scholarly journals Standardized assay for assessment of minimal residual disease in blood, bone marrow and apheresis from patients with plasma cell myeloma

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Agnieszka Blum ◽  
Katy Haussmann ◽  
Mathias Streitz ◽  
Stephan Schlickeiser ◽  
Carola Tietze-Buerger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Residual Disease ◽  
Plasma Cell Myeloma ◽  
Minimal Residual

Evaluation of a 5-Color Flow Cytometric Technique for Immunophenotyping and Quantitation of Plasma Cell Myeloma in Post-Therapy Bone Marrows.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5036-5036
Author(s):  
Tove Isaacson ◽  
Andrzej Jakubowiak ◽  
Lloyd Stoolman ◽  
Usha Kota ◽  
William Finn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Plasma Cell Myeloma ◽  
Color Flow ◽  
Flow Cytometric ◽  
Minimal Residual

Abstract Multiparameter flow cytometry is a useful tool for comprehensive immunophenotyping of plasma cell myeloma, and has been proposed as a sensitive method for the evaluation of minimal residual disease in patients following treatment. This study aimed to assess the value of flow cytometry in quantitation of residual disease, in comparison to routine morphologic examination of first-pull bone marrow aspirate smears, in myeloma patients post-therapy. Heparinized bone marrow aspirates were obtained from 27 treated patients with plasma cell myeloma. Cells were prepared for 5-color flow cytometric analysis within 24-hours of specimen draw. Surface membrane staining with anti-CD19, CD20, CD38, CD45, CD56, and CD138 was followed by ammonium chloride lysis of red cells. Fixed and permeabilized cells were analyzed for cytoplasmic light chains to confirm clonality. Data were acquired using an FC500 flow cytometer (Beckman-Coulter), analyzed with CXP software with plasma cells isolated based on bright CD38+ or CD138+ expression. A median of 97,639 cellular events (range 14,279 to 262,508) were collected per analysis. Flow cytometric enumeration of plasma cells was compared to 500-cell differential counts of Wright-Giemsa-stained first-pull aspirate smears from the same cases. The median plasma cell count as determined by flow cytometry was 0.5% (range 0–7.9%). The median plasma cell count estimated by morphologic review was 8.0% (range 0–84.4%). Flow cytometry underestimated the plasma cell content in all but one case. Clonal plasma cells expressed CD38 and CD138 in all cases; 87.5% (21/24) coexpressed CD56, 25% (6/24) coexpressed CD45, and 4.2% (1/24) coexpressed CD19. None was positive for CD20. Although detection of minimal residual disease after therapy for acute leukemia is routinely achieved by flow cytometric analysis, successful quantitation of minimal residual disease in treated myeloma patients using flow cytometry remains limited as it usually underestimates the plasma cell content of bone marrow samples compared to routine morphology of first-pull aspirates. We have observed that this holds true for both pre-treatment and post-treatment specimens. Causes for the discrepancy may include hemodilution of second-pull aspirates used for flow cytometry, fragility and loss of plasma cells during preparation for flow cytometry, and incomplete disaggregation of plasma cells from bone marrow spicules. With improved outcome of treatments, better and more reliable methods of detection of minimal residual disease are needed for optimal prognostic stratification. We are currently validating alternative methods, which may offer more sensitivity while at the same time allow more objectivity, for assessing the amount of minimal residual disease in myeloma patients.

scholarly journals Marked Variability in Reported Minimal Residual Disease Lower Level of Detection of 4 Hematolymphoid Neoplasms: A Survey of Participants in the College of American Pathologists Flow Cytometry Proficiency Testing Program

2015 ◽  
Vol 139 (10) ◽  
pp. 1276-1280 ◽  
Author(s):  
Michael Keeney ◽  
Jaimie G. Halley ◽  
Daniel D. Rhoads ◽  
M. Qasim Ansari ◽  
Steven J. Kussick ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
Plasma Cell Myeloma ◽  
Minimal Residual

