In chronic lymphocytic leukemia (CLL), there is a growing interest for minimal residual disease (MRD) monitoring, due to the availability of drug combinations capable of unprecedented complete ...clinical responses. The standardized and most commonly applied methods to assess MRD in CLL are based on flow cytometry (FCM) and, to a lesser extent, real-time quantitative PCR (RQ-PCR) with allele-specific oligonucleotide (ASO) primers of immunoglobulin heavy chain genes (IgH). Promising results are being obtained using droplet digital PCR (ddPCR) and next generation sequencing (NGS)-based approaches, with some advantages and a potential higher sensitivity compared to the standardized methodologies. Plasma cell-free DNA can also be explored as a more precise measure of residual disease from all different compartments, including the lymph nodes. From a clinical point of view, CLL MRD quantification has proven an independent prognostic marker of progression-free survival (PFS) and overall survival (OS) after chemoimmunotherapy as well as after allogeneic transplantation. In the era of mechanism-driven drugs, the paradigms of CLL treatment are being revolutionized, challenging the use of chemoimmunotherapy even in first-line. The continuous administration of ibrutinib single agent has led to prolonged PFS and OS in relapsed/refractory and treatment naïve CLL, including those with
TP53
deletion/mutation or unmutated
IGHV
genes, though the clinical responses are rarely complete. More recently, chemo-free combinations of venetoclax+rituximab, venetoclax+obinutuzumab or ibrutinib+venetoclax have been shown capable of inducing undetectable MRD in the bone marrow, opening the way to protocols exploring a MRD-based duration of treatment, aiming at disease eradication. Thus, beside a durable disease control desirable particularly for older patients and/or for those with comorbidities, a MRD-negative complete remission is becoming a realistic prospect for CLL patients in an attempt to obtain a long-lasting eradication and possibly cure of the disease. Here we discuss the standardized and innovative technical approaches for MRD detection in CLL, the clinical impact of MRD monitoring in chemoimmunotherapy and chemo-free trials and the future clinical implications of MRD monitoring in CLL patients outside of clinical trials.
The GIMEMA phase II LLC1518 VERITAS trial investigated the efficacy and safety of front-line, fixed-duration venetoclax and rituximab (VenR) in combination in young (≤65 years), fit patients with ...chronic lymphocytic leukemia and unmutated IGHV and/or TP53 disruption. Treatment consisted of the venetoclax ramp-up, six monthly courses of the VenR combination, followed by six monthly courses of venetoclax as a single agent. A centralized assessment of minimal residual disease (MRD) was performed by allele-specific oligonucleotide polymerase chain reaction assay on the peripheral blood and bone marrow at the end of treatment (EOT) and during the follow-up. The primary endpoint was the complete remission rate at the EOT. Seventy-five patients were enrolled; the median age was 54 years (range, 38-65), 96% had unmutated IGHV, 12% had TP53 disruption, and 4% had mutated IGHV with TP53 disruption. The overall response rate at the EOT was 94.7%, with a complete remission rate of 76%. MRD was undetectable in the peripheral blood of 69.3% of patients and in the bone marrow of 58.7% of patients. The 12-month MRD-free survival in the 52 patients with undetectable MRD in the peripheral blood at the EOT was 73.1%. After a median follow-up of 20.8 months, no cases of disease progression were observed. Three patients had died, two due to COVID-19 and one due to tumor lysis syndrome. The first report of the VERITAS study shows that front-line VenR was associated with a high rate of complete remissions and durable response with undetectable MRD in young patients with chronic lymphocytic leukemia and unfavorable genetic characteristics. ClinicalTrials.gov identifier: NCT03455517.
Minimal/measurable residual disease (MRD) evaluation has resulted in a fundamental instrument to guide patient management in acute lymphoblastic leukemia (ALL). From a methodological standpoint, MRD ...is defined as any approach aimed at detecting and possibly quantifying residual neoplastic cells beyond the sensitivity level of cytomorphology. The molecular methods to study MRD in ALL are polymerase chain reaction (PCR) amplification-based approaches and are the most standardized techniques. However, there are some limitations, and emerging technologies, such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS), seem to have advantages that could improve MRD analysis in ALL patients. Furthermore, other blood components, namely cell-free DNA (cfDNA), appear promising and are also being investigated for their potential role in monitoring tumor burden and response to treatment in hematologic malignancies. Based on the review of the literature and on our own data, we hereby discuss how emerging molecular technologies are helping to refine the molecular monitoring of MRD in ALL and may help to overcome some of the limitations of standard approaches, providing a benefit for the care of patients.
