Molecular Monitoring Of Minimal Residual Disease Research Articles

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Recommendations for laboratory testing of UK patients with acute myeloid leukaemia.

Allogeneic hematopoietic stem cell transplantation for pediatric acute myeloid leukemia in first complete remission: a meta-analysis

Identification of pediatric patients with acute myeloid leukemia (AML) candidates to receive allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1) is still a matter of debate. Currently, transplantation is reserved to patients considered at high risk of relapse based on cytogenetics, molecular biology, and minimal residual disease (MRD) assessment. However, no randomized clinical trial exists in the literature comparing transplantation with other types of consolidation therapy. Here, we provide an up-to-date meta-analysis of studies comparing allo-HSCT in CR1 with chemotherapy alone as a post-remission treatment in high-risk pediatric AML. The literature search strategy identified 10 cohorts from 9 studies performing as-treated analysis. The quantitative synthesis showed improved overall survival (OS) (relative risk, 1.15; 95% confidence interval [CI], 1.06–1.24; P = 0.0006) and disease-free survival (relative risk, 1.31; 95% CI, 1.17–1.47; P = 0.0001) in the allo-HSCT group, with increased relapse rate in the chemotherapy group (relative risk, 1.26; 95% CI, 1.07–1.49; P = 0.006). Sensitivity analysis including prospective studies alone and excluding studies that reported the comparison only on intermediate-risk patients confirmed the benefit of allo-HSCT on OS. Further research should focus on individualizing allo-HSCT indications based on molecular stratification and MRD monitoring.

Molecular Monitoring of NPM1 Is a Useful Tool for Detecting Early-Stage Relapse and to Guide Pre-Emptive Treatment Approaches in Patients with NPM1-Mutated Acute Myeloid Leukemia

Molecular Monitoring of NPM1 Is a Useful Tool for Detecting Early-Stage Relapse and to Guide Pre-Emptive Treatment Approaches in Patients with NPM1-Mutated Acute Myeloid Leukemia

Assay Specificity and Sensitivity Evaluation of Cepheid GeneXpert® NPM1 Prototype for Mutation Detection in Acute Myeloid Leukemia

Background: Mutations in thenucleophosmin1 (NPM1) are considered as founder mutations in acute myeloid leukemia (AML). NPM1 mutations in exon 12 occur in about 60% adult AML patients with normal karyotype. Among all NPM1 subtypes, type A (69.15%), type B (7.41%) and type D (7.41%) are found to be most frequent. The evaluation of minimal residual disease (MRD) has been considered critical to more precisely define AML response to clinical treatment, thereby refining disease risk stratification. Currently, AML with NPM1 gene mutations is recognized as a distinct entity, due to its unique clinical features. A large body of evidence demonstrated a significant independent prognosis factor of molecular MRD monitoring in NPM1-mutated AML. Objectives: Here we present GeneXpert NPM1 prototype, a fast and promising test for use in AML mutation detection (this prototype is for research use only, not for use in diagnostic procedures, and not reviewed by any regulatory body). NPM1 prototype is an easy-to-use-sample in/results out, and highly sensitive assay. The turnaround time is as fast as less than 3 hours. NPM1 prototype is designed for the quantitative detection of NPM1 mutant mRNA transcripts (types A, B and D in exon 12) and the ABL endogenous control mRNA transcript in peripheral blood of AML patients. This test is also designed for use as an aid in the monitoring of mutant NPM1 mRNA transcript levels in patients diagnosed with NPM1-mutated AML. This test does not differentiate between A, B or D type mutant NPM1 transcripts and does not detect or monitor other rare types of mutant NPM1. Results: Through assay optimization, we were able to reach limit of detection (LoD) within 0.05% for three NPM1 subtypes among all screened EDTA donor blood samples. Ct-copy number regression curve aligns well among three subtypes, within detection range. Our assay specificity has been above 95% among 30 screened EDTA donor blood samples. Conclusion: In summary, GeneXpert NPM1 prototype shows promise as a fast test for use in AML mutation detection.The presented NPM1 prototype has a great advantage covering most prevalent NPM1 mutation types up to 94% of all NPM1 subtypes. Our results demonstrated that our prototype shows satisfactory assay specificity and sensitivity. Clinical samples will be included for our clinical validation in the future. Disclosures No relevant conflicts of interest to declare.

