ROR-1 Is a Highly Discriminative Marker in Flow Cytometric Minimal Residual Disease (MRD) Detection in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3197-3197 ◽  
Author(s):  
Michaela Patz ◽  
Barbara Pentok ◽  
Kathrin Cremer ◽  
Stefanie Linnartz ◽  
Esther Lilienweiss ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Research Funding ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Cell Populations ◽  
Discrimination Analysis ◽  
Flow Cytometric ◽  
Correct Discrimination ◽  
Minimal Residual

Abstract Introduction:With the advent of new potent therapies for chronic lymphocytic leukemia (CLL) minimal residual disease (MRD) detection becomes increasingly important to assess remission depth. While molecular MRD detection for CLL remains laborious and time consuming flow cytometry is a fast, economic and sensitive method in detecting low frequencies of CLL cells. The usefulness of the antigens CD81, CD5, CD20, CD43 and CD79b has been previously described for this purpose. ROR-1 has recently been identified as a signature gene in CLL and mantle cell lymphoma. The potential utility of ROR-1 in flow cytometric minimal residual cell analysis has not been evaluated yet. Methods: 10 normal samples and 77 remnants of randomly selected samples from diagnosed patients undergoing CLL therapy were analyzed by flow cytometry. A customized dry formulation of an antibody panel was used, comprising antibodies directed against CD5, CD19, CD20, CD43, CD45, CD79b, CD81 and ROR-1 (DuraClone RE CLB). Linearity, repeatability and inter-operator variability of data analysis of the method were examined. B cell populations comprising at least 50 positive events (46 normal B cell populations, 25 CLL populations, paired and unpaired) were analyzed for their expression profile as assessed by respective mean fluorescence intensities of the antibody labels within classified populations. The expression profiles were subject to supervised discrimination analysis (DA). Results: Between124,000 and 2,122,000 (683,000 ± 450,000) CD45+ events were acquired from the 87 samples. The background of cells with a CLL-like phenotype in the normal samples was determined as <0.001% of CD45+ events. Linearity was confirmed in the range from 1% to 0.0025%. The Repeatability analysis and the inter-operator variability showed concordance with typical Poisson distribution characteristics. The 46 populations with a typical normal B cell phenotype ranged from 0.014% to 9.592% with an average of 2.45% ± 2.75 of CD45+ events. The 25 populations with a classical or non-classical CLL phenotype ranged from 0.007% to 5.459% with an average of 1.41% ± 1.65 of CD45+ events. Posterior discrimination analysis revealed 100% correct discrimination for CLL populations and 96% correct discrimination for normal populations when relying on ROR-1 expression alone in CD19+CD45+ B cells. This result was only surpassed by the complete antibody combination (100% / 100%) but not by any other of the markers, neither in single use nor in combination Conclusion: The 8-color dry flow cytometry panel comprising CD5, CD19, CD20, CD43, CD45, CD79b, CD81 and ROR-1 demonstrated sensitive, linear and specific detection of residual CLL cells in a relevant low range of frequency. ROR-1 revealed to be a highly discriminative marker in the analysis of residual CLL cells by flow cytometry. Utilizing this flow cytometry approach, MRD detection showing sensitivity comparable to molecular techniques can be achieved in CLL. Disclosures Hallek: AbbVIe: Consultancy, Honoraria; Mundipharma: Consultancy, Honoraria; Glaxo-SmithKline: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria; Roche: Consultancy, Research Funding, Speakers Bureau. Kreuzer:Gilead Sciences: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche Pharma GmbH and Mundipharma GmbH: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Combined ROR1 and CD160 Detection For Improved Minimal Residual Disease In Patients With Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2572-2572 ◽  
Author(s):  
Timothy W. Farren ◽  
Fengting Liu ◽  
Marion G. Macey ◽  
Thomas J. Kipps ◽  
Noel Warner ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Research Funding ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Flow Cytometric Assay ◽  
Single Tube ◽  
Flow Cytometric ◽  
Sequential Gating ◽  
Minimal Residual