Circulating tumor DNA (ctDNA) has become the most investigated analyte in blood. It is shed from the tumor into the circulation and represents a subset of the total cell‐free DNA (cfDNA) pool ...released into the peripheral blood. In order to define if ctDNA could represent a useful tool to monitor hematologic malignancies, we analyzed 81 plasma samples from patients affected by different diseases. The results showed that: (i) the comparison between two different extraction methods Qiagen (Hilden, Germany) and Promega (Madison, WI) showed no significant differences in cfDNA yield, though the first recovered higher amounts of larger DNA fragments; (ii) cfDNA concentrations showed a notable inter‐patient variability and differed among diseases: acute lymphoblastic leukemia and chronic myeloid leukemia released higher amounts of cfDNA than chronic lymphocytic leukemia, and diffuse large B‐cell lymphoma released higher cfDNA quantities than localized and advanced follicular lymphoma; (iii) focusing on the tumor fraction of cfDNA, the quantity of ctDNA released was insufficient for an adequate target quantification for minimal residual disease monitoring; (iv) an amplification system proved to be free of analytical biases and efficient in increasing ctDNA amounts at diagnosis and in follow‐up samples as shown by droplet digital PCR target quantification. The protocol has been validated by quality control rounds involving external laboratories. To conclusively document the feasibility of a ctDNA‐based monitoring of patients with hematologic malignancies, more post‐treatment samples need to be evaluated. This will open new possibilities for ctDNA use in the clinical practice.
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Introduction. Minimal residual disease (MRD) is the most powerful prognostic factor in acute lymphoblastic leukemia (ALL). Currently, real-time quantitative PCR (RQ-PCR) is the most widely used ...molecular method for MRD assessment, rigorously standardized within the EuroMRD consortium. According to the EuroMRD guidelines (Van der Velden et al. Leukemia 2007), a non-negligible fraction of patients with very low MRD levels are classified as positive not-quantifiable (PNQ), a definition that may result problematic in the clinical practice. Digital-droplet-PCR (ddPCR) allows an absolute quantification without the need of a standard curve and has the potential to overcome some limitations of RQ-PCR. High degrees of efficiency, sensitivity and accuracy have been reported for ddPCR compared to RQ-PCR, but no established guidelines for ddPCR MRD analysis and interpretation have so far been defined and its ability to correctly evaluate very low MRD levels is still under investigation.
In the present study, we assessed MRD by ddPCR in pediatric ALL cases classified as PNQ and/or negative by RQ-PCR at days +33 and/or +78 of the AIEOP-BFM ALL 2000 trial, to evaluate the potential of ddPCR for low MRD quantification and patients' risk stratification.
Patients and Methods. A total of 211 pediatric ALL patients enrolled in the AIEOP-BFM ALL 2000 trial were included in the study. We analyzed 124 B-lineage ALL patients defined as intermediate risk (IR) who had high positive MRD at day +33 and at day +78 were either PNQ (n=45, Slow Early Responders (SER)) or negative (n=79). A case-control design was applied to 36 B- and T-lineage relapsed ALL patients (cases) who at day +33 had PNQ MRD (n=12, IR) or were negative (n=24, standard risk (SR)) and to matched controls (21 and 30 patients who did not present a relapse). ddPCR analysis was performed as previously published (Della Starza et al, BJH 174, 541-9, 2016), by using 1.5 μg and 3.0 μg DNA of the follow-up samples. In the absence of an international consensus, data have been analyzed using two alternative guidelines; results are reported according to Della Starza et al (BJH 2016).
Results. Among 45 SER patients, ddPCR performed on 1.5 μg DNA of PNQ samples at day +78 revealed that 13 were quantifiable (Q), 16 PNQ and 16 negative (NEG) . When 3.0 μg of DNA were used (41/45 samples due to material availability), 12 were Q, 19 PNQ and 10 NEG. Event-free survival (EFS) curves are shown in Fig. 1a.
Among the 79 patients with high positive MRD at day +33 but who were negative at day +78, ddPCR on 1.5 μg DNA of day +78 identified 5 as Q, 17 PNQ and 57 NEG. When 3.0 μg DNA was used (77/79 samples), 9 patients were Q, 27 PNQ and 41 NEG. EFS curves are reported in Fig. 1b.
When ddPCR was applied to 33 PNQ samples at day +33, 2 were Q, 9 PNQ and 22 NEG; when using 3.0 μg of DNA, 1 was Q, 15 were PNQ and 17 NEG. EFS curves are shown in Fig. 1c.
Lastly, ddPCR on 1.5 μg of day +33 DNA of 54 SR patients showed 5 PNQ and 49 NEG, whilst by using 3.0 μg on 53 sample, 7 were PNQ and 46 NEG.
