As spring blossoms and nature revives, the 15th Experimental Diagnostic Hematology Conference and the 1st Conference on Rare Blood Diseases, hosted by the Chinese Medical Association, were grandly held in the city of Gusu on March 23-24, 2024. The conference invited renowned experts and scholars from the field of hematology both domestically and internationally to deliver enlightening academic presentations and engage in thematic discussions. The aim was to foster academic exchange and cooperation in the fields of experimental diagnostic hematology and rare blood diseases, to share the latest research findings, discuss industry trends, and collectively promote the prosperity of hematology in China. At the conference, Professor Yujie Wu from Jiangsu Provincial People’s Hospital (The First Affiliated Hospital of Nanjing Medical University) delivered an excellent presentation titled “Standardized Flow Cytometry Detection of CLL-MRD,” which is specially compiled here for our readers’ enjoyment.
Chronic Lymphocytic Leukemia (CLL) is an indolent lymphoid tumor characterized by the accumulation of mature B lymphocytes in the peripheral blood, bone marrow, and lymphatic organs, with an incidence that has been rising annually. CLL primarily affects elderly patients, with the peak incidence occurring between the ages of 60 and 80. The advent of novel targeted therapies such as Bruton’s tyrosine kinase inhibitors (BTKis), phosphoinositide 3-kinase inhibitors (PI3Kis), and B-cell lymphoma 2 inhibitors (BCL2is) has ushered in an era of chemotherapy-free treatment for CLL, significantly improving patient prognosis. However, minimal residual disease (MRD) remains a root cause of disease recurrence, closely associated with progression-free survival (PFS) and overall survival (OS). It accurately reflects the disease burden post-treatment and effectively evaluates the depth of remission, thus becoming a critical criterion for treatment endpoints.
To advance the detection and application of MRD in CLL patients, the Chinese Anti-Cancer Association Hematology Oncology Committee, the Hematology Branch of the Chinese Medical Association, and the China Chronic Lymphocytic Leukemia Working Group convened experts to develop the “Chinese Expert Consensus on the Detection and Clinical Interpretation of Minimal Residual Disease in Chronic Lymphocytic Leukemia (2023 Edition)” (referred to as the Chinese Expert Consensus). After two rounds of discussion by the European Research Initiative on CLL (ERIC), with contributions from Professor Yujie Wu among others, this consensus plays a significant role in standardizing CLL-MRD detection methods and enhancing the quality and reproducibility of MRD results in clinical practice.
01 Definition of CLL-MRD
Even if patients with Chronic Lymphocytic Leukemia (CLL) achieve complete remission (CR) after treatment, it does not imply a cure. CLL-MRD (Minimal Residual Disease in CLL) refers to the detection of very low levels of CLL cells in the body using advanced diagnostic technologies. There are two potential scenarios for MRD negativity: one is the actual absence of CLL cells in the patient’s body; the other is the inability of current technologies to detect minute quantities of CLL cells.
According to the standards set by the European Research Initiative on CLL (ERIC) and the International Workshop on Chronic Lymphocytic Leukemia (iwCLL), a threshold of 10-4 is used to clinically determine the positive or negative status of CLL-MRD. A residual CLL cell proportion of <10-4 is defined as u-MRD (undetectable MRD), with thresholds distinguished as detectable (d) or undetectable (u). Terms like MRD4 and MRD5 indicate the upper limits, where MRD4 represents a residual disease level of <0.01% or 10-4.
02 Sample Requirements for CLL-MRD
Current methods for detecting CLL-MRD include flow cytometry (FCM) and next-generation sequencing (NGS). The samples used are peripheral blood (PB) or bone marrow (BM). The MRD detection rate in PB is often lower than that in BM; it is recommended to first test PB, and if MRD is negative, then proceed with BM testing; for treatments aimed at achieving deep remission, simultaneous testing of PB and BM is recommended. For FCM, samples should be anticoagulated with heparin or EDTA and completed within 72 hours; for NGS, EDTA anticoagulation is recommended, with priority given to fresh samples, although frozen samples are also acceptable. For BM samples, the first tube after smear preparation is recommended.
03 FCM Testing for CLL-MRD—Applicable to All Patients
3.1 Typical Immunophenotypic Features of CLL
The intensity of antigen expression should be compared with that of normal B cells at the corresponding stage. If the mean fluorescence intensity (MFI) is higher than that of normal B cells, it is considered strong expression; otherwise, it is considered weak. The expression patterns of CD43, CD81, and CD20 in mature small B-cell lymphomas are shown in the figure below.
