scholarly journals Persistence of Minimal Residual Disease By Multiparameter Flow Cytometry Can Hinder Recovery of Organ Damage in Patients with AL Amyloidosis Otherwise in Complete Response

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3261-3261 ◽  
Author(s):  
Giovanni Palladini ◽  
Margherita Massa ◽  
Marco Basset ◽  
Francesca Russo ◽  
Paolo Milani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Cells ◽  
Residual Disease ◽  
Complete Response ◽  
Organ Damage ◽  
Multiparameter Flow Cytometry ◽  
Al Amyloidosis ◽  
Proof Of Concept ◽  
Organ Response ◽  
Minimal Residual

Abstract Introduction. In multiple myeloma, Minimal Residual Disease (MRD) demonstrated by multiparameter flow cytometry (MFC) identifies subjects with significantly shorter survival among those who attain complete response (CR). Patients with AL amyloidosis generally have a lower clonal burden than subjects with multiple myeloma, and, despite a higher rate of early death due to advanced organ damage, if they respond to therapy they have a better long term outcome and are less likely to relapse. The role of MRD in AL amyloidosis has not been assessed so far. In the present proof-of-concept study, we assessed the MRD by MFC in patients with AL amyloidosis who attained CR. Methods. Complete response was defined as per current criteria (negative serum and urine immunofixation and normal free light chain ratio). Immunofixation was performed with both a commercial semi-automated method (Hydragel, Sebia, Lisses, France) and our home-made high-resolution method in all cases and had to be negative by both techniques. Circulating free light chains were measured by the Freelite assay. For flow cytometry studies bone marrow samples were processed following the Euro Flow Bulk Lysis Standard Operating Protocol and stained with the EuroFlow-IMF MM MRD panel (Tube 1: CD138BV421 / CD27BV510 / CD38FITC / CD56PE / CD45PerCP-Cy5.5 / CD19PE-Cy7 / CD117APC / CD81APC-C750, and Tube 2: identical to Tube 1 except for CyKappaAPC / CyLambdaAPC-C750). At least 5x106 events were measured using a FACSCanto II (BD Biosciences, San Jose, USA) instrument. Data were analyzed using the Infinicyt software (version 1.7; Cytognos SL, Salamanca, Spain). Patients were identified as having residual disease if a discreet population of clonal plasma cells comprising ≥ 50 events was identified (0.001% limit of detection). Patients exposed to different treatment types, to different numbers of treatment lines, and at different points in time after achievement of CR were tested, in order to assess the possible impact of these variables on MRD. Differences in variables between patients with and without MRD were tested for significance by mid p Fisher exact test. Results. Seventeen patients were tested. Six subjects were found to have relapsed at the time of MRD assessment with monoclonal components detectable at immunofixation and/or abnormal FLC ratio. All of them also had detectable MRD. Eleven patients satisfied current criteria for CR. All of them had renal and 6 (54%) had cardiac involvement at diagnosis. Two and 3 lines of therapy were required to achieve CR in 3 and 1 subjects, respectively. Median time to CR was 8 months (range 3-23 months). Two patients underwent autologous stem cell transplant and 9 received bortezomib. Three patients (50%) had achieved cardiac response and 5 (45%) renal response at the time of attainment of CR. The median time from CR to MRD evaluation was 20 months (range 6-36 months). Flow cytometry identified MRD in 5 patients (45%). A median of 1089 (range 600-2500) corresponding to 0.03% (range 0.02-0.15%) plasma cells with abnormal phenotype were detected in patients with MRD. No differences in organ involvement, cardiac and renal stage, type of therapy (with 1 transplanted patient in each group), number of treatments, and organ response at the time of CR was found between patients with and without MRD. However, a further improvement of cardiac function compared to the time of CR attainment was observed in all 4 evaluable patients without MRD and in none of the 2 patients with MRD, with borderline statistical significance (P=0.067). Compared to the time of CR achievement, renal response was obtained in 5 subjects without MRD (83%) and in 1 (20%) with MRD (P=0.069). Overall, further improvement of cardiac or renal function after CR was significantly associated with absence of MRD (P=0.002). Interestingly, 1 patient with MRD had otherwise unexplained increase in proteinuria while still in CR, and anti-clone therapy was started based on MRD results. Conclusion. This proof-of-concept study indicates that almost 50% of patients with AL amyloidosis satisfying current criteria for CR have MRD detectable by MFC. MRD makes further organ improvement less likely and can explain organ progression. A validation study in a larger sample is ongoing at our center. The possible impact of MRD should be considered in trials aiming at increasing organ response rate in patients achieving CR. Disclosures Palladini: Prothena: Honoraria. Merlini:Takeda and Janssen-Cilag: Honoraria.