Abstract Introduction Over the past decade, treatments for patients with chronic lymphocytic leukemia (CLL) have produced complete remissions (CR) without evidence for minimal residual disease (MRD), particularly for younger and/or fitter patients. In this setting, achieving an MRD-negative CR has prognostic implications, yielding longer progression free survival (PFS) and overall survival (OS) than for patients who achieve a CR with persistent MRD. Complicating efforts to incorporate testing for MRD in clinical practice has been the lack of a defined consensus on the methods of MRD detection. In this study, we report on a novel combination of mAbs for MRD detection by flow cytometry based on two antigens: the NK-cell receptor and tumor specific antigen, CD160; and the tumor associated antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR-1). Objective To compare a novel single-tube, tumor-specific (CD160+ROR1) targeted approach to MRD detection against the previously published CD160 flow cytometric assay (CD160FCA) (Farren et al, 2011) and the new, single-tube 8-color ERIC assay. Methods Between October 2012 and July 2013, prospective assessment of MRD was performed on peripheral blood in 56 patients (86 samples). We developed a flow cytometric assay using mAbs specific for CD160 or ROR1 (Fukuda et al, 2008). For this we used the following mAb from BD Biosciences: CD2 FITC, CD5 Pe-Cy7, CD19 PerCP5.5, CD45V500, CD160PE, ROR-1 AF647 (“ROR-160FCA”) and a sequential gating strategy. This was compared with CD160FCA and the 8-color ERIC consortium protocol (unpublished). Light chain analysis (LCR) was performed in all cases and reported where detectable. A proof of concept spiking experiment simulating MRD was prepared by mixing CLL and normal peripheral blood leukocytes in a serial dilution to a level of 10-5(n=3). Statistical analysis was performed using Spearman Rank correlation coefficients, Mann-Whitney t-test, and Bland-Altman method comparison. Significance was set at <0.05%. Results To establish the proof of principle, MRD levels ranging from 0.001% to 100% (Neat CLL) were prepared by serial dilutions, in which MRD levels could be established by the ROR-160FCA to 10-5 (n=3). Assessment of the observed incidence against expected incidence of CLL MRD demonstrated a highly significant correlation (R2=0.96, p=0.01). In the study, the range of detectable disease went from <0.01% to 38.59%, of which 37% of samples had levels below <0.01%. Analyzing all flow cytometric methods, a highly significant correlation was observed between all three: CD160FCA vs ERIC: Spearman R=0.96 (95%CI: 0.93 - 0.97, p<0.001); CD160FCA vs ROR-160FCA: Spearman R=0.97 (95%CI: 0.96 – 0.99, p<0.001); ROR-160FCA vsERIC: Spearman R=0.97 (95%CI: 0.94 – 0.98, p<0.001). 54 samples had levels of disease <1%. Bland-Altman assay comparison in these patients again demonstrated significant associations between the assays (CD160FCA vs ERIC: mean 0.08 ±0.15; CD160FCA vs ROR-160FCA: mean 0.04 ±0.19; ROR-160FCA vs ERIC: mean 0.03 ±0.25). Light chain restriction was detectable in 24 patients (size of the restricted population ranged from 0.2% to 47% of all cellular events). This sub-group of patients also demonstrated excellent correlation between level of LCR and detectable disease by CD160FCA (Spearman R=0.96, 95%CI: 0.92-0.98, p<0.001), ERIC (R=0.95, 95%CI: 0.92-0.98, p<0.01) and ROR-160FCA (R=0.97, 95%CI 0.93-0.98, p<0.001). Conclusion Monitoring minimal residual disease in CLL is a key focus for clinical trials, as MRD is an important prognostic marker in CLL in terms of PFS and OS. Here we provide a single tube assay, ROR-160FCA, which is unique by targeting two antigens restricted to malignant B-cells, CD160 and ROR1. ROR-160FCA is equivalent in MRD detection compared to both CD160FCA and the current ERIC assay under development. The two tumor-specific antigens give ROR-160FCA the potential for improved sensitivity, particularly where limited sample is available. Furthermore, it only requires a simple sequential gating strategy, is rapid, and appears more cost effective than other Methods. References Farren TW, Giustiniani J, Liu FT et al. Blood. 2011;118 (8):2174-2183. Fukuda T, Chen L, Endo T et al. Proc Natl Acad Sci U S A. 2008;105 (8):3047-3052. Disclosures: Farren: BD Biosciences: Research Funding. Warner:BD Biosciences: Employment, Research Funding. Agrawal:BD Biosciences: Research Funding.