Conclusions. Our data demonstrate that ddPCR is a very promising tool for the evaluation of MRD in ALL cases with very low or negative RQ-PCR MRD results. In particular, among SER patients most relapses occurred in cases with quantifiable ddPCR MRD at day +78, while patients with negative or PNQ MRD by ddPCR at day +78 had a better outcome. Based on these results, high-risk treatment could be offered only to ddPCR quantifiable cases. Among patients with highly positive MRD at day +33 and negative at day +78, the small number of cases with quantifiable disease by ddPCR at present does not allow to establish the impact of quantification; consistently with SER patients, the outcome was similar for patients with negative or PNQ MRD by ddPCR at day +78. Similarly, among patients with PNQ MRD by RQ-PCR at day +33, a similar outcome was observed for cases negative or PNQ by ddPCR. Lastly, in most SR patients ddPCR confirmed the negative results of RQ-PCR at day +33, associated with an extremely good kinetics of disease reduction, independently of the MRD PCR method. Overall, our data indicate that ddPCR is as sensitive as RQ-PCR and can provide a potentially more accurate prognostic stratification for cases defined as PNQ MRD by RQ-PCR, in view of its ability to quantify without a standard curve. The application of ddPCR in a prospective clinical protocol with international guidelines is needed to define whether it can result in an overall improvement of pediatric ALL patients' stratification and outcome.
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Foà:AMGEN: Other: ADVISORY BOARD; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD; INCYTE: Other: ADVISORY BOARD; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; NOVARTIS: Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; GILEAD: Speakers Bureau.
Minimal residual disease (MRD) is the most powerful prognostic factor in pediatric acute lymphoblastic leukemia (ALL). Real‐time quantitative polymerase chain reaction (RQ‐PCR) represents the gold ...standard for molecular MRD assessment and risk‐based stratification of front‐line treatment. In the protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP) and the Berlin‐Frankfurth‐Munschen (BFM) group AIEOP‐BFM ALL2009 and ALL2017, B‐lineage ALL patients with high RQ‐PCR‐MRD at day+33 and positive at day+78 are defined slow early responders (SERs). Based on results of the AIEOP‐BFM ALL2000 study, these patients are treated as high‐risk also when positive MRD signal at day +78 is below the lower limit of quantification of RQ‐PCR (“positive not‐quantifiable,” POS‐NQ). To assess whether droplet digital polymerase chain reaction (ddPCR) could improve patients’ risk definition, we analyzed MRD in 209 pediatric B‐lineage ALL cases classified by RQ‐PCR as POS‐NQ and/or negative (NEG) at days +33 and/or +78 in the AIEOP‐BFM ALL2000 trial. ddPCR MRD analysis was performed on 45 samples collected at day +78 from SER patients, who had RQ‐PCR MRD ≥ 5.0 × 10–4 at day+33 and POS‐NQ at day+78 and were treated as medium risk (MR). The analysis identified 13 of 45 positive quantifiable cases. Most relapses occurred in this patients’ subgroup, while ddPCR NEG or ddPCR‐POS‐NQ patients had a significantly better outcome (P < 0.001). Overall, in 112 MR cases and 52 standard‐risk patients, MRD negativity and POS‐NQ were confirmed by the ddPCR analysis except for a minority of cases, for whom no differences in outcome were registered. These data indicate that ddPCR is more accurate than RQ‐PCR in the measurement of MRD, particularly in late follow‐up time points, and may thus allow improving patients’ stratification in ALL protocols.
Summary
BCL2/IGH rearrangements were analysed by polymerase chain reaction (PCR) at diagnosis in paired peripheral blood (PB) and bone marrow (BM) samples from 67 patients with stage I/II follicular ...lymphoma (FL). Real time quantitative PCR (RQ‐PCR) and digital droplet PCR (ddPCR) were performed in cases with a major breakpoint region (MBR+) at diagnosis and after localized radiotherapy and rituximab administration in order to investigate the applicability of ddPCR. The overall ddPCR/RQ‐PCR concordance was 81·9% (113/138 samples) and 97·5% in the 40/138 with quantifiable disease (RQ‐PCR≥10−5). At baseline, ddPCR allowed the recovery of a MBR+ marker in 8/18 (44·4%) samples that resulted MBR‐negative/minor cluster region‐negative/minor BCL2‐negative by qualitative PCR. Moreover, the tumour burden at diagnosis significantly predicted progression‐free survival (PSF) only when quantified by ddPCR. Paired PB and BM samples analysis demonstrated a high concordance in the detection of BCL2/IGH+ cells by qualitative and quantitative methods; in particular, 40/62 samples were positive by ddPCR (25 PB+/BM+; 9 PB+/BM−; 6 PB−/BM+), with 34/40 (85%) identified by the study of PB only. In conclusion, in localized FL, ddPCR is a promising tool for monitoring minimal residual disease (MRD) that is at least comparable to RQ‐PCR and potentially more accurate. PB is a suitable source for serial BCL2/IGH MRD assessments, regardless of the methodology utilized.
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