HCT, and Alb (p < 0.05). The ROC curve analysis showed that RDW > 13.5 had 94.5% sensitivity and 63.7% specificity for SMM, and 92.3% sensitivity and 63.7% specificity for RMM. CONCLUSIONS: Elevated RDW in MM patients was associated with the stage of the disease.

Comparison of Sebia Free Light Chain Assay With Freelite Assay for the Clinical Management of Diagnosis, Response, and Relapse Assessment in Multiple Myeloma.

BACKGROUND: Serum free light chain (FLC) measurement has become an important marker for the management of multiple myeloma (MM). However, several analytical challenges remain unresolved. We compared the clinical performances of the Sebia FLC assay in MM to the Freelite assay. PATIENTS AND METHODS: A total of 177 patients from the IFM DFCI 2009 trial were enrolled onto this study, with a total of 368 samples analyzed. At baseline, concordance of the involved to noninvolved FLC ratio (iFLC/niFLC) was evaluated. During therapy, comparison of the disease response assessments according to International Myeloma Working Group criteria was performed. RESULTS: Compared to Freelite, the Sebia FLC assay demonstrated lower results, with a proportional bias with increased values. We demonstrated that the Sebia equivalent of the iFLC/niFLC ratio of 100 was 16. During follow-up, agreement in response assessment was moderate (for light chains MM) to good (for intact immunoglobulin MM). In the context of relapse, the concordance was moderate, but longitudinal follow-up showed a similar kinetics. CONCLUSION: The Sebia FLC assay provides inequivalent absolute results from the Freelite assay. Despite lower absolute FLC values, the kinetics of response and relapse is exactly the same. As with other FLC assays available, follow-up of MM with the same method is advisable.

Laboratory assessment of multiple myeloma.

Laboratory testing plays an essential role in the diagnosis and management of patients with multiple myeloma. A variety of chemistry and molecular assays are routinely used to monitor patient progress, response to treatment and relapse. Here, we have reviewed current literature and core guidelines on the details of laboratory testing in myeloma-related investigations. This includes the use and value of protein electrophoresis, serum free light chain and cytogenetic testing. Furthermore, we discuss other traditional chemistry assays essential to myeloma investigation, and potential interferences that may arise due to the disease nature of myeloma, that is, the presence of a monoclonal immunoglobulin. Finally, we discuss the importance of communication in protein electrophoresis results, where laboratorians are required to relate clinically relevant myeloma-relevant information to the ordering physician on the background of a complex pattern of serum or urine proteins. Laboratory testing in myeloma-related investigation relies on several traditional chemistry assays. However, we anticipate new tests and technologies to become available in the future with improved analytical sensitivity, as well as improved clinical sensitivity in identifying patients who are at high risk of progression to multiple myeloma.

Rapid assessment of hyperdiploidy in plasma cell disorders using a novel multi-parametric flow cytometry method.

Trisomies of odd numbered chromosomes are seen in nearly half of patients with multiple myeloma (MM) and typically correlate with a hyperdiploid state and better overall survival (OS). We compared DNA ploidy of monoclonal plasma cells (as a surrogate for the presence of trisomies) assessed simultaneously by PCPRO (plasma cell proliferative index), a novel method that estimates DNA index by multi-parametric flow cytometry to fluorescence in situ hybridization (FISH) in 1703 patients with plasma cell disorders. The distribution of ploidy was hyperdiploid: 759 (45%), diploid 765 (45%), hypodiploid: 71 (4%), tetraploid/near-tetraploid: 108 (6%). FISH identified trisomies in 82% (621/756) of patients with hyperdiploidy by PCPRO and no trisomy by FISH was observed in 88% (730/834) of patients without hyperdiploidy. 95% (795/834) of patients without hyperdiploidy on PCPRO had one or less trisomy by FISH. Sensitivity and specificity of PCPRO for detecting hyperdiploidy was 86% (621/725) and 84% (730/865), respectively. Sensitivity increased to 94% (579/618) for patients with more than one trisomy. Newly diagnosed MM patients with hyperdiploidy on PCPRO (147/275) had better OS compared to nonhyperdiploid patients (median not reached vs 59 months, P = 0.008) and better progression free survival (median: 33 vs 23 months, P = 0.03). Within the hyperdiploidy group, patients with high-hyperdiploidy (DNA index: 1.19-1.50) versus those with low-hyperdiploidy (DNA index: 1.05-1.18) had superior OS (3 year OS of 88% vs 68% P = 0.03). Ploidy assessment by flow cytometry can provide rapid, valuable prognostic information and also reduces the number of copy number FISH probes required and hence the cost of FISH.

Nucleic acid based risk assessment and staging for clinical practice in multiple myeloma.