scholarly journals Orphan receptor ROR1 for detection of minimal residual disease in chronic lymphocytic leukemia

2020 ◽  
pp. 19-24
Author(s):  
Yu. V. Mirolyubova ◽  
N. S. Timofeeva ◽  
V. A. Bart ◽  
V. M. Solovyov ◽  
E. V. Tolstopyatova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
High Expression ◽  
Color Flow ◽  
B Cell Precursors ◽  
Minimal Residual

Background and Aims. The detection of minimal residual disease (MRD) of chronic lymphocytic leukemia (CLL) using multicolor flow cytometry has been widely used in clinical studies to evaluate the effectiveness of treatment. The method is being improved by searching for the most sensitive and specific markers for use in panels for 6–8 color cytometers. According to published data, ROR1 shows high expression on CLL cells, and lack of expression on mature lymphocytes, which distinguishes it from other markers used to detect MRD in CLL.Aim: to determine a significance of ROR1 for detection of MRD CLL by flow cytometry in a 4-color panel.Materials and Methods. We analyzed 64 bone marrow samples of 37 patients with a verified diagnosis of CLL after the 3rd and 6th cycles of therapy according to bendamustine and rituximab (BR) regimen – 15 MRD-negative and 49 MRD-positive. Quantitative determination of MRD was carried out by the standardized method of 4-color flow cytometry according to the recommendations of ERIC (European research initiative on CLL) with the inclusion of ROR1 in the diagnostic panel. A discriminatory analysis of the differentiating properties of diagnostic markers was performed using Statistica 10.Results. ROR1 has demonstrated high differentiating properties on CLL cells and mature lymphocytes. All the analyzed samples showed a bright monomorphic expression of ROR1 on CLL cells and B-cell precursors, and the absence of its expression on mature lymphocytes.Conclusion. ROR1 is a highly specific and sensitive marker for the detection of CLL cells among mature lymphocytes. The high expression of ROR1 on normal B-cell precursors requires the use of ROR1 in combination with a marker that differentiates CLL cells from progenitor cells (CD81).

scholarly journals A 10-Color Flow Cytometry Panel for Both Diagnosis and Minimal Residual Disease Measurement in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5451-5451
Author(s):  
Alexandre Bazinet ◽  
Ryan N Rys ◽  
Claudia M Wever ◽  
Amadou Barry ◽  
Celia Greenwood ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Residual Disease ◽  
Cost Savings ◽  
Lymphocytic Leukemia ◽  
Potential Cost ◽  
Advisory Committees ◽  
Color Flow ◽  
Minimal Residual