Context Flow cytometry is often applied to minimal residual disease (MRD) testing in hematolymphoid neoplasia. Because flow-based MRD tests are developed in the laboratory, testing methodologies and lower levels of detection (LODs) are laboratory dependent. Objectives To broadly survey flow cytometry laboratories about MRD testing in laboratories, if performed, including indications and reported LODs. Design Voluntary supplemental questions were sent to the 549 laboratories participating in the College of American Pathologists (CAP) FL3-A Survey (Flow Cytometry—Immunophenotypic Characterization of Leukemia/Lymphoma) in the spring of 2014. Results A total of 500 laboratories (91%) responded to the supplemental questions as part of the FL3-A Survey by April 2014; of those 500 laboratories, 167 (33%) currently perform MRD for lymphoblastic leukemia, 118 (24%) for myeloid leukemia, 99 (20%) for chronic lymphocytic leukemia, and 91 (18%) for plasma cell myeloma. Other indications include non-Hodgkin lymphoma, hairy cell leukemia, neuroblastoma, and myelodysplastic syndrome. Most responding laboratories that perform MRD for lymphoblastic leukemia reported an LOD of 0.01%. For myeloid leukemia, chronic lymphocytic leukemia, and plasma cell myeloma, most laboratories indicated an LOD of 0.1%. Less than 3% (15 of 500) of laboratories reported LODs of 0.001% for one or more MRD assays performed. Conclusions There is major heterogeneity in the reported LODs of MRD testing performed by laboratories subscribing to the CAP FL3-A Survey. To address that heterogeneity, changes to the Flow Cytometry Checklist for the CAP Laboratory Accreditation Program are suggested that will include new requirements that each laboratory (1) document how an MRD assay's LOD is measured, and (2) include the LOD or lower limit of enumeration for flow-based MRD assays in the final diagnostic report.

scholarly journals Novel Single Panel Method for Minimal Residual Disease Detection for Plasma Cells By Flow Cytometry

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
David Kimmel ◽  
Mohammed A Aljama ◽  
Stephen Ronan Foley ◽  
Hira S Mian ◽  
Catherine A Ross
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Large Data ◽  
Final Analysis ◽  
Distinct Advantage ◽  
Limit Of Quantitation ◽  
Minimal Residual

Introduction: Advancement in myeloma therapy has significantly improved outcomes including minimal residual disease (MRD) negativity that may be a surrogate for overall survival. We describe an assay using a single 10-colour panel to detect minimal residual disease (MRD) in plasma cell neoplasms to a level < 10-5. Methodology: Bone marrow aspirate specimens for this MRD assay must be from the first pull of 1.0 to 1.5 mL of bone marrow. A 1 in 10 dilution of the bone marrow is prepared and run on the instrument using a WBC count from the following calculation to determine the volume of specimen to process: For the MRD method, a corrective factor of 4 has been empirically determined to provide a sufficient number of cells to achieve the goal of 10-13 million viable cells during analysis. The required volume of bone marrow is added directly to Versalyse (Beckman Coulter) with 10% bovine serum albumin (BSA) in a bulk RBC lysis step. The cells are incubated for 15 minutes at room temperature while rocking. The cells are centrifuged and the following antibodies are added (Table 1): The cells and surface antibodies are incubated for a total of 20 minutes, specimen is gently vortexed at 10 minutes. Intracellular staining is achieved using the IntraPrep Kit (Beckman Coulter) using our standardized laboratory process. Cells are suspended in approximately 2mL of RPMI 1640 with 10% FCS. The specimen is loaded on to the Navios EX flow cytometer (Beckman Coulter)and data is acquired at approximately 5000 to 10000 events per second. The Navios EX is not capable of collecting more 1 700 000 events at acquisition when all 10 fluorescent detectors are in use plus light scatter detectors, so the specimen is repeatedly reloaded a total of 7 or 8 times. All data files are opened in Kaluza (Beckman Coulter) and merged in to a single file. This large data file is then imported in to the analysis template and analyzed for plasma cells. Analysis: A pilot of 20 specimens from patients with varying plasma cell disorders have been analyzed. Half of these specimens contained populations of monoclonal plasma cells. Ranked in order of smallest to largest (Figure 1): The smallest clone detected at 10x10E-4 is comprised of 1311 events. If a theoretical lower limit of quantitation of 50 events or 5x10E-6 in 10 000 000 total cells analyzed is required, this method will meet this criteria .Notably, all bone marrow specimens of adequate quality (not clotted, non-hemodilute) required less than 1.5mL of bone marrow to achieve > 10 000 000 nucleated cells in the final analysis. Analysis is complex using several dozen plots. Plasma cells are identified using CD38 and CD138. Gated plasma cells are analyzed for the immunophenotype of CD56, CD117, CD27, CD45, CD81 and cytoplasmic light chains simultaneously using n-dimensional radar plots (Figures 2, 3a, 3b): Qualitative results can be calculated from adjusted gates (Table 2): Conclusion: This rapid, high-sensitivity assay for immunophenotypically abnormal and clonal plasma cells requires low volumes of bone marrow. Results are ready in approximately 4 hours which is a distinct advantage and sensitivity can be shown to reach 5 X10-6. Disclosures Foley: Amgen,CelgeneJanssen: Honoraria. Mian:Takeda: Consultancy, Honoraria; Sanofi: Consultancy; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy.

