IwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL

Minimal Residual Disease following Allogeneic Hematopoietic Stem Cell Transplantation

3.2 Evolution of High-Sensitivity, Multi-Color Flow Cytometric Immunophenotyping for Minimal Residual Disease Detection in Chronic Lymphocytic Leukemia: Peripheral Blood versus Bone Marrow?

In this issue of Clinical Cytometry, Durrieu et al. on behalf of GEIL-GOELAMS present evidence that circulating levels of CD19+ CD5+ chronic lymphocytic leukemia (CLL)-like cells can be used to define a threshold for the detection of minimal residual disease (MRD) in CLL (this issue: page 346). This study was performed in 16 different hematology laboratories or services, and the data is analyzed centrally. The panel consisted of five tubes of four reagents each with CD19 and CD5 being present in each tube. They determined that the level of two CLL-like immunophenotypes CD19+/CD5+/CD43int/CD79blo and CD19+/ CD5+/CD81lo/CD22lo were less than 4 × 10−4 per WBC in unwashed, lyzed normal whole blood. Levels greater than this found in post treatment CLL samples were taken to signify the presence of MRD. The author further state that they are unable to determine monoclonality using light chain restriction due to their polyclonal nature in normal blood donors and the presence of cytophilic antibodies being present in the unwashed, lyzed method. And that bright CD5+ B cells are often lambda positive, and polyclonal. I have several preferences (biases) in this matter, and they include the use of CD20 rather than CD19 or preferably both CD20 and CD19 in combination to define B cells and the clone, demonstration of monoclonality or monotypia, and that reports be based on the absolute cell-count (WBC or absolute lymphocyte count ALC) and not just the percentage < or > 10−4. If the WBC is used, it should be used in conjunction with the ALC and even a B cell absolute count. I sympathize with the problem of cytophilic antibodies but in monoclonal B cell lymphocytosis studies, values of 5–50 light chain restricted B cells per microliter can be routinely detected (1). I was also surprised to see that the lower range of 1–3% CD5+ B cells seems to be the norm, whereas values of 10–25% seem to be more the rule. The European Research Initiative on CLL (ERIC) has published a standardized four-color approach to MRD detection in CLL (2). Both the authors of that study and this study find a WBC threshold of 10−4 below which is considered MRD negativity. The ERIC study allows for the primary role of kappa lambda tube for historical purposes but favors the combination of CD20/CD38/CD19/CD5 over the combinations of CD43/CD79b and CD81/CD22. They further point out the need for the evaluation of gate contamination, number of total events collected (500,000), and operator training. Certain precautions are required for differentiating between blood and marrow involvement in the setting of antibody therapies. However, due to the correlation of MRD between blood and marrow, blood should be the first site tested for MRD. The complete absence of B cells (B cell aplasia) might also be taken as a clinical measure of MRD. If ascertainment of marrow involvement in this setting were considered important, a bone marrow study would be indicated. Conversely, the timing of the blood MRD analysis could be postponed. The German CLL Study Group have also standardized three four tube panels of four-color reagents using a larger volume of blood, and where appropriate 2 million leucocytes were collected (3). This paper also shows that the overall ability of MRD flow is to detect and quantify residual CLL cells and is not affected by the addition of rituximab to FC chemotherapy (3). In a 2005 editorial, Montserrat (4) called for standardization and revision of 1996 NCI response guidelines to include MRD negative status as a goal. In 2008, Hallek et al. (5) revised the NCI guidelines to include not only a definition of MRD but also encouraged that the MRD detection become part of future CLL clinical trials because MRD negative status has prognostic impact. The GCLLSC, ERIC, and GEIL studies have all demonstrated that multicolor flow cytometry can meet the needs of a robust MRD assay. All three groups used a four-color, five tube panel and a sequential gating strategy. Newer clinical flow cytometers now allow for the use of 6–8 colors. The Salamanca group has developed an automated method for doing MRD detection by merging files on the same patient with the addition normal donor virtual 1:1 dilution as a control (6). Knowledge of the original CLL immunophenotype would be useful. One could envision an integration of the GCLLSC, ERIC, and GEIL methods combining CD19/CD20/CD5/CD38/CD79b/ CD22/CD41/CD81 and CD19/CD20/CD5/CD45/CD3/CD14/kappa/lambda. Del Poeta et al. (7) concluded that the addition of rituximab consolidation and maintenance therapy prolonged response duration significantly in patients with MRD + CLL. MRD detection is indicated in the investigation, care, and treatment of the CLL patient. Clinical trials will be required to answer the question of overall survival in CLL. Regardless, CLL remains a model disease for the appropriate treatment of the elderly with progressive disease.

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

Early Detection of Minimal Residual Disease in Biological Prognostic Risk Groups in Chronic Lymphocytic Leukaemia (CLL) Utilizing the Novel CD160FCA Assay

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).

