Minimal Residual Disease Monitoring Research Articles

The EHA Research Roadmap: Malignant Lymphoid Diseases.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

Lab validation of an ultrasensitive ctDNA pan-cancer MRD assay using whole-genome sequencing.

e13582 Background: Minimal residual disease (MRD) monitoring using liquid biopsy for solid tumors requires a highly sensitive and specific assay that can overcome the limitation of low abundance cfDNA in a standard blood draw. We developed a whole-genome sequencing (WGS)-based assay to detect the presence of circulating tumor DNA (ctDNA) in plasma. The C2i assay is a tumor-informed assay that uses personalized tumor signature, advanced noise models, and artificial intelligence (AI) modalities to interrogate plasma for the presence of ctDNA longitudinally. Methods: The C2i test was developed in accordance with CAP/CLIA and New York state validation principles. We used contrived samples to establish analytical validation of the assay performance, which was then validated with a large clinical cohort of early-stage patients across various cancer types. Briefly, aggregated tumor signatures derived from cancer cell lines were fragmented and spiked into a contrived healthy plasma pool; the mixed samples were used to assess the presence of tumor DNA signature down to tumor fractions of 10e-4. Positive samples are identified by tumor-derived variants detected above the noise levels. Noise modeling was established using a panel of normal (PON) approach. We assessed the analytical sensitivity, specificity, and accuracy using 348 contrived samples derived from five different cancer cell lines. Reproducibility and precision were assessed with multiple replicates, and statistical concordance was reported. This validation was complemented by a cohort of 200 patients and ̃1000 plasma samples across a variety of cancer types including, NSCLC, MIBC, CRC, GBM, Breast Cancer, and a mixture of other cancer types. Results: Cancer cell lines, representing the five most prevalent disease indications, used for determining analytical sensitivity are as follows: CRC HT-29, Breast SK-BR3, Bladder HT-1376, Lung HCI-H526, and Prostate LNCaP. The cell line DNA was enzymatically fragmented and size-selected to mimic ctDNA. This ctDNA was spiked into cfDNA extracted from healthy volunteers at various dilution levels, varying from 10e0 to 10e-4. The 95% probability of detecting ctDNA was established at 10e-4. The reproducibility of tumor signature between replicates was assessed to be greater than 90%. The assay was performed using both normal and maximum input amounts. These performance estimates were then validated on a cohort of plasma collected from early-stage (stage I-III) patients across various cancer types. Conclusions: C2i MRD test is an ultrasensitive pan-cancer MRD monitoring assay used in several clinical trials across the world. We present an extensive analytical and clinical validation of the assay supporting its high performance.

Clinical utility of urine DNA for noninvasive detection and minimal residual disease monitoring in urothelial carcinoma.

4583 Background: Current methods for the early detection and minimal residual disease (MRD) of urothelial carcinoma (UC) are often invasive and/or possess sub-optimal sensitivity, especially in upper tract urothelial carcinoma (UTUC). Methods: We developed an efficient workflow named the urine tumor DNA multidimensional bioinformatic predictor, utLIFE, by using low-coverage whole-genome sequencing and targeted deep sequencing. We identified the UC specific mutations and large CNV in the discovery cohort. The utLIFE-UC model was trained in a bladder cancer retrospective cohort (n = 150) and validated in an bladder cohort(n = 674) and UTUC cohort(n = 22). 31 patients established diagnosis of BC who are on neoadjuvant were also enrolled, including a MRD training cohort (n = 16) with serial urine samples at baseline, during treatment and before surgery; a independent MRD validation cohort (n = 15) with urine samples before surgery. Results: The utLIFE-UC model could discriminate UC with high accuracy (94.3%), sensitivity (92.8%), and specificity (96.0%). Furthermore, compared to cytology, the assay achieved a great improvement in sensitivity in the detection of non–muscle-invasive bladder cancer (NMIBC, 94.7% vs. 31.6%,p = 0.0002), and muscle-invasive bladder cancer(MIBC, 82.6% vs. 69.6%, p = 0.4894). The utLIFE-UC model was also validated in independent BC cohort (sensitivity 94.3%, specificity 92.6%) and UTUC cohort (sensitivity 90.9%, specificity 90.9%). utLIFE-UC score also showed outstanding potential on dynamic surveillance of residual disease in UC as the score showed dramatically decreased in pCR patients. As expected, utLIFE-UC score could classify pCR and non-pCR(PR, SD, PD) with NPV 100% in MRD training and validation cohorts, which showed superior sensitivity over that of urine cytology (100% vs.37.5%, p = 0.0070) and FISH (100 vs. 42.9%, p = 0.0125). Conclusions: utLIFE-UC diagnostic model for early diagnosis, residual disease detection in UC is a cost-effective, rapid, high-throughput, noninvasive, and promising approach, which may reduce the burden of cystoscopy and blind surgery.

