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Leukemia Research 40 (2016) 1–9

Contents lists available at ScienceDirect

Leukemia Research

journa l h om epage: www.elsev ier .com/ locate / leukres

inimal residual disease evaluation by flow cytometry is aomplementary tool to cytogenetics for treatment decisions in acuteyeloid leukaemia

aría-Belén Vidrialesa,b, Estefanía Pérez-Lópeza,∗, Carlota Pegenautea,arta Castellanosa, José-Juan Péreza, Mauricio Chandíaa, Joaquín Díaz-Mediavillac,

onsuelo Rayónd, Natalia de las Herase, Pascual Fernández-Abellánf, Miguel Cabezudog,lfonso García de Cocah, Jose Ma Alonso i, Carmen Olivier j, Jesús Ma Hernández-Rivasa,b,au Montesinosk, Rosa Fernández l, Julio García- Suárezm, Magdalena Garcían,aría-José Sayaso, Bruno Paivaa,b, Marcos Gonzáleza,b, Alberto Orfaob,p,

esús F. San Miguela,b, For PETHEMA Programa para el Estudio de la Terapéutica enemopatías Malignas Cooperative Study Group

Department of Hematology, Hospital Universitario de Salamanca, Instituto de Investigación Biomédica de Salamanca (IBSAL), SpainInstituto de Biología Molecular y Celular del Cáncer (CIC-CSIC), Salamanca, SpainDepartment of Hematology, Hospital Universitario de San Carlos, Madrid, SpainDepartment of Hematology, Hospital Central de Asturias, Oviedo, SpainDepartment of Hematology, Complejo Hospitalario de León, León, SpainDepartment of Hematology, Hospital Universitario Alicante, Alicante, SpainDepartment of Hematology, Complejo Asistencial de Ávila, Ávila, SpainDepartment of Hematology, Hospital Clínico Universitario, Valladolid, SpainDepartment of Hematology, Hospital Río Carrión, Palencia, SpainDepartment of Hematology, Complejo Asistencial, Segovia, SpainDepartment of Hematology, Hospital Universitario La Fe, Valencia, SpainDepartment of Hematology, Hospital Materno Insular, Las Palmas de Gran Canaria, SpainDepartment of Hematology, Hospital Universitario Príncipe de Asturias, Alcalá de Henares, SpainDepartment of Hematology, Hospital Virgen de la Concha, Zamora, SpainDepartment of Hematology, Hospital Universitario Dr. Peset, Valencia, SpainDepartment of Cytometry, Universidad de Salamanca, Spain

r t i c l e i n f o

rticle history:eceived 8 July 2015eceived in revised form 7 September 2015ccepted 7 October 2015vailable online 22 October 2015

eywords:low cytometryinimal residual disease

cute myeloid leukaemia

a b s t r a c t

The clinical utility of minimal residual disease (MRD) analysis in acute myeloid leukaemia (AML) is not yetdefined. We analysed the prognostic impact of MRD level at complete remision after induction therapyusing multiparameter flow cytometry in 306 non-APL AML patients. First, we validated the prognosticvalue of MRD-thresholds we have previously proposed (≥0.1%; ≥0.01–0.1%; and <0.01), with a 5-yearRFS of 38%, 50% and 71%, respectively (p = 0.002). Cytogenetics is the most relevant prognosis factor inAML, however intermediate risk cytogenetics represent a grey zone that require other biomarkers forrisk stratification, and we show that MRD evaluation discriminate three prognostic subgroups (p = 0.03).Also, MRD assessments yielded relevant information on favourable and adverse cytogenetics, sincepatients with favourable cytogenetics and high MRD levels have poor prognosis and patients with adverse

rognosis cytogenetics but undetectable MRD overcomes the adverse prognosis. Interestingly, in patients withintermediate or high MRD levels, intensification with transplant improved the outcome as comparedwith chemotherapy, while the type of intensification therapy did not influenced the outcome of patientswith low MRD levels. MultivariMoreover, a scoring system, ea

∗ Corresponding author at: Department of Hematology, Hospital Universitario de SalamE-mail address: estefipe83@hotmail.com (E. Pérez-López).

ttp://dx.doi.org/10.1016/j.leukres.2015.10.002145-2126/© 2015 Elsevier Ltd. All rights reserved.

ate analysis revealed age, MRD and cytogenetics as independent variables.

sy in clinical practice, was generated based on MRD level and cytogenetics.

© 2015 Elsevier Ltd. All rights reserved.

anca, Paseo de San Vicente 58-182, Salamanca 37007, Spain. Fax: +34 23 29 46 24.

