Multilineage Dysplasia as Assessed by Immunophenotype in Acute Myeloid Leukemia: A Prognostic Tool in a Genetically Undefined Category

Simple Summary The prognostic role of multi-lineage dysplasia is still debated in acute myeloid leukemia. The aim of our work was to study dysplasia by a technique alternative to the conventional morphological method, which is multi-parameter flow cytometry. To this end, we used an immune-phenotypic score (IPS), able to estimate dysplasia by the extent of deviation from normal profile, obtained in a control group. IPS provided no insight into prognosis when considered overall nor within well-defined genetic categories. Of interest, IPS-related dysplasia conveyed significant prognostic information when we focused on genetically undefined patients, triple-negative for NPM1, FLT3 and CEBPA. This category still represents a non-negligible fraction of patients, that lack specific molecular features either for targeted drugs or for proper risk assessment. In this context, our data could help address the relative unmet needs in treatment strategy, and provide insight into response prediction in the rapidly evolving therapeutic scenario of AML. Abstract Acute myeloid leukemia (AML) “with myelodysplasia-related changes (MRC)” is considered a separate entity by the World Health Organization (WHO) classification of myeloid neoplasms. While anamnestic and cytogenetic criteria provide objective attribution to this subset, with clear unfavorable prognostic significance, the actual role of multi-lineage dysplasia (MLD) as assessed by morphology is debated. The aim of our work was to study MLD by a technique alternative to morphology, which is multiparameter flow cytometry (MFC), in a large series of 302 AML patients intensively treated at our Center. The correlation with morphology we observed in the unselected analysis reiterated the capability of the MFC-based approach at highlighting dysplasia. MLD data, estimated through an immune-phenotypic score (IPS), provided no insight into prognosis when considered overall nor within well-defined genetic categories. Of interest, IPS-related dysplasia conveyed significant prognostic information when we focused on genetically undefined patients, triple-negative for NPM1, FLT3 and CEBPA (TN-AML). In this context, the lack of dysplastic features (IPS_0) correlated with a significantly higher CR rate and longer survival compared to patients showing dysplasia in one or both (neutrophil and erythroid) cell lineages. The impact of IPS category maintained its validity after censoring at allogeneic HSCT and in a multivariate analysis including baseline and treatment-related covariates. In a subgroup featured by the lack of genetic determinants, our data could help address the relative unmet needs in terms of risk assessment and treatment strategy, and provide insight into prediction of response in the rapidly evolving therapeutic scenario of AML.


A. Supplemental Materials and Methods
S4-S6. Outcome according to immunophenotypic score (IPS) in WHO-defined subsets S7-S11. Outcome according to immunophenotypic score (IPS) in genetically defined subsets S12-S13. Outcome according to IPS in triple-negative AML with censoring at allogeneic transplant S14-S15. Effect of IPS on outcome in triple-negative AML as depicted by Forest plot S16. Outcome according multi-lineage dysplasia as assessed by morphology in triple-negative AML. S17-S19. Effect of treatment-related covariates (induction and allogeneic transplant) on outcome in IPS-related groups as depicted by Forest plot C. Supplemental Tables S1. Characteristics of patients according to induction regimen (SDAC vs HDAC) S2. Characteristics of patients according to immunophenotypic score (IPS)