scholarly journals Assessment of minimal residual disease using multiparametric flow cytometry in patients with AL amyloidosis

2020 ◽  
Vol 4 (5) ◽  
pp. 880-884 ◽  
Author(s):  
Andrew Staron ◽  
Eric J. Burks ◽  
John C. Lee ◽  
Shayna Sarosiek ◽  
J. Mark Sloan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Residual Disease ◽  
Complete Response ◽  
Cardiac Response ◽  
Al Amyloidosis ◽  
Multiparametric Flow Cytometry ◽  
Organ Response ◽  
Minimal Residual

Abstract Despite achieving a hematologic complete response after treatment, many patients with AL amyloidosis do not attain recovery of organ function and/or experience hematologic relapse. A persistent plasma cell clone producing amyloidogenic light chains at levels below the detection threshold of traditional serologic methods is hypothesized to impede organ response in some patients. Assessment of minimal residual disease (MRD) may therefore have clinical importance as a more stringent treatment response tool for patients in a hematologic complete response. We used 2-tube, 10-color combination multiparametric flow cytometry to assess for MRD at a minimum sensitivity of 1 in 105 nucleated cells. Of 65 patients in hematologic complete response, 36 (55%) were found to have a residual clonal plasma cell population in the bone marrow. Comparing the MRD-negative and MRD-positive groups, renal response was observed in 88% vs 64% (P = .06), cardiac response in 75% vs 59% (P = .45), and any organ response in 90% vs 75% (P = .20) of patients. Depth of organ response as measured by the percent decrease in 24-hour proteinuria and brain natriuretic peptide was 96% vs 91% (P = .16) and 55% vs 46% (P = .66), respectively. These data suggest a possible correlation between MRD negativity and higher probability of organ response after treatment in AL amyloidosis. Future prospective studies with a larger cohort are needed to determine the clinical relevance of these improvements. This trial was registered at www.clinicaltrials.gov as #NCT00898235.

scholarly journals Comparison of minimal residual disease detection in multiple myeloma between the DuraClone and EuroFlow methods

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takeshi Yoroidaka ◽  
Kentaro Narita ◽  
Hiroyuki Takamatsu ◽  
Momoko Fujisawa ◽  
Shinji Nakao ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Plasma Cells ◽  
Residual Disease ◽  
Total Cell ◽  
Multiparameter Flow Cytometry ◽  
Single Tube ◽  
The Cost ◽  
Minimal Residual

AbstractIn this study, the minimal residual disease (MRD) levels in patients with multiple myeloma (MM) were assessed by comparing the new 8-color single-tube multiparameter flow cytometry method (DuraClone), which reduces the cost of antibodies and labor burden of laboratories, with the EuroFlow next-generation flow (NGF) method. A total of 96 samples derived from 69 patients with MM were assessed to determine the total cell acquisition number (tCAN), percentages of total and normal plasma cells (PCs), and MRD levels using two methods. We found that the tCAN was significantly higher with EuroFlow-NGF than with DuraClone (median 8.6 × 106 vs. 5.7 × 106; p < 0.0001). In addition, a significant correlation in the MRD levels between the two methods was noted (r = 0.92, p < 0.0001). However, in the qualitative analysis, 5.2% (5/96) of the samples showed discrepancies in the MRD levels. In conclusion, the DuraClone is a good option to evaluate MRD in multiple myeloma but it should be used with caution.

scholarly journals Comparison of Minimal Residual Disease Detection in Autografts of Patients with Multiple Myeloma between 8-Color Multiparameter Flow Cytometry (EuroFlow) and Next-Generation Sequencing