Molecular MRD Monitoring Is Feasible in the Majority of Children with AML and Is Highly Predictive of Outcome: Results from the International MyeChild01 Study

Introduction Most children with acute myeloid leukaemia (AML) harbour fusion genes which are ideal targets for molecular minimal residual disease (MRD) monitoring. However, evidence of prognostic significance is currently lacking and consequently most current paediatric AML treatment protocols rely on flow cytometric (FCM) evaluation to allocate treatment. Molecular MRD techniques provide significantly greater sensitivity and specificity and could allow more accurate outcome prediction, and consequently more personalised therapy, which is highly relevant in a disease where treatment related mortality, morbidity and relapse remain significant. Methods Between June 2016 and February 2019, MyeChild01 enrolled 170 children aged 0-18y with newly diagnosed AML who were randomly assigned to induction therapy with liposomal daunorubicin or mitoxantrone with cytarabine with or without gemtuzumab ozogamicin. Consolidation treatment was determined by karyotype, mutational profile and MRD status. Comprehensive centralised diagnostic assessment consisted of: Karyotype and fluorescence in-situ hybridisation (FISH) using a custom panel of probes to detect paediatric AML-associated gene fusions.PCR based screening for mutations in FLT3, NPM1 and CEPBA.Targeted capture of known fusion loci and paired end sequencing.RNA-seq using the Illumina TruSight fusion panel. Where a fusion gene was identified, RT-qPCR assays were designed and optimised for each patient. NPM1 mutation was also used as an MRD target if present. Paired PB and BM samples were requested after each cycle of treatment. Patients could have sequential monitoring after completion of therapy although this was not mandated. For this analysis, patients with core-binding factor (CBF) leukaemias i.e. inv(16)(p13q22) or t(8;21)(q22q22) with transcript levels above previously defined thresholds (Yin et al, 2012) were considered MRD positive. For all other targets, amplification in at least 2/3 replicates at <40 PCR cycles in either PB or BM was considered MRD positive. For patients with CBF or NPM1 mutation, both molecular and FCM MRD status contributed to treatment allocation. Otherwise, FCM MRD was used unless there was no FCM target. Allogeneic stem cell transplantation (HSCT) was recommended for all patients with poor risk cytogenetics, those with intermediate risk cytogenetics who were MRD positive after cycle 2, and those with favourable risk cytogenetics who were MRD positive at the end of cycle 3. After completion of treatment, no specific advice was given regarding management of positive MRD results. Results We identified fusion genes in 126/170 patients (74%). We designed 55 unique RT-qPCR assays to specifically amplify 27 fusions with calculated sensitivity between 1:104 and 1:106. 62/126 children provided a complete set of samples: 31 (60%) with favourable, 18 (29%) with intermediate and 13 (21%) with high risk cytogenetic / molecular profiles. We analysed the effect of molecular MRD status at the end of protocol specified treatment, which included SCT in 17/62 (27%). One year event-free survival from diagnosis was 70% (95% confidence interval 52-82%) in patients who tested MRD negative compared to 11% (1-39%) in patients who tested MRD positive at the end of treatment (p<0.0001, figure 1a). Estimated cumulative incidence of relapse was 17% (7-30%) in patients who tested MRD negative vs 78% (28-95%) in those testing positive (p<0.0001 figure 1b). MRD status at earlier time points had less impact, possibly due to treatment intensification for MRD positivity. Four patients received non-protocol specified SCT due to physician / family preference based on persistently positive or serially rising transcript levels after completion of treatment. At last follow up 4/4 were alive in complete remission. Rising transcript levels were observed in a further 8 patients to whom no pre-emptive treatment was given due to individual preference or lack of appropriate therapy. Haematological relapse subsequently occurred in 8/8. Conclusion Molecular MRD monitoring is feasible in the majority of children with AML, permitting refinement of response-adapted therapy. Molecular MRD status at the end of treatment is highly predictive of outcome, identifying molecular complete remission as a treatment goal for these children. Serially rising MRD levels reliably predict relapse in the absence of therapeutic intervention. Disclosures Dillon: Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; TEVA: Consultancy, Honoraria. Vyas:Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Forty Seven, Inc.: Research Funding; Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; Daiichi Sankyo: Speakers Bureau. Amrolia:UCLB: Patents & Royalties. Baruchel:Bellicum: Consultancy; Servier: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Long-Term Outcome of Relapsed Acute T-Lymphoblastic Leukemia (T-ALL) in Children and Adolescents

