Minimal Residual Disease Group Research Articles

Relationship between subtype-specific minimal residual disease level and long-term prognosis in children with acute lymphoblastic leukemia.

Minimal residual disease (MRD) based risk stratification criteria for specific genetic subtypes remained unclear in childhood acute lymphoblastic leukemia (ALL). Among 723 children with newly diagnosed ALL treated with the Chinese Children Leukemia Group CCLG-2008 protocol, MRD was assessed at time point 1 (TP1, at the end of induction) and TP2 (before consolidation treatment) and the MRD levels significantly differed in patients with different fusion genes or immunophenotypes (P all < 0.001). Moreover, the prognostic impact of MRD varied by distinct molecular subtypes. We stratified patients in each molecular subtype into two MRD groups based on the results. For patients carrying BCR::ABL1 or KMT2A rearrangements, we classified patients with MRD < 10-2 at both TP1 and TP2 as the low MRD group and the others as the high MRD group. ETV6::RUNX1+ patients with TP1 MRD < 10-3 and TP2 MRD-negative were classified as the low MRD group and the others as the high MRD group. For T-ALL, We defined children with TP1 MRD ≥ 10-3 as the high MRD group and the others as the low MRD group. The 10-year relapse-free survival of low MRD group was significantly better than that of high MRD group. We verified the prognostic impact of the subtype-specific MRD-based stratification in patients treated with the BCH-ALL2003 protocol. In conclusion, the subtype-specific MRD risk stratification may contribute to the precise treatment of childhood ALL.

Distinct Prognosis of Minimal Residual Disease According to Breast Cancer Subtype in Patients with Breast or Nodal Pathologic Complete Response After Neoadjuvant Chemotherapy.

Few studies have reported on patient prognosis according to residual cancer burden after neoadjuvant chemotherapy (NAC). Herein, we evaluated the survival of patients based on residual disease after NAC to identify subpopulations with distinct prognoses. We retrospectively reviewed 728 patients treated with NAC from 2010 to 2017. Patients were divided into four subgroups depending on post-surgical residual disease according to the staging system: pathological complete response (pCR) (ypT0/TisN0), minimal residual disease (MRD) (ypT1mi/T1aN0 or ypT0/Tis ypN0i+/N1mic), node-only pCR (≥ ypT1b ypN0), and breast-only pCR (ypT0/Tis ≥ ypN1a). Clinicopathological characteristics and survival outcomes were analyzed by adjusting for factors affecting survival. Overall, 50.4% (n = 367) of patients achieved pCR, with the MRD group accounting for 16.5% (n = 120). Although age and clinical stage were not different among the study groups, histologic grade, subtypes, chemotherapy response, and local treatment showed differences. Event-free survival (EFS) and overall survival (OS) demonstrated no significant difference between the pCR and MRD groups. In the multivariate analysis, pCR status was the only significant factor in EFS, and no statistical difference was noted between the pCR and MRD groups. However, clinical stage, pCR status, and subtype significantly affected the OS. MRD showed favorable outcomes in terms of both EFS and OS in all subtypes, except for those with triple-negative breast cancer (TNBC). Patients with MRD showed outcomes comparable to those of patients who achieved pCR and may be candidates for de-escalation of post-NAC treatment, except for those with a TNBC subtype.

Longitudinal Monitoring of Circulating Tumor DNA From Plasma in Patients With Curative Resected Stages I to IIIA EGFR-Mutant Non–Small Cell Lung Cancer

IntroductionFor patients with early stage EGFR-mutant–positive (EGFR-M+) NSCLC, curative surgery followed by adjuvant chemotherapy is considered the standard of care. This study evaluated the feasibility and efficacy of longitudinal monitoring of circulating tumor DNA (ctDNA) as a valuable biomarker for early detection of minimal residual disease (MRD) and provides identification of the group at high risk for recurrence in resected stages I to IIIA EGFR-M+ NSCLC. MethodsBetween August 2015 and October 2017, a total of 278 patients with curative resected, stages I to IIIA (American Joint Committee on Cancer seventh version) common EGFR-M+ NSCLC were analyzed. Radiological follow-up was accompanied with longitudinal monitoring of ctDNA using a droplet-digital polymerase chain reaction from baseline (preoperative), 4 weeks after curative surgery, and follow-up per protocol until 5 years. The primary outcomes were disease-free survival (DFS) according to the status of ctDNA positivity at landmark points and the sensitivity of longitudinal monitoring of ctDNA. ResultsAmong 278 patients, preoperative baseline ctDNA was detected in 67 (24%) patients: 23% (stage IA), 18% (IB), 18% (IIA), 50% (IIB), and 42% (IIIA) (p = 0.06). Of patients with baseline ctDNA, 76% (51 of 67) had clearance at 4 weeks after surgery (postoperative). Patients were classified into the following three groups; group A, baseline ctDNA negative (n = 211) versus group B, baseline ctDNA positive but postoperative MRD negative (n = 51) versus group C, baseline ctDNA positive and postoperative MRD positive (n = 16). The 3-year DFS rate was significantly different among the three groups (84% for group A, 78% for group B, and 50% for group C, p = 0.02). After adjusting for clinicopathologic variables, ctDNA still remains an independent risk factor for DFS along with stage (p < 0.001) and micropapillary subtype (p = 0.02). With longitudinal monitoring of ctDNA, MRD was detected before radiological recurrence in 69% of patients with exon 19 deletion and in 20% with L858R mutation. ConclusionsThese results suggest that patients with baseline ctDNA-positive or MRD-positive status were associated with poor DFS in curative resected stages I to IIIA EGFR-M+ NSCLC and that longitudinal monitoring of ctDNA, a noninvasive method, might be useful to detect early recurrence before radiological recurrence.

Abstract P4-07-14: Long-term prognosis of minimal residual disease in breast cancer patients with breast or nodal pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC)

Abstract Background: Achieving pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC) is known important for predicting survival. However, few studies have reported prognosis of minimal residual disease. In this study, we evaluated the long term survival of the patients with breast or nodal pCR and compare patients with minimal residual disease to the breast and node pCR group. As it is difficult to analyze residual cancer burden and tumor-infiltrating lymphocytes in the retrospective study, we assessed the residual tumor or nodal status according to the pathologic report after surgery based on the AJCC 8th edition staging system. Methods: This study retrospectively reviewed 753 patients who achieved pCR of breast or node after NAC from 2010 to 2017 at Severance Hospital. We divided patients into five groups based on the residual disease; breast and node pCR (ypT0/TisN0), minimal residual disease (ypT1mi/T1aN0, ypT0/TisN0i+/N1+), breast-only pCR (ypT0/TisN1+), node-only pCR with residual tumor size no larger than 20mm (ypT1b/T1cN0), and node-only pCR with residual tumor size larger than 20mm (ypT2+N0). The basic characteristics and the 10-year event free survival (EFS) were evaluated. EFS was defined as time from surgery to last follow-up date without any events. Results: We analyzed the data until May 26, 2021, and 50.2% (n=378) of the patients achieved breast and node pCR. Minimal residual disease, defined as residual tumor size no larger than 5mm and residual nodal size 2mm, included 126 patients (16.7%). Overall, there was significant difference in 10-year EFS among five groups (ypT0/TisN0; 94.7%, ypT1mi/T1aN0, ypT0/TisN0i+/N1+; 93.7%, ypT1b/T1cN0; 86.8%, ypT0/TisN1+; 87.9%, ypT2+N0; 71.9%, p&amp;lt;0.001). As the variables such as age, subtypes, clinical T and N stage, regimen of chemotherapy, response to the chemotherapy and operation type were adjusted, the minimal residual disease group showed no significant difference with the breast and node pCR group (HR 1.410, 95% CI 0.615 - 3.235, p=0.417), yet the other groups showed poorer outcomes (ypT1b/T1cN0; HR 3.183, 95% CI 1.687- 6.004, p&amp;lt;0.001, ypT0/TisN1+; HR 3.186, 95% CI 1.047 - 9.694, p=0.041, ypT2+N0; HR 6.089, 95% CI 3.019- 12.281, p&amp;lt;0.001). Conclusion: Patients with minimal residual tumor 5mm or less, or residual metastatic lymph node with a maximal diameter of 2mm have shown equivalent 10-year EFS compared to the patients who achieved breast or node pCR after NAC. Keywords: Neoadjuvant chemotherapy, Pathologic complete response, Minimal residual disease, Long-term survival, Breast cancer Citation Format: Jieon Go, Jee Hyun Ahn, Jung Min Park, Soon Bo Choi, Jee Ye Kim, Hyung Seok Park, Seung Il Kim, Byeong-Woo Park, Seho Park. Long-term prognosis of minimal residual disease in breast cancer patients with breast or nodal pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-14.