Background: Chronic lymphocytic leukemia (CLL) has a classic immunophenotype, consisting of light chain restriction, CD5+, CD19+, dim CD20, CD23+, CD43+, CD200+, CD10- and CD79b-. This distinguishes it from normal B cells and other lymphoproliferative disorders (LPDs). Antibodies targeting these antigens are included in two 8-color flow cytometry panels developed by the Euroflow consortium for the work up of B cell LPDs. Combining these antibodies into one 10-color panel would be more cost-effective. Furthermore, new CLL therapies can induce deep remissions, creating an increasing demand to measure minimal residual disease (MRD), defined as having over 1 residual leukemic cell per 10,000 leukocytes (10-4). The current international standardized approach for measuring MRD established by the European Research Initiative on CLL (ERIC) uses a panel of antibodies targeting CD3, CD5, CD19, CD20, CD22, CD43, CD79b and CD81. However, these antibody-fluorochrome combinations are different than those used by the Euroflow diagnostic panels. Thus laboratories considering implementing MRD testing would need to purchase antibodies for 3 different panels (2 diagnostic and 1 MRD). We expanded the Euroflow 8-color lymphocyte screening tube (LST) to include CD200 and CD23, such that CLL can be detected in one 10-color tube, at levels as low as 0.01%. The goal of this study was to determine the potential cost savings in implementing this new panel and to determine if it is sensitive enough to detect MRD. Methods: We calculated the number of samples analyzed with our modified 10-color LST tube (mLST1, obtained lyophilized) from April 2018 to March 2019 to rule out an LPD and the number of antibody aliquots saved using this approach compared to the standard 2-tube Euroflow method. We also created a version of the above-mentioned panel (mLST2) using liquid antibodies to increase the generalizability of our results, substituting CD38 with CD43 to see if this improved MRD detection (see panels below). For MRD testing, we used CLL samples from 24 different patients to produce 60 MRD samples at various concentrations of leukemic cells. Samples were prepared by spiking CLL cells into suspensions of normal leukocytes at approximate concentrations of 0.1%, 0.01% and 0.001%. Each sample was aliquoted and stained with the three panels: ERIC, mLST1 and mLST2. Data was acquired using a BD FACSCanto II or a BD FACSAria Fusion and analysed using BD FACSDiva software. CLL cells were identified based on differential expression of key markers and MRD was calculated as the number of CLL cells/total leukocytes. MRD positivity was defined as ≥ 0.01%. Agreement between the panels was assessed using the Bland-Altman plot method. We also calculated the percentage agreement between the panels in identifying MRD positivity. Results: In 1 year, mLST1 was performed on 474 samples, of these 220 had an LPD and 123 (56%) had a classic CLL phenotype, obviating the need for further testing. This resulted in the net savings of 476 antibody aliquots. For MRD assessment, differential expression of CD5 and CD20 were the most significant contributors in distinguishing CLL from normal B cells using the mLST1 and mLST2. We identified one CLL case with an atypical immunophenotype (dim CD5, bright CD20) which proved difficult to gate using a mLST panel. There was agreement in MRD results obtained with the mLST panels and the ERIC panel. For values above the limit of quantification, the 95% limit of agreement was ±0.3369 log for the ERIC vs mLST1 comparison and ±0.3485 log for the ERIC vs mLST2 comparison. Thus, variability in MRD levels between the panels was less than 2-fold the majority of the time, which we considered clinically acceptable as MRD is measured on an exponential scale. The ERIC panel and the mLST1 had 88.3% agreement in distinguishing MRD-positive versus MRD-negative samples. Agreement was 93.3% between the ERIC panel and the mLST2. Conclusions: Using a modified 10-color LST panel for both diagnosis and MRD measurement of CLL is feasible. The advantages are increased familiarity with the antibodies and potential cost savings, making MRD accessible to more cytometry laboratories. Atypical CLL cases without the usual CD5 positivity and dim CD20 are very difficult to gate using an LST panel. In these cases, the ERIC panel is clearly superior as CD22, CD79b, CD81 and CD43 can still provide separation between the malignant and normal lymphocytes. Disclosures Bazinet: BD Biosciences: Other: Provided a significant amount of the antibodies used in this project free of cost.. Wever:Teva Canada Innovation: Employment. Gimmig:BD Biosciences: Employment. Johnson:Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Merck: Consultancy, Honoraria; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; BMS: Consultancy, Honoraria; Seattle Genetics: Honoraria.