The recently introduced Revised International Staging System (R-ISS) for multiple myeloma (MM) integrates albumin, ß2 microglobulin, lactate dehydrogenase (LDH) with high-risk cytogenetic aberrations (CA), i.e., t(4;14) and t(14;16) and del17p using fluorescent in situ hybridization (FISH). We evaluated utility of nucleic acid-based tests of multiplex ligation-based probe amplification (MLPA) and quantitative real-time polymerase chain reaction (qRT-PCR) to define the CA and the R-ISS categories as per this approach were evaluated for their ability to predict outcome in terms of response, progression-free (PFS), and overall survival (OS). In this study (n = 180), 17 (9.4%), 118 (65.6%), and 45 (25%) patients were assigned to R-ISS1, R-ISS2, and R-ISS3 categories with statistically significant differences in median PFS (p = 0.02) and OS (p < 0.001).On univariate analysis, serum creatinine, LDH, 17p deletion, chromosome 1q gain, and response after first induction therapy were associated with statistically significant differences (p < 0.05) in PFS and in addition, age> 65 years and use of triplet therapy with OS. On multivariate analysis, only serum creatinine, LDH, and response after first induction therapy retained significance for predicting PFS and in addition, use of triplet therapy retained significance for the OS. The proposed nucleic acid-based algorithm using qRT-PCR and MLPA for R-ISS is resource-effective in terms of small quantities of sample required; feasibility of batch processing and reduced overall cost for the total number of regions evaluated and retained the prognostic significance of R-ISS, making it suitable for clinical practice for molecular characterization of MM.

Using Both Lactic Dehydrogenase Levels and the Ratio of Involved to Uninvolved Free Light Chain Levels as Risk Factors Improves Risk Assessment in Patients With Newly Diagnosed Multiple Myeloma.

BACKGROUND: This study aimed to evaluate the prognostic value of the ratio of involved to uninvolved free light chain (rFLC) levels and lactic dehydrogenase (LDH) levels in the risk stratification of patients with multiple myeloma (MM). MATERIALS AND METHODS: Clinical data of 283 patients with newly diagnosed MM were retrospectively analyzed. RESULTS: In the traditional chemotherapy group, patients with an rFLC < 100 had a better prognosis than those with an rFLC ≥ 100 (40 months versus 6 months, P = 0.022), as did patients with an LDH ≤ upper limit of normal (ULN) compared to those with an LDH > ULN (29 months versus 6 months, P = 0.023). In patients who underwent novel drug-combined therapy, no significant difference was observed between the rFLC < 100 group and the rFLC ≥ 100 group (54 months versus median not reached, P = 0.508). However, patients with an LDH ≤ ULN had a better prognosis than those with an LDH > ULN (60 months versus 21 months, P = 0.004). Using an rFLC ≥ 100 and an LDH ≥ ULN as adverse risk factors, patients were classified into 3 groups: group 1 (no adverse risk factors), group 2 (1 adverse risk factor) and group 3 (2 adverse risk factors). The median overall survival (OS) of groups 1, 2 and 3 was 52 months, 34 months and 15 months, respectively (P = 0.001). CONCLUSIONS: rFLC and LDH levels were sensitive prognostic factors in MM patients, combining them could improve the risk stratification and treatment choice of patients in clinical practice.

N-glycosylation of serum proteins for the assessment of patients with IgD multiple myeloma.

BACKGROUND: Because glycosylation is one of the most common post-translational modifications of proteins and because changes in glycosylation have been shown to have a significant correlation with the development of many cancer types, we investigated the serum N-glycome used to diagnose, stage and evaluate the pathological outcomes in IgD multiple myeloma. METHODS: Serum samples were available for 20 patients with IgD multiple myeloma, 41 patients with light chain multiple myeloma and 42 healthy control subjects. Serum N-glycans were released and analysed using DNA sequencer-assisted fluorophore-assisted capillary electrophoresis. RESULTS: Characteristic changes were revealed in the serum N-glycome of IgD myeloma. In particular, three N-glycans (NG1(6)A2F, Peak3; NG1(3)A2F, Peak4; NA2FB, Peak7) showed increased clinical value. The best area under the ROC curve of NG1(6)A2F to diagnose IgD myeloma was 0.981, with a 95.0% sensitivity and 95.2% specificity, and that of NG1(3)A2F was 0.936, with a 95.0% sensitivity and 78.6% specificity. The best area under the ROC curve of NA2FB/NG1(3)A2F to differentially diagnose IgD myeloma versus light chain myeloma was 0.744, with a 95.3% sensitivity and 50.0% specificity. The level of NG1(3)A2F was correlated with the international staging system, while the higher abundance of NA2FB presented in IgD myeloma was predictive of a shorter progression-free survival. CONCLUSIONS: The advent of serum N-glycan signatures may play a role in the diagnosis, staging and prognosis of IgD myeloma and will serve as the foundation for a precision medicine approach to this rare subtype of multiple myeloma.