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 258-258 ◽  
Author(s):  
Hiroyuki Takamatsu ◽  
Naoki Takezako ◽  
Rachel K Wee ◽  
Takeshi Yoroidaka ◽  
Takeshi Yamashita ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Research Funding ◽  
Prognostic Value ◽  
Plasma Cells ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry ◽  
Next Generation ◽  
Generation Sequencing ◽  
Minimal Residual

Abstract Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognosis of patients with multiple myeloma (MM). However, most patients with MM are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper responses are required. Next-generation sequencing (NGS) and multiparameter flow cytometry (MFC) methods have been used to assess MRD. However, the lack of standardization of conventional MFC approaches has had a negative impact on its reproducibility. Recently, a next-generation MFC method (EuroFlow, NGF) has been developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for a highly sensitive and standardized detection of MRD in MM. Aims: To compare the prognostic value of MRD detection in autografts in MM between NGS (Adaptive) and 8-color MFC method (EuroFlow, NGF), and also MRD levels between fresh and cryopreserved autografts. Methods: A total of 39 newly-diagnosed MM patients who underwent ASCT were enrolled in this study. Median age 60 at ASCT (range 41-69); males 22, females 17; ISS 1 (n=10), 2 (n=19), 3 (n=10). 10 patients showed high-risk chromosomal abnormalities (t(4;14) (n=9), del17p & t(4;14) (n=1)). The induction regimen was bortezomib-based chemotherapy. All patients received melphalan 200 mg/sqm as conditioning regimen before ASCT. 34 of 39 (87%) patients received maintenance therapy until progressive disease. The best response post-ASCT was as follows: 23sCR, 2CR, 12VGPR, 2PR. 39 autografts, one from each MM patient, were analyzed using NGF and NGS methods. The NGF method was based on a standardized lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of cells and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC) (J Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD Assay (Seattle, WA) (Martinez-Lopez et al., Blood 2014). To assess the correlation of MRD levels between fresh and cryopreserved autografts using NGF, 6 additional MM patients' autografts were used. Results: MRD levels in all 39 autografts were assessed using EuroFlow, while those in 32 of 39 (82%) were assessed with NGS due to limited availability of material for calibration. We identified abnormal plasma cells (aPC) in autografts based on multivariate analysis of individual cells from each patient (e.g. CD56+, CD19-, CyIgκ+, CD117+). Since there was a good correlation in MRD levels between fresh and thawed frozen autografts detected by EuroFlow (R=0.943, P=0.02), we assessed the MRD levels in thawed frozen autografts. For the MM MRD in autografts, the events from tube 1 and tube 2 were combined and a median of 7.3×106 (range: 2.2×106-37.6×106) events was acquired. The sensitivity of EuroFlow was 1×10-5-2×10-6 while that of NGS was 10-7 due to the high number of DNA derived from autografts (Takamatsu et al., Ann Oncol 2017). 21 of 39 (54%) cases were MRD positive by 8-color MFC while 22 of 32 (69%) cases were MRD positive by NGS. The correlation of MRD levels between 8-color MFC and NGS was relatively high (Fig. 1A). MRD negative by NGF (MRDMFC (-)) cases tended to show better PFS than MRDMFC (+) cases (P=0.145) (Fig. 1B) while MRD negative by NGS (MRDNGS (-)) cases showed significantly better PFS than MRDNGS (+) cases (P=0.03) (Fig. 1C). Furthermore, MRDMFC (-) MRDNGS (-) cases showed significantly better PFS than MRDMFC (-) MRDNGS (+) cases (P=0.01), but the PFS of MRDMFC (-) MRDNGS (+) cases was not different from that of MRDMFC (+) MRDNGS (+) cases (P=0.70). MRDMFC (-) and MRDNGS (-) cases showed better OS than MRDMFC (+) (P=0.14) and MRDNGS (+) (P=0.08) cases, respectively. Conclusions: Although EuroFlow is a fast and accurate method for detecting MRD of MM in autografts, in this study the NGS platform had a higher sensitivity and prognostic value than EuroFlow. The homogenous nature of the mobilized autograft relative to the focal nature of myeloma in bone marrow might provide a better sample to assess MRD. Figure 1. Figure 1. Disclosures Takamatsu: Celgene: Honoraria, Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