Real-World Experience with Minimal Residual Disease Testing with Next Generation Flow Cytometry and Functional Imaging in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
David Böckle ◽  
Paula Tabares Gaviria ◽  
Xiang Zhou ◽  
Janin Messerschmidt ◽  
Lukas Scheller ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
High Risk ◽  
Functional Imaging ◽  
Real World ◽  
Research Funding ◽  
Residual Disease ◽  
Focal Lesions ◽  
High Risk Disease ◽  
Minimal Residual

Background: Minimal residual disease (MRD) diagnostics in multiple myeloma (MM) are gaining increasing importance to determine response depth beyond complete remission (CR) since novel agents have shown to induce high rates of deep clinical responses. Moreover, recent reports indicated combining functional imaging with next generation flow cytometry (NGF) could be beneficial in predicting clinical outcome. This applies in particular to the subset of patients suffering from relapsed/refractory multiple myeloma (RRMM) who tend to show a higher incidence of residual focal lesions despite serological response. Here, we report our institutions experience with implementing both functional imaging and NGF-guided MRD diagnostics in clinical practice. Methods: Our study included patients with newly diagnosed multiple myeloma (NDMM) and RRMM achieving VGPR, CR or sCR. Bone marrow aspirates were obtained for MRD-testing according to IMWG 2016 criteria. Samples were collected between July 2019 and July 2020 and analyzed with NGF (according to EuroFlowTM guidelines) at a sensitivity level of 10-5. Results were compared to functional imaging obtained with positron emission tomography (PET) and diffusion-weighted magnetic resonance imaging (DW-MRI). High-risk disease was defined as presence of deletion 17p, translocation (14;16) or (4;14). Results: We included 66 patients with NDMM (n=39) and RRMM (n=27) who achieved VGPR or better. In patients with RRMM the median number of treatment lines was 2 (range 2-11). Fifteen patients suffered from high-risk disease. Median age at NGF diagnostics was 64 years (range 31-83). Among patients achieving VGPR (n=27), CR (n=10) and sCR (n=29) seventeen (26%) were MRD-negative by NGF testing. CR or better was significantly associated NGF MRD-negativity (p=0.04). Notably, rates of NGF MRD-negativity were similar among patients with NDMM (28%) and RRMM (26%). Even some heavily pretreated patients who underwent ≥ 4 lines of therapy achieved MRD-negativity on NGF (2 of 9). Functional imaging was performed in 46 (70%) patients with DW-MRI (n=22) and PET (n=26). Median time between NGF and imaging assessment was 2 days (range 0-147). Combining results from imaging and NGF, 12 out of 46 (26%) patients were MRD-negative with both methods (neg/neg). Three patients displayed disease activity as measured with both, imaging and NGF (pos/pos). Twenty-nine of the remaining patients were MRD-positive only according to NGF (pos/neg), while two patients were positive on imaging only (neg/pos). More patients demonstrated combined MRD-negativity on NGF and imaging (neg/neg) in the NDMM setting than in RRMM (32% versus 19%). We also observed that 30% of the patients with high-risk genetics showed MRD-negativity on both imaging and NGF. Of note, none of the patients with very advanced disease (≥4 previous lines) was MRD-negative on both techniques. Conclusion In the clinical routine, MRD diagnostics could be used to tailor maintenance and consolidation approaches for patients achieving deep responses by traditional IMWG criteria. Our real-world experience highlights that MRD-negativity can be achieved in patients suffering from high-risk disease and also in late treatment lines, supporting its value as endpoint for clinical trials. However, our data also support MRD diagnostics to be combined with functional imaging at least in the RRMM setting to rule out residual focal lesions. Future studies using MRD for clinical decision-making are highly warranted. Disclosures Einsele: Takeda: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. Rasche:Celgene/BMS: Honoraria; GlaxoSmithKline: Honoraria; Oncopeptides: Honoraria; Skyline Dx: Research Funding; Janssen: Honoraria; Sanofi: Honoraria.

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.