Molecular disease monitoring in patients with relapsed/refractory B-cell non-Hodgkin lymphoma receiving anti-CD19 CAR T-cell therapy.

7531 Background: Chimeric antigen receptor (CAR) T-cell therapy has improved the historically poor outcomes for relapsed and refractory (R/R) B-cell non-Hodgkin’s lymphoma (NHL). However, nearly 60% of patients will either fail to respond or relapse after CAR T-cell therapy. Currently, PET/CT scans are used to assess response. Cell-free circulating tumor DNA (ctDNA) is released by tumor cells into the peripheral blood and can be measured for minimal residual disease (MRD) assessment. Methods: In this retrospective, IRB approved pilot study, archived lymphoma tissues and ctDNA from peripheral blood samples on day 0, 14, 28, 56, 80, 180, and 365 after CAR T-cell infusion from 10 patients with R/R NHL were collected for next-generation sequencing (NGS) of clonal variable-diversity-joining (VDJ) rearrangements [Adaptive biotechnologies (Seattle, WA)]. Response was assessed by PET/CT on days 30, 60, 90, 180 and 365 and graded according to the Lugano 2014 criteria. The primary endpoint was to determine the feasibility of detecting ctDNA to monitor disease response after anti-CD19 CAR T-cells. The secondary endpoint was to compare the sensitivity/specificity of MRD assessment from ctDNA to PET/CT imaging. Results: Nine out of 10 patients with a trackable sequence [median age 69 (range: 56-76); 55.6% male; median LDH 224], were included in this study. Each received tisagenlecleucel CAR T-cell therapy after median two prior treatments (range: 2-4). 7/9 patients had R/R diffuse large B-cell lymphoma (DLBCL), and 2/9 had transformed follicular lymphoma. At a median follow up of 12.7 months (range: 1.5-30 months), four patients were alive. By day 90, three patients (33.3%) achieved a radiographic complete response (CR) whilst six patients (66.6%) had progressive disease (PD). MRD and radiographic response are outlined in the Table. Detectable MRD on day 14 or day 28 had 83% sensitivity and 100% specificity for radiographic progression at any time before one year. For patients with PD, the median (interquartile range) MRD at day 0, 14, and 28 were 17.31 (1.01, 96.84), 9.12 (0.30, 18.8), and 23.77 (8.01, 137.53) copies per milliliter (mL), respectively. For patients with detectable MRD at day 28, mOS and mPFS were 205 and 38 days, respectively. Conclusions: Our pilot study demonstrated that monitoring MRD was a sensitive and specific method to detect disease response and relapse. In addition, MRD can be monitored more frequently, thus allowing for earlier detection of relapse. MRD and PET/CT identified progressive disease (PD) or complete response (CR) by day 90 in nine patients (A-I) with R/R NHL post CAR T-cell infusion.[Table: see text]

Detection of NPM1 Mutation in Acute Myeloid Leukemia by Droplet Digital PCR and Its Clinical Application Value

To establish the droplet digital PCR (ddPCR) assay for the detection of NPM1 type A mutation in patients with acute myeloid leukemia (AML), and to evaluate its specificity, sensitivity and its value in clinical application. NPM1 mutant and wildtype plasmids were used to verify the performance of ddPCR. Both ddPCR and Sanger sequencing were used to detect the bone marrow samples of 87 AML patients, which were confirmed by next generation sequencing (NGS). Moreover, NPM1 mutation burden was dynamically monitored in five patients by ddPCR. The limit of blank (LOB) of ddPCR established for NPM1 mutation detection was 1.1 copies/μl, and the limit of detection (LOD) was 2.43 copies/μl, which had good linearity. Among the 87 newly diagnosed AML patients, ddPCR identified seventeen cases positive for NPM1 mutation (19.5%)， which was consistent with Sanger sequencing. NGS confirmed 12 positive cases, including 8 of type A mutations, 2 of type D mutations, and 2 of rare type mutations. The results of dynamic monitoring of NPM1 mutation burden in 5 patients showed that the NPM1 mutation burden decreased obviously even close to 0, when patients achieve complete remission after chemotherapy. However, the mutation burden was increased again at the time of relapse. In this study, we established a ddPCR method for detection of NPM1 mutation with good sensitivity and repeatability, which can be used for screening NPM1 mutation in newly diagnosed AML patients and for minimal residual disease monitoring after remission in positive AML patients to guide treatment.