Combination of NCCN Risk Stratification System and Pre-Transplant Minimal Residual Disease Levels for the Prognosis of Acute Myeloid Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation

BackgroundNumerous studies have confirmed that National Comprehensive Cancer Network (NCCN) risk stratification or pre-transplant minimal residual disease (MRD) levels can predict the risk of recurrence and survival after transplantation. But it is unclear whether combining these two parameters can more accurately predict prognosis.MethodsWe retrospectively analyzed 85 patients with acute myeloid leukemia (AML) who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) and constructed a new risk stratification tool combining NCCN risk stratification and pre-MRD levels. All patients were grouped by NCCN risk stratification (favorable/intermediate prognosis and poor prognosis group), pre-MRD levels (MRD (-) group (<0.1%) and MRD (+) group (≥0.1%)) and a combination of the above (low, intermediate and high risk groups), and graft-versus-host disease (GVHD) and prognosis were compared between groups.ResultsRelative to the favorable/intermediate prognosis group, OS and RFS were poorer in the poor prognosis group (71% vs 82%, P= .156; 60% vs 74%, P= .101) and CIR (29% vs 20%, P= .229) and NRM (23% vs 14%, P= .200) were better. The incidence of aGVHD and cGVHD was slightly lower in the favorable/intermediate prognosis group than in the poor prognosis group (38% vs 46%, P= .415; 10% vs 11%, P=. 572). Relative to the MRD (+) group, the MRD (-) group had significantly better OS and RFS (89% vs 59%, P= .002; 79% vs 50%, P= .003), lower CIR and NRM (15.1% vs 37.5%, P= .011; 11.3% vs 28%, P= .040), and a lower incidence of cGVHD (6% vs 19%, P= .022). The new risk stratification tool stratified patients into low, intermediate and high risk groups. Patients in the high-risk group had the highest incidence of aGVHD and cGVHD (42% vs 35% vs 53%, P= .606; 6% vs 11% vs 20%, P= .157). The difference in cGVHD between the low- and high-risk groups was significant (P= .038). Three-year OS was 93.9%, 70% and 60% (P= .011) and RFS was 85%, 62% and 46.7% (P= .009) for low-, intermediate- and high-risk patients, respectively. The differences in OS and RFS between the low- and intermediate-risk groups were statistically significant (P= .010; P= .025), as were the differences in OS and RFS between the low- and high-risk groups (P= .001; P= .001). Patients in the high-risk group had the highest CIR and NRM relative to those in the low- and intermediate-risk groups (9% vs 32% vs 33.3%, P= .027; 6% vs 24.3% vs 26.7%; P= .059). The differences in CIR (P= .012) and NRM (P= .028) were statistically significant in both the low-risk and intermediate-risk groups, as well as in the low- and high-risk groups (CIR: P= .028; NRM: P= .021). Multivariate analysis indicated that time to ANC recovery, time from diagnosis to transplantation, and novel risk stratification were independent prognostic factors.ConclusionsBoth pre-MRD levels and NCCN risk stratification predict AML prognosis after allo-HSCT, and combining the two can more accurately predict post-transplant prognosis. DisclosuresNo relevant conflicts of interest to declare.

An Artificial Intelligence Approach for B Lymphoblastic Leukemia Minimal Residual Disease Detection and Clinical Prognosis Prediction Using Flow Cytometry Data

IntroductionDetection of minimal residual disease (MRD) of B-cell acute lymphoblastic leukemia (B-ALL) measured by flow cytometry (FC) has been shown to be an important prognostic indicator in B-ALL. However, the current analysis is based on the manual gating by the reviewers; therefore, objectivity could be compromised, and decentralization of the B-ALL MRD detection is difficult to be established. We then aimed to develop an objective diagnostic tool via artificial intelligence (AI) technique for FC data interpretation in order to provide efficient and objective B-ALL MRD detection.MethodsRetrospective FC data of patients with adult B-ALL from 2009 to 2016 in National Taiwan University Hospital were enrolled. A total of 1,073 unique bone marrow FC samples were collected for analysis. Each of the samples had a 10-tube test done on 100,000 cells - each cell was measured in 6 fluorescent channels (FSC, SSC, FITC, PE, PerCP, APC) within one tube.The flow conclusion of each FC sample was categorized into “positive MRD” and “no MRD” groups according to previous manual interpretation, and the whole dataset was randomly divided into 3 groups: Training set (n=718), Accuracy set (n=141) and Validation set (n=178). The Training set was used to train the algorithm, and the Accuracy set was used to evaluate the accuracies achieved and optimize algorithmic parameters. The final optimized algorithm was then evaluated blindly in Validation set, with the concordance rate comparing the manual and the AI conclusions in distinguishing “positive MRD” from “no MRD”.For the algorithm development, the recorded numerical values of the 6 fluorescent channels (FSC, SSC, FITC, PE, PerCP, APC) of each tube (100,000 cells) were considered as raw feature attributes. The probabilistic distributions of these attributes were first modeled as multivariate Gaussian mixture model using a sub-dictionary learning approach. Then a probabilistic derivation was exploited to compute per-sample L2-normalized vectorized representations. Lastly, representations of each tube were then concatenated to be the final feature input to the supervised machine learning classifier, in this case, a support vector machine with linear kernel. In addition, ANOVA-based feature selection was also conducted throughout the experiments.For outcome correlation analysis, we included 35 adult B-ALL patients with available FC data after induction therapy, record their clinical parameters and measured their overall survival (OS) and progression-free survival (PFS), with median follow-up 40.0 months. The Kaplan-Meier curves were constructed to estimated OS and RFS.ResultsThe primary algorithm concordance estimated using the Accuracy set was 90.8% and the area under the receiver operating characteristic curve (AUC) is 0.944 (Table1). The calibrated algorithm, adjusted with training in Accuracy set, can produce a concordance rate of 88.2% with manual interpretation on Validation set, and the AUC is 0.919 (Table 1).It is noteworthy that it only took 7 seconds to analyze one FC sample using the AI algorithm while it took approximately 15 to 30 minutes for manual gating by trained professionals. Furthermore, the concordance of the algorithm developed from 10 tube-data remained almost unchanged when the tube number decreased. In fact, algorithm from 1 tube still yields an AUC of 0.874 (vs. 0.919 from 12 tubes), indicating that AI algorithm may reduce laboratory testing currently applied in standard FC processing.For outcome correlation, B-ALL patients with no MRD by AI algorithm had significant longer OS compared to those with positive MRD (NR vs 20.0 months, p=0.0051). Moreover, patients with no MRD also had significant longer RFS compared to those with positive MRD (8.9 vs 5.2 months, p=0.019), (Figure.1).ConclusionsThis is the first study using AI approach to develop FC data diagnostic algorithms with prognosis implications for B-ALL, demonstrating the advantages of reliability, high efficacy and time saving. Incorporating other test results into AI algorithm may lead to more precise prognostic prediction in the future and potentially guide therapeutic strategy for B-ALL. [Display omitted] DisclosuresKo:Celgene International Sàrl: Research Funding. Li:Celgene International Sàrl: Research Funding. Tien:Celgene International Sàrl: Research Funding. Tang:Celgene International Sàrl: Research Funding.