Prognosis Impact of Positive Minimal Residual Disease By Flow Cytometry Prior to Transplant According to the Cut-Off Threshold in Patients with Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-16
Author(s):  
Claudia Nunez-Torron ◽  
Fernando Martin Moro ◽  
Juan Marquet Palomanes ◽  
Miguel Piris-Villaespesa ◽  
Ernesto Roldan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Residual Disease ◽  
Allogeneic Transplant ◽  
Group 3 ◽  
Baseline Characteristics ◽  
Group 2 ◽  
Minimal Residual ◽  
Group 1

Introduction: Patients with Acute Myeloid Leukemia (AML) and positive Minimal Residual Disease (MRD) prior to allogeneic transplant are currently considered to be a group at high risk of relapse. Multiparameter flow cytometry is a standard technique to measure MRD, and generally we use a 0.1% threshold for positivity. The clinical significance of those patients with an MRD levels &gt;0% but &lt;0.1% is uncertain and it is recommended to define the prognosis of this subgroup. Material and methods: We performed a single-center retrospective analysis of 88 patients transplanted between 2012 and 2020. All patients achieved complete remission (CR) with or without hemoperipheral recovery prior to allogeneic transplant. We have divided our cohort into three groups according to MRD state by flow cytometry: Group 1 patients with negative MRD, Group 2 patients with MRD level &gt;0% but &lt;0.1% and Group 3 patients with MRD ≥ 0.1%. The baseline characteristics of each group were compared using the Chi2 test. The survival analysis was performed through Kaplan-Meier method and the risk was calculated with Cox regression. The Overall Survival (OS) was defined as the time from transplantation to death and the Relapse-Free Survival (RFS) as the time from transplantation to either relapse or death. P&lt;0.05 was defined as statistically significant difference. Results: The baseline characteristics of our cohort are reflected in Table 1. We did not find statistical significant differences except for the response to induction. The median follow-up of the entire cohort was 13.5 months (range 6-43.5). The 4-year RFS (4y-RFS) was 47% and the 4-year OS (4y-OS) 50%. The 4y-RFS was 52.5% in Group 1 vs 59% in Group 2 vs 30% in Group 3. The 4y-OS was 60% in Group 1 vs 60% in Group 2 vs 31% in Group 3 (Image 1). The Hazard Ratio (HR) for RFS and OS comparing Group 1 vs Group 2 was 0.9 [95% CI ((0.3-2.5)] and 1.1 [95% CI (0.4-3)] respectively. The HR for the RFS and OS comparing Group 1 vs 3 was 1.2 [95% CI (0.9-1.7)] and 1.2 [95% CI (0.8-1.6)]. We have stratified patients according to the European LeukemiaNet risk classification. In Group 1, the 4y-RFS was 79% in patients with Favorable Risk (FR) vs 55% in those with Intermediate Risk (IR) and 53% in patients with Adverse Risk (AR) [HR 1.2, 95% CI (0.6-2.3)] and the 4y-OS was 79% vs 54% vs 53% respectively [HR 1.3, 95% CI (0.6-2.5)]. In Group 2, the 4y-RFS was 100% in those with FR vs 83% in IR vs 33% in AR [HR 3.9, 95% CI (0.4-30)] and the 4y-OS was 100% vs 82% vs 36% respectively [HR 4, 95% CI (0.5-32%)]. In Group 3, the 4y-RFS in patients with FR was 82% vs 0% in IR vs 0% in AR [HR 2.1, 95% CI (1.1-4.1)] and the 4y-OS was 82% vs 0% vs 0% respectively [HR 1.6, 95% CI (0.8-3.3)] (Image 2). Conclusions: In our cohort, positive MRD &gt;0.1% prior to transplant identified a group with worse RFS and OS compared to those with negative MRD or positive MRD level &gt;0% but &lt;0.1%. Positive MRD &gt;0.1% is especially relevant in the IR and AR groups of the European LeukemiaNet risk classification. In the AR subgroup even any detectable level of positive MRD could identify patients with unfavorable post-transplant OS and RFS outcomes. We must establish post-transplant strategies in these patients to improve survival. Disclosures Garcia-Gutiérrez: Pfizer: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Incyte: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Bristol-Myers Squibb: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Novartis: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding.