S1. Treatment Protocols
Protocol-1: since April 2004 to March 2007, patients received induction according to standarddose cytarabine (SDAC) based course, namely "3+7" (Cytarabine 100 mg/sqm bid on days 1-7; Idarubicin 12 mg/sqm on days 1-3). From 2006 on, etoposide 100 mg/sqm on days 1-5 was added (ICE course). High-dose Cytarabine (3000 mg/sqm bid days 1, 3, 5) was used as first consolidation in patients aged < 61 years attaining complete remission (CR) after ICE. Patients with persistent disease (i.e. > 5% BM blasts at hematopoietic recovery) after first course received a salvage regimen (FLA-Ida). In an intention-to-treat approach, patients aged < 55 years with high-risk karyotype, FLT3-ITD or adverse clinical features (secondary AML, CR after second course, hyperleukocytosis) were assigned to undergo allogeneic stem cell transplantation (SCT) from matched related or unrelated donor. Patients with intermediate cytogenetic risk in the absence of FLT3-ITD and adverse clinical features were allocated to allogeneic SCT if a related donor was available. Autologous SCT was offered to patients aged < 61 y with low-risk cytogenetics, intermediate-risk cytogenetics without sibling donor and high-risk disease not eligible to allogeneic SCT. Peripheral blood (PB) stem cells for autologous SCT were collected after a DIA course (Cytarabine 500 mg/sqm bid on days 1-6; Daunorubicin 50 mg/sqm on days 4-6). Patients who failed mobilization received two additional courses with high dose cytarabine.
Protocol-2: since April 2007 to April 2014, patients were treated according to Northern Italy Leukemia Group (NILG) AML 02-06 protocol. Until March 2012, patients were recruited within the NILG AML 02/06 trial [(ClinicalTrials.gov Identifier: NCT00495287] [1]. From April 2012, after closure of NILG AML 02/06 trial, patients were treated according to the standard arm provided by the protocol. The protocol provided a randomization at induction between a standard ICE induction versus an experimental intensified one. Patients aged > 65 y were treated according to standard arm. Upon CR achievement, patients received standard doses cytarabine consolidation and were divided into standard and high risk cases (SR, HR): SR: favorable or intermediate risk cytogenetics (according to SWOG criteria) without any adverse clinical factor (secondary AML, FLT3-ITD, CR after cycle 2, persistence of pre-existing cytogenetic abnormality despite morphological CR; total WBC count >50 x10 9 /L); HR: all non-SR cases. HR patients were assigned to undergo allogeneic SCT. Provided sufficient CD34+ cells were previously collected (>2x10 6 /kg) upon recovery from high doses cytarabine, SR patients and HR patients excluded from allo-SCT and aged 65 years or less were randomized between autologous SCT and high doses consolidation therapy (R2). HR/SR patients unable to be randomized in R2 because of inadequate blood stem cell yield received intermediatedose consolidation. Patients randomized to experimental arm were excluded from outcome analysis.
Protocol-3: since May 2014 to April 2017, patients received induction according to Ida-FLA course, (Cytarabine 2000 mg/sqm on days 1-4; Fludarabine 30 mg/sqm on days 1-4; Idarubicin 10 mg/sqm on days 2-4). High-dose Cytarabine (3000 mg/sqm bid days 1, 3, 5) was used as first consolidation in patients aged < 61 years attaining complete remission (CR). Patients with persistent disease (i.e. > 5% BM blasts at hematopoietic recovery) after first course received a salvage regimen (Clofarabine-based). In post CR phase, patients were stratified according to European Leukemia Net 2010 guidelines [2]. Patients in adverse-risk category were allocated to allogeneic HSCT from matched related or unrelated donor. Patients in intermediate category were allocated to allogeneic SCT if a related donor was available. Patients in favorable-risk ELN category and high-risk disease not eligible to allogeneic SCT received up to two additional courses with high dose cytarabine.

S2. Assessment of Multi-Lineage Dysplasia by Multiparameter Flow Cytometry
A minimum of 250,000 cells per tube was acquired. Instrument setup, calibration and quality control were performed in order to assure measures' stability [3]. For data analysis, Infinicyt (Cytognos SL, Salamanca, Spain) software was used. Some major BM cell compartments were identified on the basis of forward (FSC) and sideward (SSC) light scatter characteristics and their reactivity for CD45 as in the Figure below: These subsets were: (i) blasts; (ii) maturing neutrophil compartment; (iii) mature monocytic compartment; (iv) mature erythroid compartment. In order to estimate dysplasia, we considered neutrophil and erythroid compartments only; monocytic compartment was excluded from IPS because often clonally involved in AML and thus inadequate to assess MLD. As for neutrophil compartment, it was identified on the basis of CD45 dim expression with high SSC signal. Consistently with WHO criteria for morphological dysplasia, the analysis was then focused on the more mature stage, by gating cells co-expressing CD13 and CD11b or expressing CD16. The expression of CD65 and cyMPO were evaluated on whole granulocytic compartment. As for erythroid compartment, it was selected by negativity for CD45 and CD34 with low SSC signal. With respect to MDS-related approach [4]. CD235a was substituted by CD105. Dysplasia was appraised for neutrophil and erythroid compartments through an immuno-phenotypic score (IPS) including 17 parameters (13 for neutrophil and 4 for erythroid compartment) as in the Cell compartments were considered not assessable for dysplasia when not detectable as at least 0.01% of total BM cells. Parameters included by IPS were expressed as percentage of positive cells for an antigen within a cell compartment and/or its mean fluorescence intensity (MFI; arbitrary relative linear units, scaled from 0 to 10 4 , normalized upon a control). BM samples from healthy donors were used to define normal phenotypic profile as the interval between mean value ± two standard deviations (SD) for each parameter. A score was calculated for each parameter: 0.5, 1 or 2 was assigned when the value was between the mean ± 2 SD and the mean ± 3 SD, between the mean ± 3 SD and the mean ± 4 SD and, over the mean ± 4 SD of the normal profile, respectively.