FLT3-ITD in Children with Early T-cell Precursor (ETP) Acute Lymphoblastic Leukemia: Incidence and Potential Target for Monitoring Minimal Residual Disease (MRD).

Simple SummaryThe prevalence of FLT3-ITD among children with ETP-ALL must be determined. MRD monitoring in ETPs is hampered by the lack of Immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements. We determined the incidence of FLT3-ITD among children with ETP and performed MRD monitoring using FLT3-ITD sequences, successfully testing a new method of MRD detection. Moreover, we highlighted that the FLT3 pathway could represent a therapeutic target for precision therapy in patients with ETP. Early T-cell precursor (ETP) is an aggressive form of acute lymphoblastic leukemia (ALL), associated with high risk of relapse. This leukemia subtype shows a higher prevalence of mutations, typically associated with acute myeloid leukemia (AML), including RAS and FLT3 mutations. FLT3-ITD was identified in 35% cases of adult ETP-ALL, but data in the pediatric counterpart are lacking. ETPs frequently lack immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements, used for minimal residual disease (MRD) monitoring. Among 718 T-ALL enrolled in Italy into AIEOP-BFM-ALL2000, AIEOP-ALLR2006, and AIEOP-BFM-ALL2009 consecutive protocols, 86 patients (12%) were identified as ETP and 77 out of 86 children were studied for the presence of FLT3-ITD. A total of 10 out of 77 (13%) ETP cases were FLT3-ITD positive. IG/TR MRD monitoring was feasible only in four cases. FLT3-ITD MRD monitoring was performed using real-time PCR in all FLT3-ITD positive ETP cases. A comparison between IG/TR and FLT3-ITD resulted in comparable findings. Our study demonstrated that the FLT3-ITD prevalence in children was lower (13%) than that reported in adult ETP-ALL. FLT3-ITD can be used as a marker for sensitive molecular MRD monitoring in ETP-ALL when IG/TR markers are not available, potentially selecting those patients who should spare allogeneic hematopoietic stem cell transplantation (HSCT). Finally, the FLT3 pathway is a robust druggable target in this aggressive form of leukemia.

The translational challenges of precision oncology.

The translational challenges in the field of precision oncology are in part related to the biological complexity and diversity of this disease. Technological advances in genomics have facilitated large sequencing efforts and discoveries that have further supported this notion. In this review, we reflect on the impact of these discoveries on our understanding of several concepts: cancer initiation, cancer prevention, early detection, adjuvant therapy and minimal residual disease monitoring, cancer drug resistance, and cancer evolution in metastasis. We discuss key areas of focus for improving cancer outcomes, from biological insights to clinical application, and suggest where the development of these technologies will lead us. Finally, we discuss practical challenges to the wider adoption of molecular profiling in the clinic and the need for robust translational infrastructure.

Optical Genome Mapping as a Diagnostic Tool in Pediatric Acute Myeloid Leukemia.