Pre-Transplant Exhausted Marrow Infiltrating T Lymphocytes in MRD Negative Patients with Acute Leukemia Predict Early Relapse Following Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplant (alloSCT) is curative for patients with high-risk acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Despite this, relapse remains the leading cause of treatment failure. The use of flow cytometry to identify aberrant leukemia-associated immunophenotypes is an established method of detecting minimal residual disease (MRD). MRD status immediately prior to transplant remains one of the single best predictors of post-transplant outcome. Unfortunately, not all MRD(-) patients maintain remission post-transplant. Therefore, we sought to find additional predictors of post-transplant relapse. T cell exhaustion occurs following continuous exposure to antigen and leads to sustained up-regulation of multiple inhibitory receptors, progressive loss of function, and expression of a distinct transcriptional state. Bachireddy et al recently reported that exhausted CD8+ marrow infiltrating lymphocytes (MILs) were a novel predictor of response to donor lymphocyte infusion (DLI) for patients with relapsed chronic myelogenous leukemia (CML). Based on this principle, we hypothesized that exhausted CD8+MILs may also be predictive of outcome for MRD(-) patients prior to alloSCT.We matched pre-transplant samples from 17 MRD(-) patients who maintained remission for greater than 2 years post-transplant to 11 MRD(-) patients who suffered relapse within 2 years of transplant. Patients had either ALL or AML and did not differ by demographic details. MILs were evaluated for exhaustion using 2 multicolor flow cytometry panels which collectively included a viability dye, T cell markers (CD3 and CD8) surface inhibitory receptors (PD-1, Tim-3, and TIGIT), transcription factors (T-bet and Eomes), and intracellular cytokine staining (IL-2, TNF-α, and IFN-γ) in response to CD3/CD28 bead stimulation.There was no difference in the percentage of total T cells (CD3), the CD8+ T cells (CD3+CD8+), or CD8+ T cell subsets between the 2 groups. While there was also no difference in the mean fluorescent intensity (MFI) of Tim-3, both PD-1 and TIGIT were significantly elevated on the CD8+ MILs of MRD(-) patients that eventually relapsed (p=0.01 and p=0.03, respectively). Among CD8+ MILs, cells with the highest PD-1 expression (PD-1High) were significantly elevated in the relapse group (p=0.006), as was co-expression of multiple inhibitory receptors on single cells (p=0.01) (Figure A,B). In contrast, the proportion of CD8+ MILs lacking all 3 of these inhibitory receptors was significantly higher among the MRD(-) group that maintained remission (p=0.01) (Figure C). Interestingly, CD8+ MILs in the remission group with dual PD-1+TIGIT+ expression or expressing PD-1High were more likely to be central memory cells (both p=0.05), while these same immunophenotypes were significantly more often effector memory cells in patients who went on to relapse (p=0.03 and p=0.02, respectively).As noted above, inhibitory receptor expression alone does not define exhaustion. We further assessed the CD8+ inhibitory receptor expressing MILs in the relapse group for other phenotypic evidence of exhaustion by evaluating their function and transcription factor expression. Cells with either PD-1+TIGIT+ dual expression or PD-1High were directly compared to cells that lacked inhibitory receptors. The dual PD-1+TIGIT+ cells had impaired ability to produce TNF-α and IFN-γ (both with p<0.0001), while PD-1High cells produced significantly less TNF-α (p<0.0001), IFN-γ (P<0.0001) and IL-2 (p=0.0001). Moreover, both sets of MILs had significantly decreased polyfunctional cytokine production compared to cells without these surface receptors (p < 0.0001) (Figure D). Furthermore, transcription factor expression was also altered among inhibitory receptor expressing cells. They exhibited both increased early exhaustion as evidenced by high T-bet expression in PD-1 expressing cells as well as terminal exhaustion indicated by significantly elevated Eomes expression on PD-1High expressing cells (Figure E,F).Collectively, this study suggests that the presence of exhaustion in CD8+ MILs in acute leukemia patients who are MRD(-) prior to transplant may be at higher risk for post-transplant relapse. If validated, this data may be used to further risk-stratify patients and/or direct pre- or post-transplant therapy, including the potential use of checkpoint blockade. [Display omitted] DisclosuresBachanova:Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Wagner:Novartis: Research Funding; Magenta Therapeutics: Research Funding. Miller:Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding; Celegene: Consultancy.

Rapid Capture NGS Identifies Genomic Rearrangements in BCP-ALL for MRD Diagnostics