scholarly journals Comparison of the Performance of Surface Alone or Surface Plus Cytoplasmic Approaches for the Assessment of Minimal Residual Disease in Multiparameter Flow Cytometry in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1799-1799
Author(s):  
Marie C Bene ◽  
Nelly Robillard ◽  
Philippe Moreau ◽  
Soraya Wuilleme
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Plasma Cells ◽  
Residual Disease ◽  
Cell Loss ◽  
Molecular Techniques ◽  
Light Chains ◽  
Multiparameter Flow Cytometry ◽  
Minimal Residual

Background. During the follow-up of treated myeloma patients, the assessment of minimal residual disease (MRD) is gaining an increasing importance. The detection of remaining abnormal plasma-cells (PC) may rely on molecular techniques investigating immunoglobulin rearrangements of the malignant clone or on multiparameter flow cytometry (MFC). The latter allows to obtain a rapid response by dealing with fresh cells. It also focuses on cells still alive, since dead cells are discarded as debris. Numerous publications have reported that the most reliable markers of PC in MFC are CD38 and CD138 their co-expression being a good way to select the population of PC in a bone marrow (BM) or, more rarely tested, blood sample. Malignant PC often but not always differ from normal PC by the loss of CD19 expression and the acquisition of CD56. Other immunophenotypic alterations are related, among others, to the expression of CD20, CD27, CD28, CD33, CD45, CD81 or CD117. Malignant PC also display the monotypic usage of light chains by the myelomatous immunoglobulin, which can readily be assessed in MFC after permeabilization of the PC, although this induces an additional technical step that could induce some cell loss. Here we compared the two panels proposed by the Euroflow consortium (Flores Montero, 2018) which use the same backbone of antibodies with a "surface" strategy associating CD81 and CD117 or a "cytoplasmic" strategy investigating for the expression of kappa and lambda immunoglobulin light chains. Methods. From a cohort of patients for whom MRD had been assessed in our MFC platform, 100 samples were retrospectively selected as displaying detectable MRD in the cytoplasmic strategy. All BM samples had first been submitted to bulk lysis to increase the PC concentration. Between 5 and 10x106 nucleated cells were used for surface staining, premeabilization and intracytoplasmic staining. Another aliquot of the same suspension, with 3 to 5x106 nucleated cells, was used for the "surface" tube. Briefly, both samples were surface stained with antibodies to CD45 (Ozyme), CD19 (Beckman Coulter), CD38 (Cytognos), CD138 (BD Biosciences) and CD27 (Ozyme). The "surface tube" also contained antibodies to CD81 (Clinisciences) and CD117 (BD Biosciences). After this incubation, the "cytoplasmic tube" was submitted to permeabilization (Intrastain® Dako) and cells further incubated with antibodies to kappa and lambda chains (Dako and Clinisciences). All samples were acquired on the same day. Listmodes of the "cytoplasmic tubes" were analyzed and data provided to the clinician within 24 hours. For this study, the listmodes of the "surface tube" were analyzed blindly using the Kaluza® software. Data were then compared to those of the "cytoplasmic tube" Results. A good linear correlation was observed between the two results, with a R2 coefficient of 0.73. The global difference between both tubes was usually a lower MRD level detected with the "cytoplasmic tube", seen in 68% of the cases (median -0.0113; range -0.0001 to -1.4). Of note higher levels (0.0007 to 1.44) were observed in 32%, ruling out a systematic loss of cells that could have been responsible for this difference. The gating strategy adopted (Robillard 2013) delineated four populations on a CD19/CD56 bivariate histogram. Monotypy was then investigated in each of the four subsets thus identified. The same strategy was applied for the "surface tube" looking at the coexpression profile of CD81 and CD117 in each subset. Globally, 58% of the samples were CD56 positive among which 43 were CD19-. CD19 was also absent in 40 CD56- samples. All configurations of CD117 and CD81 coexpression were seen, making each patient a challenging case. In about 10% of the cases, two suspect subsets were seen in the "surface tube" while monotypy was seen in only one in the "cytoplasmic tube". Conclusion. Although this study shows a good correlation between the two panels, it was found that a greater confidence could be attributed to the "cytoplasmic tube", where data are comforted by identification of a monotypic population with the same light chain as the monoclonal peak. Moreover, although confirmation of the abnormal subset was required in numerous cases with the "surface tube", the reverse was never observed. Single use of the "cytoplasmic" combination can thus be recommended as a robust method of MRD assessment in multiple myeloma. Disclosures Moreau: AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.

