BackgroundFlow‐cytometric minimal residual disease (FC‐MRD) monitoring is a well‐established risk‐stratification factor in B‐lymphoblastic leukemia/lymphoma (‐B‐ALL) and is being considered as a basis for deintensification or escalation in treatment protocols. However, currently practiced standard FC‐MRD has limited sensitivity (up to 0.01%) and higher false MRD‐negative rate. Hence, a highly sensitive, widely applicable, and easily reproducible FC‐MRD assay is needed, which can provide a reliable basis for therapeutic modifications.MethodsA 10‐color high‐event analysis FC‐MRD assay was studied for the evaluation of MRD status at postinduction, (PI; day‐35), postconsolidation, (PC; day‐78), and subsequent follow‐up time‐points (SFU) in bone marrow samples from pediatric B‐ALL.ResultsOne‐thousand MRD samples (PI‐62.2%; PC‐26.5%; and SFU‐11.3%) from 622 childhood B‐ALL patients were studied. High‐event analysis was performed with median 4,452,000 events (range, 839,000 to 8,866,000 events) and >4 million events in 71% samples. MRD was measurable in 43.2% of PI‐samples, in 29.4% PC‐samples, and in 32.7% SFU‐samples. To simulate comparison with standard FC‐MRD, we reanalyzed MRD results gating only first 500,000 and first 1000,000 events in 122 PI‐MRD positive samples with MRD levels <0.02%. Of these samples gated for 500,000 events and 1000,000 events, 32% and 21.3% were found to be falsely MRD‐negative, respectively.ConclusionsWe report an easily reproducible high‐sensitivity 10‐color FC‐MRD assay with the sensitivity of 2‐in‐106 (0.0002%). It allowed the detection of low‐level MRD in samples, which could have been reported negative using the standard FC‐MRD with limited event analysis. Thus, this high‐sensitivity MRD‐methodology can provide a reliable basis for therapeutic modifications in B‐ALL. © 2019 International Clinical Cytometry Society

We accrued 201 patients of adult AML treated with conventional therapy, in morphological remission, and evaluated MRD using sensitive error-corrected next generation sequencing (NGS-MRD) and multiparameter flow cytometry (FCM-MRD) at the end of induction (PI) and consolidation (PC). Nearly 71% of patients were PI NGS-MRD+ and 40.9% PC NGS-MRD+ (median VAF 0.76%). NGS-MRD+ patients had a significantly higher cumulative incidence of relapse (p = 0.003), inferior overall survival (p = 0.001) and relapse free survival (p < 0.001) as compared to NGS-MRD− patients. NGS-MRD was predictive of inferior outcome in intermediate cytogenetic risk and demonstrated potential in favorable cytogenetic risk AML. PI NGS-MRD− patients had a significantly improved survival as compared to patients who became NGS-MRD− subsequently indicating that kinetics of NGS-MRD clearance was of paramount importance. NGS-MRD identified over 80% of cases identified by flow cytometry at PI time point whereas FCM identified 49.3% identified by NGS. Only a fraction of cases were NGS-MRD− but FCM-MRD+. NGS-MRD provided additional information of the risk of relapse when compared to FCM-MRD. We demonstrate a widely applicable, scalable NGS-MRD approach that is clinically informative and synergistic to FCM-MRD in AML treated with conventional therapies. Maximum clinical utility may be leveraged by combining FCM and NGS-MRD modalities.