Introduction Despite improvements in first-line treatment of childhood T-ALL, relapse remains the main cause of treatment failure. Patients with T-ALL experiencing leukemia recurrence have a dismal outcome, mostly due to a failure to achieve a second complete remission (CR) or to subsequent relapse. Assessment of minimal residual disease (MRD) may allow identifying subsets of patients with different prognosis. We analyzed the outcomes of 141 Italian children and adolescents who experienced T-ALL relapse after frontline multicenter AIEOP-BFM ALL treatment trials. Methods: This retrospective study aimed at describing the outcomes of Italian patients with T-ALL who were enrolled in the AIEOP-BFM ALL2000, 2006 and 2009 trials and subsequently experienced disease relapse. Among the 750 T-ALL Italian patients enrolled in the above-mentioned trials, 141 had a disease recurrence (70/251 patients in ALL2000 trial, 29/190 in ALL2006 trial, and 42/309 in ALL2009 trial). Molecular MRD monitoring after induction therapy for leukemia relapse was available for 38 patients. Results: The 141 patients, 113 males and 28 females, had a median age of 9 years at diagnosis (range 1-17) and 10 at relapse (range 2-19). At diagnosis, 7 patients were allocated to the Standard (SR), 45 to the Medium (MR), and 89 to the High Risk (HR) group, respectively. Thirty-four (4 MR and 30 HR patients) of these children were given allogeneic hematopoietic stem cell transplantation (allo-HSCT) in 1st CR and 41 had received central nervous system (CNS) radiotherapy. Relapses were classified as very early (occurring <18 months from diagnosis), early (occurring ≥18 months from diagnosis and <6 months after completion of primary therapy), and late (occurring >6 months after the end of the frontline protocol) in 92, 36, and 13 patients, respectively. Site of relapse was isolated bone marrow (BM) in 74 patients, combined BM/extramedullary (EM) in 23 patients, and isolated EM in 44 patients (involving a single EM site in 37 cases and >1 EM site in 7 cases; CNS was involved in 29 patients). At the time of relapse, 7 patients were treated according to AIEOP ALL REC98 Trial, 57 according to AIEOP ALL REC2003 Trial, while the remaining 77 patients received other combination of chemotherapy. After reinduction/consolidation treatment, 58 patients were given allo-HSCT from either a matched family donor (16) or an unrelated donor (UD, 31 patients, of which 21 from a matched UD and 10 from a mismatched UD) or an HLA-partially matched relative (11). With a median follow-up of 7.5 years (range 2-14), 34 patients are currently alive; 17 of these patients had EM isolated relapse, which was very early in 13 cases, and 17 had BM involvement at relapse, with late relapse in 8 cases. The 8-year overall survival (OS) of the whole cohort was 23.3% (95% CI 16.5-30.7) (Figure 1A). Isolate EM relapse (Figure 1B) and late relapses were associated with a better OS probability. Both factors remained statistically significant in multivariable analysis. Moreover, in univariable analysis a better prognosis was observed in patients treated more recently (i.e., after year 2008) (8-year OS 31.8% vs 16.2%, p=0.01). After allo-HSCT, 29 patients are currently alive, while the remaining 29 patients died from progressive disease (21) or transplant-related causes (8), leading to a 8-year OS for patients given allo-HSCT in 2nd RC of 48.5% (95% CI 34.7-61.0). Of the 83 children given sole chemotherapy, 5 are currently alive, while the remaining 78 died due to progressive disease (53), organ-failure (7), infection (9), or other causes (9). MRD monitoring was performed for 38/97 patients with BM involvement (5 with late, 13 with early, and 20 with very early relapse, respectively). Patients with a post-induction MRD <10-3 showed a reduced incidence of relapse (Figure 1C), resulting into better probability of 8-year OS (Figure 1D) in comparison to patients with higher MRD levels. Conclusions: Our results confirm that T-ALL relapse, especially if involving BM and occurring within 6 months from treatment discontinuation is still associated with poor prognosis, although in the last 10 years a higher proportion of children with relapsed T-ALL has been rescued by second line therapy, including transplantation. In this Italian retrospective cohort, a MRD value >10-3 at the end of induction therapy predicts a miserable outcome, this finding allowing to identify patients eligible to experimental therapies. Figure 1 Disclosures Merli: Novartis: Honoraria; Amgen: Honoraria; Bellicum: Consultancy; Sobi: Consultancy. Rizzari:JazzPharma, Shire, medac , Sigma-Tau, Baxalta, Shire and Servier: Consultancy, Honoraria; Together with study group from Shire, medac , Sigma-Tau, Baxalta, Shire and Servier: Research Funding. Parasole:Servier: Honoraria; Baxalta: Honoraria; Eusapharma: Honoraria. Locatelli:bluebird bio: Consultancy; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Quantitative Analysis of MLL Fusion Transcripts By Droplet Digital PCR to Monitor Minimal Residual Disease in MLL-Rearranged Acute Myeloid Leukemia