scholarly journals High-risk cytogenetics and persistent minimal residual disease by multiparameter flow cytometry predict unsustained complete response after autologous stem cell transplantation in multiple myeloma

Blood ◽  
2012 ◽  
Vol 119 (3) ◽  
pp. 687-691 ◽  
Author(s):  
Bruno Paiva ◽  
Norma C. Gutiérrez ◽  
Laura Rosiñol ◽  
María-Belén Vídriales ◽  
María-Ángeles Montalbán ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Stem Cell ◽  
High Risk ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Complete Response ◽  
Multiparameter Flow Cytometry ◽  
Minimal Residual

Abstract The achievement of complete response (CR) after high-dose therapy/autologous stem cell transplantation (HDT/ASCT) is a surrogate for prolonged survival in multiple myeloma; however, patients who lose their CR status within 1 year of HDT/ASCT (unsustained CR) have poor prognosis. Thus, the identification of these patients is highly relevant. Here, we investigate which prognostic markers can predict unsustained CR in a series of 241 patients in CR at day +100 after HDT/ASCT who were enrolled in the Spanish GEM2000 (n = 140) and GEM2005 < 65y (n = 101) trials. Twenty-nine (12%) of the 241 patients showed unsustained CR and a dismal outcome (median overall survival 39 months). The presence of baseline high-risk cytogenetics by FISH (hazard ratio 17.3; P = .002) and persistent minimal residual disease by multiparameter flow cytometry at day +100 after HDT/ASCT (hazard ratio 8.0; P = .005) were the only independent factors that predicted unsustained CR. Thus, these 2 parameters may help to identify patients in CR at risk of early progression after HDT/ASCT in whom novel treatments should be investigated.

scholarly journals Minimal residual disease negativity by next-generation flow cytometry is associated with improved organ response in AL amyloidosis

2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Giovanni Palladini ◽  
Bruno Paiva ◽  
Ashutosh Wechalekar ◽  
Margherita Massa ◽  
Paolo Milani ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Organ Dysfunction ◽  
Light Chain ◽  
Residual Disease ◽  
Cell Clone ◽  
Cardiac Response ◽  
Al Amyloidosis ◽  
Next Generation ◽  
Organ Response ◽  
Minimal Residual

AbstractLight chain (AL) amyloidosis is caused by a small B-cell clone producing light chains that form amyloid deposits and cause organ dysfunction. Chemotherapy aims at suppressing the production of the toxic light chain (LC) and restore organ function. However, even complete hematologic response (CR), defined as negative serum and urine immunofixation and normalized free LC ratio, does not always translate into organ response. Next-generation flow (NGF) cytometry is used to detect minimal residual disease (MRD) in multiple myeloma. We evaluated MRD by NGF in 92 AL amyloidosis patients in CR. Fifty-four percent had persistent MRD (median 0.03% abnormal plasma cells). There were no differences in baseline clinical variables in patients with or without detectable MRD. Undetectable MRD was associated with higher rates of renal (90% vs 62%, p = 0.006) and cardiac response (95% vs 75%, p = 0.023). Hematologic progression was more frequent in MRD positive (0 vs 25% at 1 year, p = 0.001). Altogether, NGF can detect MRD in approximately half the AL amyloidosis patients in CR, and persistent MRD can explain persistent organ dysfunction. Thus, this study supports testing MRD in CR patients, especially if not accompanied by organ response. In case MRD persists, further treatment could be considered, carefully balancing residual organ damage, patient frailty, and possible toxicity.

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