Simple SummaryTreatment of pediatric acute myeloid leukemia (AML) is stratified according to multiple recurrent genetic aberrations, which require for detection of different diagnostic methods such as karyotyping and fluorescence in situ hybridization (FISH). The aim of this study was to analyze whether optical genome mapping (OGM), as a new all-in-one methodological approach, can identify all stratification-relevant genetic aberrations that were described by karyotyping. Therefore, frozen bone marrow and blood cells from 24 pediatric patients with AML, bi-lineage leukemia, and mixed-phenotype acute leukemia collected at diagnosis were analyzed by OGM. The results of OGM were compared with routine diagnostic results from karyotyping and FISH. We show that OGM has much potential to address limitations of cytogenetics and even identify new structural aberrations that can be useful for monitoring minimal residual disease (MRD) in patients without an MRD marker.Pediatric AML is characterized by numerous genetic aberrations (chromosomal translocations, deletions, insertions) impacting its classification for risk of treatment failure. Aberrations are described by classical cytogenetic procedures (karyotyping, FISH), which harbor limitations (low resolution, need for cell cultivation, cost-intensiveness, experienced staff required). Optical Genome Mapping (OGM) is an emerging chip-based DNA technique combining high resolution (~500 bp) with a relatively short turnaround time. Twenty-four pediatric patients with AML, bi-lineage leukemia, and mixed-phenotype acute leukemia were analyzed by OGM, and the results were compared with cytogenetics. Results were discrepant in 17/24 (70%) cases, including 32 previously unknown alterations called by OGM only. One newly detected deletion and two translocations were validated by primer walking, breakpoint-spanning PCR, and DNA sequencing. As an added benefit, in two cases, OGM identified a new minimal residual disease (MRD) marker. Comparing impact on risk stratification in de novo AML, 19/20 (95%) cases had concordant results while only OGM unraveled another high-risk aberration. Thus, OGM considerably expands the methodological spectrum to optimize the diagnosis of pediatric AML via the identification of new aberrations. Results will contribute to a better understanding of leukemogenesis in pediatric AML. In addition, aberrations identified by OGM may provide markers for MRD monitoring.

Post-Transplantation Day +100 Minimal Residual Disease Detection Rather Than Mixed Chimerism Predicts Relapses after Allogeneic Stem Cell Transplantation for Intermediate-Risk Acute Myelogenous Leukemia Patients Undergoing Transplantation in Complete Remission

Chimerism and minimal residual disease (MRD) are suggested to be prognostic for post-transplantation relapse in patients with acute myelogenous leukemia (AML). Nevertheless, the predictive value of both approaches in homogeneous populations remains underinvestigated. Here we suggest that MRD may have greater predictive value for relapse than mixed chimerism (MC) in intermediate-risk AML patients. Seventy-nine patients with intermediate-risk AML (40 males; median age, 57 years [range, 19 to 77 years]) were included. MRD detection on day +100 was performed in bone marrow via multiparameter flow cytometry (MFC) and quantitative real-time PCR (qPCR) for patients with an NPM1 mutation. Chimerism analysis was performed in peripheral blood. MC was defined as the persistence of <99.9% of donor alleles. The area under the receiver operating characteristic curve was highest for qPCR-MRD (.93), followed by MFC-MRD (.80) and MC (.65). The highest rate of relapse at 3 years was observed in day +100 qPCR-MRD-positive patients (100%), followed by MFC-MRD-positive patients (55%; P < .001). No patients with MC and without detectable MRD experienced relapse. The median 3-year overall survival (OS) and leukemia-free survival (LFS) for patients with MC without detectable MRD were both 86% (range, 61% to 96%), significantly higher than the values in day +100 MFC-MRD-positive patients (OS, 61% [range, 36% to 84%]; LFS: 30% [range, 11% to 59%]) and with day +100 qPCR-MRD-positive patients (OS: 17% [range, 3% to 56%], P = .001; LFS: 0%, P < .001). In patients with intermediate-risk AML, the qPCR-MRD on day +100 was highly predictive for relapse and long-term survival after allogeneic stem cell transplantation, followed closely by MFC-MRD. In contrast, chimerism status had limited predictive potential. Thus, molecular and flow cytometry MRD monitoring rather than MC in the first several months post-transplantation can identify patients at increased risk of relapse who may benefit from early post-transplantation preemptive intervention.

Pretransplantation minimal residual disease monitoring by multiparameter flow cytometry predicts outcomes of AML patients receiving allogeneic hematopoietic stem cell transplantation

