Background: Whereas, in most patients with multiple myeloma (MM), achieving undetectable MRD anticipates a favorable outcome, some others relapse shortly afterwards. Although one obvious explanation ...for this inconsistency is the use of nonrepresentative marrow samples due to hemodilution, there is no guidance on how to evaluate this issue. Methods: Since B-cell precursors, mast cells and nucleated red blood cells are normally absent in peripheral blood, we analyzed them in 1404 bone marrow (BM) aspirates obtained in numerous disease settings and in 85 healthy adults (HA). Results: First, we confirmed the systematic detection of the three populations in HA, as well as the nonreduced numbers with aging. Pairwise comparisons between HA and MM patients grouped according to age and treatment showed significant variability, suggesting that hemodilution should be preferably evaluated with references obtained from patients treated with identical regimens. Leveraging the MRD results from 118 patients, we showed that a comparison with HA of similar age could also inform on potential hemodilution. Conclusions: Our study supports the routine assessment of BM cellularity to evaluate hemodilution, since reduced BM-specific cell types as compared to reference values (either treatment-specific or from HA if the former are unavailable) could indicate hemodilution and a false-negative MRD result.
Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited.
To standardize the performance of the LymphoTrack next-generation ...sequencing (NGS) assays (Invivoscribe), targeting clonal immunoglobulin rearrangements, in order to reproduce the detection of tumor clonotypes and MRD quantitation in myeloma.
The quantification ability of the assay was evaluated through serial dilution experiments. Paired samples from 101 patients were tested by LymphoTrack, using Sanger sequencing and EuroFlow's next-generation flow (NGF) assay as validated references for diagnostic and follow-up evaluation, respectively. MRD studies using LymphoTrack were performed in parallel at 2 laboratories to evaluate reproducibility.
Sensitivity was set as 1.3 tumor cells per total number of input cells. Clonality was confirmed in 99% and 100% of cases with Sanger and NGS, respectively, showing great concordance (97.9%), although several samples had minor discordances in the nucleotide sequence of rearrangements. Parallel NGS was performed in 82 follow-up cases, achieving a median sensitivity of 0.001%, while for NGF, median sensitivity was 0.0002%. Reproducibility of LymphoTrack-based MRD studies (85.4%) and correlation with NGF (R2 > 0.800) were high. Bland-Altman tests showed highly significant levels of agreement between flow and sequencing.
Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures.
Summary
Immunoparesis is the suppression of normal polyclonal immunoglobulins and is present in most patients with newly diagnosed multiple myeloma (MM). The association of immunoparesis at ...diagnosis, and particularly its recovery along with treatment, with survival in patients ineligible for autologous stem‐cell transplantation (ASCT) has not been well established. This retrospective study evaluated the impact of immunoparesis in 431 patients diagnosed with MM, ineligible for ASCT, with a median overall survival of 36 months 95% confidence interval (CI): 31–40. Immunoparesis was present in 81.2% of patients at diagnosis and was associated with a trend to a worse overall response rate (ORR: 84.8% vs. 74.9%; OR 1.88 (95% CI: 0.97–3.63), shorter progression‐free survival (PFS) 22.0 vs. 18.2 months; hazard ratio (HR) 0.775; 95%CI: 0.590–1.018; p = 0.066, and overall survival (OS) (45.9 vs. 34.2 months; HR 0.746; 95% CI: 0.551–1.010; p = 0.057). Twenty‐four per cent of patients who had immunoparesis at diagnosis recovered polyclonal immunoglobulins in the follow‐up period. Interestingly, these patients had a better ORR (96.3% vs. 68.2%; OR 12.29 (95% CI: 3.77–40.06), PFS (HR 0.703; 95CI%: 0.526–0.941; p = 0.018) and OS (HR 0.678; 95 CI%: 0.503–0.913; p = 0.011) than patients who did not recover it. In summary, restoring a healthy immune system along with first‐line treatment in patients with MM, not receiving ASCT, is associated with better outcomes.