BackgroundImmunoglobin heavy chain (IGH), T-cell receptor δ (TRδ) rearrangements and translocations have frequently been described in patients with B-cell precursor (BCP) ALL. In order to detect the various types of fusions we used a high-throughput sequencing (HTS) assay based on direct capturing and sequencing of enriched genomic DNA fragments. Our approach focused on enrichment and direct paired-end sequencing of IG/TR rearrangements to analyze clonality and to detect kinase- and/or cytokine-receptor fusion breakpoints. We selected the corresponding chromosomal regions spanning exonic as well as intronic genomic areas from IGH-JH, TRδ and from several “Ph-like”-associated genes (ABL1, ABL2, CSF1R, CRLF2, EPOR, JAK2, PDGFRß). For the identification of clonality and fusion breakpoints we used Vidjil (www.Vidjil.org) and “segemehl” bioinformatics algorithms, respectively. Clonal rearrangements and genomic breakpoints were subsequently compared for their use in detection of minimal residual disease (MRD).Patients and methodsFor combined clonality and breakpoint analysis, 162 BCP ALL patients (MLL-; TEL-AML1-, BCR-ABL1-) were selected due to the MRD status at the end of induction (EOI) therapy, which include a substantial proportion of ALL samples (n = 68) with a high MRD burden (≥ 1 x 10-2). To test the feasibility of the analytical approach, additional BCR-ABL1+ ALLsamples (n=19) were included for genomic breakpoint analysis. Captured genomic DNA from eighty ALL samples was sequenced on a HiSeq2500 Illumina device, the remaining samples were subjected to several MiSeq (Illumina) runs multiplexing up to 12 samples using MiSeq Reagent Kit v3 (600 cycle) chemistry.ResultsFor TRδ, a total of 267 clonal rearrangements were characterized in 142/162 samples (88%). Beside clonal Vδ2-Dδ3, Dδ2-Dδ3, Vδ2-Jα, we identified a novel group of recurrent Dδ-Jα rearrangements in 46 samples (28%), comprising Dδ2 or Dδ3 segments fused predominantly to Jα29. For IGH-JH, 304 clonal rearrangements were identified in 152/162 samples, including several novel VDJ joinings usually not detectable by standard PCR-based approaches. In BCR-ABL1+ ALL, genomic breakpoints were identified in 17/19 cases. In BCP-ALL lacking a classical rearrangement, 50 gene fusions were detected, between IGH-JH and either DUX4, EPOR or CRLF2 in 21 of 162 cases. In addition, EBF1-PDGFRß or PAR1 deletions were detected in 5 and 10 cases, respectively. Prolonged MRD was observed in ALL carrying CRLF2rearr, EPORrearr -, PDGFR-ßrearr and as an unanticipated finding in 8/10 DUX4rearr patients who mainly underwent hematopoietic stem cell transplantation in first remission (n=5) or had a relapse (n=2). MLPA revealed a non-deleted ERG status in these 8 DUX4rearr ALL samples. A distinct subgroup of MRD high-risk patients were characterized by a complete lack of IGH-VDJ rearrangements, but they exhibited frequent IKZF1del (7/10) and a variety of other genomic rearrangements (n=3 EPORrearr, n=3 DUX4rearr, n=1 CRLF2rearr, n=1 EBF1-PDGFRß or n=1 FOXP1-ABL1). Among gene-fusion positive ALLs, breakpoint- specific real-time quantitative MRD assays at EOI were established, which demonstrated equivalent or superior sensitivity and specificity compared to conventional TR/IG targeted MRD measurements.ConclusionsThe HTS approach presented here yields classical IGH/TRδ-related clonal MRD targets and genomic breakpoints from gene fusions at a time, the latter of which can subsequently serve as robust markers for follow-up monitoring. The MRD high-risk group (≥ 1 x 10-2 at EOI) revealed an enrichment of aberrant gene fusions/rearrangements in 31 of 68 (46%) patients compared to 19 of 94 (20%) in the moderate-risk MRD group (>1 x 10-4 < 1 x 10-2). Rapid capture sequencing in a clinical diagnostics setting allows for specific ABL or JAK-STAT pathway directed early intervention in the majority of gene-fusion positive patients. DisclosuresNo relevant conflicts of interest to declare.

Clinical significance of minimal residual disease in B-lineage acute lymphoblastic leukemia pediatric patients with different fusion gene backgrounds

Objective To study the clinical significance of minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (B-ALL) pediatric patients with different fusion gene backgrounds. Methods A retrospective analysis was performed on the medical data of 441 B-ALL children who were treated from January 2008 to April 2015. Among the 441 children, 336 had negative fusion gene, 79 had positive ETV6-RUNX1 fusion gene, and 26 had positive E2A-PBX1 fusion gene. Flow cytometry was used to detect MRD, and the influence of MRD on day 15 (TP1), day 33 (TP2), and week 12 (TP3) of induction therapy on prognosis was analyzed. Results In patients with negative fusion gene, the positive MRD group had significantly lower overall survival (OS) rate and event-free survival (EFS) rate (P 0.05). Conclusions MRD has the most definite prognostic significance in pediatric B-ALL patients with negative fusion gene, while it has unsatisfactory prognostic significance in those with positive ETV6-RUNX1 or positive E2A-PBX1.

Minimal Residual Disease Assessment and Allogeneic Transplantation As Consolidation Therapy in Acute Lymphoblastic Leukemia

Minimal Residual Disease Assessment and Allogeneic Transplantation as Consolidation Therapy in Acute Lymphoblastic Leukemia Abstract Introduction: Detectable minimal residual disease (MRD) after therapy for acute lymphoblastic leukemia (ALL) is the strongest predictor of hematologic relapse (1-4). Colombian reports of adults with ALL showed poor survival rates, the median OS was 12 months and the median PFS was only 7 months (5,6). Most reports from Colombia as well as Latin America did not include patients that had a transplant as an initial consolidation strategy and used MRD as prognostic factor. (7-9). The objective of the study was to assess progression free survival (PFS) and overall survival (OS) of patients with ALL according with MRD status at end of induction therapy. Methods: A retrospective cohort comparing PFS and OS in adults with de novo ALL, according to the MRD status at the end of induction chemotherapy, and the type of post-induction consolidation strategy used. This research work had the endorsement of the ethics and research committees of the institutions where it was carried out. Results: Were included 165 adults with ALL in the MRD investigational group, the basal characteristics of the population are described in table 1.There were 73 patients in the MDR negative group and 92 in the MDR positive group. Median PFS for the MRD positive group was 11 months (CI 95% 11.7 - 22.2), and not reached for the MRD negative group (p&amp;lt;0.001). At three-years PFS was 18% and 55 %, respectively (p&amp;lt;0,001). Fig 1. The median OS for MRD positive patients was 16 months (CI 95%, 8.8 - 23.15) and was not reached in the MRD negative group. At three-years, OS was 26 % and 51 % for the former and latter group, respectively. Fig 2. Among subjects that were not transplanted, median PFS was 21 months for MRD negative and 9 months MRD positive patients (p&amp;lt;0.001). Fig 3A. Median of PFS was not reached in either group, whereas 3-year PFS was 64% for MRD negative and 70% for MRD positive patients when transplantation in first remission was used (p=0.861). Fig 3B. For the patients with only chemotherapy for consolidation treatment, the median OS were 16 months (IC 95% 11.32 to 20.67) for MRD positive, and 27 months IC 95% (20.91 to 33.09) in negative MRD. (p=0.004). The 3-year OS 34% for MRD negative and 22% for MRD positive patients. Fig 4A. The individuals with allogeneic transplantation during first remission overcome de risk of death in the positive MRD status. The median OS were 16 months for positive MRD and transplantation and not reached in negative MRD group, The 3 year OS were 74% for MRD negative and 49% for MRD positive patients. (p=0.187). Fig 4B. Multivariate analysis At multivariate analysis, the Negative MRD status and transplantation in first remission were associated with better PFS and OS, in these patients with ALL de novo, Table 2. Conclusion: MRD status at end of induction is an independent prognostic factor for PFS and OS in adult ALL. Allogeneic transplantation in first remission may overcome the adverse prognostic impact MRD. This is the first report that verifies the impact of MRD, on progression free survival and overall survival in the Colombian population. Disclosures No relevant conflicts of interest to declare.

