Post-consolidation Minimal Residual Disease Research Articles

In Adults with Ph-Negative Acute Lymphoblastic Leukemia (ALL), Age-Adapted Chemotherapy Intensity and MRD-Driven Transplant Indication Significantly Reduces Treatment-Related Mortality (TRM) and Improves Overall Survival - Results from the Graall-2014 Trial

Background During the 2005-2014 period, the GRAALL conducted the GRAALL-2005 trial in patients (pts) with Philadelphia chromosome (Ph)-negative ALL aged 18-59y. In this trial, all pts received a pediatric-inspired chemotherapy, whatever their age. Post-hoc analysis revealed an unacceptable TRM in pts aged 45-59y (Huguet et al. J Clin Oncol 2018; 36:2514-23). Allogeneic hematopoietic stem cell transplantation (HSCT) was offered in first complete remission (CR) to most CR pts, defined at high-risk (HR) by at least one baseline clinical or biological HR factor (CNS involvement, complex karyotype [≥5 abns.], low hypodiploidy/near triploidy, poor early blast clearance, late CR, as well as WBC ≥ 30x109/L, lack of CD10 expression, KMT2A rearrangement or TCF3::PBX1 fusion for B-cell precursor [BCP] ALL). Minimal residual disease (MRD) response was not considered for HSCT indication at that time. In the next GRAALL-2014 trial conducted in a similar population, we introduced two major changes in the treatment strategy. First, chemotherapy intensity (steroids, anthracycline and L-asparaginase) was reduced in pts aged 45-59y to decrease excessive TRM. Secondly, the indication for HSCT was modified, relying on IG/TR MRD response only (post-induction MRD ≥10-3 and/or post-consolidation MRD ≥10-4) while not on any former baseline HR criterion. Here, we compare the outcomes of the two trials, focusing on the impact of these two major evolutions. Patients & Methods A total of 743 pts treated in the GRAALL-2014 trial between 2015 and 2020 were compared to the 787 pts from the historical GRAALL-2005 trial, in terms of CR and induction death rates, rate of HSCT in CR1, cumulative incidence of TRM (CITRM) and relapse (CIR), relapse-free (RFS) and overall (OS) survival. It should be noted that two nested Phase 2 trials were introduced by amendment during the GRAALL-2014 course (in 2017 and 2018, respectively), aiming to evaluate post-remission addition of nelarabine and blinatumomab in 87 T-ALL and 94 BCP-ALL selected pts, respectively. Results Main patient characteristics, including age, gender, BCP/T-ALL, WBC, and historical baseline HR features, did not significantly differ between the two trial cohorts, even if CNS disease at diagnosis was more frequent in the 2014 cohort (12 vs 7%, p= 0.001). Outcome comparisons are shown in Table 1. Overall, the induction death rate was significantly reduced in the GRAALL-2014 (3 vs 6%, p= 0.005). As expected, this was only observed in pts aged 44-59y (3 vs 11%, p= 0.001), in whom dose-intensity reduction also translated into a higher need for second induction course (9 vs 5%, p= 0.05) eventually resulting in a higher CR rate (92 vs 86%, p= 0.05). Due to the newly introduced MRD-based stratification, the rate of pts with HSCT indication was markedly reduced (30 vs 65%, p<0.001), especially in pts aged 18-44y (21 vs 40%, p<0001). Consequently, the rate of pts transplanted in CR1 dropped down from 38 to 23% (p<0.001). The GRAALL-2014 strategy also yielded a significant reduction in CITRM (5 vs 11% at 3 years; p<0.001; Figure 1B) after CR achievement. This reduction was more pronounced in pts aged 45-59y (7 vs 17%, p<0.001 compared to 4 vs 8%, p= 0.02 in 18-44y pts). This was associated with an increased CIR (35% vs 28% at 3 years; p= 0.01), with more late relapses as depicted in Figure 1A. Even if the resulting RFS was similar in both cohorts (59 vs 62% at 3 years; p= 0.77), OS was significantly longer in the GRAALL-2014 (71 vs 64%; p= 0.002) (Figure 1C) likely due to better post-relapse outcomes. When censoring those GRAALL-2014 pts who received nelarabine or blinatumomab in CR1, observations were basically unchanged, even if the difference in CIR was even more marked. Finally, using a 3-month RFS landmark in the 211 GRAALL-2014 pts eligible for HSCT in CR1 based on their poor early MRD response, HSCT significantly prolonged RFS (HR= 0.46 [95% CI, 0.27-0.78]; p= 0.004). Conclusions In adults with Ph-negative ALL enrolled in the GRAALL-2014, age-adapted chemotherapy intensity and MRD-driven indication for HSCT significantly reduced induction and post-remission TRM. This translated into a prolonged OS, indicating that this strategy was safe, despite a higher incidence of late relapses. Newly available salvage options along with HSCT in CR2 might also have played a role. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effectiveness and Security of a Modified CALGB10403 in Adolescents and Young Adults with Acute Lymphoblastic Leukemia in Low-and-Middle-Income Countries: A Multicentric Experience in Central America