Summary Chimeric antigen receptor (CAR) T‐cell therapies have increased the patients with relapsed/refractory multiple myeloma (RRMM) in whom standard electrophoretic techniques fail to detect the ...M‐protein. Quantitative immunoprecipitation mass spectrometry (QIP‐MS) can accurately measure serum M‐protein with high sensitivity, and identify interferences caused by therapeutic monoclonal antibodies. Here, we investigate the outcome of QIP‐MS in 33 patients treated with the academic BCMA‐directed CAR T‐cell ARI0002h (Cesnicabtagene Autoleucel). QIP‐MS offered more detailed insights than serum immunofixation (sIFE), identifying glycosylated M‐proteins and minor additional peaks. Moreover, the potential interferences owing to daratumumab or tocilizumab treatments were successfully detected. When analysing different assay platforms during patient's monitoring after ARI0002h administration, we observed that QIP‐MS showed a high global concordance (78.8%) with sIFE, whereas it was only moderate (55.6%) with bone marrow (BM)‐based next‐generation flow cytometry (NGF). Furthermore, QIP‐MS consistently demonstrated the lowest negativity rate across the different timepoints (27.3% vs. 60.0% in months 1 and 12, respectively). Patients with QIP‐MS(+)/BM‐based NGF(−) showed a non‐significant shorter median progression free survival than those with QIP‐MS(−)/BM‐based NGF(−). In summary, we show the first experience to our knowledge demonstrating that QIP‐MS could be particularly useful as a non‐invasive technique when evaluating response after CAR T‐cell treatment in MM.
The clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. >20years ago, the International Union of ...Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clones for the detection of IgG and IgA subclasses for a broad range of laboratory assays, except for flow cytometry. Here we analyzed the performance of commercially available Ab clones to detect IgG and IgA subclasses in memory B-cells and plasma cells (PCs) by flow cytometry. In a first step, 28 Ab clones were evaluated in peripheral blood from healthy donors. Only 17/28 clones showed reactivity against IgG and IgA subclasses expressed on the B-cell and PC surface membrane, including Ab clones for IgG1 (SAG1, HP6188, HP6001 and HP6186), IgG2 (SAG2, HP6014 and HP6002), IgG3 (SAG3, HP6095 and HP6050), IgG4 (SAG4), IgA1 (SAA1, H69-11.4 and B3506B4) and IgA2 (SAA2, 2E2, and A9604D2). In a second step, for each Ig subclass a single clone was selected according to its specificity and fluorescence intensity (resolution power), for further more detailed validation (SAG1, SAG2, SAG3, SAG4, SAA1 and SAA2). This validation process was carried out in 4 different laboratories by testing the selected Ab clones in human peripheral blood, bone marrow and tonsil samples, using different staining protocols (e.g. surface membrane and/or cytoplasmic staining). All selected Ab clones displayed strong positivity, high specificity and optimal resolution between negative and positive cells. Alternative Ab clones were also validated. Thus, our results show the feasibility of using the validated Ig subclass Ab clones in combination with other B cell-associated markers for detailed dissection of the memory B-cell and PC compartments that express distinct Ig subclasses in different human tissues.
•IgG1–4 and IgA1–2 memory B-cell and PC subsets can be dissected by flow cytometry.•Both smIgH and cyIgH subclasses can be evaluated in any human tissue.•Antibodies used for serum IgH quantitation should be validated for cytometry.