scholarly journals The Use of Next Generation Gene Sequencing to Measure Minimal Residual Disease in Patients with AL Amyloidosis and Low Plasma Cell Burden: A Feasibility Study

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4353-4353 ◽  
Author(s):  
Shayna Sarosiek ◽  
Vaishali Sanchorawala ◽  
Mariateresa Fulcinti ◽  
Allison P. Jacob ◽  
Nikhil C. Munshi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Plasma Cells ◽  
Residual Disease ◽  
Al Amyloidosis ◽  
Next Generation ◽  
Minimal Residual

Background: AL amyloidosis is a bone marrow disorder in which clonal plasma cells produce light chains that misfold and deposit in vital organs, such as the kidneys and heart, leading to organ failure and eventual death. Treatment is directed towards the clonal plasma cell population in an effort to halt the production of toxic light chains and recuperate organ function. Pallidini et al. demonstrated that almost 50% of patients with AL amyloidosis who achieved a complete hematologic response to prior therapy had minimal residual disease (MRD) detectable in their bone marrow by multiparametric flow cytometry (MPF).1. Next generation gene sequencing (NGS) has been a successful tool in measuring MRD among patients with multiple myeloma2 though the data regarding its use in AL amyloidosis are limited. AL amyloidosis is a disease with a much smaller plasma cell burden at baseline (typically 5-10%), making the task of isolating an initial clonal sequence even more challenging. We sought to evaluate NGS as a method of isolating a clonal population of plasma cells among patients with systemic AL amyloidosis in a first-ever feasibility study. Methods: Patients were eligible if they had systemic AL amyloidosis and no clinical evidence of concurrent active multiple myeloma. In this study, feasibility was deemed successful if discovery of a clone could be achieved in 3 out of 10 of patients. Approximately five cc's of peripheral blood and bone marrow aspirate were collected from each patient and processed for CD138 selection and DNA isolation/purification. De-identified samples were sent to Adaptive Biotech Inc. (Seattle, WA) for initial clonal identification using the ClonoSEQ immunoglobulin heavy chain (IGH) assay. Genomic DNA was amplified by implementing consensus primers targeting the IGH complete (IGH-VDJH) locus, IGH incomplete (IGH-DJH) locus, immunoglobulin κ locus (IGK) and immunoglobulin l locus (IGL). The amplified product was sequenced and a clone identified based on frequency. After proof of feasibility in the first 10 patients an additional 27 patients had initial clonal identification via the same process mentioned above. Results: In total, 37 patient samples underwent NGS via the ClonoSEQ IGH assay method. The median patient age was 66 years old (range: 44 to 83), 24% of which were female. All 37 patients had measurable disease based on serum electrophoresis and immunofixation and/or serum free light chain assay (Table 1). Four patients had no monoclonal protein detected on SIFE or UIFE and 13 patients had a normal sFLC ratio. Of the 33 patients with monoclonal disease on immunofixation, 12 patients had only a free lambda monoclonal protein and the remaining 21 patients had a clonal heavy chain with an associated light chain. Bone marrow biopsies demonstrated clonal plasmacytosis of 40% or lower. ClonoSEQ IGH assay identified trackable clones in 31 of 37 patients (84%) (see Table 1). Four patients had at least one trackable sequence (range: 1 to 5 sequences) in the peripheral blood and 29 patients had at least one trackable sequence in the bone marrow aspirate (range: 1 to 7 sequences). No correlation was seen between the detection of a clone and standard measures of plasma cell tumor burden (SIFE, SPEP, UIFE, UPEP, and sFLCs). Conclusion: NGS was successful in identifying an initial clone in 29 of 37 patients with systemic AL amyloidosis, four of which were detectable in the peripheral blood. Due to the low clonal burden in patients with AL amyloidosis, it is often difficult to assess disease status, especially post-treatment. These encouraging results may enhance disease monitoring and improve patient care in this rare disease. We are currently tracking MRD in the patients with identifiable clones as they receive systemic treatment, the results of which will be available for presentation in December 2019. REFERENCES 1. Palladini G, Massa M, Basset M, Russo F, Milani P, Foli A, et al. Persistence of Minimal Residual Disease By Multiparameter Flow Cytometry Can Hinder Recovery of Organ Damage in Patients with AL Amyloidosis Otherwise in Complete Response. Abstr 3261. 2016; 2. Ladetto M, Brüggemann M, Monitillo L, Ferrero S, Pepin F, Drandi D, et al. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders. Leukemia. 2014;28:1299-307. Table 1 Disclosures Sarosiek: Acrotech: Research Funding. Sanchorawala:Proclara: Consultancy, Honoraria; Takeda: Research Funding; Caelum: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Prothena: Research Funding; Celgene: Research Funding. Jacob:Adaptive Biotechnologies: Employment, Other: shareholder. Munshi:Amgen: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy.