▪Background:Mexico and Central America have a high incidence of acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA). Chemotherapy with Hyper-CVAD has been widely used with poor outcomes, with a 3-year overall survival (OS) of 25.7% in this group of age.In low-and middle-income countries (LMIC), limitations in supportive care such as low access to neutrophil stimulant agents, antifungal prophylaxis and limited intensive care access, may increase treatment-related mortality. On the other hand, reports suggest that specific high-risk subgroups may be more frequent in Hispanic patients from Mexico and Central America.We hypothesize that the use of a less-myeloablative regimen, based on L-asparaginase could overcome the bad outcomes previously reported.MethodsWe modified the original CALGB 10403 based on local drug-access. We include patients with newly diagnosed Philadelphia-negative B- or T-cell ALL between 14-49 years from 4 centers in Mexico and one in Guatemala.We modified the regimen as following: replaced pegaspargase by E. Coli asparaginase, thioguanine by 6-mercapatopurine and incorporate rituximab 375mg/m2 for 6 doses in CD20 positive patients. After the first interim analysis (October 2019), we replaced the prednisone by dexamethasone during induction. Minimal residual disease (MRD) was assessed by flow cytometry after induction and after first consolidation. We considered high-risk karyotype if MLL-rearrangements, complex or hypodiploid and high-white blood cell count (WBC) if >30 x10 3/mcL for B-ALL or >100 x10 3/mcL for T-ALL.The main objective was to evaluate OS and as secondary objectives to evaluate complete response (CR) rate, relapse-free survival (RFS) and to assess the safety of this regimen.ResultsFrom January 2017 to December 2020, 95 patients have been enrolled with a median age of 23 years (range 14-49). One third (34.6%) had overweight and 11.7% were obese.The majority (92.6%) had a B-cell ALL and a normal karyotype (81.2%). The median WBC was 18.4 x10 3/mcL (0.2-427.7) and 40.9% had a high-WBC.During induction, adverse events (AE) included grade 3/4 elevated bilirubin (21.1%), transaminases (14.7%), hyperglycemia (14.7%), hypofibrinogenemia (44.2%), thrombosis (10.5%), hypersensitivity (2.2%) and pancreatitis (2.1%). During consolidation, AE included grade 3/4 hepatic toxicity (18.9%), hypertriglyceridemia (14.8%), thrombosis (5.3%) and pancreatitis (2.1%).Neutropenic fever occurred in 55.8% during induction (grade 4: 31.5%), and in 32.9% during consolidation (grade 4/5: 5.3%).A dose adjustment due to AE was required in 22.1% during induction and in 23.2% during consolidation. The induction related-mortality (IRM) rate was 7.4%The CR rate was 87.8%. After-induction, MRD was <0.01% in 39.1%, 0.01-0.1% in 39.1% and > 0.1% in 24.6%. Post-consolidation MRD was only measured in 43 patients and was <0.01% in 37.2%. During follow-up, 26.7% relapsed: 62.5% bone marrow (BM) relapses, 25.0% central nervous system (CNS) relapses and 12.5% CNS + BM relapses. Eight patients (8.4%) received an allogeneic-stem cell transplant (HSCT) as consolidation.The 2-year OS was 72.1%. The post-induction MRD <0.1% was associated with a better OS (figure 1A) (HR: 0.17 (95%CI: 0.06-0.55), p=0.003) and a high-WBC with an inferior OS (figure 1B) (HR: 4.13 (95%CI: 1.68-10.14), p=0.002).The 2-year RFS was 65.2%. The post-induction MRD <0.1% was associate with a better RFS (figure 1C) (HR: 0.19 (95%CI: 0.07-0.50), p=0.001) and a high-WBC and overweight / obesity with an inferior RFS (HR: 4.08 (95%CI: 1.71-9.73), p=0.001 and 2.50 (95%CI: 1.06-5.86), p=0.036 respectively) (figure 1D).Conclusions:The adoption of modified CALGB10403 regimen in Central America based on local resources is feasible. It is associated with a significant improvement in the OS and decrease in IRM when compared with previous reports. Despite a very high-rate of hepatic and metabolic toxicities, these were manageable. As reported by other groups, MRD, high-WBC and overweight/obesity are associated with poor outcomes.Despite being encouraging results, a significant number of patients persist with positive MRD and the main cause of dead is disease progression. Access to cellular therapies, and BiTes is cost restricted in LMIC. Hence, we should generate strategies to intensify treatment in MRD positive patients and expand transplant access to overcome outcomes. [Display omitted] DisclosuresRangel-Patiño: Bristol: Consultancy; Abbvie: Speakers Bureau. Ceniceros: Amgen: Speakers Bureau. Espinosa: Amgen: Speakers Bureau; Janssen: Consultancy; Pfizer: Consultancy. Amador: Abbvie: Consultancy, Speakers Bureau; Bristol: Consultancy. Cabrero Garcia: Takeda: Speakers Bureau; Abbvie: Speakers Bureau; Roche: Speakers Bureau; Janssen: Speakers Bureau; Astellas: Consultancy; BD: Speakers Bureau. Inclan-Alarcon: Janssen: Speakers Bureau; Boehringer: Speakers Bureau. Neme Yunes: Janssen: Consultancy, Speakers Bureau; Bristol: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Bristol: Consultancy, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau. Meillon-García: Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Roche: Speakers Bureau; Astellas: Consultancy. Apodaca: Sanofi: Consultancy; Asofarma: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau. Demichelis: Bristol/Celgene: Consultancy, Speakers Bureau; Astellas: Consultancy; Gilead: Consultancy; ASH: Research Funding; Abbvie: Consultancy, Speakers Bureau; AMGEN: Consultancy, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau; Jazz: Consultancy.

Prognostic Significance of Minimal Residual Disease in Patients with Secondary Acute Myeloid Leukemia

BACKGROUNDSecondary acute myeloid leukemia (AML) is defined as AML that develops from a prior myelodysplastic or myeloproliferative disorder, or from exposure to chemotherapy, radiation, or environmental toxins. Patients with secondary AML have inferior prognosis compared to those with de novo AML,1 and are more likely to be elderly with increased comorbidities and worse performance status.2 The detection of minimal residual disease (MRD) also has independent prognostic value in patients with AML.3-6 In elderly AML patients, MRD predicts worse outcomes independent of the poor treatment risk factors associated with increased age.7Due to the similar disease pathogenesis between elderly AML and secondary AML patients, it is likely that MRD testing will have similar implications. However, there is limited data on the prognostic role of MRD testing in secondary AML. METHODSWe retrospectively analyzed de novo and secondary AML patients at Washington University Medical Center who received post-induction, post-consolidation, pre-transplant, or post-transplant MRD testing during their treatment. MRD testing was performed with multiparameter flow cytometry testing using the “different from normal” method at one reference laboratory,8,9 with positive MRD defined as detection of an abnormal myeloblast population above 0.1%. A total of 180 patients were included. Any patient with at least one positive MRD was considered “MRD positive.” Baseline pretreatment characteristics were compared between groups with Mann-Whitney, Chi-square, or Fisher's exact test, when appropriate. We evaluated overall survival (OS) and relapse rates in de novo and secondary AML patients stratified by MRD status using Kaplan-Meier curves and the log rank test, or cumulative incidence function, as appropriate. Multivariate Cox proportional hazards model was also fitted to determine if MRD testing has similar prognostic value across de novo and secondary AML patients. RESULTSOur analysis included 133 patients with de novo and 47 patients with secondary AML. Secondary AML patients were significantly older at age of diagnosis (mean age 59.85 versus 51.17, p<0.001), however had similar rates of MRD positivity (28% versus 29%, p=0.830). There was a trend toward poorer cytogenetic risk profiles and worse Karnofsky Performance Status in secondary AML (Table 1). In all patients, we confirmed that MRD positivity confers significantly worse OS (45.5% versus 70.8%, p=0.020) and relapse rates at 2 years (53% versus 25.5%, p=0.029) (Figure 1). In de novo AML patients, MRD positivity continued to demonstrate significantly worse 2-year OS (44.1% versus 76.3%, p=0.006) and relapse rates (57.6% versus 20.7%, p=0.008). However, in secondary AML patients, there were no differences in OS (p=0.69) or relapse rates (p=0.98) between MRD positive and negative patients. Multivariate analysis demonstrated significant interaction (p=0.042) between MRD status and AML subtypes. MRD positivity emerged as an independent prognostic indicator of worse OS in de novo patients (HR 2.8, 95% CI 1.524-5.405) but not in secondary patients (HR 1.5, 95% CI 0.188-2.336). DISCUSSIONWe confirm that MRD status is a poor prognostic indicator in all AML patients, and this also holds true in patients with de novo AML. However, we found that MRD status has less prognostic value in secondary AML patients. This may be due to the overall poor prognosis seen in secondary AML patients, suggesting that MRD monitoring in this group of patients may be less useful for risk stratification. However, our study is limited by the small sample size, particularly in the number of secondary AML patients who displayed MRD positivity (N=13). In addition, we defined MRD positivity across all time points of testing, which may decrease patient homogeneity. A larger study with increased follow-up is necessary to confirm our findings, as these results have important implications in risk-stratification and treatment of patients with secondary AML. [Display omitted] DisclosuresDiPersio:BMS, Asterias, Amphivena, Bluebird: Other: Travel, Accomodations, Expenses; Hemedicus, DAVA Oncology: Speakers Bureau; Celgene, Bioline, Vasculox, Cellworks, Rivervest: Consultancy; Magenta Therapeutics: Equity Ownership.