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. Introduction

Currently, the two main criteria for risk-adapted treatmentn acute myeloid leukaemia (AML) are the presence of adverseytogenetic or molecular features, and the response to induc-ion treatment assessed by morphology. In the case of the latter,nly failure to respond is truly informative, since relapses fre-uently occur among patients who achieve morphological CRmCR). Therefore, more sensitive techniques are needed to evaluatehe response, aimed to identify small number of residual leukemicells that are undetectable by conventional morphology and haveeen called “minimal residual disease” (MRD).

At present, MRD detection in AML is based on molecular (RQ-CR) and multiparameter flow cytometry (MFC) techniques. Theatter relies on the presence of aberrant phenotypes in leukemiclast cells (LAP; “leukaemia-associated phenotype”) at diagnosis,hich are either absent or present at very low frequencies inormal bone marrow (BM). Although molecular techniques areore sensitive, the applicability of MFC is higher. Several groups

ave reported the clinical utility of the MRD analysis in acuteymphoblastic leukaemia (ALL) for defining risk-adapted treat-

ent protocols [1–5]. By contrast, in AML the information is stillcarce, and MRD status is not considered routinely in clinical set-ings to decide consolidation therapy. This is probably because themmunological characterisation of myeloid leukemias is techni-ally more challenging, since it requires the use of large panels ofoAbs to cover different myeloid lineages and the multiple AML

last cell populations frequently coexisting at diagnosis. The sensi-ivity of flow-MRD in AML ranges between 10−3 and 10−4, withn applicability of >80% when 4 colours are applied [6–10], orven 100% if a very large panel of monoclonal antibodies (MoAbs)11] is employed, or if more than 4 markers are simultaneouslysed [12–16]. Although phenotypic changes are frequent at relapse,everal studies have demonstrated that at least one LAP remainsonstant in 75–100% of cases [17–19]. These data have been vali-ated in the clinical setting, showing the prognostic value of MRDnalysis using MFC [6–11,15,20–33].

The potential value of MRD detection within different cytoge-etic subgroups in AML has not yet been fully defined. This raiseshe question of whether a negative MRD result could counter-alance the adverse effect of poor-risk cytogenetics, or, to put itnother way, whether high MRD levels after induction modify theutcome of patients with otherwise favourable features (mCR andood or intermediate cytogenetics). In addition, it is not known forertain whether the modality of intensification therapy (high-dosehemotherapy, autologous or allogeneic transplantation) modifieshe influence of the level of MRD assessed after induction therapy.

In the present study we analysed the prognostic impact of MRDevel on the bone marrow (BM) at the time of mCR achievementsing MFC in a series of 306 non-APL AML patients. Our resultshow that early immunophenotypic evaluation of MRD identi-es different patient risk-group categories and may contributeo post-induction treatment stratification. Interestingly, the flow-

RD analysis is valuable for each of the cytogenetic risk groups andodalities of intensification therapy.

. Material and methods

.1. Patients

Three-hundred and six patients were included in this study,

ll of whom fulfilled the following eligibility criteria: (1)nequivocal de novo non-APL AML diagnosis based on mor-hological, cytochemical and immunophenotypic criteria; (2)resence of immunophenotypic aberrancies detected in blast

Research 40 (2016) 1–9

cells at diagnosis, suitable for MRD monitoring during follow-up[6–9,11,24,27,34,35]; (3) achievement of morphological completeremission (mCR) after induction therapy [36] being candidates forintention-to-cure therapy, and (4) availability of the correspondingmCR BM sample for flow-MRD investigation. From the total seriesof patients in which the multiparameter FC study was available atdiagnosis and at the time of mCR (n = 352), we detected an aberrantimmunophenotype in 87% of cases (n = 306), these patients com-prising the body of the present study. Eighty-two of the 306 patientshad been included in a previously published series [7]. Of these306 patients, 154 were males and 152 females, with a mean age of47 ± 16 years (median 48 years), and only 39 patients were olderthan 65 years. The distribution of the sample by FAB class was asfollows: MO, 9%; M1, 27%; M2, 25%; M4, 19%; M5, 17%; M6, 1%; M7,1%; unclassifiable, 0.8%. All patients were uniformly treated accord-ing to the Spanish Pethema Cooperative Group’s 1996 and 1999protocols. Remission-induction therapy included one or two 3/7courses of anthracycline (12 mg/m2 of idarubicin) and cytosine ara-binoside (ARA-C) (200 mg/m2); 248 cases (80%) achieved mCR afterone induction cycle, 49 cases (16%) needed two courses to achievemCR, while the remaining 9 patients (4%) achieved mCR after res-cue chemotherapy (Cheson criteria [36], were used to define mCR).Subsequently, all patients received one identical cycle. Accordingwith the design of the study, the time point for the MRD evaluationby flow cytometry was at the achievement of morphologycal CR.Afterwards, 83 patients underwent allogeneic transplantation, 118patients received one intensification cycle (ARA-C 3 g/m2/12 h fora total of 8 doses plus idarubicin 12 mg/m2 for 3 days) plus high-dose therapy (BEA regimen: busulfan 0,8 mg/kg/6 h iv on days −8to −5, etoposide 20 mg/kg/day i.v. on days −4 and −3, and cytosinearabinoside 3 g/m2/12 h i.v. on days −3 and −2) with autologousstem cell rescue, and 105 patients received one or two courses ofintensification therapy ARA-C 3 g/m2/12 h for a total of 8 doses plusidarubicin 12 mg/m2 for 3 days. The mean follow-up of the totalseries was 64 months (5 years).