Rearrangements of the mixed lineage leukemia gene (MLL, re-named KMT2A) result in aggressive leukemia. Current risk stratification of MLL-rearranged (MLL-r) leukemia is directed by the fusion partner gene and, increasingly, by minimal residual disease (MRD) assessment after induction therapy. The clinical significance of quantifying fusion transcript levels in leukemia patients is firmly established in chronic myeloid leukemia and acute promyelocytic leukemia but is less well studied in MLL-r patients. Real-time quantitative PCR (RQ-PCR) is the standardized assay for molecular MRD monitoring in patients with MLL-rearranged leukemia. However, this method is less precise when few leukemic cells are present, thus limiting its application for highly sensitive MRD monitoring. Droplet digital PCR (ddPCR) allows for absolute quantification of fusion transcripts when multiple copies of fusion transcripts are present per cell. Therefore, we aimed to evaluate whether determining MLL fusion transcript levels by ddPCR could improve the sensitivity of MRD monitoring in MLL-r leukemia.A total of 44 diagnostic and follow-up samples obtained from paediatric MLL-r leukemia patients (26 ALL, 18 AML) were subjected to targeted next-generation sequencing to obtain patient-specific fusion sequences. MLL fusion transcripts were quantified by ddPCR in a total of 17 samples obtained from 4 paediatric AML patients with MLL fusions involving MLLT3 (n = 3) and MLLT10 (n = 1). Fusion-specific probe assays were designed from each of the patient specific fusion sequences for MRD assessment by ddPCR. To determine the detection limit of this method in quantifying MLL fusion transcripts, two MLL-r AML cell lines (MV4-11 and THP-1), and one MLL-wt cell line (Kasumi-1) were used. MLL fusion transcript level of detection of ddPCR was determined by serially diluting MLL-r cDNA into MLL-wt cDNA (Kasumi-1). Using 20ng of MLL-r cDNA in 200ng diluent as the highest concentration, a 10-fold dilution series was performed to make concentrations ranging from 10−2 to 10−7. Each ddPCR reaction mixture contained 11ul of cDNA mix as template with 1X Supermix no dUTP (Bio-Rad), 500 nM of both F/R primers and 250 nM of 5'-FAM labelled probe (IDT). Droplets were generated using a QX200 Droplet Generator (Bio-Rad). A general thermal cycler protocol with annealing at 61°C for 1 minute was performed and positive fluorescence droplets were read using QX200 Droplet Reader (Bio-Rad). MRD of patient samples, derived from ddPCR, was then compared to MRD derived from DNA-based RQ-PCR, following the guidelines established by the EuroMRD group.Our ddPCR method showed high reliability and sensitivity, with the detection limit determined to be 10-5 for a cell line with low MLL fusion transcript expression (THP-1), and 10-6 for a cell line with high MLL fusion transcript expression (MV4-11). Comparison of results obtained by RQ- PCR and ddPCR in a total of 17 diagnostic and follow-up samples from 4 AML patients showed excellent/good concordance between methods for 13 samples with moderate MRD levels. The 4 samples with low levels of MRD (10-4 to 10-5) below the quantitative range as defined by EuroMRD for RQ-PCR were all detectable by ddPCR, highlighting that ddPCR could provide robust and highly sensitive MRD assays compared to the standardized RQ-PCR assays.In conclusion, ddPCR is a promising technique that can reproducibly and reliably quantify MLL-r transcripts for MRD monitoring of MLL-r leukemia. Highly sensitive and robust molecular MRD monitoring by ddPCR holds promise for improving response-based therapeutic stratification and prediction of molecular relapse before overt hematological relapse. DisclosuresNo relevant conflicts of interest to declare.

Clinical and Biological Characterization of Children with FLT3ITD Mutated Acute Myeloid Leukemia (AML): A Report from the AIEOP AML-2002 Study Group