Minimal Residual Disease and Survival Outcomes in Patients with Mantle Cell Lymphoma: a systematic review and meta-analysis

Background: Minimal residual disease (MRD) has shown the prognostic value in mantle cell lymphoma (MCL). To quantify the relationships between progression free survival (PFS) and overall survival (OS) with MRD status in MCL, we conducted this meta-analysis.Methods: We searched databases including Pubmed, Embase, Web of Science and the Cochrane Library up to July 15th, 2020. Data of patients' characteristics, MRD assessment and survival outcomes were extracted and analyzed.Results: Ten articles were included. For the impact of post-induction MRD status on survival outcomes, MRD positive status was associated with worse PFS (HR=1.44; 95%CI 1.27-1.62; P<0.00001) and OS (HR=1.30; 95%CI 1.03-1.64; P=0.03) compared with MRD negative status. Regarding the impact of post-consolidation MRD status on survival outcomes, MRD positivity predicted shorter PFS (HR=1.84; 95%CI 1.49-2.26; P<0.00001) and OS (HR=2.38; 95%CI 1.85-3.06; P<0.00001) than MRD negativity.Conclusions: This study indicated that MRD positivity after induction and consolidation treatments was associated with worse PFS and OS for MCL. MRD-based treatment strategies should be further explored in clinical trials and real-world practice.

Minimal residual disease in childhood B Lymphoblastic Leukemia and its correlation with other risk factors.

Objective:To determine frequency of post induction and post consolidation minimal residual disease (MRD) in pediatric B-lymphoblastic leukemia (B-ALL) patients and its association with clinical risk factors.Methods:This is a retrospective, cross sectional study carried out at the Indus Hospital on paediatric patients (1-17 years) was performed from May 2015 to January 2018. On day 35, MRD testing was done on bone marrow aspirate using four color flow cytometer with 0.01% cut off. Positive cases were retested at post consolidation. Data was collected for demographics, total leukocyte count (TLC), central nervous system status (CNS), Cytogenetics for BCR-ABL, MLL, TEL-AML by FISH and prophase response then analyzed in association to MRD status.Results:Out of 362 patients, 133 (37%) were post induction MRD positive, with no statistically significant association to age, gender, TLC, CNS status, prophase response, BCR-ABL and TEL-AML1. However, MLL showed closely significant association (p-value=0.05). Post consolidation, 49 (44%) were MRD positive; age, National cancer institute (NCI) risk groups and CNS status showed statistical significance (p-value <0.05).Conclusion:Despite high frequency of MRD positivity, significant association is not observed between post induction MRD and risk factors. However, post consolidation MRD has a significant association with NCI risk groups, age and CNS status.

T-ALL Minimal Residual Disease Using a Simplified Gating Strategy and Its Clinico-hematologic Correlation: A Single Center Experience from North India.

The presence of minimal residual disease (MRD) is one of the strong predictors of disease outcome in various hematological malignancies including B-ALL and T-ALL, independent of pre-therapeutic risk factors. There is scant Indian data on MRD by flowcytometry in T-ALL including gating strategies, clinical correlation etc. The primary aim of this retrospective observational study was to define the clinico-hematologic characteristics and prognostic significance of patients with ETP/near-ETP versus non-ETP immunophenotype, especially in terms of minimal residual disease at different time points as well as event-free survival (1year). Baseline hematologic characteristics along with post-induction (Day-35) and post-consolidation (Day-78) MRD in bone marrow samples from newly diagnosed T-ALL patients were studied. 14.3% patients had ETP-ALL immunophenotype, 11.4% were near-ETP ALL patients and the remaining 74.5% were of non-ETP subtype. The ETP/near ETP patients was significantly associated with higher risk of MRD positivity ( > 0.01%) at the end of induction in comparison to the non-ETP patients (p = 0.033). Also, these patients showed a trend towards proclivity to anemia (p = 0.06) and higher rates of induction failure (p = 0.07) However, no difference was observed between the two subgroups in terms of age, high TLC, thrombocytopenia, adverse cytogenetics, steroid responsiveness on Day + 8 of induction, MRD-positivity > 0.01% at the end of consolidation and EFS-1year. Through this preliminary study, it can be stated clearly that ETP status is associated with MRD > 0.01% post-induction but has no significant impact on long-term survival of these patients.

Phase 3, randomized, placebo-controlled trials evaluating glasdegib in combination with intensive or nonintensive chemotherapy in patients with untreated acute myeloid leukemia.

TPS7073 Background: Glasdegib is an oral Hedgehog pathway inhibitor with clinical activity in patients (pts) with untreated AML or higher-risk MDS, and improved survival when combined with low-dose cytarabine (AraC) in unfit pts with AML. BRIGHT AML1019 comprises two Phase 3, randomized (1:1), double-blind trials evaluating glasdegib 100 mg once daily (QD) or placebo (PBO) + chemotherapy in untreated adult AML (NCT03416179). The protocol includes 2 parallel, simultaneous trials: 1 with intensive chemotherapy (IC) and 1 with nonintensive chemotherapy (nIC). Methods: Both trials include pts aged ≥18 y with untreated AML (WHO 2016), including AML evolved from MDS or antecedent hematologic disease, or secondary AML. Key exclusions: inadequate organ function, acute promyelocytic leukemia, active CNS leukemia. Assignment to IC or nIC trial is per Investigator. In the IC trial, 400 pts are randomized (1:1) to glasdegib 100 mg QD or PBO on Day 1 and continue for up to 2 y or until post consolidation minimal residual disease (MRD)-negative status. Glasdegib or PBO are administered with 7+3 induction (AraC 100 mg/m2 IV for 7 days + daunorubicin 60 mg/m2 for 3 days); induction 2, if needed, will use 7+3 or 5+2. Consolidation consists of single-agent AraC 1 or 3 g/m2 IV over 3 h twice daily on Days 1, 3 and 5 every 28 days for ≤4 cycles; eligible patients may receive hematopoietic stem cell transplant. In the nIC trial, 320 pts are randomized (1:1) to glasdegib 100 mg QD or PBO, each with azacitidine 75 mg/m2 daily SC or IV for 7 days, in 28-day cycles. Treatments continue until disease progression, unacceptable toxicity, withdrawal or death. In both trials, glasdegib and PBO continue regardless of chemotherapy dose modifications/delays. The primary endpoint is overall survival. Secondary endpoints include response, time to and duration of response, event-free survival, safety, patient-reported outcomes and pharmacokinetics. Clinical trial information: NCT03416179.

Minimal residual disease negativity in elderly patients with acute myeloid leukemia may indicate different postremission strategies than in younger patients.

In the present analysis, we evaluated whether in elderly acute myeloid leukemia (AML) patients (>60years), minimal residual disease (MRD) assessed by flow cytometry may have a role in guiding choice of postremission strategies. We analyzed 149 young and 61 elderly adults who achieved morphological CR after induction course of EORTC/GIMEMA protocols. Elderly patients reached a postconsolidation MRD negative status less frequently than younger ones (11 vs 28%, p = 0.009). MRD negativity resulted in a longer 5-year disease-free survival (DFS) both in elderly (57 vs 13%, p = 0.0197) and in younger patients (56 vs 31%, p = 0.0017). Accordingly, 5-year cumulative incidence of relapse (CIR) of both elderly (83 vs 42%, p = 0.045) and younger patients (59 vs 24% p = NS) who were MRD positive doubled that of MRD negative ones. Nevertheless, CIR of MRD negative elderly patients was twofold higher than that of younger MRD negative ones (42 vs 24%, p = NS). In conclusion, elderly patients in whom chemotherapy yields a MRD negative CR have duration of DFS and rate of CIR significantly better than those who remain MRD positive. Nonetheless, the high CIR rate observed in the elderly suggests that MRD negativity might have different therapeutic implications in this population than in the younger counterpart.