Detection of measurable residual disease (MRD) by mutation specific techniques has prognostic relevance in NPM1 mutated AML (NPM1mut AML). However, the clinical utility of next generation sequencing (NGS) to detect MRD in AML remains unproven. We analysed the clinical significance of monitoring MRD using ultradeep NGS (NGS-MRD) and flow cytometry (FCM-MRD) in 137 samples obtained from 83 patients of NPM1mut AML at the end of induction (PI) and consolidation (PC). We could monitor 12 different types of NPM1 mutations at a sensitivity of 0.001% using NGS-MRD. We demonstrated a significant correlation between NGS-MRD and real time quantitative PCR (RQ-PCR). Based upon a one log reduction between PI and PC time points we could classify patients as NGS-MRD positive (<1log reduction) or negative (>1log reduction). NGS-MRD, FCM-MRD as well as DNMT3A mutations were predictive of inferior overall survival (OS) and relapse free survival (RFS). On a multivariate analysis NGS-MRD emerged as an independent, most important prognostic factor predictive of inferior OS (hazard ratio, 3.64; 95% confidence interval [CI] 1.58 to 8.37) and RFS (hazard ratio, 4.8; 95% CI:2.24 to 10.28). We establish that DNA based NPM1 NGS MRD is a highly useful test for prediction of relapse and survival in NPM1mut AML.

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BackgroundMeasurable residual disease (MRD) assessment using multicolor flow cytometry (MFC) has become the center point of pediatric B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) risk stratification and therapeutic management. The addition of new markers can improve the accuracy and applicability of MFC‐based MRD assay further. Herein, we evaluated the utility of a new marker, CD304/neuropilin‐1, in the assessment of MFC‐based MRD.MethodsExpression patterns of CD304 were studied in leukemic blasts from BCP‐ALL patients and in normal precursor B cells (NPBC) from uninvolved non‐BCP‐ALL bone marrow samples using 10‐color MFC. MRD was monitored at end‐of‐induction (EOI; Days 35–40) and end‐of‐consolidation (Day 78–80) time points.ResultsWe studied CD304 expression in 300 pediatric BCP‐ALL patients and found it positive in BCP‐ALL blasts in 41.7% of diagnostic samples. It was significantly associated with ETV6‐RUNX1 (p < .001) as well as BCR‐ABL1 (p = .019) and inversely associated with TCF3‐PBX1 fusion gene (p = .0012). It was found clearly negative in NPBC. EOI‐MRD was detectable in 152/300 (50.7%; ≥0.01% in 35.33% and <0.01% in 15.33%) samples, in which CD304 was positive in 72/152 (47.4%) diagnostic and 63/152 (41.4%) MRD samples. It was positive in 45.7% (21/46) of low‐level (<0.01%) MRD samples. In comparison with diagnostic samples, its expression was retained in 68.06% (49/72), lost in 31.94% (23/72), and gained in 14/80 (17.5%) of EOI‐MRD samples.ConclusionsCD304 is commonly expressed in leukemic blasts of BCP‐ALL. It is very useful in distinguishing residual disease from hematogones and is a fairly dependable marker. Hence, it is a valuable addition for enhancing the sensitivity and applicability of MFC‐based MRD assay in BCP‐ALL.

PURPOSE Infections remain a major challenge in the treatment of acute myeloid leukemia (AML). Induction-related mortality reported in the literature is approximately < 5% in clinical trials. However, the real-world scenario is different, especially in developing countries, given the high incidence of multidrug-resistant (MDR) organisms, high incidence of fungal pneumonia at baseline, and significant delay before initiation of chemotherapy. We aimed to look at contemporary infections and infection-related mortality and analyze the patterns of infections. MATERIALS AND METHODS This retrospective study was conducted at a large tertiary care oncology center in India. Patients with newly diagnosed AML who were older than age 15 years, considered fit for intensive therapy, and treated in the general wards of the adult hematolymphoid unit from March 1, 2014, until December 31, 2015, were included. RESULTS One hundred twenty-one patients were treated during the study period. The most common presenting complaint was fever (85%). The focus of infection at presentation was found in 63% of patients, with respiratory infection being the most common (47%). MDR organisms were isolated in 55% of patients during induction from various foci. Klebsiella pneumoniae was the most common blood culture isolate (42.9%). Fungal pneumonia was diagnosed in 55% of patients during induction despite antifungal prophylaxis. Treatment-related mortality was 10.7% in all phases, with an induction mortality rate of 7.4%. Complete remission was attained in 69% of patients. Of all patients who received induction chemotherapy, 74% completed all three consolidation cycles. The 121 patients were followed up for a median period of 53 months. Four-year event-free survival was 35.8%, and 4-year overall survival was 41.5%. CONCLUSION Infections and infection-related mortality are major challenges during AML induction. Gram-negative MDR and fungal infections are particularly common in our region.