Flow Cytometry-Based Pre-Transplant Minimal Residual Disease Detection Strongly Impacts on the Outcome of Haploidentical Transplantation with Αβ T Cell Depletion in Pediatric Acute Lymphoblastic Leukemia

Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is indicated for patients with relapsed or refractory acute lymphoblastic leukemia. Patients with persistence of minimal residual disease (MRD) before HCT are at increased risk of disease relapse. Multiparameter flow cytometry (MFC) is the most commonly used method of MRD detection in clinical practice. This study aimed to evaluate MRD status before HCT on outcome of ALL patients receiving allogeneic HSCT from haploidentical donors with TCRαβ+/CD19+ depletion of the graft. Materials and methods A total of 120 pts with ALL (T-lineage ALL (T-ALL)- 37, B cell precursor (BCP)-ALL-83, 45 female, 75 male, median age 8.7 years (0.5-20) underwent allogeneic HSCT between June 2013 and June 2019. All pts received Haplo graft and were in morphologic remission. Disease status at transplant was CR1 in 35 pts, CR2 in 68 pts and CR&gt;2 in 17 pts. Transplantation in CR1 was performed according to risk stratification scheme in the current institutional ALL protocol (Moscow-Berlin 2008, 2015). MRD detection in the bone marrow prior to НSСТ was performed in all pts by MFC according the AIEOP BFM FLOW Network SOP. MRD negativity was defined as &lt;0.001% of all bone marrow nucleated cells. Seventy-nine patients were MRD negative before HSCT, 41 were MRD-positive. The median MRD level (among MRD-positive patients) prior HCT was 0.025%. Thirty (25%) pts received treosulfan-based myeloablative preparative regimen, while TBI-based regimen was used in 90 (75%) pts. Two regimens of GvHD prophylaxis were used. Regimen 1 (n=27): thymoglobulin 5mg/kg, rituximab 200 mg/m2 and bortezomib on day +2, +5; regimen 2 (n=93): tocilizumab at 8 mg/kg on day -1 and post-transplant bortezomib, 89 pts receive additional abatacept at 10 mg/kg on day +2, +7, +14, +28. TCR αβ+/CD19+ depletion of HSCT with CliniMACS technology was implemented in all cases. The median dose of CD34+ cells was 9.3 x106/kg (range 4.3-19.8), αβ T cells - 30x103/kg (range 1-361). Median time of follow-up for survivors was 1.6 years (range: 0.13 - 4.8). Results Primary engraftment was achieved in 116 of 120 pts (3 pts died before engraftment due to septic events, one relapsed early), the median time to neutrophil and platelet recovery was 13 and 14 days, respectively. All engrafted pts had verified morphologic remission and achieved sustained complete donor chimerism by day +30, seven of them had detectable MRD (5 of them were MRD-positive before HCT). Transplant-related mortality was 5 % (95% CI: 2-11). The cumulative incidence (CI) of relapse at 1.5 years was 25%(95%CI:18-35) for the whole cohort. Among patients, who had MRD-negative remission prior to HSCT, CI of relapse was 14 % (95%CI:8-26) with median time of relapse of 0.54 months, as compared to MRD-positive cohort, with CI of relapse of 44 % (95%CI:31-63) with median time to relapse 0.29 months, p=0.0004. pEFS (event=death or relapse) was 70% (95%CI: 61-78) for the whole cohort, in MRD (-) group pEFS was 79% (95%CI: 68-88), as compared to 56%(95%CI:40-72) in the MRD(+) group, p=0.025. CI of relapse in BCP-ALL pts, who had MRD-negative remission prior to HCT was 12 %(95%CI:6-25), in MRD(+) group 49% (95%CI:33-72), p=0.0002, in T-ALL pts in MRD (-) and MRD (+) groups CI of relapse was 17 % (95%CI:6-51) and 38 % (95%CI:20-76), respectively, p=0.14. Conclusion These results suggest that MRD detection by multiparameter flow cytometry prior to HSCT is a highly significant prognostic factor in the setting of haploidentical HSCT on the platform of ab T cell depletion. We expect that further improvement of the outcome can be achieved based on the combination of current safe haplo HSCT platform and novel targeted immunotherapy approaches. Figure Disclosures Maschan: Miltenyi Biotec: Other: lecture fee.

Quantitative Analysis of Minimal Residual Disease (MRD) Shows High Rates of Undetectable MRD after Fixed-Duration Chemotherapy-Free Treatment and Serves As Surrogate Marker for Progression-Free Survival: A Prospective Analysis of the Randomized CLL14 Trial

Fischer: Roche: Other: travel grants; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ritgen:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding. Al-Sawaf:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: travel support; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support. Tandon:Roche: Equity Ownership; Roche Products Ltd: Employment. Fink:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Roche: Other: travel grants. Stübig:Hexal: Speakers Bureau. Brüggemann:Amgen, Celgene, Janssen: Honoraria, Speakers Bureau; Amgen, Janssen: Membership on an entity's Board of Directors or advisory committees; affimed, Amgen, Celgene, Regeneron: Research Funding; Amgen, Incyte, PRMA: Consultancy. Jiang:Genentech: Employment, Equity Ownership; F. Hoffman-La Roche: Equity Ownership. Schary:Abbvie: Employment, Equity Ownership. Eichhorst:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BeiGene: Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; ArQule: Membership on an entity's Board of Directors or advisory committees. Wendtner:MorphoSys: Consultancy, Honoraria, Research Funding; GILEAD Science: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Janssen-CILAG: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding. Tausch:AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau. Kreuzer:Roche and Abbvie: Honoraria, Other: Expert testimony. Langerak:F. Hoffmann-La Roche Ltd: Research Funding; Gilead: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Genentech, Inc.: Research Funding. Goede:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants, speaker fees, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: speaker fees, Speakers Bureau; janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants, speaker fees, Speakers Bureau. Böttcher:AbbVie: Honoraria, Other: Travel grants, Research Funding; Celgene: Research Funding; Janssen-CILAG: Honoraria, Other: Travel grants, Research Funding; Roche: Honoraria, Research Funding; Genentech: Research Funding; Becton Dickinson: Research Funding; Novartis: Research Funding. Stilgenbauer:AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau. Hallek:Roche, Gilead Sciences, Inc., Mundipharma, Janssen, Celgene, Pharmacyclics, AbbVie: Honoraria, Research Funding, Speakers Bureau.

Subtypes of minimal residual disease, association with Gleason score, risk and time to biochemical failure in pT2 prostate cancer treated with radical prostatectomy.

IntroductionThe Gleason score is a strong prognostic factor for treatment failure in pathologically organ-confined prostate cancer (pT2) treated by radical prostatectomy (RP). However, within each Gleason score, there is clinical heterogeneity with respect to treatment outcome, even in patients with the same pathological stage and prostate-specific antigen (PSA) at diagnosis. This may be due to minimal residual disease (MRD) remaining after surgery. We hypothesise that the sub-type of MRD determines the risk of and timing of treatment failure, is a biological classification, and may explain in part clinical heterogeneity. We present a study of pT2 patients treated with RP, the subtypes of MRD for each Gleason score and clinical outcomes.Patients and methodsPatients with Gleason ≤6 (G6) or Gleason 7 (G7) pT2 cancer participated in the study. One month after surgery, blood was taken for circulating prostate cell (CPCs); mononuclear cells were obtained by differential gel centrifugation and identified using immunocytochemistry with anti-PSA. The detection of one CPC/sample was defined as a positive test. Touch-preparations from bone-marrow biopsies were used to detect micro-metastasis using immunocytochemistry with anti-PSA. Biochemical failure was defined as a PSA >0.2 ng/mL. Patients were classified as: Group A MRD negative (CPC and micro-metastasis negative), Group B (only micro-metastasis positive) and Group C (CPC positive). Biochemical failure-free survival (BFFS) using Kaplan–Meier and time to failure using Restricted Mean Survival Time (RMST) after 10 years of follow-up were calculated for each group based on the Gleason score.ResultsOf a cohort of 253 men, four were excluded for having Gleason 8 or 9 prostate cancer, leaving a study group of 249 men of whom 52 had G7 prostate cancer. G7 patients had a higher frequency of MRD (69% versus 36%) and worse prognosis. G6 and G7 patients negative for MRD had similar BBFS rates, 98% at 10 years, time to failure 9.9 years. Group C, G6 patients had a higher BFFS and longer time to failure compared to G7 patients (19% versus 5% and 7 versus 3 years). Group B showed similar results up to 5 years, thereafter G6 had a lower BFFS 63% versus 90%.ConclusionsG7 and G6 pT2 patients have different patterns of MRD and relapse. Risk stratification using MRD sub-types may help to define the need for adjuvant therapy. This needs confirmation with large randomised long-term trials.