ROR1 (Receptor Tyrosine Kinase-like Orphan Receptor 1) is a highly specific tumor marker that mediates the Wnt5a signaling pathway in cancer stem cells and is highly expressed in Chronic Lymphocytic Leukemia (CLL). A study published in Leukemia Research shows that, on average, 96.8% of CLL cells are ROR1 positive (ranging from 80.1% to 99.8%). Professor Yujie Wu, as the corresponding author of another study, demonstrated that ROR1 can serve as an auxiliary diagnostic marker for CLL. It is particularly valuable in the diagnosis and differential diagnosis of atypical immunophenotypic CLL, Mantle Cell Lymphoma (MCL), and other types of B-cell Chronic Lymphoproliferative Disorders (B-CLPD). The expression characteristics of ROR1 in normal bone marrow (BM) B cells are shown in the figure below.
3.2 FCM Detection of CLL-MRD: Basis and Protocol
The basis for detecting CLL-MRD using Flow Cytometry (FCM) involves identifying CLL cells through the characteristic expression of a series of cell surface antigens. The typical immunophenotype for CLL includes: CD19+, CD5+, CD23+, CD10-, FMC7-, CD81-, CD43+, ROR1+, CD200++, CD45+, reduced expression of CD20, CD22 and/or CD79b, and weak monoclonal expression of kappa or lambda.
In 2016, ERIC introduced an eight-color single-tube standardized approach in a Leukemia publication, featuring core antibodies including CD81, CD20, CD22, CD43, CD5, CD79b, CD19, and CD3, notably omitting light chains. Addressing issues with flow cytometry standardization in China, Professor Yujie Wu conducted studies comparing detection protocols with and without light chains. In tests of 257 instances, only 31 cases (12%) showed discrepancies. Analysis of these inconsistencies revealed that the no-light-chain combination had higher sensitivity. This discrepancy may be due to the inability to detect one-quarter of CLL cases that are kappa/lambda negative and the significant impact of detection procedures and the sample itself on light chains. The study suggests that light chain detection is not essential.
Furthermore, the ERIC expert group further proposed that: (1) CD3 is not always necessary, and it might be superfluous if extremely high precision is required in the 0.0010 to 0.010% range; (2) CD20 and CD22 are also not essential when ⩾2 typical CLL phenotypes like CD5, CD79b, CD43, and CD81 are present; (3) A core antibody combination including six markers (CD19, CD20, CD5, CD43, CD79b, CD81) is defined as the CLL core antibody combination.
The Chinese Expert Consensus recommends an eight-color or more single-tube scheme for CLL-MRD, aiming for simplicity while sufficiently distinguishing between normal mature B cells and CLL cells. It focuses on eight core antigens that are frequently abnormally expressed in CLL cells and remain stable before and after treatment, including: CD19, CD20, CD79b, CD5, CD81, CD43, ROR1, and CD45. Note that CD22 and CD3 are omitted, with ROR1 and CD45 added. Using CD45 to gate and leukocyte counts as the denominator for calculating MRD can significantly enhance the consistency and accuracy of results across different laboratories, which is especially important in multi-center clinical drug trials. The process for FCM detection of CLL-MRD is detailed in the figure below.
To validate the reproducibility of the FCM-MRD protocol from the Chinese Expert Consensus, Jiangsu Provincial People’s Hospital conducted tests on the same sample at 24 hours and 48 hours using the same flow cytometer in the same laboratory by the same technician. The results showed MRD levels of 1.21% at 24 hours and 1.23% at 48 hours. Another study involved two laboratories analyzing and recording data from 10 samples using a blind method. The results showed no significant difference in MRD levels between the two laboratories (paired T-test, P=0.018, SD=0.29), with a strong statistical correlation (R=0.99, P<0.0001, 95% CI: 0.959–0.998). Additionally, the variation between laboratories was minimal (bias 0.135; 95% CI: -0.439 to 0.709), indicating that the antibody combination including CD45 and ROR1 yields highly reproducible results, minimally affected by sample preparation and analyst experience. The absence of light chains still allowed for the differentiation between CLL-MRD and B1 cells, as shown in the figure below.
