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Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL), B-cell chronic lymphocytic leukemia (CLL), and multiple myeloma and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. Real-time quantitative PCR (RQ-PCR) targeting circulating lymphoma cells is still the gold standard for MRD detection in indolent B-NHL and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, CLL, and FL with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy (IGH) and light chain genes. Complete and incomplete IGH and additionally IG kappa light chain rearrangements can be used as targets for MRD. Next-generation sequencing (NGS) of IG-rearrangements (IG-NGS) as new sequencing-based technology can overcome the limitation of PCR-based approaches and has a potential for higher sensitivity. Chromosomal translocations like the t(14;18)(q32;q21) translocation associated with IGH::BCL2 fusion in FL and t(11;14)(q13;q32) translocation in MCL leading to the IGH::CCND1 fusion can be used as MRD target in selected lymphoma subtypes. In patients with CLL, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10 -4 is achieved.MRD diagnostics targeting the IG loci is complex and requires extensive knowledge and experience because the junctional regions of each clonal rearranged gene have to be identified before the patient-specific PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation within the rearranged IGH gene occurring during B-cell development of germinal center and post-germinal center B-cell lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. PCR-based methods using allele-specific primers can reach a high sensitivity of up to 10 -5 . This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IG gene rearrangements and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results. This is available by the EuroMRD network ( https://euromrd.org ), a subgroup of ESHLO ( https://eslho.org )., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Minimal/measurable residual disease (MRD) diagnostics using real-time quantitative PCR analysis of rearranged immunoglobulin and T-cell receptor gene rearrangements are nowadays implemented in most treatment protocols for patients with acute lymphoblastic leukemia (ALL). Within the EuroMRD Consortium, we aim to provide comparable, high-quality MRD diagnostics, allowing appropriate risk-group classification for patients and inter-protocol comparisons. To this end, we set up a quality assessment scheme, that was gradually optimized and updated over the last 20 years, and that now includes participants from around 70 laboratories worldwide. We here describe the design and analysis of our quality assessment scheme. In addition, we here report revised data interpretation guidelines, based on our newly generated data and extensive discussions between experts. The main novelty is the partial re-definition of the "positive below quantitative range" category by two new categories, "MRD low positive, below quantitative range" and "MRD of uncertain significance". The quality assessment program and revised guidelines will ensure reproducible and accurate MRD data for ALL patients. Within the Consortium, similar programs and guidelines have been introduced for other lymphoid diseases (e.g., B-cell lymphoma), for new technological platforms (e.g., digital droplet PCR or Next-Generation Sequencing), and for other patient-specific MRD PCR-based targets (e.g., fusion genes)., (© 2024. The Author(s).)

Liquid biopsy is a novel diagnostic approach at first developed to characterize the molecular profile of solid tumors by analyzing body fluids. For cancer patients, it represents a noninvasive way to monitor the status of the solid tumor with respect to representative biomarkers. There is growing interest in the utilization of circulating tumor DNA (ctDNA) analysis also in the diagnostic and prognostic fields of lymphomas. Clonal immunoglobulin (IG) gene rearrangements are fingerprints of the respective lymphoid malignancy and thus are highly suited as specific molecular targets for minimal residual disease (MRD) detection. Tracing of the clonal IG rearrangement patterns in ctDNA pool during treatment can be used for MRD assessment in B-cell lymphomas. Here, we describe a reproducible next-generation sequencing assay to identify and characterize clonal IG gene rearrangements for MRD detection in cell-free DNA., (© 2022. The Author(s).)

We report assessment of minimal residual disease (MRD) status and its association with outcome in rituximab-refractory follicular lymphoma (FL) in the randomized GADOLIN trial (NCT01059630). Patients received obinutuzumab (G) plus bendamustine (Benda) induction followed by G maintenance, or Benda induction alone. Patients with a clonal marker (t[14;18] translocation and/or immunoglobulin heavy or light chain rearrangement) detected at study screening were assessed for MRD at mid-induction (MI), end of induction (EOI), and every 6-24 months post-EOI/discontinuation by real-time quantitative PCR. At MI, 41/52 (79%) patients receiving G-Benda were MRD-negative vs. 17/36 (47%) patients receiving Benda alone (p = 0.0029). At EOI, 54/63 (86%) patients receiving G-Benda were MRD-negative vs. 30/55 (55%) receiving Benda alone (p = 0.0002). MRD-negative patients at EOI had improved progression-free survival (HR, 0.33, 95% CI, 0.19-0.56, p < 0.0001) and overall survival (HR, 0.39, 95% CI, 0.19-0.78, p = 0.008) vs. MRD-positive patients, and maintained their MRD-negative status for longer if they received G maintenance than if they did not. These results suggest that the addition of G to Benda-based treatment during induction can significantly contribute to the speed and depth of response, and G maintenance in MRD-negative patients potentially delays lymphoma regrowth.

Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0-14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0-14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL) and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. MRD diagnostics performed by real-time quantitative PCR (RQ-PCR) is still the gold standard and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy chain gene (IGHV). Chromosomal translocations like the t(14;18) translocation in FL and t(11;14) translocation in MCL can be used as MRD target in selected lymphoma subtypes. In patients with B-cell chronic lymphocytic leukemia, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10 -4 shall be achieved.MRD diagnostics targeting the IGHV gene is complex and requires extensive knowledge and experience because the junctional regions of each lymphoma have to be identified before the patient-specific RQ-PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation (SHM) within the rearranged IG heavy variable (IGHV) gene occurring as during B-cell development of germinal center and post-germinal center lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. RQ-PCR-based methods can reach a good sensitivity (≤10 -4 ). This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IGHV gene rearrangement and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results.

In this study, we compared immunoglobulin heavy-chain-gene-based minimal residual disease (MRD) detection by real-time quantitative PCR (RQ-PCR) and next-generation sequencing (NGS) to assess whether NGS could overcome some limitations of RQ-PCR and further increase sensitivity, specificity, accuracy and reproducibility. In total, 378 samples from 55 patients with acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) or multiple myeloma (MM) were investigated for clonotype identification, clonotype identity and comparability of MRD results. Forty-five clonotypes were identified by RQ-PCR and 49 by NGS. Clonotypes identified by both tools were identical or >97% homologous in 96% of cases. Both tools were able to routinely reach a sensitivity level of 1 × E-05. A good correlation of MRD results was observed (R=0.791, P<0.001), with excellent concordance in 79.6% of cases. Few discordant cases were observed across all disease subtypes. NGS showed at least the same level of sensitivity as allele-specific oligonucleotides-PCR, without the need for patient-specific reagents. We conclude that NGS is an effective tool for MRD monitoring in ALL, MCL and MM. Prospective comparative analysis of unselected cases is required to validate the clinical impact of NGS-based MRD assessment.

Long-term clinical and molecular remissions in patients with mantle cell lymphoma (MCL) following high-dose therapy (HDT) and autologous stem cell transplantation (ASCT) have been evaluated in only a few studies. Thirty-six patients with MCL received ASCT in our institution (27 patients undergoing first-line therapy, 8 patients undergoing second-line therapy, and 1 patient undergoing third-line therapy). In the case of long-term remission (≥5 years; n = 8), peripheral blood was tested for minimal residual disease (MRD) by t(11; 14) polymerase chain reaction (PCR) and immunoglobulin heavy-chain (IGH) PCR at the last follow-up. Ten-year overall survival (OS), progression-free survival (PFS), and freedom from progression (FFP) after first-line ASCT were 42 %, 43 %, and 54 %; after second-line ASCT, these were all 0 %. Four-year OS, PFS, and FFP for the first-line cohort were 75 %, 48 %, and 61 %, respectively. Four-year OS, PFS, and FFP after second-line ASCT were 55 %, 30 %, and 30 %, respectively. Treatment-related mortality (3 months after ASCT) was 0 %. The only prognostic factor for OS, PFS, and FFP was treatment line (p = 0.011, p = 0.046, and p = 0.023, respectively). No relapses occurred after 5 years following ASCT. So far, eight patients developed sustained long-term clinical and molecular complete remissions of up to 14.6 years following ASCT in the first treatment line. Sustained long-term clinical and molecular remissions can be achieved following ASCT in the first treatment line and apparently less frequent in the second treatment line.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell Non-Hodgkin lymphomas (B-NHL) and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. MRD diagnostics performed by real-time quantitative PCR (RQ-PCR) is the gold-standard and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). RQ-PCR analysis of the junctional regions of the rearranged immunoglobulin heavy-chain gene (IgH) serves as the most broadly applicable MRD target in B-NHL (∼80%). Chromosomal translocations as t(14;18) translocation in FL and t(11;14) translocation in MCL can be used in selected lymphoma subtypes. In patients with B-cell chronic lymphocytic leukemia, both flow-cytometry as well as RQ-PCR are equally suitable for MRD assessment as long as a sensitivity of ≤10(-4) shall be achieved.MRD diagnostics targeting the IgH gene is complex and requires extensive knowledge and experience because the junctional regions of each lymphoma have to be identified before the patient-specific RQ-PCR assays can be designed for MRD monitoring. Furthermore, somatic mutations of the IgH region occurring during B-cell development of germinal center and post-germinal center lymphomas may hamper appropriate primer binding leading to false negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IgH joining region of chromosome 14. RQ-PCR-based methods can reach a good sensitivity (≤10(-4)). This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IgH gene rearrangement and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results.