PB2324 LONG TERM OUTCOME OF AUTOLOGOUS STEM CELL TRANSPLANTATION IN ADULT ACUTE MYELOID LEUKEMIA PATIENTS: THE ROLE OF MINIMAL RESIDUAL DISEASE

Background:The role of autologous stem cell transplantation (ASCT) as consolidation therapy in acute myeloid leukemia (AML) remains unclear although most studies comparing ASCT with repeated cycles of intensive chemotherapy demonstrated a significantly lower rate of relapse following ASCT. Evaluation of minimal residual disease (MRD) can better identify patients who benefit from ASCT.Aims:We report our single center experience to evaluate long term outcome in terms of overall survival (OS) and disease free survival (DFS) of AML patients who consecutively received ASCT and to retrospectively analyze the correlation between MRD status pre ASCT and OS in patients with molecularly detectable MRD.Methods:From 1998 to 2018, 65 adult patients with de novo (61/65) or secondary (4/65) AML underwent ASCT at Seràgnoli Hematology Institute. Median age was 50 (range 16‐68 years). Most patients had favorable (23/65) or intermediate (35/65) cytogenetic‐molecular profile according to ELN2017 risk classification. 24 patients (MRD marker group) had any of the following detectable leukemia‐specific molecular markers at diagnosis evaluated through qPCR: 9 NPM1, 5 RUNX1/RUNX1T1, 10 CBFB/MYH11. All the patients received intensive induction chemotherapy: 24 patients received a “3+7” like induction therapy, 23 were treated with fludarabine‐based induction and 18 received a gemtuzumab ozogamicin‐containing regimen.Results:Sixty‐two out of 65 patients (95.4%) obtained complete remission (CR) after induction chemotherapy. In the MRD group, 13 obtained MRD negativity after induction, 2 after first and 8 after the second consolidation. The median number of consolidation cycles before ASCT was two. All patients received myeloablative busulphan based‐conditioning regimen in association to melphalan (74%) or cyclophosphamide (26%). 35 patients received bone marrow stem cells and 30 peripheral blood stem cells. Before ASCT five patients were MRD+ (2 NPM1, 3 CBFs). With a median follow up of 10 years, median OS of the population was not reached and median DFS was 2470 days. OS estimates at 3, 5 and 10 years were respectively 68%, 61% and 54%. Better outcome was observed in patients receiving more than one consolidation cycle in terms of DFS (p = 0.006) and OS (p = 0.03). MRD marker group patients had estimated OS at 3 and 5 years of 90% and 85% respectively. Of 30 patients who relapsed after ASCT, 16 received a rescue therapy and were successfully candidate to allogeneic stem cell transplantation (alloSCT) in second CR with a long survival rate of 50%. In the MRD marker group, 2/2 NPM1 MRD+ relapsed and 3/3 CBFs MRD+ (all with CBFB/MHY11 fusion transcript) obtained molecular remission and are still alive in CR. Pre‐ASCT MRD positivity do not impact prognosis in term of OS (10‐yr OS 80% for MRD+, 88% for MRD‐) or DFS (2‐yr DFS 70% and 61% for MRD‐ and MRD+ respectively). Of note, NPM1 MRD‐ patients who underwent ASCT remained in CR for a long time after ASCT (median OS not reached and median DFS of 1156 in NPM1‐ and 249 NPM1+).Summary/Conclusion:our study confirm that ASCT is a feasible and effective strategy for adult AML patients, mainly in those with low‐intermediate ELN2017 risk and treated with at least 2 post‐remissional cycles and it can concede long term survival mainly to NPM1 AML patients MRD‐ before ASCT. Relapse after ASCT can be successfully rescue by alloSCT. The application of prospective evaluation of MRD through more sensitive molecular methods (qPCR) on a wider population of adult AML patient may better select the patients who could benefit from ASCT.

S1612 MOLECULAR MRD STATUS AND OUTCOME AFTER TRANSPLANTATION IN NPM1 MUTATED AML: RESULTS FROM THE UK NCRI AML17 STUDY

Background:Relapse remains the primary cause of treatment failure for patients with acute myeloid leukaemia (AML) who undergo allogeneic stem cell transplantation (alloSCT) and carries a grave prognosis. Multiple studies have identified the presence of minimal residual disease (MRD) assessed by flow cytometry or next‐generation sequencing prior to alloSCT as a strong predictor of relapse, but it is not clear how these findings apply to patients who test positive in molecular MRD assays which have far greater sensitivity.Aims:We aimed to correlate pre‐transplant molecular MRD status with outcome in patients with NPM1 mutant AML to identify clinically relevant MRD thresholds, evaluate interaction with established prognostic factors and examine the effect of transplant type.Methods:We analysed pre‐transplant blood and bone marrow samples by reverse‐transcription polymerase chain reaction (RT‐qPCR) in 107 patients with NPM1 mutant AML enrolled in the UK NCRI AML17 study for younger adults eligible for intensive chemotherapy.Results:After a median follow up of 4.9 years from transplant, estimated 5‐year overall survival (5y‐OS) was 65% for patients transplanted in 1st remission and 54% for those transplanted after a molecular or morphological relapse (p = 0.3). Evaluable pre‐SCT PB and BM samples were available for 103 and 78 patients.In total, 57 (53%) patients were MRD negative prior to SCT; 5y‐OS was 76% vs 46% for patients who were MRD positive. A threshold of 200 copies/105 ABL in the PB and 1000 copies in the BM provided maximal discrimination. Patients with negative, low (positive below the defined thresholds) and high levels of MRD had 2y‐OS of 83%, 63% and 13% respectively (p &lt; 0.0001).Presence of a FLT3 ITD at diagnosis was associated with a poorer outcome only in patients with a low level of MRD (hazard ratio 7.14, p = 0.04, fig 1a). There was no impact in patients who were MRD negative or who had high levels of MRD. Peripheral blood MRD status after second induction cycle (PC2, previously identified as a powerful prognostic factor) was also associated with poor outcome in the MRD low group (5y OS 82% vs 45%, p = 0.033) but had no effect in patients who had negative or high levels of MRD. These variables retained prognostic power in multivariate analysis and combining these could divide patients into two groups with 2y OS of 10% and 81% (adjusted hazard ratio 7.70, p &lt; 0.0001, figure 1b).We investigated the effect of donor source, conditioning regimen and T‐depletion on outcome. In the MRD negative group, none of these factors correlated with outcome. In the MRD positive group, T‐depletion was significantly associated with inferior survival (34% vs 100% for patients who did not undergo T‐depleted transplant, p = 0.002). No effect of donor source or conditioning protocol was detected.Summary/Conclusion:In contrast to patients who are MRD positive by flow cytometry or NGS, patients who test positive for NPM1 mutant transcripts prior to alloSCT do not have a universally poor outcome. Adverse outcome is predicted in patients with high levels of MRD (above 200 copies / 105 ABL in the PB or 1000 copies in the BM). For patients with MRD positivity below these levels, FLT3 ITD and post‐induction PB status are strongly associated with poor outcome. We observed no effect of transplant conditioning regimen for any MRD group however use of T‐depletion was associated with adverse outcome in patients who were MRD positive. These findings may help inform peri‐transplant management and the design of future interventional studies.image