3.3 FCM and NGS MRD Detection and Considerations
FCM and NGS MRD technologies show high consistency in detecting MRD levels. In an iFCR cohort study, 60 peripheral blood samples and 57 bone marrow samples underwent paired FCM and NGS MRD testing. The results showed:
94.2% (49/52) of the peripheral blood samples were simultaneously identified by both FCM and NGS as MRD4 negative, with NGS detecting 40.8% (20/49) as MRD6, 16.3% (8/49) as MRD5, and 42.9% (21/49) as MRD4.
93.2% (41/44) of the bone marrow samples were simultaneously identified by both FCM and NGS as MRD4 negative, with NGS detecting 43.9% (18/41) as MRD6, 12.2% (5/41) as MRD5, and 43.9% (18/41) as MRD4.
Patients who achieved MRD6 negativity early in the study consistently maintained MRD6 negativity during long-term dynamic monitoring; however, patients with intermediate MRD levels >1% did not benefit from additional chemotherapy.
Considerations for CLL-MRD Testing: (1) For the rare cases of ROR1-negative CLL with atypical immunophenotypes, the inclusion of kappa and lambda light chains may assist in diagnosis. (2) Treatment with CD20 monoclonal antibodies may lead to reduced or even absent CD20 expression. (3) After CD19-CAR-T cell therapy, ROR1 combined with CD24 can be used as alternative gating markers. (4) For clinical trials aiming for maximal disease remission, treatments like CAR-T, and hematopoietic stem cell transplantation, choosing a threshold between 10^-5 and 10^-6 may offer more clinical value. Next-Generation Flow (NGF) can be utilized, achieving a sensitivity of above 10^-5 when more than 2 million cells are obtained.
3.4 Cell Count and Reporting Method for FCM Detection
Internationally, the minimum limit of detection (LOD) for reproducibly identifying MRD cell populations is 20 clustered cells, i.e., LOD = 20/total number of leukocytes obtained × 100%. Accordingly, at least 200,000 leukocytes need to be acquired to reach a sensitivity of 10^-4. The minimum limit of quantitation (LOQ) for identifying MRD cell clusters is 50 cells, i.e., LOQ = 50/total number of leukocytes obtained × 100%, thus requiring at least 500,000 leukocytes. The FDA requires that the MRD detection threshold should be at least tenfold lower than the clinical decision threshold (MRD definition).
Reporting method for CLL-MRD by FCM: If fewer than 20 CLL-MRD cells are found, the report should state MRD-negative or not detected; if MRD-positive, it should report the percentage of MRD cells among leukocytes, along with a description of the immunophenotypic characteristics of the cell group. For example, if fewer than 20 CLL cells are detected, the report should state: MRD not detected (LOD:0.01%); if 60 CLL cells are detected in 1 million cells, the report should state: CLL-MRD=0.006% (LOQ:0.005%); if 30 CLL cells are detected in 1 million cells, the report should state: CLL-MRD detected, below LOQ (0.002-0.005%).
3.5 Timing of CLL-MRD Testing
For patients undergoing chemotherapy, CLL-MRD testing should be conducted concurrently with treatment response assessment, including mid-treatment efficacy evaluation and at least two months after the last treatment. For long-term treatment modalities with limited depth of remission, CLL-MRD testing should be done when the best clinical effect is achieved. For patients participating in clinical trials, CLL-MRD testing should be carried out at fixed times as per study design. MRD should also be tested in patients who achieve partial remission.
Expert Profile
Deputy Director of the Department of Hematology, Jiangsu Provincial People’s Hospital
Professor Yujie Wu holds a PhD in Internal Medicine Hematology, serves as a researcher, and mentors master’s students. She is recognized as a high-level talent under the Six Peaks Project in Jiangsu Province. Professor Wu is an expert for the National Medical Products Administration’s Medical Device Evaluation Center, a member of the Experimental Diagnostics Committee of the Chinese Medical Association’s Hematology Branch, a standing committee member of the Cell Analysis Committee of the Chinese Society of Biotechnology, a standing committee member of the Clinical Flow Cytometry Group of the Immunology Branch of the Chinese Immunology Society, the Vice Chair of the Flow Cytometry Diagnostic Expert Committee of the Integrated Traditional Chinese and Western Medicine Society, a standing committee member of the Medical Testing Quality Management Committee of the China Association for Quality Management in Pharmaceuticals, and a member of the CLL Work Group of the Chinese Anti-Cancer Association. She specializes in the clinical application of flow cytometry, the diagnosis of blood diseases, and basic research. Professor Wu has led multiple national and provincial natural science projects, holds four patents for inventions, and has authored and contributed to several expert consensus documents.