Purpose. While most of the recurrent molecular marker of AML have been already discovered and used for risk stratification in clinical protocols, post-treatment monitoring of these abnormalities can be useful in the clinical management of patients. In particular, monitoring of minimal residual disease (MRD), largely used in acute lymphoblastic leukemia, is gaining popularity also in AML, in the perspective of optimizing risk stratification of patients in terms of subsequent clinical relapse. While MRD monitoring in AML is mainly based on flow-cytometry approaches, molecular MRD measurements are not currently routinely used for taking clinical decision. Here we examine if the molecular MRD and Allelic Ratio (AR) levels monitoring may influence the survival of FLT3 internal tandem duplication (ITD)-mutated AML patients.Patients and methods. We retrospectively analyzed 507 children with de novo AML for FLT3ITD mutation by RT-PCR. Mutation was sequenced and the AR was calculated by Genescan. Bone marrow samples after induction treatment were analyzed for MRD levels by Real-Time PCR. We correlated these parameters with both patient event-free survival (EFS) and gene expression profile (GEP) findings.Results. 54/507 patients (10.6%) harboured FLT3ITD mutation. AR was calculated both at cDNA and DNA levels showing a reliable correlation (R=0.68), even if only AR measured on cDNA was found to be a significant poor prognostic feature. This latter observation supports the concept that expression of the mutation is more important than genetic bulk architecture at diagnosis. Patients with high AR showed a significant worse EFS as compared to those with low AR (19.2% for AR>0.51, vs 63.5% for AR<0.51), mainly because of a failure to achieve complete remission. Molecular monitoring of MRD showed that a low MRD reduction (<2 logs) after I induction course was an independent prognostic factor predicting both a high risk of incidence of relapse (cumulative incidence of relapse, CIR=68.7% vs 24.3, p <0.05), and a lower EFS (22.2% vs 59.4%, p<0.05). After the second induction course, a low MRD reduction also predicted a worse EFS. Through GEP analysis, we showed that high ITD-AR patients had a different transcriptional profile that those with low ITD-AR. Gene ontology revealed that patients with high AR had a negative regulation of genes involved in H3K27 tri-methylation tightly associated with inactivation of gene promoters as compared to those with low ITD-AR, suggesting that methylation might play a crucial role in this subgroup of FLT3ITD patients. Furthermore, we found that the cyclinA1 was significantly overexpressed. We hypothesised that cyclinA1-CDK2 complex is driving B-MYB phosphorylation, which, in turn, over-activates its own expression and sustains the B-MYB transcriptional oncogenic program in FLT3ITD patients with high AR. Support to this hypothesis is given by the observation that B-MYB signature was found to discriminate patients with low and high ITD-AR. GEP was also performed by comparing poor or good responders patients, showing that the former have a significant enrichment in stemness, in resistance to drugs and histone acetylation pathways.Conclusion. Our results show that ITD-AR and MRD status are important independent prognostic factors for the management of of FLT3ITD patients, to be taken into consideration in planning the post-induction treatment. The high expression of the FLT3ITD mutation and the persistence of the mutated clone confer an aberrant hyperactivation of the downstream oncogenic FLT3 pathway influencing the outcome within these AML. Transcriptional profiles opens for further consideration of epigenetic targeting for FLT3ITD patients. DisclosuresNo relevant conflicts of interest to declare.

Quantitative Monitoring By RT-PCR of Molecular Markers on Day +100 after Allogeneic Stem Cell Transplantation Predicts Outcome in Patients with Acute Myeloid Leukemia

Introduction:Monitoring of minimal residual disease (MRD) is commonly used after allogeneic stem cell transplantation (SCT) in acute myeloid leukemia (AML) patients. Flow cytometry of leukemia-associated aberrant immunophenotypes (LAIP) and chimerism analyses are frequently used tools to monitor MRD. These methods have lower sensitivities and lower relapse detection rates in contrast to molecular markers monitored by quantitative RT-PCR. However, their clinical implications are still limited and only little evidence of single molecular markers is available. We report on a retrospective study of 144 patients with molecular MRD markers transplanted at the Ludwig-Maximilans-University Hospital of Munich-Grosshadern, and analyzed the prognostic values of molecular MRD Monitoring after SCT, baseline risk factors, and follow-up (FU) markers (e.g. chimerism analyses and LAIP).Patients and Methods:Between January 2000 and August 2012, 495 AML patients underwent SCT at our institution. 164 patients had molecular MRD markers and 144 patients had at least one sample with quantitative results prior and/or after SCT, and were included into the analyses. At transplantation, patients had a median age of 48 years (range, 18 -73), and 49 patients were in complete remission, while 95 patients were in a more advanced status of their disease (>CR2). 106 patients received SCT from a HLA matched (10/10) and 38 patients from a HLA mismatched donor. Grafts were obtained either from related (n=60) or unrelated (n=84) donors. Most patients received reduced intensity conditioning (n=142). In 338 bone marrow samples MRD was monitored prior to SCT, on day +30 and on day +100. During the FU period after day +100, MRD was monitored at individual intervals in 429 peripheral blood samples. Quantitative RT-PCR was performed for NPM1 mutation (n=52), MLL-PTD (n=31), RUNX1-RUNX1T1 (n=12), CBFß-MYH11 (n=14), MLL rearrangements (n=20), MDS-EVI1/EVI1 (n=10), and DEK-CAN (n=5). Sensitivities of the different RT-PCRs assays ranged between 10-4 and 10-6.Results:After a median FU of 41 months (range, 4 – 115), 43 patients (30%) relapsed. The MRD levels monitored by RT-PCR prior to SCT and on day +30 after SCT showed no significant impact on relapse-free survival (RFS) and overall survival (OS). At day +100 after SCT, MRD positivity was strongly associated with worse RFS (HR 3.1, p=0.001) and OS (HR 3.2, p=0.004). This was also reflected in a cumulative two-year incidence of relapse of 61% for MRD positive patients versus 15% for MRD negative patients (p<0.0001). In addition, a change to MRD positivity during the FU period was strongly associated with a worse RFS (Mantel-Byar, p<0.0001). Furthermore, in univariate regression models we analyzed SCT baseline factors and FU markers. The remission status prior to SCT, the NPM1 mutation status, a HLA matched donor, the platelet count at day +100 (cut-off <50 G/l), and the European LeukemiaNet risk stratification (favorable versus others) were prognostic relevant risk factors. In the multivariate model only MRD positivity on day +100 (HR 3.7, p=0.013) and a low platelet count on day +100 (HR 5.0, p=0.005) were significantly associated with worse RFS. LAIP analyses (3-colors flow-cytometry, cut-off 0.1%) and a change in chimerism (STR-PCR and XY-FISH, cut-off 80% and 90%) of sorted (CD3+) and unsorted cells at the respective time points had no influence on RFS and OS.Conclusions:Molecular MRD monitoring after SCT might be a useful tool to identify AML patients at high risk for relapse, in contrast to other FU markers, e.g. chimerism analyses and LAIP. In particular, MRD positivity on day +100 after SCT and the switch to MRD positivity during the FU period were significantly associated with worse RFS. As perspective, the individual molecular MRD course might be used as prognostic factor for the guidance of treatment post SCT. Patients with MRD positivity on day +100 after SCT or with a switch to MRD positivity in the FU period may be considered for donor lymphocyte infusions (DLI) or chemotherapeutic interventions, such as hypomethylating agents. DisclosuresSubklewe:AMGEN Inc.: Research Funding.