Prognostic Value of Minimal Residual Disease before Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia

IntroductionSeveral studies have shown that the minimal residual disease (MRD) in acute myeloid leukemia (AML) patients has a prognostic value after induction and consolidation therapy. Nevertheless the relapse is the most important cause of treatment failure in these patients, although they achieved a negative MRD, and even after an allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nowadays, the value of the MRD before allogeneic BMT is still controversial.MethodMulticentric study where we have studied correlative AML patients who went under an allo-HSCT in a situation of complete response, between 2012 and April'18. The MRD was analyzed by 8-coloured multiparametric flow cytometry, at least with 2 tubes per patient and 1,000,000 events per tube. We evaluated the prognostic value of the MRD before allo-HSCT.ResultsBetween January'12 and April'18 we have gathered 90 allogeneic BMT in AML patients who were in CR, with a median age of 45 years old (17 - 66). The pre-HSCT situation was 1st complete remission (CR) in 75 patients and 2nd CR in 15. In 45 patients the conditioning regimen was myeoablative. In the group of patients (67) where we could know the risk group at diagnosis, the distribution was: low risk 18%, intermediate risk 59.7% and high risk 22.4%. The 46.7% of the donors were not related. In the last follow-up after allo-HSCT 24 patients have suffered a relapse (26.7%) and 41 (45.5%) have died (17 cases of mortality related to the transplant and 24 not related). In the global analysis the median follow-up of the overall survival (OS) was 37.5 months. Among the 90 patients, MRD was valuable in 86. Ten of 59 patients (16.9%) with negative MRD relapsed vs 12/27 (44.4%) with positive MRD, p= 0.016. If we consider only patients in 1st CR, 9/50 (18%) patients with negative MRD relapsed vs 10/22 (45.5%) with positive MRD, p= 0.02. This statistically significant difference does not exist if we consider only patients in 2nd CR. The median follow-up of OS and event free survival (EFS) was not reached in the negative MRD group and 571 days and 299 days in the positive MRD group. OS and EFS at 2 years after transplantation were 65% and 64% in the negative MRD group and 42% and 37% in the positive MRD group, p= 0.03 and p= 0.008 respectively (figure 1).ConclusionsThe detected MRD by 8-colour multiparametric flow cytometry previous an allo-HSCT in patients with AML in 1st CR is a prognostic factor in terms of relapse. Patients with a positive MRD before the allo-HSCT have a poorer OS and EFS than the patients with a negative MRD. DisclosuresNo relevant conflicts of interest to declare.

The Frequency of Occurrence of Minimal Residual Disease into Different Prognostic Groups of Patients with Chronic Lymphocytic Leukemia

Background. It is known, that genetic factors and the absence of minimal residual disease (MRD) are strongly affecting prognosis of chronic lymphocytic leukemia (CLL).Aim. To determine the influence of genetic abnormalities (GA) on achieving MRD-negative remissions in patients with CLL.Methods. Twenty-four adult pts (median age 57 years, range 35-67; male 14, female 10) with newly diagnosed CLL were included. The CLL was diagnosed according to the standard basic examination (complete blood count with differential, multicolor flow cytometry (MFC) of blood and bone marrow (BM), lymph node and BM immunohistochemistry (IHC), computered tomography). Cytogenetic studies were performed on blood samples using standard GTG-method. Interphase FISH analyses were performed according to the manufacturer's protocol using DNA probes: LSI 13(RB1)13q14, LSI ATM (11q22), CEP12, LSI TP53 (17p13.1) (Abbott). Immunophenotype (IFT) of CLL cells assessed with combinations: CD3/CD19, CD19/CD5, CD19/CD11c, CD19/CD20, CD19/CD22, CD19/CD23, CD19/CD25, CD19/CD38, CD19/CD43, CD19/CD81, CD19/HLA-DR, and CD19/CD5/CD23. We have used NCI revised guidelines (Hallek M, et al., 2008) for treatment initiation and assessment of response after completion of primary therapy with rituximab-based regimens (FCR or RB). All patients treated subsequently with rituximab maintenance. MRD was detected by MFC.Results. The frequency of GA in CLL was 50.0% (12/24): 15.0% (3/20) - by conventional karyotyping, 47.8% (11/23) - by FISH analyses and 9.5% (2/21) - using both methods. Stratification of patients into prognostic groups based on identified GA. Favorable prognosis (Group 1) - patients with del(13q) (n = 5); neutral prognosis (Group 2) - normal karyotype (n = 12) or trisomy of chromosome 12 (n = 3); unfavorable prognosis (Group 3) - del(11q) (n = 3) or the complex karyotype (n = 1).Statistically significant differences in the frequency of achieving MRD-negative remissions between FCR (5/11) vs. RB (5/13) were not detected (p<0.05).Complete remissions (CR) were reached in 37.5% (9/24) pts, partial remissions (PR) - 62.5% (15/24). The MRD-negative remissions were reached in 10 patients: in Group 1 - 2/5 (40%; CR - 1), in Group 2 - 5/15 (33.3%, CR - 6), in Group 3 - 75.0% (3/4; CR - 2).Statistically significant differences in PFS were detected between MRD-negative vs. MRD-positive groups (p=0.03). Median PFS in MRD-negative has not been reached. Median PFS MRD-positive was 33.1 month.Conclusions. Further researches aimed at examining the relationship between the presence or absence of MRD and genetic prognostic groups, will help to understand the most important factors affecting the overall and progression-free survival. DisclosuresNo relevant conflicts of interest to declare.

Integration of Minimal Residual Disease with Other Patient Risk Factors Identifies a Population with Very Poor Overall Survival in Pediatric ALL: Results from the UKALL 2003 Trial