Background and purposeIs minimal residual disease (MRD) monitoring by multiparameter flow cytometry (MFC) prognostic for acute myeloid leukemia (AML) patients before allogeneic hemopoietic stem cell transplantation (allo-HSCT)? And if so, what level of MRD eradication can be used to help guide the timing of HSCT? Can haplo-HSCT improve the prognosis of AML patients with MRD positive? To figure out these questions, we initiated this retrospective study. Methods96 AML patients were included retrospectively and divided into 5 groups, according to pre-transplantation MRD levels (from 5 × 10−2 to <1 × 10−4), to analyze the overall survival (OS), disease-free survival (DFS) and cumulative incidence of relapse (CIR). Secondly, we compared the prognosis of MRD-negative (MRDneg) and MRD-positive (MRDpos) AML patients (cutoff value = 1 × 10−3) who underwent allo-HSCT, and further analyzed the prognosis of MRDpos patients after received different transplantation modalities. ResultsIt is found that the 2-year OS and DFS of MRD negative group were better than the MRD positive group, and that the deeper the eradication of MRD before transplantation, the better the prognosis of patients. The CIR in patients received HLA-identical transplantation, was higher in the MRDpos than in the MRDneg. Haploid transplantation reduced the CIR disparity between MRDpos and MRDneg group. Subsequently, in AML patients who remain MRD positive before HSCT, we show that haplo-HSCT offered a better prognosis than HLA-identical transplantation (MSDT and MUDT). ConclusionIt is suggested that achieving MFC-MRD <10−3 (10−4 or even better) before allo-HSCT could reduce the relapse of AML and improve OS and DFS significantly, while haplo-HSCT may be preferred for patients not achieving MRD negativity.

PEAC: An Ultrasensitive and Cost-Effective MRD Detection System in Non-small Cell Lung Cancer Using Plasma Specimen.

Circulating tumor DNA (ctDNA), a tumor-derived fraction of cell-free DNA (cfDNA), has emerged as a promising marker in targeted therapy, immunotherapy, and minimal residual disease (MRD) monitoring in postsurgical patients. However, ctDNA level in early-stage cancers and postsurgical patients is very low, which posed many technical challenges to improve the detection rate and sensitivity, especially in the clinical practice of MRD detection. These challenges usually include insufficient DNA input amount, limit of detection (LOD), and high experimental costs. To resolve these challenges, we developed an ultrasensitive ctDNA MRD detection system in this study, namely PErsonalized Analysis of Cancer (PEAC), to simultaneously detect up to 37 mutations, which account for 70–80% non-small cell lung cancer (NSCLC) driver mutations from low plasma sample volume and enables LOD of 0.01% at a single-site level. We demonstrated the high performance achieved by PEAC on both cfDNA reference standards and clinical plasma samples from three NSCLC patient cohorts. For cfDNA reference standards, PEAC achieved a specificity of 99% and a sensitivity of 87% for the mutations at 0.01% allele fraction. In the second cohort, PEAC showed 100% concordance rate between ddPCR and Next-generation sequencing (NGS) among 29 samples. In the third cohort, 22 of 59 patients received EGFR TKI treatment. Among them, three in four patients identified low level actionable gene mutations only by PEAC had partial responses after targeted therapy, demonstrating high ctDNA detection ability of PEAC. Overall, the developed PEAC system can detect the majority of NSCLC driver mutations using 8–10 ml plasma samples, and has the advantages of high detection sensitivity and lower costs compared with the existing technologies such as ddPCR and NGS. These advantages make the PEAC system quite appropriate for ctDNA and MRD detection in early-stage NSCLC and postsurgical recurrence monitoring.

Molecular characterization of a Tunisian B-lineage leukemia using Kde gene rearrangements

Background: Molecular approaches applied to the study of B-lineage leukemia have made significant contributions to understanding and to characterizing the disease. The Kde rearrangement was targeted as clonal marker to characterize Tunisian B-chronic lymphocytic leukemia (B-CLL) and B-acute lymphoblastic leukemia (B-ALL). Objective: We used a multiplex PCR to amplify deletional Kde rearrangements and Southern blot assay with Cκ and Kde probes in seven B-CLL and two B-ALL. Direct sequencing was performed in order to determine the n region. Results: An interesting RSS-Kde biallelic rearrangement was found in one adult male patient with B-ALL, since only Vκ-Kde rearrangement has been reported in adult B-ALL. In B-CLL, one case with two clonal cell proliferations in favor of oligoclonality which is very infrequent in B-CLL was identified and might be associated with bad outcome. These first results need more investigation to establish correlation between molecular characteristic and the poor outcome. Conclusion: The molecular approach was successfully improved and applied for clonality assessment of B-lineage leukemia for a better diagnosis and performing monitoring minimal residual disease.

