Circulating Tumor DNA–Based MRD Assessment in Patients with CLL Treated with Obinutuzumab, Acalabrutinib, and Venetoclax

Abstract

Purpose: With the advent of highly efficacious time-limited combination treatments of targeted agents in chronic lymphocytic leukemia (CLL), minimal residual disease (MRD) assessment has gained importance as a measure for therapeutic success and as a surrogate for progression-free survival. The currently most widely used method is multicolor flow cytometry, which detects circulating CLL cells in the peripheral blood. However, it seems to be less sensitive for the detection of MRD in the lymph node compartment. Patients and Methods: To evaluate whether a cell-free approach can overcome this limitation, we performed serial assessments of circulating tumor DNA (ctDNA) in patients with CLL treated with obinutuzumab, acalabrutinib, and venetoclax in the phase II CLL2-BAAG trial. Patient-specific variability, diversity, joining (VDJ) rearrangements as well as somatic driver mutations were tracked before, during and after treatment by digital droplet PCR in blood plasma. Furthermore, these were systematically compared to matched flow cytometry data. Results: In the 381 sample pairs, ctDNA and flow cytometry yielded highly concordant results. However, clone-specific ctDNA was detected in 44 of 152 samples (29%) that were assessed as undetectable MRD (uMRD) by flow cytometry (defined as less than one CLL cell in 10,000 normal leukocytes). 29 ctDNA-negative samples showed detectable MRD >10–4 by flow cytometry. Also, somatic driver mutations were detected with a similar sensitivity compared with patient-specific VDJ rearrangements in plasma. In patients with predominantly nodal residual disease, ctDNA compared favorably with 4-color flow cytometry and seemed to more accurately reflect the entire disease burden across compartments. Conclusions: On the basis of these findings, ctDNA-based MRD assessment appears to be a promising method to complement cell-based MRD approaches like flow cytometry that focus on circulating CLL cells in the peripheral blood.

Noninvasive minimal residual disease assessment in relapsed/refractory large B‐cell lymphoma using digital droplet PCR

“…Taqman assays were designed using the Integrated DNA Technologies (IDT) primerquest tool (https://www.idtdna.com) deliberately spanning one or more V(D)J segment boundaries (determined by the IMGT tool) to increase specificity. Digital PCR assays were performed in triplicates using 4 μL containing a maximum amount of 80 ng cell‐free DNA per reaction as previously described 19 . Of note, each assay was tested with a set of negative controls containing no template, DNA obtained from peripheral blood mononuclear cells (pBMC) of a healthy donor, DNA obtained from a CLL sample, and DNA obtained from another rrDLBCL sample of the present study.…”

“…Digital PCR assays were performed in triplicates using 4 μL containing a maximum amount of 80 ng cell-free DNA per reaction as previously described. 19 Of note, each assay was tested with a set of negative controls containing no template, DNA obtained from peripheral blood mononuclear cells (pBMC) of a healthy donor, DNA obtained from a CLL sample, and DNA obtained from another rrDLBCL sample of the present study. In line with our previous study in chronic lymphocytic leukemia 19 and guidelines for the application of ddPCR in lymphoproliferative disorders, 22 samples were considered positive only when a fluorescence signal in the FAM channel was similar to the FAM signal in the positive control, a minimum number of three DNA fragments were supporting the variant (the dominant V(D)J-clone or the MYD88 p.L265P mutation), and the negative controls confirmed no signal.…”

“…Digital droplet polymerase chain reaction (ddPCR) is an ultrasensitive and fast method for the detection of specific DNA sequences in a sample and has been demonstrated to provide improved sensitivity as compared to real‐time quantitative (q)PCR in hematologic malignancies such as mantle cell lymphoma 18 . Here, we modified a ddPCR‐based assay that we have recently developed for chronic lymphocytic leukemia 19 to detect tumor‐specific V(D)J rearrangements of the immunoglobulin heavy chain locus in ctDNA and present proof‐of‐principle for noninvasive MRD detection within short time intervals in rrDLBCL.…”

Chronic Lymphocytic Leukemia: Time-Limited Therapy in the First-Line Setting and Role of Minimal Residual Disease

“…Several studies have confirmed that CR or PR of the bone marrow is not very relevant in the presence of uMRD, and the outcome is not entirely clear in patients with inconsistent MRD results in PB and BM. To address these issues, attempts are being made to use cell-free DNA from plasma to measure MRD to provide information about residual disease in different compartments [ 65 , 66 ]. This assay also allows tracking and responding to the emergence of new clones and potential mutations.…”

Challenging the Value of Minimal Residual Disease in Predicting Outcome of Patients With Chronic Lymphocytic Leukemia

“…Assessing the cell-free DNA (cfDNA) in plasma for CLL-associated VDJ rearrangements is a newer method that has the advantage of being able to analyze for residual disease across multiple compartments and, in theory, could assess the total body MRD after therapy with a sensitivity that appears at least similar, if not greater, than that of flow cytometry. 6 However, some of the limitations noted for detecting clonal VDJ rearrangements in DNA extracted from leukocytes also apply to assays for MRD in cfDNA; moreover, for unexplained reasons, detection of MRD in some patients (eg, those with del(11q)) appeared more readily achieved using flow cytometry than through analysis of cfDNA for idiosyncratic clonal VDJ rearrangements; this did not appear to be the case for patients lacking del(11q), suggesting that the sensitivity of this approach for monitoring MRD may be influenced by factors associated with the leukemia cell's cytogenetics.…”