Background:Overall survival (OS) for children with ALL treated on contemporary protocols is now > 90%. To further improve outcomes, stratification must direct intensive treatment to those patients with the highest risk of relapse. Whilst minimal residual disease (MRD) has emerged as the most powerful predictor of outcome, its use in isolation is insufficient to identify those patients with the greatest risk. To address this, we performed a detailed analysis of the entire patient cohort from the recently reported large multi-center randomized controlled trial, UKALL 2003. Integration of end of induction (EOI) MRD with other predictive factors - cytogenetics, age and white cell count (WCC) - allowed the identification of very high risk groups that are likely to benefit from early treatment intensification or novel therapies.Methods:The UKALL 2003 trial recruited 3126 patients aged 1-25 years with Philadelphia-negative ALL between Oct 1, 2003, and June 30, 2011. Treatment was initially stratified based on NCI risk and cytogenetic results. Subsequent treatment was directed by EOI MRD measured using EuroMRD approved real-time quantitative PCR for immunoglobulin and T-cell receptor gene rearrangements. Analysis was performed on 2666 patients with available MRD results and focused on the systematic integration of MRD results with other patient characteristics to identify subgroups with a very poor OS (<75% at 5 years). MRD was considered positive at a level ≥0.005% and low risk if <0.005% unless otherwise stated.Results:Both MRD and cytogenetic risk group were independently predictive of survival. In particular, patients with high risk (HR) genetic abnormalities - low hypodiploidy, near haploidy, KMT2A (MLL) rearranged, iAMP21 and t(17;19)(q23;p13) (n=100) had a poor outcome with 5 yr OS of 76.0%. Combining these factors showed that MRD could stratify the HR cytogenetic subgroup with a significant difference in survival between those with low risk MRD and positive MRD (5yr OS 93.0 vs. 63.1%, p=0.002, n=43 vs. 57). Crucially, this demonstrates that HR cytogenetics are only truly poor risk in the context of poor treatment response. Importantly, there were almost no survivors following relapse in the HR cytogenetic/MRD positive group, whilst 50% of those with low risk MRD could be salvaged after relapse. In addition, whilst t(1;19)(q23;p13)was not considered a HR cytogenetic factor in this trial, combination with positive EOI MRD identified an additional subgroup of 20 patients with a 5yr OS of only 74.7% (vs. 96.7% in MRD low risk patients, p=0.003).Whilst WCC at presentation was also predictive of outcome, this difference was significant only in those with B-ALL and not T-ALL. In B-ALL, WCC>100 x109/L was associated with significantly poorer survival (5yr OS 95.1 vs. 84.0%, p<0.0001) whilst high WCC in T-ALL had little effect on survival, even at WCC >200 x109/L (5yr OS 90.2 vs. 86.7%, p=0.283). Integration of MRD results allowed the identification of a very high risk group with B-ALL, WCC>100 and positive EOI MRD (n=80) which had a 5yr OS of only 71.3%. Further breakdown by cytogenetic risk group showed those patients with good risk cytogenetics (ETV6-RUNX1, high hyperdiploidy) (n=19) had a remarkably good survival despite WCC>100 (5yr OS 94%). In contrast, those with intermediate (n=34) or HR cytogenetics (n=15) had a very poor 5yr OS (65.7% and 60.0% respectively).Older age (≥10yr) was also associated with poorer outcomes compared to those <10yr (5yr OS 95.8 vs. 87.4%, p<0.0001). Integration with MRD results further separated these groups but did not identify a very high risk subgroup. However, combining older age with a higher EOI MRD cut-off of 1% successfully identified a subgroup (n=96) with a very poor 5yr OS of 51.3% (vs. 91.4% in those with MRD<1%, p<0.0001). Interestingly, this was independent of cytogenetic risk group.Conclusions:Through the integration of EOI MRD with other risk factors in a large pediatric ALL cohort we have identified a population of patients with a very poor OS. When combined, these criteria select only 6% of all patients, but capture 35% of all deaths suggesting current treatment is suboptimal in this group. This information is essential for the planning of future trials to ensure that treatment intensification and novel therapies are targeted at those patients at greatest risk, whilst avoiding unnecessary treatment in those patients that already experience good outcomes. DisclosuresNo relevant conflicts of interest to declare.

Predictive Value of Minimal Residual Disease: Analysis By Flow-Cytometry in Children with Relapsed Acute Lymphoblastic Leukemia

Introduction: Despite the advances observed in the outcome of pediatric acute lymphoblastic leukemia (ALL) treatment during the last 20 years, relapse remains the most common cause of treatment failure in childhood ALL. Several factors have been associated to the prognosis of these patients; however, minimal residual disease (MRD) emerges as a relevant predictor of outcome.Objectives: The aims of this study were to assess MRD by flow-cytometry in relapsed ALL and to evaluate its prognostic impact as a predictor factor of outcome at the end of the induction therapy and prior to hematopoietic stem cell transplantation (HSCT).Patients and Methods: From Aug'10 to Jun'15, 123 ALL patients were treated at our center. MRD determination at least at two time-points during relapse treatment was a requirement for considering a patient eligible for the present study. Sixty-six cases were excluded due to the following causes: 10 patients died during induction, 2 died early in complete remission (CR), 29 did not respond to chemotherapy, in 13 patients MRD determination was not performed: 4 did not have clinical data available, 4 patients were Down Syndrome and 4 children received treatment for relapse in other centers. Thus, fifty-seven patients achieved CR and were evaluated for MRD at two time points. Of them, 56 patients belonged to S4 and S3 and 1 patient to S1 group as defined by the Berlin-Frankfurt-Münster stratification for relapsed ALL. MRD was analyzed by multiparametric flow-cytometry following ALL-IC 2009 guidelines. Negative MRD was defined as disclosing less than 0.1% of blasts. For this analysis, patients were stratified based on MRD levels at two different time points: after end of induction, before HSCT or at any other time point during the follow-up for patients who did not undergo HSCT. Three groups were defined: Group-1: negative at both time points (n= 23), Group-2: positive at 1 time point (n= 13) and Group-3: positive at both time points (n= 21). Patients who relapsed before receiving HSCT were considered Group-3. Twenty-five patients underwent HSCT: 13 of them from Group-1, 9 from Group-2 (2 had positive MRD previous to receive HSCT) and 3 patients from Group-3. HSCT was performed with matched familiar donor in 16 cases and matched unrelated donor in 9 cases.Results: The distribution of events according to receiving or not HSCT was: 5 died due to transplant related mortality (TRM), 9 relapsed after receiving HSCT and 16 during treatment with chemotherapy. With a median follow-up of 16 (range: 6-67) months, overall 3-year EFS probability (EFSp) (SE) was 32 (8)%. The 3-year EFSp was 75 (11)% for Group-1, 24 (14)% for Group-2 and 0% for Group-3 (p-value <0.00001). Comparing patients who did not receive HSCT vs. patients who did, EFSp (SE) was 32 (12)% and 29 (11)% respectively (p-value: non-significant). The EFSp (SE) according to MRD groups in patients who underwent HSCT was: Group-1: 53 (19)%, Group-2: 14 (13)% and 0% for Group-3 (p-value: 0.06).Conclusions: MRD quantification by flow-cytometry demonstrated to be a significant prognostic factor for relapsed ALL. Both, TRM and death in CR rates, were high and should be decreased by improving supportive measures. MRD determination by flow-cytometry in patients who underwent HSCT showed a trend to achieve a better EFSp, thus representing a relevant tool for stratifying relapsed ALL patients in order to achieve a better selection of patients to receive HSCT. DisclosuresNo relevant conflicts of interest to declare.

Increase in myeloid-derived suppressor cells (MDSCs) associated with minimal residual disease (MRD) detection in adult acute myeloid leukemia.

Myeloid-derived suppressor cells (MDSCs) are thought to help provide a cellular microenvironments in many solid tumors, in which transformed cells proliferate, acquire new mutations, and evade host immunosurveillance. In the present study, we found that MDSCs (CD33 + CD11b + HLA-DR(low/neg)) in bone marrow were significantly increased in adult acute myeloid leukemia (AML) patients. MDSCs levels in newly diagnosed AML patients correlated well with extramedullary infiltration and plasma D-dimer levels. Remission rates in the MDSCs > 1500 group and MDSCs < 1500 group were 72.73 and 81.25 %, respectively. No significant differences were found between the two groups. MDSC levels in the complete remission group were significantly decreased after chemotherapy, while in the partial remission and non-remission groups, there were no significant differences. The level of MDSCs in the high minimal residual disease (MRD) group was significantly higher than that in the middle and low MRD groups. High levels of Wilms' Tumor-1 (WT-1) protein were strongly correlated with higher bone marrow MDSC levels. In conclusion, we report here a population of immunosuppressive monocytes in the bone marrow of patients with AML characterized by the CD33(high)CD11b + HLA-DR(low/neg) phenotype. These cells appear to impact the clinical course and prognosis of AML. This data may provide potentially important targets for novel therapies.