Minimal residual disease (MRD) detection has become increasingly important for the assessment of therapy response in chronic lymphocytic leukemia (CLL). However, current MRD analysis methods, both molecular genetic and flow cytometric, are time-consuming and require experienced laboratory staff. To reduce the demands of flow cytometric MRD detection in CLL, we have introduced a novel flow cytometric 8-color protocol. The MRD analysis results using this protocol were then compared with the commonly employed 4-color protocol and the molecular genetic (real-time quantitative allele-specific oligonucleotide IGH polymerase chain reaction; RQ-ASO IGH PCR) approach. Forty-two CLL patient samples were repeatedly analyzed after allogeneic stem cell transplantation (n=20) or after fludarabine-based therapy (n=22), and 100% concordance was found using both flow cytometric protocols. Furthermore, there was a strong correlation (r=0.94) between flow cytometric and RQ-ASO IGH PCR results in MRD detection. Flow cytometry is less time-consuming, less financially demanding, and moreover, MRD assessment using our novel 8-color protocol is less complicated than the 4-color approach and molecular methods.

Sequencing-Based Detection of Minimal Residual Disease Is Associated with Outcomes after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Lymphoid Malignancies

Background: Circulating tumor (ctDNA) for detection of minimal residual disease (MRD) in colorectal cancers is prognostic, however, many cancers are not detected prior to clinical recurrence. Chemotherapy and surgery may limit detection of ctDNA due to increased shedding of DNA. We are exploring the use of an ultra-sensitive MRD assay (NeXT Personal®) in a prospective study of colorectal cancers (VICTORI) undergoing resection, with the aim to determine the optimal timepoint post-surgery to detect MRD. Methods: Patients enrolled undergo whole genome sequencing on tissue samples to generate a personalized panel of up to ∼1800 somatic variants for MRD detection, enabling detection of ctDNA down to ∼1 part per million (PPM). Blood draws are taken prior to surgery, within the MRD landmark window (weeks 2, 4, 6 and 8), and on follow-up thereafter every 3 months for 3 years. Results: We present preliminary results on 474 samples from the first 68 patients (N=40 rectal [59%], N=28 colon [41%]; N= 51 stage I-III [75%], N=17 stage IV [25%]) with a median follow up of 387 days. Pre-surgery positivity in treatment-naïve patients was 93.8% (n=30/32; negative are both stage I), and 72.4% in patients that received neoadjuvant therapy (n=21/29). Sixty-seven patients were evaluable for clinical outcomes, of which 23 had a recurrence. Of these, all evaluable patients were ctDNA-positive prior to recurrence (100%, 21/21; 2 patients were excluded due to lack of plasma samples prior to recurrence). ctDNA detection preceded clinical relapse determined by standard of care imaging by a median of 194 days (range 1-416 days). Most recurrences had ctDNA detected in the MRD landmark window (86%, 18/21). Of the three detected after the landmark window, one (stage IV) had neoadjuvant therapy prior to surgery, and was detected at the first follow-up timepoint (month 3). Another (stage III) was detected at month 6, and the third (stage II) at month 9. The median level of detection for the first post-surgical MRD positive sample was 54.9 PPM (range 2.45-111,120 PPM), and the ctDNA level at the first detection was correlated with disease-free survival (r=-0.47, p=0.05, Spearman). Detection of MRD at each individual timepoint in the MRD landmark window was associated with reduced disease-free survival (week 2 HR 4.75 [95% CI:1.63-13.84], p=0.0043; week 4 HR 11.72 [3.87-35.47], p=1.33x10-5; week 6 HR 7.73 [2.79-21.41], p=8.4x10-5; week 8 HR 16.70 [4.85-57.49], p=8.08x10-6). Conclusions: The VICTORI study is an ongoing study to understand the prognostic value of ultra-sensitive ctDNA detection and determine the optimal timepoint for detecting MRD post-surgery. Preliminary results indicate NeXT Personal detects MRD at ultra-low levels and is prognostic as early as two weeks post-surgery for recurrences. Citation Format: Emma Titmuss, Joao Paulo Solar Vasconcelos, Fabio C. Navarro, Neeraja Ravi, Charles Abbott, Brendan Chia, James T. Topham, Gale Ladua, Daniela Hegebarth, Sophie C. Chuang, Howard J. Lim, Karamjit Gill, Sharlene Gill, Carl J. Brown, Amandeep Ghuman, Adam Meneghetti, David F. Schaeffer, Richard O. Chen, Sean M. Boyle, Jonathan M. Loree. Detection of post-surgical minimal residual disease (MRD) in colorectal cancer; preliminary results from the VICTORI study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3774.