Predictive role of minimal residual disease and log clearance in acute myeloid leukemia: a comparison between multiparameter flow cytometry and Wilm’s tumor 1 levels

In acute myeloid leukemia (AML), the detection of minimal residual disease (MRD) as well as the degree of log clearance similarly identifies patients with poor prognosis. No comparison was provided between the two approaches in order to identify the best one to monitor follow-up patients. In this study, MRD and clearance were assessed by both multiparameter flow cytometry (MFC) and WT1 expression at different time points on 45 AML patients achieving complete remission. Our results by WT1 expression showed that log clearance lower than 1.96 after induction predicted the recurrence better than MRD higher than 77.0 copies WT1/10(4) ABL. Conversely, on MFC, MRD higher than 0.2% after consolidation was more predictive than log clearance below 2.64. At univariate and multivariate analysis, positive MRD values and log clearance below the optimal cutoffs were associated with a shorter disease-free survival (DFS). At the univariate analysis, positive MRD values were also associated with overall survival (OS). Therefore, post-induction log clearance by WT1 and post-consolidation MRD by MFC represented the most informative approaches to identify the relapse. At the optimal timing of assessment, positive MRD and log-clearance values lower than calculated thresholds similarly predicted an adverse prognosis in AML.

Age-Stratified Analysis Of The Prognostic Role Of Minimal Residual Disease Detection By Flow Cytometry, In Adult Patients With Acute Myeloid Leukemia

▪Although the therapy of acute myeloid leukemia (AML) has remarkably improved over the last 2-3 decades, two thirds of young adults still die of their disease. In elderly adults, who represent the majority of patients with AML, the results are even more unsatisfactory with less than 10% of patients being long-term survivors. Based on this, age is universally recognized as a critical prognosticator affecting outcome and therefore treatment choice. In consecutive series of adult patients with de novo AML, we have repeatedly demonstrated the prognostic role of minimal residual disease (MRD) as detected by flow cytometry. In particular, we have found that a level of MRD ≥ 3.5x10e-4 residual leukemic cells (RLC) at the end of consolidation is associated with a relapse rate of 70-80%. In the present study we evaluated whether the prognostic impact of MRD assessment after consolidation remained unaltered even in age-stratified (< 60 and > 60 years) populations of adult patients with de novo AML. To this end, we analyzed 149 young (median age 46, range 18-60) and 61 elderly adults (median age 67, range 61-78). All patients under study achieved complete remission after an induction therapy of the EORTC/GIMEMA protocols AML10, LAM99P and AML12 (for patients < 60 years) or AML13, AML15A and AML17 (for patients > 60 years). The two cohorts were well balanced in terms of frequency of FLT3-ITD and NPM1 mutated cases. A lower frequency of favorable-risk karyotypes was observed in elderly versus young patients (4% vs 19%, p=0.024). Of 149 younger patients, 105 (70%) underwent stem cell transplantation (SCT) (45 allogeneic, 60 autologous) as compared to 7 (11%) in the older age group (1 allogeneic, 6 autologous), (p=<0.0001). The frequency of MRD negative measurements was lower among elderly patients as compared to the younger ones [7/61 (11%) vs 42/146 (28%), p=0.009]. The median value of MRD after induction was 2.9x10e-3 RLC (range 0-170) and 3.1x10e-3 RLC (range 0-220) in younger and older patients (p=NS), respectively. Conversely, post-consolidation MRD levels were significantly lower in the younger cohort (1.7x10e-3 RLC, range 0-360) as compared to the older one (3.3 x10e-3 RLC, range 0-72), (p=0.018). In both age groups, disease-free survival (DFS) (Fig. 1) and cumulative incidence of relapse (CIR) were significantly longer and lower for patients who achieved MRD negativity at the post-consolidation time-point (p=0.0003 and <0.0001, respectively). In multivariate analysis, MRD at the post-consolidation time-point (p=0.0013) and karyotype (favorable vs adverse, p=0.0073; intermediate vs adverse, p=0.06) confirmed an independent prognostic role affecting DFS. Even SCT procedure (both autologous or allogeneic), analyzed as a time-dependent covariate, was significantly associated to DFS (p<0.001). Age did not score as a significant variable in any prognostic model. In conclusion, elderly adults with AML infrequently become flow-MRD negative after consolidation therapy (11% vs 29%, p=0.008), likely due to a less intensive approach. However, those who succeed to enter MRD negativity have significantly superior DFS and CIR – comparable to the ones of young patients - that than those who remain positive. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Risk-Adapted, MRD-Refined Therapeutic Approach for the Treatment of Acute Myeloid Leukemia: From a Single Center Experience to the Cooperative Gimema Protocol AML1310

AbstractAbstract 1422The outcome of young adult (< 60 years) with acute myeloid leukemia (AML) still remains unsatisfactory. In fact, in spite of complete remission (CR) rates ranging from 60 to 80%, only 30–40% of young patients will be long-term survivors. Advances in biologic characterization of AMLs are expected to enhance a more realistic assessment of disease aggressiveness so that therapies will be delivered in the context of a stratified approach. Cytogenetic/genetic profile is the most relevant prognostic factor established at diagnosis. Nevertheless, it is well recognized that it cannot always reliably predict outcome in individual patients. Minimal residual disease (MRD) detection promises to be an efficient tool to establish on an individual basis the leukemia’s susceptibility to treatment and guide delivery of risk-tailored therapies. A further element underlying the dismal long-term outcome of young patients with AML pertains the chance to get access to allogeneic stem cell transplantation (ASCT) when carrying high-risk features. The extensive use of ASCT option is precluded by the paucity of full matched family donor (25–30%). These premises are the background to the risk-adapted approach, developed at the Institute of Hematology, University Tor Vergata, based on the following strategies: 1) combination of upfront cytogenetics/genetics and MRD status (< or ≥3.5×10−4 residual leukemic cells as counted by flow cytometry) at the end of consolidation to determine risk assignment; 2) once a given patients was categorized as high-risk (due to the expression of an unfavorable karyotype, FLT3-ITD positivity or post-consolidation positive MRD status) and therefore selected as candidate for ASCT, the transplant procedure was given whatever the source of stem cells. The present analysis includes 30 high-risk patients treated according to this design (prospective cohort = PC) and, for comparative purposes, 55 consecutive high-risk patients treated in an “old fashion” design based on donor availability (retrospective cohort = RC). The PC included 4 patients with favorable-karyotype (FK) and a MRD+ status, 12 with intermediate kayotype (IK) and a MRD+ status, 5 with unfavorable karyotype (UK) and 9 with FLT3-ITD mutation. The RC included 8 FK/MRD+, 34 IK/MRD+, 1 UK and 12 with FLT3-ITD mutation. In the PC, 22 (73%) of 30 patients received ASCT (8 matched family donor, 7 matched unrelated donor, 7 haploidentical related donor), 8 did not due to relapse (6) or because too early (2). In the RC, 12 (22%) received ASCT (11 matched family donor, 1 haploidentical related donor) whereas 24 (44%) autologous SCT (AuSCT); 19 were not transplanted at all due to relapse (13) or mobilization failure (6). Therefore, using the risk-adapted approach, 73% of high-risk patients in the PC received ASCT versus 22% of those in the RC (p <0.001). With a median follow-up of 30 and 50 months for the PC and RC, respectively, DFS is 73% vs 15% (p=0.011), OS 69% vs 20% (p=0.020), CIR 21% vs 76% (p<0.001). Based on these results, the GIMEMA Group has activated a clinical trial (AML1310, ClinicalTrials.gov.IdentifierNCT01452646) of “risk-adapted, MRD directed therapy for young adult with AML”. The trial relies on a stringent disease characterization at diagnosis in terms of cytogenetic/genetic definition and identification of “leukemia associated immunophenotype” for MRD assessment at the post-consolidation time-point. The 2 parameters are exploited to qualify the category of risk which the patients belong to: low vs intermediate vs high. All patients will receive induction and consolidation according to the previous GIMEMA LAM99P protocol. After the first consolidation, patients belonging to the low-risk category (CBF+ AML without c-Kit mutations, NPM1+FLT3-ITD- AML) will receive AuSCT and those with high-risk features (UK, FLT3-ITD mutations) ASCT. Patients with FLT3-TKD mutations or c-Kit mutated CBF+ AML and those belonging to the IK category will be stratified according to the post-consolidation MRD status and will receive AuSCT or ASCT. All patients who meet the criteria for high-risk definition will be offered ASCT regardless of the availability of a HLA identical sibling, therefore all the other sources of hematopoietic stem cells will be considered. Applying this strategy, we expect a 10% survival advantage at 24 months as compared to the historical control (LAM99P protocol) where OS at 2 years was 50%. Disclosures:No relevant conflicts of interest to declare.

