Validation of Minimal Residual Disease Flow Cytometry Method for Residual Disease Monitoring in Chronic Lymphocytic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4196-4196
Author(s):  
Shabnam Tangri ◽  
Annalee Estrellado ◽  
Julie Ranuio ◽  
Elisa Romeo ◽  
Jonathan Lawson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Light Chain ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Normal Cells ◽  
Chain Analysis ◽  
Minimal Residual

Abstract Monitoring Minimal Residual Disease (MRD) of Chronic Lymphocytic Leukemia (CLL) patients who achieved complete remission has been difficult and challenging using flow cytometry detection methods in peripheral blood Earlier flow cytometry methods described for detection of MRD relied on the assessment of CD20 expression that maybe compromised during rituximab therapy (CD5/19/20/79B and light chain analysis) or were done using the classic CLL staining panel (CD5/19/23 and light chain analysis) that had a low sensitivity. To facilitate the development of methods that would be suitable for rituximab containing regimens and have better sensitivity, a panel of antibody combinations was identified for use in an internationally standardized flow cytometric approach for MRD detection in CLL-treated patients (Rawstron et al., 2007). This panel consists of 5 combinations of antibodies specific for 1-sIgKappa/sIgLambda/CD19/CD5, 2-CD45/CD14/CD19/CD5, 3-CD43/CD79b/CD19/CD5, 4-CD22/CD81/CD19/CD5 and 5-CD20/CD38/CD19/CD5 antigens. The first two combinations are utilized for confirmation of clonality and assessment of Tcell contamination rate within the B cell gate, whereas combinations 3, 4 and 5 are specific for MRD detection. This standardized 5-combination panel was technically validated by Genoptix Medical Laboratory for use in BiogenIdec’s clinical trials in CLL with Lumiliximab. To validate this panel assay, selected CLL samples were mixed with normal donor blood or “disease-free” bone marrow specimens, to achieve 1%, 0.1%, 0.05% and 0.01% of CLL cells in “non-CLL” leukocytes. Our results show that it is possible to identify up to 1 CLL cell in 10,000 normal cells in some but not all cases, and that on a consistent basis our analysis is able to identify 1 CLL cell in 1000 normal cells. In comparison, the classic screening panel commonly used to diagnose CLL has a limit of detection (LOD) of about 1% (1 leukemic cell in 100 normal cells); thus this new method provides a 10 to 100 fold improvement in sensitivity. The enhanced sensitivity and LOD of the new assay is mainly due to the reduction of the non-CLL cell background in peripheral blood and bone marrow Furthermore, to make analysis guidelines consistent among analysts, an internal semi-quantitative clustering scoring analysis system was developed for this assay. The cluster scoring scheme assigns a negative score to scattered events with no clearly visible clone; well grouped events with tight visible clones are assigned positive scores and those gated events are considered for MRD final call. To reliably define the presence of MRD, at least 2 of the 3 MRD specific antibody combinations must have a positive clustering score. Due to the small number of cells to be analyzed, collection of 500,000 events is recommended. Finally, the results of additional studies showed that the anticoagulant employed may impact the stability of the antigens over a period of 7 days, and for accurate MRD determination blood specimens should be drawn into Heparin vacutainer tubes when long shipping times are expected.

Safety and Efficacy Of Abbreviated Induction With Only 4 Cycles Of Fludarabine (F), Cyclophosphamide (C) and Rituximab (R) In Physically Fit Patients With Chronic Lymphocytic Leukemia (CLL) Who Achieve Early Complete Remission (CR) With Undetectable Minimal Residual Disease (MDR)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2886-2886
Author(s):  
Carolina Pavlovsky ◽  
Astrid Pavlovsky ◽  
Isolda Fernandez ◽  
Adriana Galeano ◽  
Francisco Lastiri ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Minimal Residual Disease ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Clinical Stage ◽  
Progression Free Survival ◽  
Lymphocytic Leukemia ◽  
Blue Laser ◽  
Minimal Residual