Value of Minimal Residual Disease (MRD) Negative Status at Response Evaluation in Chronic Lymphocytic Leukemia (CLL): Combined Analysis of Two Phase III Studies of the German CLL Study Group (GCLLSG)

A Complementary Role of High Throughput Sequencing and Multiparameter Cytometry for Minimal Residual Disease (MRD) Detection in Chronic Lymphocytic Leukemia (CLL):an European Research Initiative (ERIC) Study

Over the past 2 decades, dramatic improvements in the efficacy of treatments for chronic lymphocytic leukemia have led to progressively higher percentages of clinical complete remissions. A molecular eradication of the leukemia has become not only a desirable, but also an achievable, end point that needs to be evaluated within clinical trials. The assessment of complete remission only at the clinical and morphological level is insufficient, at least for physically fit patients. The detection of minimal residual disease (MRD) in chronic lymphocytic leukemia has become feasible using PCR-based or flow cytometric techniques that reproducibly allow reaching the detection level of less than 1 leukemic cell per 10 000 leukocytes (10(-4)), the level currently defined as MRD(-) status. Emerging data indicate that the MRD status during and at the end of treatment is one of the most powerful predictors of progression-free and overall survival. This predictor appears to be independent of clinical response, type or line of therapy, and known biological markers. For these reasons, the time is ripe to test the use of MRD as a surrogate marker of clinical end points and as a real-time marker of efficacy and/or resistance to the administered therapies. In the near future, clinical trials will determine whether MRD assessment can be used for guiding therapy, either to improve quality of responses through consolidation or to prevent relapses through preemptive therapies based on the reappearance of MRD.

Different Treatment Regimens, Optimal Time Points and Threshold Level While Minimal Residual Disease Evaluation in AML Patients

Detection of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) is becoming increasingly important as treatments improve. An internationally harmonised four-colour (CLR) flow cytometry MRD assay is widely used but has limitations. The aim of this study was to improve MRD analysis by identifying situations where a less time-consuming CD19/CD5/κ/λ analysis would be sufficient for detecting residual CLL, and develop a six-CLR antibody panel that is more efficient for cases requiring full MRD analysis. In 784 samples from CLL patients after treatment, it was possible to determine CD19/CD5/κ/λ thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/κ/λ analysis was unsuitable for predicting iwCLL/NCI response status or identifying cases with no detectable MRD. For the latter cases requiring a full MRD assessment, a six-CLR assay was designed comprising CD19/CD5/CD20 with (1) CD3/CD38/CD79b and (2) CD81/CD22/CD43. There was good correlation between four-CLR and six-CLR panels in dilution studies and clinical samples, with 100% concordance for detection of residual disease at the 0.01% (10(-4)) level (n=59) and good linearity even at the 0.001-0.01% (10(-5)-10(-4)) level. A six-CLR panel therefore provides equivalent results to the four-CLR panel but it requires fewer reagents, fewer cells and a much simpler analysis approach.

Minimal residual disease (MRD) detection based on the standardized molecular monitoring of the t(9;22)-related BCR-ABL1 fusion transcript is well established for patients with chronic myeloid leukemia (CML). The levels of BCR-ABL1 serve as a guide to tailor treatment of the CML patient. In acute myeloid leukemia (AML) MRD detection based on polymerase chain reaction (PCR) approaches targeted towards the acquired molecular abnormalities is less well established. MRD measurement of the CBFB-MYH11 and RUNX1-RUNX1T1 fusion transcripts after induction therapy has been shown to be of some clinical importance. However, these transcripts can persist during long term complete remission, without having an effect on treatment outcome. In contrast, sequential MRD monitoring of the PML-RARA fusion transcript in acute promyelocytic leukemia (APL) is a strong predictor of relapse. Initial molecular MRD studies were limited to these favorable AML subtypes. Due to the discovery of novel recurrent abnormalities in AML the potential of molecular MRD detection has increased substantially. Although, certain acquired mutations, such as those in NPM1, are known for a number of years, only recently the application of these molecular abnormalities for MRD detection has been investigated in larger clinical trials. By NPM1 mutant MRD detection we can now recognize patients with higher risk of relapse. Highly sensitive targeted detection of the hotspot mutations in AML subsets is feasible by means of real-time PCR, but detection of patient specific mutations with this technology is still challenging. Next generation sequencing (NGS) revealed that AML is an extremely heterogeneous disease, as illustrated by the multitude of acquired mutations, but this technology has also opened possibilities for detection of MRD in virtually every patient. With NGS there is no need for patient specific assays since practically all mutations are detected. These molecular abnormalities, as single marker or in combination, will most certainly improve MRD monitoring of AML. However, it remains yet to be determined how MRD levels are assessed and which combination of markers in a MRD detection result in clinically relevant information, requiring extensive validation in large clinical AML trials. Smaller studies already demonstrated the variable dynamics of MRD during treatment and associations between somatic mutations persistence and risk of relapse. However, clonal hematopoiesis of undetermined potential, i.e., preleukemic mutations that may persist after treatment, provides an extra layer of complexity to the applicability of MRD detection. For example, the clinical applicability of MRD detection in the setting of mutant DNMT3A and IDH mutations is likely less effective due to the persistent DNMT3A and IDH mutant preleukemic cells following treatment. However, should all mutations be cleared after treatment or can preleukemic mutations in otherwise normal hematopoiesis persist without resulting in relapse? Taken together, there is need for molecular approaches to understand the dynamics of residual disease in AML during treatment. DisclosuresNo relevant conflicts of interest to declare.