Augmentation of Sensitivity of FLT3/ITD Assay Allows Detection of Minimal Residual Disease In Stem Cell Transplant Recipients-Correlation with Flow Cytometric MRD Assessment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1717-1717
Author(s):  
Maya Danielle Hughes ◽  
Rong Zeng ◽  
Kristen L. Miller ◽  
Soheil Meshinchi
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Residual Disease ◽  
Flow Cytometric ◽  
Detection Assay ◽  
Minimal Residual

Abstract Abstract 1717 FLT3 internal tandem duplication (FLT3/ITD) is a somatic mutation that is associated with therapy resistance in acute myeloid leukemia (AML). Early data demonstrated low sensitivity for this assay, thus limiting its utility to the evaluation of diagnostic specimens, and precluding its utility in remission samples. We inquired whether the standard FLT3/ITD assay can be modified to enable its utility to detect presence of residual disease in remission specimens. Enhanced FLT3/ITD assay sensitivity was accomplished by altering annealing temperature, increasing the number of cycles as well as amount and concentration of the product that was subjected to capillary electropheresis. To assess the sensitivity of the enhanced assay, FLT3/ITD positive cells M4V11 were serially diluted in a population of ITD negative cells (HL60). The concentration of M4V11 cells in each sample ranged from 10% to 0.0001%. PCR product was subjected to capillary electropheresis and the appropriate region of the electropherogram was examined for the presence of the appropriate mutant product length. Appropriate FLT3/ITD signal was detected in dilutions down to 0.01%, validating our ability to detect extremely low levels of FLT3/ITD. We subsequently examined the remission marrows from patients with a history of FLT3/ITD who had undergone stem cell transplantation. Available bone marrow specimens (N = 51) from patients who underwent stem cell transplantation for FLT3/ITD-positive AML were analyzed and the result was correlated with the available standard PCR as well as the available MRD assessment by muti-dimensional flow cytometry; samples negative for FLT3/ITD by standard assay (N=11) were then subjected to the enhanced PCR methodology. Available ITD length for each patient was used for examination of the appropriate region of the electropherogram in each case. Of the available 51 bone marrow specimens analyzed, 23 specimens had FLT3/ITD detectable by standard PCR protocol. Using our modified PCR method and capillary electrophoresis, an additional 13 specimens had identifiable FLT3/ITD. In 6/11 patients, where initial FLT3/ITD was negative by standard methodology, enhanced assay identified FLT3/ITD signal. In each case, detection of FLT3/ITD by the enhanced assay was followed by morphologic or immunophenotypic emergence of disease, prompting therapeutic intervention. We further evaluated the ability to detect FLT3/ITD in patients with minimal residual disease by flow cytometry. 33 of the bone marrow specimens analyzed had a less than 5% abnormal blast population as detectable via flow cytometry. Among these samples, 7 had FLT3/ITD detectable using standard detection techniques. An additional 11 samples had detectable FLT3/ITD when our modified protocol was employed. Of the specimens that had less than 1% abnormal blast population as detectable via flow cytometry (N = 27), 4 had FLT3/ITD detectable using the standard detection assay; when our modified protocol was employed, an additional 6 samples had detectable FLT3/ITD. 17 bone marrow specimens had no abnormal blast cells detectable via flow cytometry; of these samples 1 had detectable FLT3/ITD using the standard detection assay, while an additional 3 had detectable FLT3/ITD using our modified assay. In four patients, FLT3/ITD was detected in bone marrow specimens found to have flow cytometric MRD of 0% (N=2), 0.1% (N=1) and 0.4% (N=1). In two patients with no detectable disease by MDF, both had emergence of morphologic (60% blast) or immunophenotypic disease by MDF (1.1%) within 4–6 weeks of detection of FLT3/ITD by enhanced assay. In this study, we demonstrate that simple modifications to the FLT3/ITD genotyping assay significantly increases its sensitivity and provides a highly sensitive and very specific assay for identifying this disease associated mutation in remission specimens. The enhanced assay can be incorporated into the standard evaluation of remission status for patients with FLT3/ITD. Disclosures: No relevant conflicts of interest to declare.

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