Immunoglobulin M (IgM) monoclonal gammopathies show considerable variability, involving three different stages of presentation: IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), ...asymptomatic Waldenström’s macroglobulinemia (AWM), and symptomatic WM (SWM). Despite recent findings about the genomic and transcriptomic characteristics of such disorders, we know little about the causes of this clinical heterogeneity or the mechanisms involved in the progression from indolent to symptomatic forms. To clarify these matters, we have performed a gene expression and mutational study in a well-characterized cohort of 69 patients, distinguishing between the three disease presentations in an attempt to establish the relationship with the clinical and biological features of the patients. Results showed that the frequency of genetic alterations progressively increased from IgM-MGUS to AWM and SWM. This means that, in contrast to
MYD88
p.L265P and
CXCR4
WHIM mutations, present from the beginning of the pathogenesis, most of them would be acquired during the course of the disease. Moreover, the expression study revealed a higher level of expression of genes belonging to the Toll-like receptor (TLR) signaling pathway in symptomatic versus indolent forms, which was also reflected in the disease presentation and prognosis. In conclusion, our findings showed that IgM monoclonal gammopathies present higher mutational burden as the disease progresses, in parallel to the upregulation of relevant pathogenic pathways. This study provides a translational view of the genomic basis of WM pathogenesis.
Summary
Recommended genetic categorization of acute myeloid leukaemias (AML) includes a favourable‐risk category, but not all these patients have good prognosis. Here, we used next‐generation ...sequencing to evaluate the mutational profile of 166 low‐risk AML patients: 30 core‐binding factor (CBF)‐AMLs, 33 nucleophosmin (NPM1)‐AMLs, 4 biCEBPα‐AMLs and 101 acute promyelocytic leukaemias (APLs). Functional categories of mutated genes differed among subgroups. NPM1‐AMLs showed frequent variations in DNA‐methylation genes (DNMT3A, TET2, IDH1/2) (79%), although without prognostic impact. Within this group, splicing‐gene mutations were an independent factor for relapse‐free (RFS) and overall survival (OS). In CBF‐AML, poor independent factors for RFS and OS were mutations in RAS pathway and cohesin genes, respectively. In APL, the mutational profile differed according to the risk groups. High‐risk APLs showed a high mutation rate in cell‐signalling genes (P = 0·002), highlighting an increased incidence of FLT3 internal tandem duplication (ITD) (65%, P < 0·0001). Remarkably, in low‐risk APLs (n = 28), NRAS mutations were strongly correlated with a shorter five‐year RFS (25% vs. 100%, P < 0·0001). Overall, a high number of mutations (≥3) was the worst prognostic factor RFS (HR = 2·6, P = 0·003). These results suggest that gene mutations may identify conventional low‐risk AML patients with poor prognosis and might be useful for better risk stratification and treatment decisions.
Bortezomib plus melphalan and prednisone (VMP) and lenalidomide plus low-dose dexamethasone (Rd) are 2 standards of care for elderly untreated multiple myeloma (MM) patients. We planned to use VMP ...and Rd for 18 cycles in a sequential or alternating scheme. Patients (233) with untreated MM, >65 years, were randomized to receive 9 cycles of VMP followed by 9 cycles of Rd (sequential scheme; n = 118) vs 1 cycle of VMP followed by 1 cycle of Rd, and so on, up to 18 cycles (alternating scheme; n = 115). VMP consisted of one 6-week cycle of bortezomib using a biweekly schedule, followed by eight 5-week cycles of once-weekly VMP. Rd included nine 4-week cycles of Rd. The primary end points were 18-month progression free survival (PFS) and safety profile of both schemes. The 18-month PFS was 74% and 80% in the sequential and alternating arms, respectively (P = .21). The sequential and alternating groups exhibited similar hematologic and nonhematologic toxicity. Both arms yielded similar complete response rate (42% and 40%), median PFS (32 months vs 34 months, P = .65), and 3-year overall survival (72% vs 74%, P = .63). The benefit of both schemes was remarkable in patients aged 65 to 75 years. In addition, achieving complete and immunophenotypic response was associated with better outcome. The present approach, based on VMP and Rd, is associated with high efficacy and acceptable toxicity profile with no differences between the sequential and alternating regimens. This trial was registered at www.clinicaltrials.gov as #NCT00443235.
•The sequential and alternating administration of VMP and Rd have equal efficacy and toxicity.•The greatest benefit of this total therapy approach was observed for patients aged 65 to 75 years.
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