Detection of minimal residual disease in patients with multiple myeloma using clonotype-specific PCR primers designed from DNA extracted from archival bone marrow slides

Polymerase chain reaction (PCR)-negative molecular complete remission (mCR) can be induced by stem cell transplantation in some patients with multiple myeloma (MM) and is associated with long-term progression-free survival (PFS). The detection of molecular minimal residual disease (MRD), however, requires fresh or frozen materials for designing clone-specific primers, which are not always readily available. In this study, we used DNA extracted from archival bone marrow (BM) slides for PCR to detect MRD in 50 patients with MM who received various induction therapies and autologous peripheral blood stem cell transplantation (ASCT). Clonotype-specific immunoglobulin (Ig) H PCR primers were prepared for 32 of 50 cases (64%) using BM slides, and for 9 of 14 cases (64%) using fresh BM cells. DNA in peripheral blood stem cell autografts of the 22 patients who achieved at least a partial response after ASCT was subjected to PCR to amplify clonotype-specific rearranged IgH gene sequences. The median PFS of the eight patients with MRD-positive autografts was 18 months, whereas that of 14 patients with MRD-negative autografts was not reached at a median follow-up of 27 months (p = 0.012). Post-ASCT PFS of the four patients who achieved mCR was 100% at a median follow-up of 47 months. These results indicate that archival BM slides can serve as a source of DNA for preparing clonotype-specific primers for MRD monitoring in patients with MM whose cryopreserved myeloma cells are not available for DNA preparation. Our results also suggest that patients with MM who received MRD-negative autografts and achieved mCR have a long PFS.

Flow cytometry and IG/TCR quantitative PCR for minimal residual disease quantitation in acute lymphoblastic leukemia: a French multicenter prospective study on behalf of the FRALLE, EORTC and GRAALL

Minimal residual disease (MRD) quantification is widely used for therapeutic stratification in pediatric acute lymphoblastic leukemia (ALL). A robust, reproducible, sensitivity of at least 0.01% has been achieved for IG/TCR clonal rearrangements using allele-specific quantitative PCR (IG/TCR-QPCR) within the EuroMRD consortium. Whether multiparameter flow cytometry (MFC) can reach such inter-center performance in ALL MRD monitoring remains unclear. In a multicenter study, MRD was measured prospectively on 598 follow-up bone marrow samples from 102 high-risk children and 136 adult ALL patients, using IG/TCR-QPCR and 4/5 color MFC. At diagnosis, all 238 patients (100%) had at least one suitable MRD marker with 0.01% sensitivity, including 205/238 samples (86%) by using IG/TCR-QPCR and 223/238 samples (94%) by using MFC. QPCR and MFC were evaluable in 495/598 (83%) samples. Qualitative results (<0.01% or ≥0.01%) concurred in 96% of samples and overall positivity (including <0.01% and nonquantifiable positivity) was concurrent in 84%. MRD values ≥0.01% correlated highly (r(2)=0.87) and 69% clustered within half-a-log(10). QPCR and MFC can therefore be comparable if properly standardized, and are highly complementary. MFC strategies will benefit from a concerted approach, as does molecular MRD monitoring, and will contribute significantly to the achievement of 100% MRD informativity in adult and pediatric ALL.