To evaluate the prognostic impact of minimal residual disease (MRD), quantitative real-time polymerase chain reaction (RQ-PCR) of clonal IGH rearrangements was performed in 29 patients with mantle cell lymphoma (MCL) treated with high-dose radiochemotherapy and autologous stem cell transplantation (ASCT). Fourteen of 27 patients evaluable for MRD after ASCT achieved complete clinical and molecular remission, whereas 13 patients had detectable MRD within the first year after ASCT. Molecular remission after ASCT was strongly predictive for improved outcome, with a median progression-free survival (PFS) of 92 months in the MRD-negative group compared with 21 months in the MRD-positive group (P < .001). Median overall survival (OS) was 44 months in the MRD-positive group and has not been reached in the MRD-negative group (P < .003). In multivariate analysis, molecular remission and bulky disease were independent prognostic factors for PFS (P = .001 and P = .021, respectively). While cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP)-like cytoreduction had only modest influence, ara-C-containing mobilization and myeloablative radiochemotherapy significantly reduced MRD. Quantitative MRD measured in the stem cell products of 27 patients was not predictive for molecular remission. We conclude that sequential quantitative monitoring of residual disease after ASCT is a powerful indicator for treatment outcome in MCL and defines subgroups of patients with a significantly different prognosis.

The aim of this study was to investigate if graft-versus-leukemia (GVL) activity conferred by allogeneic stem cell transplantation (allo-SCT) is effective in chronic lymphocytic leukemia (CLL) with unmutated V(H) gene status. The kinetics of residual disease (MRD) were measured by quantitative allele-specific immunoglobulin heavy chain (IgH) polymerase chain reaction (PCR) in 9 patients after nonmyeloablative allo-SCT for unmutated CLL. Despite an only modest decrease in the early posttransplantation phase, MRD became undetectable in 7 of 9 patients (78%) from day +100 onwards subsequent to chronic graft-versus-host disease or donor lymphocyte infusions. With a median follow-up of 25 months (range, 14-37 months), these 7 patients remain in continuous clinical and molecular remission. In contrast, PCR negativity was achieved in only 6 of 26 control patients (23%) after autologous SCT for unmutated CLL and it was not durable. Taken together, this study shows for the first time that GVL-mediated immunotherapy might be effective in CLL with unmutated V(H).

The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

Current MRD studies in T-cell acute lymphoblastic leukemia (T-ALL) mainly use T-cell receptor gamma, delta and SIL-TAL1 gene rearrangements as MRD-PCR targets. However, low frequency or limited diversity of these markers restricts the number of evaluable patients, particularly because two markers are recommended for MRD monitoring. Hence, we developed a new strategy implementing the TCR beta (TCRB) locus for MRD quantification. The frequency and characteristics of complete and incomplete TCRB rearrangements were investigated in 53 childhood and 100 adult T-ALL patients using the BIOMED-2 multiplex PCR assay. Clonal rearrangements were identified in 92% both childhood and adult T-ALL (Vbeta-Dbeta-Jbeta rearrangements in 80%, Dbeta-Jbeta rearrangements in 53%). Comparative sequence analysis of 203 TCRB recombinations revealed preferential usage of the 'end-stage' segment Jbeta2.7 in childhood T-ALL (27%), whereas Jbeta2.3 was most frequently involved in adult T-ALL (24%). In complete rearrangements, three downstream Vbeta segments (19-1/20-1/21-1) were preferentially used. Subsequently, a TCRB real-time quantitative PCR assay to quantify MRD with 13 germline Jbeta primer/probe combinations and allele-specific oligonucleotides was developed and applied to 60 clonal TCRB rearrangements. The assay allowed the detection of one leukemic cell within at least 10(4) polyclonal cells in 93% of cases and will be of high value for future MRD studies.

Modern treatment protocols lead to complete remission (CR) in a considerable proportion of patients with lymphoproliferative disorders. However, many of these patients ultimately relapse, implying that achievement of a clinical CR is compatible with significant amounts of residual malignant cells. Cytogenetic, molecular and immunological techniques that are more sensitive than morphology are increasingly used to assess and quantify minimal residual disease (MRD). Immunological marker analysis allows the detection of aberrant or unusual immunophenotypes, PCR techniques target fusion regions of chromosome aberrations and clone-specific immunoglobulin and T-cell receptor gene rearrangements. The rationale underlying MRD studies is to improve measurement of treatment response, to provide independent prognostic information and to optimise therapeutic strategies. In acute lymphoblastic leukemia (ALL), the MRD based evaluation of initial response to front-line therapy emerged as a highly relevant diagnostic tool, particularly in childhood ALL, where MRD has been shown to be an independent prognostic factor allowing a precise risk group classification. In patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL) the prognostic significance of MRD is still a matter of debate, as the majority of patients remain MRD positive after conventional treatment. This phenomenon has changed with the implementation of new treatment modalities, such as application of monoclonal antibodies, where a significant proportion of patients with NHL converts to MRD negativity and experiences prolonged remission. Whether this molecular remission will translate into a superior overall survival is currently the goal of ongoing prospective studies., (Copyright 2004 S. Karger AG, Basel)