scholarly journals Evaluation of Hematopoietic Stem Cell Product As a New Site for Minimal Residual Disease By Next Generation Flow in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4927-4927
Author(s):  
Herbert Henrique de Melo Santos ◽  
Glaciano Ribeiro ◽  
Allan de souza Santos ◽  
Marcos Chaves ◽  
Joanna Leal ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Stem Cell ◽  
Minimal Residual Disease ◽  
Hematopoietic Stem Cell ◽  
Plasma Cell ◽  
Residual Disease ◽  
Next Generation ◽  
Hematopoietic Stem ◽  
Minimal Residual

Abstract Introduction- Next generation flow (NGF) is one of the approaches for testing multiple myeloma (MM) minimal residual disease (MRD) over conventional response assessments. Actually, bone marrow (BM) is the preference site of evaluation because of its sensitivity. Because of its invasively technic, other possible sites for MRD evaluation outside the BM have been studied. In the present study we analyzed the MRD between the BM and the hematopoietic stem cell collected product (HSC product), once the concentration of plasma cell in the HSC product could be higher than peripheric blood sample. Aims- To compare MRD quantification of plasma cell between BM and HSC product after induction from Newly Diagnosed MM(NDMM) Transplant Eligible (TE) patients (pts) exposed to daratumumab, cyclophosphamide, thalidomide and dexamethasone (Dara-CTD) protocol. Methods- The SC product and BM samples were collected after four 28 days cycles of induction therapy from pts treated with Dara-CTd protocol described before by (Crusoe E. et al. Blood 2020; 136 (supplement 1): 17-18). MRD was evaluated by next-generation flow (NGF) based in the EuroFlow® protocol. EuroFlow standards was used to identify clonality and aberrant PC immune phenotype, consisting by EuroFlow 8-color 2-tube method (MM MRD kit, Cytognos, Salamanca), with the acquisition of 5 million events each tube and then merged into a single analysis tube on approximately 10 million events. Plasma cells were identified by CD38 multiepitope and CD138. Other markers were used to detect abnormal phenotypes. For comparison of MRD results, Bland-Altman plot comparing BM-MRD and HSC product-MRD was performed. Results- The first pts was enrolled in November 2018. A total of 24 pts were included, the median age was 60 (range 37- 67 years), 23 (92%) were non-white, 5 (21%) had an R-ISS = 1, 12 (54%) had an R-ISS = 2 and 4 (16%), an R-ISS = 3. Six (25%) pts had high-risk chromosomal abnormalities [del17p, t(4;14) or t(14;16)]. To date, all pts have completed induction and 20 have received transplant. Regarding response rates, after the end of induction (cycle 4), 19 (90%) of the pts obtained > PR and 8 (38%) obtained >VGPR, including three MRD negativity by NGF. 19 pts were analyzed for MRD. Negative MRD in sensitivity <10 -5, >=10 -5 and <10 -4, >=10 -4 evaluated in bone marrow was 4/19(21%), 4/19(21%), 11/19(58%) respectively. Negative MRD in sensitivity <10 -5, >=10 -5 and <10 -4, >=10 -4 evaluated in the HSC product was 13/19(68%), 3/19(16%), 3/19(16%) respectively. Median bone marrow sensitivity 10 -4 lower quartile 10 -5 upper quartile 10 -3. Normal distribution of the differences between BM and SC product MRD was first assessed (Kolmogorov-Smirnov's p < 0.001, n = 19). Discussion-Conclusions- The