Cloned IGH VDJ targets as tools for personalized minimal residual disease monitoring in mature lymphoid malignancies; a feasibility study in mantle cell lymphoma by the Groupe Ouest Est d'Etude des Leucémies et Autres Maladies du Sang

Molecular minimal residual disease (MRD) analysis is fast emerging as an essential clinical decision-making tool for the treatment and follow-up of mature B cell malignancies. Current EuroMRD consensus IGH real-time quantitative polymerase chain reaction RQ-PCR assays rely on flow cytometric assessment of diagnostic tumour burdens to construct 'normalized', patient-specific, diagnostic DNA-based MRD quantification standards. Here, we propose a new 'hybrid' assay that relies on plasmid-based quantification of patient-specific IGH VDJ targets by consensus IGH real time (RQ)-PCR, combined with EuroMRD guidelines, for MRD monitoring in lymphoid malignancies. This assay was evaluated for MRD assessment in a total of 273 samples from 29 mantle cell lymphoma (MCL) patients treated within a Groupe Ouest Est d'Etude des Leucémies et Autres Maladies du Sang (GOELAMS) Phase II trial and was feasible, reliable and consistently comparable to gold-standard MRD techniques (99% concordance across all samples including 32 samples within the quantitative range) when analysed in parallel (117 samples). Integrating clinical prognostic parameters and MRD status in peripheral blood at the post-induction stage was predictive of progression-free survival (P = 0·034) thus demonstrating the clinical utility of the approach. Plasmid-based standards for the quantification of IGH VDJ targets are therefore confirmed to offer new opportunities for further standardization and clinical evaluation of MRD-guided management of patients with mature B cell malignancies.

Minimal Residual Disease Assessment by Flow Cytometry In AML Patients at Time of Aplasia Post-Induction Is Highly Predictive of Outcome and Strongly Correlates with Molecular MRD In a Subgroup of NPM1 Positive Patients

AbstractAbstract 1723 Background:Quantification of minimal residual disease (MRD) by multiparameter flowcytometry (MFC) in patients with acute myeloid leukemia (AML) provides significant prognostic information. Previous analyses have shown that positivity of leukemia-associated aberrant immunophenotype (LAIP) after induction and consolidation therapy is a risk factor concerning relapse and reflects over-all survival and relapse-free survival. Albeit molecular MRD monitoring has translated into therapeutic strategies in certain AML subgroups, e.g. APL, flowcytometric MRD assessment is still not integrated into therapeutic strategies. Methods:We performed a retrospective analysis of flowcytometric MRD data of 201 adult patients with AML. Patients were treated within the clinical study of the German AML cooperation group (AMLCG99 or sHAM); patients with APL were excluded. 165 patients were in the intermediate risk group according to karyotype; NPM1 and Flt3 positivity was present in 80 and 59 patients, respectively. 51% of patients relapsed in the course of disease, the median overall survival (OS) and relapse-free survival (RFS) were 518 and 292 days, respectively. MRD assessment by 3-color-flowcytometry was performed at initial diagnosis, during bone marrow aplasia (day 16 – 18 of induction therapy), after induction, after consolidation and at time of relapse. MRD flow was compared to molecular MRD assessment in patients with mutated NPM1. Results:Data on flowcytometric MRD assessment were available in 99% of patients at initial diagnosis, 74% on day16 of induction therapy, 80% post-induction, 42% post-consolidation and 62% at time of relapse. MRD positivity was defined as percentage of LAIP positive cells > 0,15%. In addition, the post-therapeutic degree of LAIP reduction compared to initial diagnosis was assessed: an indequate degree of LAIP reduction was set at a log difference (initial diagnosis/ day16) < 2. We could confirm that flowcytometric MRD predicts OS and RFS. The absolute level of MRD as well as the relative degree of MRD reduction on day16 of induction therapy defined significantly distinct risk groups concerning OS and RFS (OS: p = 0,03, RFS: p = 0,002). Discriminating the different types of LAIPs, we could demonstrate that cross-lineage phenotypes have the best sensitivity and specificity for predicting relapse (sensitivity 64%, specificity 88% on day16). The course of LAIP and NPM1 as assessed by rtPCR, highly correlates on day16 (correlation coefficient 0,84). Conclusion:Flowcytometric MRD assessment provides significant prognostic information at a very early point in induction therapy. In a subpopulation of NPM1+ AML patients we found a strong correleation of flowcytometric MRD and molecular MRD assessment. LAIP reduction on day 16 post-induction could therefore be integrated into prognostic models and can improve risk stratification especially in the absence of molecular MRD markers. Disclosures:No relevant conflicts of interest to declare.

Correlation Between Clinical Outcome and Disease Kinetics by Quantitative PCR in Myeloma Patients Following Post-Transplant Consolidation with Bortezomib, Thalidomide and Dexamethasone.