Cytogenetic Risk Groups and Minimal Residual Disease Are Strong Prognostic Factors for Post-Transplant Outcome in Patients with Acute Myeloid Leukaemia and Myelodysplastic Syndrome

Abstract 4482Allogeneic stem cell transplantation (alloSCT) is a curative treatment option for patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Relapse after alloSCT is still a major cause for the treatment failure. Molecular genetic (FLT3, NPM1 mutations) and cytogenetic risk-categories have an important impact on the prognosis of patients undergoing alloSCT. However, it has been shown that there is a closely relationship between the level of minimal residual disease (MRD) and relapse after alloSCT in leukemia patients.We analyzed 140 AML/MDS patients transplanted at our institution. We examined the contribution of cytogenetic aberrations and molecular genetic markers detected prior alloSCT to survival, relapse, and mortality after transplantation. Furthermore, we analyzed MRD status after consolidation therapy and in post-transplant period. We classified molecular genetic/cytogenetic status before alloSCT into 2 groups: low-risk: good and intermediate-risk karyotype, FLT3-wt, NPM1-mutated cases and high-risk: adverse-risk cytogenetic, FLT3-ITD mutated cases. A good risk karyotype was present in 8 patients; an intermediate risk in 56 patients, whereas 76 patients had a poor cytogenetic risk. 51 patients were treated with standard myeloablative conditioning regiments prior to alloSCT, 73 patients received reduced intensity conditioning and 16 non-myeloablative conditioning. 40 patients had a matched-related donor and 100 patients had a matched-unrelated donor. Overall survival (OS) in the group of patients with low cytogenetic risk was 839 days as compared with 613 days in high-risk group. The low risk cohort also showed a lower relapse (33% vs. 51%, p<0,03) and mortality rate (20% vs. 71%, p<0,003). We analyzed MRD status in all patients after consolidation therapy. MRD negative patients are characterized by favorable prognosis as compared with MRD positive patients, OS 87% vs. 53%, p<0,001. We conclude that risk-stratification combining molecular genetics and cytogenetics aberrations at the time of diagnosis with post-consolidation MRD status improve identification of high-risk category of patients. New treatment strategies are needed to overcome poor outcome of high risk AML patients, for instance, immunotherapy options like prophylactic DLI, shorter and modified immunosuppression, specific reduced intensity conditioning or new drugs like tyrosine kinase inhibitors. Disclosures:No relevant conflicts of interest to declare.

Gene Mutations and Minimal Residual Disease (MRD) As Predictors of Remission Duration in Adults with Core Binding Factor (CBF) Acute Myeloid Leukemia (AML) Treated with High-Dose Cytarabine (HDAC) - First Results of the Prospective French Intergroup CBF-2006 Trial

Abstract 410 Background.CBF-AML is a favorable AML subset cytogenetically defined by t(8;21) or inv(16)/t(16;16) rearrangements, respectively responsible for RUNX1-RUNX1T1 (CBFa) or CBFB-MYH11 (CBFb) gene fusion. Nonetheless, relapse incidence may reach 30–40% in these patients. Even if not yet prospectively validated, early MRD monitoring based on chimeric fusion gene transcript quantification by RQ-PCR may help to define patients at higher risk of relapse. Detection of activating KIT and FLT3 gene mutations has also been retrospectively associated with higher relapse rate in these patients. The French CBF-2006 trial (NCT00428558) aimed to prospectively evaluate both markers as predictors of remission duration in homogeneously treated patients. Methods.Patients (18–60y) with newly diagnosed CBF-AML were eligible. All were studied for KIT exon 8/17, FLT3-ITD, FLT3-D835, N-RAS and K-RAS mutations. They were randomized to receive timed-sequential or standard induction. Timed-sequential induction was cytarabine (AraC) 500 mg/m2 CI D1-3 and daunorubicine (DNR) 60 mg/m2 D1-3, followed by AraC 1 g/m2/12h D8-10 and DNR 35 mg/m2 D8-9. Standard arm was AraC 200 mg/m2 CI D1-7 and DNR 60 mg/m2 D1-3, followed by the second sequence at D16-18 only in case of persistent bone marrow (BM) blasts at D15. Patients in first complete remission (CR1) received 3 HDAC consolidation cycles with AraC 3 g/m2/12h D1/3/5. Fusion gene expression was quantified at baseline and before each HDAC cycle (MRD1, MRD2, MRD3) by the 100 × fusion gene/cABL ratio. Patients not reaching a 3-Log ratio reduction at MRD2 were offered allogeneic stem cell transplantation (SCT) in CR1 if they had an HLA-identical donor or could enter a single-agent dasatinib sub-study (see Boissel et al., this meeting). Results.Among the planned 200 patients randomized, 198 met eligibility criteria (96 CBFa and 102 CBFb, compared in Table 1). Overall, incidences of KIT, RAS, FLT3-D835 and FLT3-ITD mutations were 21%, 29%, 10% and 6%, respectively. Patients with KIT and FLT3-D835 mutations had higher WBC (P=0.04 and 0.02) and BM blast percentage (P=0.0004 and 0.05). Those with RAS mutations had lower BM blast percentage (P=0.01) and baseline transcript ratio (P=0.001). Overall, 196 patients reached CR1 and 55 relapsed. At 3 years, probabilities of durable remission, disease-free (DFS) and overall survival from CR were 64%, 61% and 84%, respectively, without differences between the CBF subsets or induction arms. Fifty-five patients did not reach the 3-Log MRD2 reduction (18 CBFa and 37 CBFb, P=0.006), 9 receiving SCT in CR1. Poor MRD2 response correlated with age (P=0.01), WBC (P=0.04), KIT mutations (P=0.04), and initial transcript ratio (P=0.004). Among the characteristics listed in Table 1, shorter remission duration was associated with high WBC (P=0.011), high BM blast percentage (P=0.062), del(9q) (P=0.041), KIT mutation (P=0.048), FLT3-D835 mutation (P=0.077), high initial transcript ratio (P=0.053), and <3-Log MRD2 reduction (P=0.006) in univariate analysis stratified by CBF subtype and treatment arm. In multivariate analysis, the three latter factors, but not KIT mutations, remained independently predictive of shorter remission duration and DFS (P= 0.014, 0.033, 0.002 and 0.001, 0.012, 0.005, respectively). Censoring patients receiving SCT in CR1 yielded similar results. Conclusions.This prospective study of 198 CBF-AML patients homogeneously treated with HDAC revealed significant correlations between initial characteristics and response to therapy. Prognostic analysis did not find that KIT mutations had independent impact on outcome, which was rather influenced by baseline fusion transcript ratio and post-consolidation MRD reduction (reminiscent of Schnittger et al. 2003), and FLT3-D835 mutation. Ongoing pan-genomic analyses might help to further dissect CBF-AML heterogeneity.Table 1Patient characteristicsCBFa (N = 96)CBFb (N = 102)P valuesMedian age42y42y0.37Median WBC, G/L1122<0.001Median BM blast percentage52570.10Therapy-related AML1020.016Loss of Y chromosome*330<0.001Tri 22*018<0.001Tri 8*2150.002Del(9q)*141<0.001Del(7q)/–7*450.99KIT exon 8/17/both mutation**5/16/112/6/00.38FLT3 D835/ITD/both mutation**4/6/015/4/10.11N/K-RAS mutation**13/228/110.001Median baseline transcript ratio30389<0.001>3-Log ratio reduction at MRD2***71580.006*196,**194, and***184 patients studied. Disclosures:No relevant conflicts of interest to declare.