Longitudinal minimal residual disease assessment in multiple myeloma patients in complete remission \u2013 results from the NMSG flow-MRD substudy within the EMN02/HO95 MM trial

BackgroundMultiple myeloma remains an incurable disease with multiple relapses due to residual myeloma cells in the bone marrow of patients after therapy. Presence of small number of cancer cells in the body after cancer treatment, called minimal residual disease, has been shown to be prognostic for progression-free and overall survival. However, for multiple myeloma, it is unclear whether patients attaining minimal residual disease negativity may be candidates for treatment discontinuation. We investigated, if longitudinal flow cytometry-based monitoring of minimal residual disease (flow-MRD) may predict disease progression earlier and with higher sensitivity compared to biochemical assessments.MethodsPatients from the Nordic countries with newly diagnosed multiple myeloma enrolled in the European-Myeloma-Network-02/Hovon-95 (EMN02/HO95) trial and undergoing bone marrow aspiration confirmation of complete response, were eligible for this Nordic Myeloma Study Group (NMSG) substudy. Longitdudinal flow-MRD assessment of bone marrow samples was performed to identify and enumerate residual malignant plasma cells until observed clinical progression.ResultsMinimal residual disease dynamics were compared to biochemically assessed changes in serum free light chain and M-component. Among 20 patients, reaching complete response or stringent complete response during the observation period, and with ≥3 sequential flow-MRD assessments analysed over time, increasing levels of minimal residual disease in the bone marrow were observed in six cases, preceding biochemically assessed disease and clinical progression by 5.5 months and 12.6 months (mean values), respectively. Mean malignant plasma cells doubling time for the six patients was 1.8 months (95% CI, 1.4–2.3 months). Minimal malignant plasma cells detection limit was 4 × 10–5.ConclusionsFlow-MRD is a sensitive method for longitudinal monitoring of minimal residual disease dynamics in multiple myeloma patients in complete response. Increasing minimal residual disease levels precedes biochemically assessed changes and is an early indicator of subsequent clinical progression.Trial registrationNCT01208766

Assessment of Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia: A Multicenter Study From Turkey.

Assestment of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) is of utmost importance both for risk classification and tailoring of the therapy. The data of pediatric ALL patients that received treatment with Berlin-Frankfurt-Münster (BFM) protocols were retrospectively collected from 5 university hospitals in Turkey. Of the 1388 patients enrolled in the study 390 were treated according to MRD-based protocols. MRD assestment was with real time quantitative polymerase chain reaction (qPCR) in 283 patients and with multiparametric flow cytometry (MFC)-MRD in 107 patients. MRD monitoring had upstaged a total of 8 patients (2%) from intermediate risk group to high-risk group. Univariate analysis revealed age 10 years or above, prednisone poor response, PCR-MRD ≥10-3 on day 33 and on day 78 as poor prognostic factors affecting event-free survival (EFS). Detection of >10% blasts on day 15 with MFC (MFC-high-risk group) was not shown to affect EFS and/or overall survival (log-rank P=0.339). Multiple logistic regression analysis revealed PCR-MRD ≥10-3 on day 78 as the only poor prognostic factor affecting EFS (odds ratio: 8.03; 95% confidence interval: 2.5-25; P=0.000). It is very important to establish the infrastructure and ensure necessary standardization for both MRD methods for optimal management of children with ALL.

Significance of WT1 and multiparameter flow cytometry assessment in patients with chronic myelomonocytic leukemia receiving allogeneic hematopoietic stem cell transplantation.

The objective of this study was to investigate the clinical significance of minimal residual disease (MRD) monitoring through Wilms tumor 1 (WT1) gene expression and multicolor flow cytometry (FCM) in patients with chronic myelomonocytic leukemia (CMML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). For this purpose, WT1 gene expression and the CMML-related abnormal immunophenotype were examined using real-time quantitative polymerase chain reaction and FCM, respectively. In total, 59 patients with CMML who underwent allo-HSCT were enrolled in this study. Thirteen cases (22.0%) developed hematological relapse, and 15 patients (25.4%) expired during the follow-up period. Thirty-four patients (37.6%) were positive for WT1 (WT1+), and 44 patients (74.6%) were positive for FCM prior to allo-HSCT. After allo-HSCT, there were 21 WT1+ patients (35.6%) and 10 patients (16.9%) who were positive in FCM (FCM+). Post-transplant WT1+ (post-WT1 0.6+; 50.7% vs. 7.6%, p<.001) and post-transplant FCM+ (post-FCM+; 90.0% vs. 8.8%, p<.001) indicated a higher 3-year cumulative incidence of relapse (CIR) compared with the WT1- or FCM-patients. Multivariate analysis of event-free survival (EFS), overall survival (OS), and CIR showed that the FCM status after transplantation was an independent prognostic factor for relapse (p<.05). Both FCM and WT1 after HSCT were identified as important predictors of recurrence of CMML following transplantation and may be useful in guiding interventions against disease relapse.