Abstract Background Chemoimmunotherapy with 6 cycles of FCR is considered standard therapy for physically fit patients (pts) with Chronic Lymphocytic Leukemia (CLL). Many pts are unable to complete planned treatment, due to treatment related complications. Levels of minimal residual disease (MRD) have been shown to correlate with PFS in previously untreated patients with CLL (CLL8, Boettcher S et al. Leukemia, 2009). Achieving a negative MRD is therefore a mayor endpoint in treatment. Patients and methods From 4/2003, 39 physically fit pts with CLL who had IWCLL-NCI criteria for initiating treatment started therapy with FCR in our institution. Eleven pts had previously received chlorambucil/prednisone and 28 were not previously treated. Median age at start of therapy was 63 years (34-80), Binet´s clinical stage were A/B: 22 pts (56%) and C: 17 (44%). The CD38 expression was positive (>7% of cells) in 23 (59%) and negative in16 (41%) of the pts. After 4 courses of FCR response was assessed in peripheral blood (PB) or bone marrow (BM) using three colour flow Cytometry. Negative MRD was defined as < 0,1% of light chain restricted CD5+CD19+ B cells in PB and BM as assesed collecting 100000 CD19 cells in a three colour cytometer (FacsScalibur- blue laser ). All these patients stopped therapy after evaluation due to early CR with eradication of MRD. Results All patients had negative MRD in peripheral blood, 35 were also evaluated in bone marrow, 29 showed CR and 6 nodular partial remission (NPR). Neutropenia and infectious events grade 3-4 were observed in 24% and 7% of all the courses respectively. No pts died of toxicity. After a median follow-up of 81 months (4.6-120), progression free survival (PFS) and overall survival (OS) at 72 months was 51% and 75% respectively. Five pts died of progressive disease and 3 of a secondary neoplasm. Conclusion Stopping therapy in patients who achieve negative MRD after 4 cycles of FCR is safe and induces durable remission with a PFS and OS of 51% and 75% at 72 months exposing them to less chemotherapy. Large randomized trials are necessary to confirm this data. Disclosures: Pavlovsky: Novartis: Speakers Bureau; BMS: Speakers Bureau.

Bone Marrow May Be More Informative Than Peripheral Blood To Evaluate Minimal Residual Disease During 1st and 2nd Year Follow Up After First-Line Fludarabine, Cyclophosphamide, and Rituximab For Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1621-1621
Author(s):  
Paolo Strati ◽  
Michael J. Keating ◽  
Susan O'Brien ◽  
Jan A. Burger ◽  
Alessandra Ferrajoli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Response Assessment ◽  
Lymphocytic Leukemia ◽  
First Line ◽  
Final Response ◽  
Minimal Residual

Abstract Background Minimal Residual Disease (MRD) status at end of first-line chemoimmunotherapy is an independent prognostic factor for patients (pts) with chronic lymphocytic leukemia (CLL). In the CLL8 trial of the German CLL Study Group, peripheral blood (PB) was monitored for MRD during follow up. Because the microenvironment is important for CLL cell growth and survival and typically it is the last site to eliminate residual disease with chemoimmunotherapy, bone marrow (BM) might be a more reliable site to monitor MRD. Methods Two-hundred thirty-seven pts with CLL and an indication for therapy (IWCLL-WG 2008) received first-line fludarabine, cyclophosphamide, and rituximab (FCR) on protocol between 09/2008 and 09/2012. MRD was prospectively assessed in BM and/or PB by flow cytometry using the highly sensitive international standardized approach, 2 months after the last course of treatment (final response assessment) and every 3-6 months thereafter. Kaplan-Meier estimates were compared using the log-rank test. Results Sixty-one percent of pts were male, 21% were >65 years old, 40% had Rai stage III-IV, 41% had beta2-microglobulin (B2M) ≥4 mg/L, 61% had unmutated IGHV, and 21% had FISH analysis positive for deletion 11q and 7% for deletion 17p. Seventy-five percent of pts received ≥3 total courses of FCR. The complete remission (CR) and overall response (OR) rates were 65 and 97%, respectively. BM MRD negativity was achieved in 59% of pts at final response assessment. For monitoring, BM MRD was assessed in 121 pts during the 1st year and in 30 pts during the 2nd year after completion of treatment with FCR; all samples were serial. PB MRD was assessed in 106 pts during the 1st year and in 57 during the 2nd year of follow up; again all samples were serial. BM MRD negativity was observed in 63 (52%) pts during the 1st year of follow up and in 15 (50%) pts during the 2nd year. PB MRD negativity was observed at the same staging times in 81 (76%) and 29 (51%) pts, respectively. Concurrent BM and PB samples were taken during the 1st year in 51 pts, and in 6 pts during the 2nd year of follow up. We evaluated the association between MRD negativity during the 1st and 2nd year of follow-up and progression-free survival (PFS). BM MRD positive status was associated with shorter PFS when assessed during both the 1st and 2nd year of follow up (p<0.001 and p=0.001, respectively; Figure). In contrast, PB MRD positive status did not correlate with PFS for either time (p=0.15 and p=0.79, respectively; Figure). Conclusions After first-line FCR for pts with CLL, positive BM MRD may identify pts at higher risk for progression. Based on this finding, BM may be preferred to assess MRD status and pts with positive BM MRD could be considered for maintenance or consolidation strategies. Additional studies confirming these findings are warranted. Disclosures: No relevant conflicts of interest to declare.

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.

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