MRD-Directed Post-Remissional Therapy Dramatically Improves Outcome of Adult Patients with High Risk AML by Implementing Allogeneic Transplant Option

AbstractAbstract 2702Despite improvements in supportive care and wider availability of novel drugs, the outcome of young adult with AML still remains unsatisfactory with a long term survival of 30–40%. Such a dismal outlook is due to the contribution of several reasons. Among these, we believe that risk-category allocation based on the sole definition of pre-treatment biological features (genetics/cytogenetics) may fail to distinguish some high-risk patients which consequently are not timely addressed to intensive treatments. Furthermore, proper treatment of patients with poor prognosis may be hampered by the limited availability of HLA full compatible sibling donors (30% of patients). For the same reasons, some low-risk patients may be misclassified and over-treated (e.g. by giving them allotransplant for the simple reason that they have a full related compatible donor) with an excessive rate of treatment related mortality. Minimal residual disease (MRD) detection promises to be a strong predictive factor in adult AML when measured after the [induction-consolidation] phase and in fact, we have demonstrated (Venditti Blood 2000, Maurillo JCO 2008, Buccisano Blood 2010) that it refines the upfront genetic/cytogenetic risk classification. Thus, we designed a risk-directed pilot study which took into account: 1) an adjusted risk-stratification based on pretreatment genetics/cytogenetics and MRD status at the end of consolidation; and 2) a fast prospective assignment of all high-risk patients to allogeneic stem cell transplantation (alloSCT) in the form of HLA full compatible sibling, matched unrelated donor, umbilical cord blood or full haploidentical donor transplant. By applying this adjusted risk-stratification, we were able to discriminate two discrete category of patients: 1) Low-risk: good and intermediate-risk karyotype (K) that were MRD negative after consolidation (MRDneg good/int-K); and 2) High-risk: adverse-risk K, FLT3-ITD mutated cases and good/intermediate-risk K that were MRD positive after consolidation (MRDpos good/int-K). We also analyzed the outcome of a matched historical cohort of high-risk patients whose post-consolidation treatment was oriented according to a “genetic randomization”: alloSCT for those with a HLA full compatible sibling and autologous stem cell transplant (auSCT) for those lacking this option. Results achieved in this retrospective cohort were compared with those generated in the analysis of the prospective one. The prospective cohort included 25 high risk patients (4 MRDpos good-K, 9 MRDpos int-K, 4 adverse-karyotype and 8 FLT3-ITD). Five of them relapsed early being 20 evaluable for this analysis. The retrospective cohort accounted for 52 high-risk patients (8 MRDpos good-K, 33 MRDpos int-K, 1 adverse-karyotype and 10 FLT3-ITD). Ten of them relapsed early being 42 evaluable for this analysis. Even though the median follow-up of the prospective cohort is still short (18 months) survival estimates were dramatically superior as compared to the retrospective one (DFS 70% vs. 20%, p=0.00047; OS 69% vs. 24%, p=0.046). Accordingly, the prospective cohort also showed a lower relapse rate (20% vs. 52%, p=0.003). The 2 cohorts were well balanced in terms of frequency of FLT3-ITD, adverse-K and treatment related mortality rate, therefore, we believe that the improvement in survival estimates is related to the higher percentage of patients who were able to receive alloSCT. In fact, in the prospective cohort 21/20 (95%) of patients entering the post-consolidation phase underwent alloSCT as compared to 12/42 (30%) in the retrospective cohort (p<0.001). Notably, in the prospective cohort, only 8/21 patients had an HLA full compatible sibling whereas in 6 and 7 an haploidentical or MUD/UCB donor was found, respectively. We conclude that: 1) risk-assessment combining upfront genetics/cytogenetics with post-consolidation MRD status improves identification of high-risk category of patients who share a very poor prognosis if not selectively submitted to alloSCT; 2) the actual risk of disease recurrence should guide alloSCT option according to a “transplant vs no transplant” rather than “donor versus no donor” approach. This implies that once high-risk patients are identified they should receive alloSCT whatever the source of stem cell and, in our experience, this is feasible in the large majority of patients. Disclosures:No relevant conflicts of interest to declare.

Cytogenetic and molecular diagnostic characterization combined to postconsolidation minimal residual disease assessment by flow cytometry improves risk stratification in adult acute myeloid leukemia

AbstractA total of 143 adult acute myeloid leukemia (AML) patients with available karyotype (K) and FLT3 gene mutational status were assessed for minimal residual disease (MRD) by flow cytometry. Twenty-two (16%) patients had favorable, 115 (80%) intermediate, and 6 (4%) poor risk K; 19 of 129 (15%) carried FLT3-ITD mutation. Considering postconsolidation MRD status, patients with good/intermediate-risk K who were MRD− had 4-year relapse-free survival (RFS) of 70% and 63%, and overall survival (OS) of 84% and 67%, respectively. Patients with good- and intermediate-risk K who were MRD+ had 4-year RFS of 15% and 17%, and OS of 38% and 23%, respectively (P < .001 for all comparisons). FLT3 wild-type patients achieving an MRD− status, had a better outcome than those who remained MRD+ (4-year RFS, 54% vs 17% P < .001; OS, 60% vs 23%, P = .002). Such an approach redefined cytogenetic/genetic categories in 2 groups: (1) low-risk, including good/intermediate K-MRD− with 4-year RFS and OS of 58% and 73%, respectively; and (2) high risk, including poor-risk K, FLT3-ITD mutated cases, good/intermediate K-MRD+ categories, with RFS and OS of 22% and 17%, respectively (P < .001 for all comparisons). In AML, the integrated evaluation of baseline prognosticators and MRD improves risk-assessment and optimizes postremission therapy.