Revealing the Mysteries of Acute Myeloid Leukemia: From Quantitative PCR through Next-Generation Sequencing and Systemic Metabolomic Profiling.

The efforts made in the last decade regarding the molecular landscape of acute myeloid leukemia (AML) have created the possibility of obtaining patients’ personalized treatment. Indeed, the improvement of accurate diagnosis and precise assessment of minimal residual disease (MRD) increased the number of new markers suitable for novel and targeted therapies. This progress was obtained thanks to the development of molecular techniques starting with real-time quantitative PCR (Rt-qPCR) passing through digital droplet PCR (ddPCR) and next-generation sequencing (NGS) up to the new attractive metabolomic approach. The objective of this surge in technological advances is a better delineation of AML clonal heterogeneity, monitoring patients without disease-specific mutation and designing customized post-remission strategies based on MRD assessment. In this context, metabolomics, which pertains to overall small molecules profiling, emerged as relevant access for risk stratification and targeted therapies improvement. In this review, we performed a detailed overview of the most popular modern methods used in hematological laboratories, pointing out their vital importance for MRD monitoring in order to improve overall survival, early detection of possible relapses and treatment efficacy.

Monitoring of post-transplant MLL-PTD as minimal residual disease can predict relapse after allogeneic HSCT in patients with acute myeloid leukemia and myelodysplastic syndrome

BackgroundThe mixed-lineage leukemia (MLL) gene is located on chromosome 11q23. The MLL gene can be rearranged to generate partial tandem duplications (MLL-PTD), which occurs in about 5-10% of acute myeloid leukemia (AML) with a normal karyotype and in 5-6% of myelodysplastic syndrome (MDS) patients. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently one of the curative therapies available for AML and MDS with excess blasts (MDS-EB). However, how the prognosis of patients with high levels of MLL-PTD after allo-HSCT, and whether MLL-PTD could be used as a reliable indicator for minimal residual disease (MRD) monitoring in transplant patients remains unknown. Our study purposed to analyze the dynamic changes of MLL-PTD peri-transplantation and the best threshold for predicting relapse after transplantation.MethodsWe retrospectively collected the clinical data of 48 patients with MLL-PTD AML or MDS-EB who underwent allo-HSCT in Peking University People’s Hospital. The MLL-PTD was examined by real-time quantitative polymerase chain reaction (RQ-PCR) at the diagnosis, before transplantation and the fixed time points after transplantation. Detectable MLL-PTD/ABL > 0.08% was defined as MLL-PTD positive in this study.ResultsThe 48 patients included 33 AML patients and 15 MDS-EB patients. The median follow-up time was 26(0.7-56) months after HSCT. In AML patients, 7 patients (21.2%) died of treatment-related mortality (TRM), 6 patients (18.2%) underwent hematological relapse and died ultimately. Of the 15 patients with MDS-EB, 2 patients (13.3%) died of infection. The 3-year cumulative incidence of relapse (CIR), overall survival (OS), disease-free survival (DFS) and TRM were 13.7 ± 5.2, 67.8 ± 6.9, 68.1 ± 6.8 and 20.3% ± 6.1%, respectively. ROC curve showed that post-transplant MLL-PTD ≥ 1.0% was the optimal cut-off value for predicting hematological relapse after allo-HSCT. There was statistical difference between post-transplant MLL-PTD ≥ 1.0% and MLL-PTD < 1.0% groups (3-year CIR: 75% ± 15.3% vs. 0%, P < 0.001; 3-year OS: 25.0 ± 15.3% vs. 80.7% ± 6.6%, P < 0.001; 3-year DFS: 25.0 ± 15.3% vs. 80.7 ± 6.6%, P < 0.001; 3-year TRM: 0 vs. 19.3 ± 6.6%, P = 0.277). However, whether MLL-PTD ≥ 1% or MLL-PTD < 1% before transplantation has no significant difference on the prognosis.ConclusionsOur study indicated that MLL-PTD had a certain stability and could effectively reflect the change of tumor burden. The expression level of MLL-PTD after transplantation can serve as an effective indicator for predicting relapse.