use of HSC product could enhance the plasma cell concentration and may be an alternative and attractive method for MRD detection that diminished the invasiveness of repetitive bone marrow aspirations and tackling the heterogeneity distribution of MM cells. In this preliminary data the sample size did not allow to show a direct correlation between BM and HCS product. A larger sample would be needed to confirm the hypothesis. Figure 1 Figure 1. Disclosures Hungria: Amgen, BMS, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending meetings/travel ; Abbvie: Honoraria; Sanofi: Honoraria, Other: Support for attending meetings/travel ; Takeda: Honoraria. De Queiroz Crusoe: Janssen: Research Funding.

scholarly journals Participation in the College of American Pathologists Laboratory Accreditation Program Decreases Variability in B-Lymphoblastic Leukemia and Plasma Cell Myeloma Flow Cytometric Minimal Residual Disease Testing: A Follow-up Survey

2020 ◽  
Author(s):  
Meghan M. Hupp ◽  
Christine Bashleben ◽  
Jolene L. Cardinali ◽  
David M. Dorfman ◽  
William Karlon ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Proficiency Testing ◽  
Plasma Cell ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Laboratory Accreditation ◽  
Plasma Cell Myeloma ◽  
Accreditation Program ◽  
Minimal Residual

Context.— Minimal residual disease (MRD) testing by flow cytometry is ubiquitous in hematolymphoid neoplasm monitoring, especially B-lymphoblastic leukemia (B-ALL), for which it provides predictive information and guides management. Major heterogeneity was identified in 2014. Subsequently, new Flow Cytometry Checklist items required documentation of the sensitivity determination method and required lower level of detection (LLOD) inclusion in final reports. This study assesses Laboratory Accreditation Program (LAP) participation and new Checklist items' impact on flow cytometry MRD testing. Objectives.— To survey flow cytometry laboratories about MRD testing for B-ALL and plasma cell myeloma. In particular, enumerate the laboratories performing MRD testing, the proportion performing assays with very low LLODs, and implementation of new Checklist items. Design.— Supplemental questions were distributed in the 2017-A mailing to 548 flow cytometry laboratories subscribed to the College of American Pathologists FL3 Proficiency Testing Survey (Flow Cytometry–Immunophenotypic Characterization of Leukemia/Lymphoma). Results.— The percentage of laboratories performing MRD studies has significantly decreased since 2014. Wide ranges of LLOD and collection event numbers were reported for B-ALL and plasma cell myeloma. Most laboratories determine LLOD by using dilutional studies and include it in final reports; a higher proportion of LAP participants used these practices than nonparticipants. Conclusions.— Several MRD testing aspects vary among laboratories receiving FL3 Proficiency Testing materials. After the survey in 2014, new Checklist items were implemented. As compared to 2014, fewer laboratories are performing MRD studies. While LLOD remains heterogeneous, a high proportion of LAP subscribers follow the new Checklist requirements and, overall, target LLOD recommendations from disease-specific working groups are met.

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