Detection of Minimal Residual Disease by Flow-Cytometry Otimizes Risk Stratification When Combined with Cytogenetics in AML.

Cytogenetic risk assessment at diagnosis is a major determinant of outcome in adult patients with AML. Nevertheless, the impact of this up-front prognosticator may be altered by additional factors intervening during and after treatment such as toxicity, speed of blast clearance and minimal residual disease (MRD), both the latters reflecting the quality of response. We have demonstrated that the persistence of given amounts of leukemic cells after consolidation, affects the prognosis of AML, independently from cytogenetics, leading to a faster leukemic relapse. In the present study, we aimed at proving that the integrated analysis of karyotype and MRD determination by multiparametric flow-cytometry (MPFC) may improve the risk stratification process of patients belonging to established cytogenetic groups. We analyzed 127 non-M3 AML cases entered into the EORTC/GIMEMA protocols AML10/AML12 (age <61yrs) or AML13/AML15/AML17 (age >61 yrs). By applying the maximally selected log-rank statistics, the threshold discriminating MRD negative from positive cases was set at 3.5 x10−4 residual leukemic cells, a level that distinguished, at the post-consolidation time-point, discrete subsets of patients with different prognosis. According to the MRC classification, 18/127 (14%) and 7/127 (6%) were classified into the favorable and unfavorable risk categories, respectively; 102/127 (80%) patients had an intermediate risk karyotype. As expected, having a favourable, intermediate or unfavourable karyotype was associated with a significant difference in terms of relapse rate (28%, 63%, 100%; p=0.005), 5-years overall survival (OS) (67%, 27%, 14%; p<0.001) and 5-years relapse free survival (RFS) (68%, 28%, 0%; p<0.001). These three groups, also differed for the rate of MRD negativity: 78% in the favourable group, 28% in the intermediate group, whereas none among unfavourable risk patients reached a MRD− status (p<0.001). Therefore, we examined each cytogenetic group according to the MRD status and we observed that:1.patients bearing favourable karyotypes and achieving MRD negativity (good-MRD−) had a better outcome in terms of 5-years OS (81%) and RFS (76%) as compared to those remaining MRD positive (good-MRD+) (5-years OS % and RFS 25% and 43%, respectively) (p=0.06).2.also the patients with intermediate risk karyotype were dissected in two distinct categories by MRD status, MRD+ patients (Int-MRD+) with a very poor prognosis (5-yrs RFS and OS less than 20% for both) and MRD− ones (Int-MRD−) (5-yrs RFS and OS 69% and 50%, respectively) (p<0.001).Therefore, favorable karyotype does not guarantee by definition a good prognosis; in fact, good-MRD+ patients have a high relapse rate (50%) and a disappointing long term outcome; similarly, the outcome of Int-MRD+ patients was comparable to that of poor risk cytogenetics whereas Int-MRD− patients fared as favorably as those in the good karyotype category. In conclusion, an appropriate risk assessment should result from the combination of pre-treatment and delayed parameters (cytogenetics plus post-consolidation MRD determination) rather than relies on the evaluation of sole up-front prognosticators; this approach would enable to outline in a more reliable manner the outcome of adult patients with AML and the relative post-remissional therapy.

Optimal Post-Remission Therapy for Flow-Cytometry Minimal Residual Disease Positive Patients with Acute Myeloid Leukemia.

Autologous (AuSCT) and allogeneic stem cell transplantation (SCT) are well established post-remissional strategies for patients with Acute Myeloid Leukemia (AML). However, there is still a debate ongoing about the relative merit of each of these options in first CR. Minimal residual disease (MRD) may be a useful tool to stratify AML patients into categories of risk which can benefit from differentiated post-remissional approaches. To address this issue, we analysed retrospectively, a series of 123 patients affected with AML in whom flow-cytometry serial determinations of MRD were available, at established time-points (post-induction, post-consolidation, post-stem cell transplantation). All were entered into the EORTC/GIMEMA protocols AML10/AML12 (age <61 yrs) or AML13 (age>61 yrs), all consisting in intensive induction and consolidation cycles, and, for patients aged <61 years, AuSCT or SCT. Median age was 52 years (range 18–78), no APL cases were included in the study. The “Maximally Selected Rank Statistic” analysis was used to select the MRD level and the time-point of analysis achieving the best prognostic significance in terms of overall survival (OS) and relapse free survival (RFS). This test was specifically developed to find out the optimal cut-off for a given biological variable correlating with clinical parameters of interest. This approach confirmed the threshold of 3.5x10−4 residual leukemic cells after consolidation therapy, as a discriminator between MRD− and MRD+ cases with different 5-years OS (64% vs. 14%, P<.001) and RFS (68% vs. 13%, P<.001). Therefore, among these 123 patients, we enucleated 2 groups of patients which underwent stem cell transplant procedure, 53 AuSCT and 11 SCT. The two subgroups were balanced in terms of FAB categories, WBC count, karyotype and expression of MDR-1 phenotype; 34/64 (53%) were MRD+ (9 SCT, 25 AuSCT) and 30/64 (47%) were MRD− (1 SCT, 28 AuSCT). Among the 53 patients submitted to AuSCT, MRD− had a significant better outcome than those MRD+, both in terms of 5-years OS (68% vs. 24%, P=0.003) and RFS (68% vs. 11%, P<.001). In the SCT group the low numbers hampered any firm conclusion; however, when the category of post-consolidation MRD+ patients was separately analysed, the use of SCT was associated with a better RFS (49% vs. 11%, P=NS); we assume that the lack of statistical significance was merely due to the few cases in the SCT group. In conclusion,the threshold of 3.5x10−4 at post-consolidation check-point is critical to predict disease outcome;MRD− patients have an excellent outcome regardless of the post-consolidation therapy;in the MRD+ group, AuSCT seems not to improve the prognosis whereas the use of SCT is associated with a superior outcome, although a larger number of patients is required to confirm this assumption. [Display omitted]

Statistical Validation of a Threshold To Discriminate Flow-Cytometry Minimal Residual Disease (MRD) Levels with Prognostic Significance in Acute Myeloid Leukemia (AML).