The Importance of Complementary Immunological Markers in the Diagnosis of Minimal Residual Disease in Multiple Myeloma

Background. The population of non-tumor plasma cells in healthy subjects’ bone marrow is known to be fairly heterogeneous. Among them, there may be a small number of CD19-, CD56+, CD45- plasma cells which differ from the main bulk of the normal plasmacytic cells by the lack of CD19 and CD45 expression and the presence of CD56 expression. It is the fact which makes the monitoring of minimal residual disease (MRD) especially challenging in multiple myeloma (MM) since normal and aberrant plasma cells should be compared. For this reason, a study of such complementary diagnostic markers as CD27, CD28, CD117, and CD81 is extremely important. Aim. To analyze the role of complementary diagnostic markers (CD27, CD28, CD117, and CD81) of MRD in MM patients at different disease stages. Materials &amp; Methods. The present study enrolled 62 MM patients aged 31-76 years (median 58 years); 25 women and 37 men. The analysis focused on morphological and immunophenotypic properties of bone marrow plasma cells. MRD was detected by 8-color flow cytometry with the use of FACSCanto II Flow Cytometer (USA) based on EuroFlow standards. Results. At the stage of primary diagnosis of MM, the imunophenotype of plasma cells was analyzed in all 62 patients using two 8-color panels recommended by the EuroFlow Consortium (2012). In accordance with primary immunophenotyping data, MRD was evaluated on the basis of not only the main diagnostic markers of plasma cells (CD38, CD138, CD45, CD56, and CD19), but also the complementary ones, such as CD27, CD28, CD117, and CD81. The study was basically conducted after induction therapy and upon remission. With cut-off &gt; 0.01 % of aberrant plasma cells, the MRD incidence was the following: 91.0 % with CD27, 90.6 % with CD28, 87.0 % with CD117, and 96.7 % with CD81 markers. Respectively, no MRD was detected in 9.0 % with CD27, 9.4 % with CD28, 13.0 % with CD117, and 3.3 % with CD81 markers.

ROR-1 Expression in the Diagnosis and Monitoring of Minimal Residual Disease in Chronic Lymphocytic Leukemia

Background. In view of similar morphological and phenotypic characteristics of some B-cell lymphoproliferative diseases and despite the known phenotype of tumor cells, a search is currently underway for new diagnostic markers, the expression of which remains stable during chronic lymphocytic leukemia (CLL) treatment and can be used for both diagnosis and assessment of residual tumor population. One of such markers is ROR-1. Aim. To assess the expression and feasibility of the ROR-1 marker using В-lymphocytes in minimal residual disease (MRD) dynamics and monitoring in CLL. Materials &amp; Methods. Hematological and immunophenotypic analyses were performed in 110 CLL patients (50 of them with newly diagnosed disease and 60 on therapy). In addition to that, 20 patients with reactive lymphocytosis and 32 donors were examined. The ROR-1 expression in В-lym-phocytes were measured with FACS Canto II flow cytometer using the following monoclonal antibody panel: CD45, CD19, CD20, and ROR-1. Results. The analysis showed that ROR-1 is essentially not expressed in normal and reactive В-lymphocytes and is detected in 100 % of CLL tumor cells both at disease onset and on therapy. The ROR-1 expression does not change during CLL treatment and can be used not only for CLL diagnosis but also for detection of MRD. Bone marrow aspirates (п = 64) and peripheral blood samples (п = 6) were analysed for MRD assessment by two methods: according to the standardized protocol, recommended by ERIC (European Research Initiative on CLL) in 2007, with FACS Canto II flow cytometer (BD Biosciences) and using DuraClone RE CLB Tube (Beckman Coulter) with Navious flow cytometer (Beckman Coulter).

Cell-free DNA as a biomarker in cancer

Translational research of liquid biopsy is just at the edge of routine clinical application: an emerging validity of circulating tumor DNA (ctDNA) tests suggests its use for earlier cancer detection and better monitoring of minimal residual disease (MRD) and resistance development, thus offering earlier guidance for therapy choices with the intent to cure cancer. In this review, we focus on ctDNA as an advanced and standardized validated marker in liquid biopsy. We also discuss what will be needed to reach the new milestone of personalized (precision) medicine to be used as a common standard of care. We summarize recent developments of cell-free DNA (cfDNA) and its clinical use as a biomarker in cancer.