Abstract 960 Background and aims:We have recently shown that multiple myeloma (MM) patients undergoing autologous transplantation (ASCT) followed by a consolidation with Bortezomib/Thalidomide/Dexamethasone (VTD) achieve molecular remission in 17% and major tumor shrinking assessed by real time-quantitative (RQ) PCR in the majority of cases (Ladetto et al, ASH Meeting 2008). However little is known on the minimal residual disease (MRD) kinetics occurring after such an extensive cytoreduction. In this study we have investigated the disease kinetics in the post-consolidation phase with the aim of identifying the RQ-PCR patterns associated with persistent remission or increased risk of relapse. Patients and methods:Inclusion criteria and consolidation treatment for this study have been already reported and included: 1) a documented complete or very good partial remission following ASCT delivered as first line treatment; 2) no previous treatment with thalidomide and bortezomib; 3) presence of a molecular marker based on the immunoglobulin heavy chain rearrangement (IgH-R). Consolidation consisted of four courses of: a) Bortezomib 1.6 mg/m2 on days 1, 8, 15, 22; b) Thalidomide at the initial dose of 50 mg/day with increments up to 200 mg; c) Dexamethasone 20 mg/day on days 1 to 4, 8 to 11 and 15 to 18. MRD was assessed on bone marrow samples at diagnosis, study entry, after two VTD courses, at the end of treatment and then at six months intervals. Qualitative and RQ-PCR analysis were carried out using IgH-R derived patient specific primers as already described (Voena et al, Leukemia 1997; Ladetto et al, Biol Bone Marrow Transpl 2000). MRD kinetics of relapsing vs non relapsing patients has been measured using observed marginal medians of ln RQ-PCR values. For the predictive value of MRD kinetics patients were defined as having low tumor burden (TB) if they reached after VTD a MRD level <100 IgH-R/106 diploid genomes and as having active disease whenever a 10-fold tumor burden increase was recorded. Results:Feasibility, toxicity and clinical outcome of the trial have been already reported (Ladetto et al, ASH Meeting 2008). Thirty-nine patients were enrolled. Median follow-up from study entry is currently 32 months (50 from start of first line treatment). Following VTD, six patients achieved molecular remission (MR). Of these none has so far experienced a clinical relapse. MR was persistent in all patients, with an occasional PCR positive result in a patient who later reverted to PCR negativity. Among PCR positive patients 12 clinical relapses have been so far reported (50 months PFS: MR 100% vs no MR 62% - Figure 1A, p<0.001). Figure 2 shows the kinetics of MRD overtime in relapsing vs not relapsing patients (p<0.001), assessed on 230 RQ-PCR determinations at different timepoints. When RQ-PCR results of PCR positive patients were correlated with outcome we observed the following: 1) 14 patients achieved low TB and never had signs of active disease: none of them has so far relapsed except one who refused to undergo MRD monitoring and relapsed three year after the last PCR evaluation; 2) 13 patients never achieved a low TB and 8 of them relapsed; 3) 5 patients achieved a low TB but subsequently showed evidence of active disease: 3 of them relapsed. Patients in the first subgroup showed a 50 months PFS of 100% as opposed to those in group 2-3 who had a PFS of 37% (Figure 1B, p=0.001). Conclusions:Our results indicate that: 1) MRs following VTD are stable over time with no evidence of clinical and molecular relapse; 2) The vast majority of relapses occur in patients failing to achieve a low and stable TB; 3) Molecular monitoring of MRD allows to identify a large subset of patients (51% of cases) with an extremely low-risk of short-term relapse. [Display omitted] [Display omitted] Disclosures:Ladetto:CELGENE: Honoraria; JANSSEN-CILAG: Research Funding. Cavallo:CELGENE: Honoraria. Caravita:CELGENE: CONSULTANCY. Musto:JANSSEN-CILAG: Honoraria; CELGENE: Honoraria. Boccadoro:CELGENE: CONSULTANCY, ADIVISORY COMMITTEES, Research Funding; JANSSEN-CILAG: CONSULTANCY, ADIVISORY COMMITTEES, Research Funding; PHARMION: CONSULTANCY, ADIVISORY COMMITTEES, Research Funding. Palumbo:CELGENE: Honoraria; JANSSEN-CILAG: Honoraria.

Molecular Monitoring of Minimal Residual Disease in Patients with Multiple Myeloma

Improvement of transplantation strategies and a multitude of emerging novel therapies result in a better treatment outcome in patients with multiple myeloma (MM). This gives rise to the need for sensitive methods to detect minimal residual disease (MRD) in MM. Qualitative molecular monitoring using allele-specific oligonucleotide PCR for the immunoglobulin heavy chain (IgH) is well established to detect clonotypic cells after therapy or in stem cell harvests. Recently, real-time IgH PCR or limiting dilution based PCR assays offer the possibility to quantify the amount of residual tumour cells. In this review, different qualitative and quantitative IgH PCR techniques will be discussed as well as the current clinical role of molecular monitoring of MRD in patients with MM.