The current experience in the use of multiparametric flow-cytometry for immunological detection of MRD in AML, indicates that the method is feasible, rapid and relatively sensitive. In our previous studies of AML patients with aberrant phenotype (Blood 2000, Leukemia 2003 & 2006), we found that the level of MRD after consolidation therapy was the best predictor of outcome. In fact, a MRD level higher than 3.5x10−4 residual leukemic cells after consolidation was significantly correlated with a poor outcome. The major problem with using an empirically established cut-off to define thresholds is that it may not be reproducible over different populations of patients. In order to confirm the prognostic value of the threshold of 3.5x10−4 residual leukemic cells, previously selected, we used the Maximally Selected Rank Statistic analysis to determine optimal cutpoint, yielding the best separation of AML patients into two groups with different OS and/or RFS probabilities. To do this, we evaluated the trend of standardized log-rank statistics using RFS (Fig. 1, upper panels) and OS (Fig. 1, lower panels) as dependent variables, and the values of residual leukemic cells, determined both at the post-Induction (post-Ind) and post-consolidation (post-Cons) checkpoints, as independent variable (Fig. 1A and 1B, respectively). The experimental cut-off points, identified as the absolute peak in standardized log-rank statistics plots (vertical dotted lines in Fig. 1), were 3.7x10−4 and 3.4x10−4 residual leukemic cells for post-Ind and post-Cons values, respectively. According to this data we decided to utilize the 3.5x10−4 residual leukemic cells cut-off value to discriminate MRD− from MRD+ cases both after induction and consolidation. Therefore, patients with a residual leukemic cells values below the cutoff of 3.5×10−4 will be referred to as MRD−, while those with residual leukemic cells equal or exceeding the 3.5×10−4 level will be classified as MRD+. Post-Cons MRD− patients had a superior outcome in terms of relapse rate (27% vs. 78%, P<0.001), 7-years OS (64% vs. 14%, P<0.001) and RFS (68% vs. 13%, P<0.001). Moreover, post-Cons MRD− patients had a better outcome regardless of the MRD status after induction: in particular, patients entering MRD negativity only after consolidation showed the same outcome as those achieving early negativity after induction. Multivariate analysis, including karyotype, age, MDR1 phenotype, post-Ind and post-Cons MRD levels, indicated that the post-Cons MRD status independently affected relapse rate, OS and RFS (P<0.001, for all comparisons). In conclusion:the threshold of 3.5x10−4 is valid in discriminating risk categories in adult AML;post-consolidation MRD assessment is critical to predict disease outcome regardless of the MRD status after induction. [Display omitted]

The kinetics of reduction of minimal residual disease impacts on duration of response and survival of patients with acute myeloid leukemia

6533 Background: Multiparametric flow-cytometry is frequently used to assess the levels of minimal residual disease (MRD) in acute myeloid leukemia (AML) patients achieving complete remission after intensive chemotherapy. In our previous experience, MRD negativity after consolidation cycle, as defined by a level of bone marrow residual leukemic cells ≤3.5x10−4, was associated with a significantly longer relapse free survival (RFS) and overall survival (OS). The aims of the present study were to confirm, in a larger series of patients, the prognostic relevance of the post consolidation MRD assessment and to validate the threshold of 3.5x10−4. Methods: 100 patients affected with AML were entered into the EORTC/GIMEMA protocols AML10/AML12 (age &lt;61 yrs) or AML13/AML15 (age &gt;61 yrs), consisting in intensive induction and consolidation cycles. Median age was 52 years (range 18–78), all FAB subtypes were represented with the exception of APL cases. A Maximally Selected Rank Statistic analysis was used to select the best threshold. Results: The statistical test confirmed that the level of 3.5×10−4 residual leukemic cells was the most significant in discriminating categories of risk. In fact, levels of MRD measured at the post-consolidation time-point identified 2 distinct subgroups: 36 patients MRD negative and 56 MRD positive. MRD negative patients had a better outcome in terms of relapse rate (P&lt;0.001), OS (P=0.039) and RFS (P=0.008), regardless of MRD status after induction. In fact, patients entering MRD negativity only after consolidation showed the same outcome as those achieving early negativity after induction. The multivariate analysis, including karyotype, age, MDR1 phenotype and post induction and post consolidation MRD levels, indicated that the post-consolidation MRD status was an independent factor affecting relapse rate (P&lt;0.001), OS (P=0.039) and RFS (P=0.008). Conclusions: We conclude that 1) the threshold of 3.5x10−4 is valid in discriminating risk categories in AML; 2) MRD assessment at post-consolidation check-point is critical to predict disease outcome. The incorporation in clinical trials of flow-cytometric MRD determination may allow a more accurate risk stratification of AML patients. No significant financial relationships to disclose.

The Kinetic of Reduction of Minimal Residual Disease Impacts on duration of Response snd Survival of Patients with Acute Myeloid Leukemia.

In acute myeloid leukemia (AML) patients achieving complete remission, the levels of minimal residual disease (MRD) as determined by flow cytometry have been shown to impact on remission duration and survival. However, some issues such as the most suitable source (BM or PB) or the most appropriate timing (early or delayed evaluation) for MRD determination are still a subject of debate. In our experience, we observed that MRD negativity, as defined by a number of bone marrow residual leukemic cells (BMRLC) <3.5x10−4, after consolidation cycle was associated with a significantly longer disease free survival (DFS) and overall survival (OS). Based on this, the present study was designed to analyze the kinetic of MRD reduction during the post-induction phase, and therefore to determine to what extent post-induction chemotherapy might impact on the outcome of patients with AML. Eighty-nine adult patients with AML were entered into the EORTC/GIMEMA protocols AML10/AML12 (age <61 yrs) or AML13/AML15 (age >61 yrs), all consisting in intensive induction and consolidation cycles, and allogeneic or autologous stem cell transplantation for patients aged < 61 years. Median age was 53 years (range 17–78), all FAB subtypes were represented with the exception of APL cases which were not included. Eighty-one of 89 patients maintained complete remission after consolidation (8/89 had early relapse after induction) and were suitable for the analysis. After consolidation cycle, 32 of 81 (39%) patients had <3.5x10−4 BMRLC and were considered MRD neg. The remaining 49 (61%), were MRD pos since the measured levels of MRD were ≥3.5x10−4 BMRLC. Among these MRD pos patients, in 16 the levels of MRD, although still above the value of 3.5x10−4 BMRLC, were significantly reduced as compared to the post-induction levels (median reduction 6x10−4 BMRLC, range 0–643). Therefore, these 16 patients were considered as having a “chemosensitive MRD” and in fact, within this MRD pos category, they had a superior duration of DFS and OS (P=.010 and .004, respectively) as compared to MRD pos patients with “chemoresistant MRD”, namely those not showing improvement in the level of MRD between the induction and consolidation course. Therefore, we identified 3 discrete categories of patients: 1) 32 patients MRD neg at the end of consolidation therapy (BMRLC <3.5x10−4); 2) 16 patients MRD pos at the end of consolidation therapy (BMRLC ≥3.5x10−4) but with “chemosensitive MRD”; 3) 33 patients MRD pos at the end of consolidation therapy (BMRLC ≥3.5x10−4) but with “chemoresistant MRD”. These 3 groups differed significantly in terms of relapse rate (84% vs. 75% vs. 28%, respectively) both in univariate and multivariate analysis (P<.001). Accordingly, DFS and OS (at 5-years, 66%, 50 % and 12%, respectively) (P<.001) duration also differed; the multivariate analysis confirmed the independent prognostic role of MRD status at the end of consolidation (P<.001). In conclusion, 1) at variance with previous reports emphasizing the prognostic value of an early flow-cytometric determination of MRD, we have found that a delayed (post-consolidation) MRD evaluation provides the best predictive information on patients outcome; 2) the quantitative determination of MRD at specific time-points may also allow the identification of MRD pos patients with variable prognosis (chemosensitive vs chemoresistant MRD).