Introduction: One of the mainstays of chemotherapy in acute myeloid leukemia (AML) is induction with a goal to achieve morphological complete remission (CR). However, not all patients by this remission criterion achieve long-term remission and a subset relapse. This relapse is explained by the presence of measurable residual disease (MRD). Methods: We accrued 451 consecutive patients of adult AML (from March 2012 to December 2017) after informed consent. All patients received standard chemotherapy. MRD testing was done at post-induction and, if feasible, post-consolidation using 8- and later 10-color FCM. Analysis of MRD was done using a combination of difference from normal and leukemia-associated immunophenotype approaches. Conventional karyotyping and FISH were done as per standard recommendations, and patients were classified into favorable, intermediate, and poor cytogenetic risk groups. The presence of FLT3 -ITD, NPM1 , and CEBPA mutations was detected by a fragment length analysis-based assay. Results: As compared to Western data, our cohort of patients was younger with a median age of 35 years. There were 62 induction deaths in this cohort (13.7%), and 77 patients (17.1%) were not in morphological remission. The median follow-up was 26.0 months. Poor-risk cytogenetics and the presence of FLT3 -ITD were significantly associated with inferior outcome. The presence of post-induction MRD assessment was significantly associated with adverse outcome with respect to OS ( p = 0.01) as well as RFS ( p = 0.004). Among established genetic subgroups, detection of MRD in intermediate cytogenetic and NPM1 mutated groups was also highly predictive of inferior outcome. On multivariate analysis, immunophenotypic MRD at the end of induction and FLT3 -ITD emerged as independent prognostic factors predictive for outcome. Conclusion: This is the first data from a resource-constrained real-world setting demonstrating the utility of AML MRD as well as long-term outcome of AML. Our data is in agreement with other studies that determination of MRD is extremely important in predicting outcome. AML MRD is a very useful guide for guiding post-remission strategies in AML and should be incorporated into routine treatment algorithms.

Introduction: In 2016, Children Oncology Group (COG) described a new high-risk subtype of acute myeloid leukemia (AML) with a distinct immunophenotypicsignature, RAM-phenotype (RAM-AML). Data on clinical and laboratory features of RAM-AML are still limited to COG report only. Herein, we report the clinicopathological characteristics and detailed immunophenotypic features of RAM-AML patients. In COG report, 38% of RAM-AML belonged to acute megakaryoblastic leukemia (AMKL)-subtype. Hence, we further compared the immunophenotypic features RAM-AML with non-RAM-AMKL diagnosed during the same study period. Methods: We included RAM-AML and non-RAM AMKL patients diagnosed between January 2017 and December 2019. We studied their morphological, cytochemical, immunophenotyping, cytogenetic, and molecular characteristics. Mean fluorescent intensity (MFI) and expression-pattern of immunophenotypic markers of RAM-AML were compared with non-RAM AMKLs patients. Results: We identified 11 RAM-AML (1%) and 21 non-RAM AMKL (1.9%) patients in 1102 pediatric-AML patients. Seven of 11 (63.64%) patients belonged to FAB-M7subtype. CD56, CD117, and CD33 demonstrated overexpression, whereas CD45 and CD38 showed under-expression in RAM-AML patients. CD36 was consistently negative in RAM-AML, whereas moderate-bright positive in non-RAM AMKLs patients (p < 0.0001). On principle component analysis, addition of CD36 enhanced the visual-separation between RAM-AML and non-RAM AMKL clusters. Cytogenetic and molecular studies did not show any recurrent abnormality; however, RNAsequencing study revealed CBFA2T3-GLIS2-fusion in three of seven (42.8%) RAM-AML patients. Conclusion: We report the clinicopathological characteristics and the detailed immunophenotypic profile in the world's second series of RAM-AML patients. We further report a novel finding of CD36-negative expression as an additional parameter to the multidimensional immunophenotypic signature of this entity.