Relevant data concerning disease characteristics at diagnosisand during follow-up were collected and included in the databasefor further analysis. These included age, white blood cell (WBC)and platelet counts, hemoglobin (Hb) levels, percentage of blastcells in BM and absolute number in peripheral blood (PB), numberof cycles to achieve morphological CR, type of treatment, morpho-logical FAB classification and cytogenetics. Karyotypic informationwas available for 258 patients, and was classified according to inter-national criteria [37,38]; as favourable (n = 30; 12%); intermediate(n = 187; 72%) or adverse (n = 41; 16%). In the rest of the patients(n = 48; 15%) no mitosis were obtained for cytogenetics evaluation.No differences were founded in the major clinical characteristicsbetween patients with or without available cytogenetic informa-tion (Table 1).

2.2. Immunophenotypic investigation of MRD by multiparameterflow cytometry

Erythrocyte-lysed whole BM samples obtained at diagnosiswere analysed with a large panel of four-colour combinations ofMoAbs to identify phenotypic aberrancies that could potentially beused, later on, as “custom-built” probes for detecting residual blastcells displaying the same phenotypic profile, in morphological CRBM samples obtained following induction therapy [6–9,23,24,27].Antigenic expression in blast cells at diagnosis was systemati-cally analysed by multiparametric flow cytometry (FACScalibur,Becton/Dickinson, Biosciences San Jose, CA, USA) according to

previously described methods using [1,6,7,17]; quadruple-stainingwith the following fluorochrome-conjugated (FITC, PE, PerCP-Cy5,APC) combinations of MoAbs, aimed at defining leukaemic pheno-types that are absent from or extremely infrequent in normal BM

M.-B. Vidriales et al. / Leukemia Research 40 (2016) 1–9 3

Table 1Main clinical characteristics of patients with or without available cytogenetic information.

No cytogenetics information(n = 48) Cytogenetics information(n = 258)

AML subtypes (FAB)M0 5 (11%) 20 (8%)M1 11 (23%) 73 (27%)M2 6 (13%) 68 (24%)M4 11 (23%) 46 (18%)M5 14 (28%) 45 (17%)M6 0 (0%) 3 (3%)M7 1 (2%) 3 (3%)

Blast cell in BM (%) 78% ± 17 73% ± 21

Age>65 years 9 (8%) 30 (12%)≤65 years 39 (92%) 228 (88%)

WBC count≥100 × 109/L 8 (17%) 34 (13%)<100 × 109/L 40 (83%) 224 (87%)

mCRAfter 1 cycle 42 (87%) 206 (80%)After ≥ 2 cycles 6 (13%) 52 (20%)

Intensification therapyChemotherapy 23 (47%) 82 (32%)Autologous transplantation 17 (36%) 101 (39%)Allogeneic transplantation 8 (17%) 75 (29%)

No statistical differences were founded between patients with or without available cytogenetic information.

F n = 29

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ig. 1. Impact of MRD levels in the whole cohort of 306 patients, for cases with low (

amples: cyCD79a/cyMPO/CD45/CD34; CD7/cCD3/CD45/CD34;TdT/CD19/CD45/CD34; HLA-DR/CD33/CD45/CD34;D15/CD33/CD45/CD34; CD11b/CD13/CD45/CD34;D15/CD117/CD45/CD34; CD36/CD64/CD45/CD34 + CD14;D61/GlycophorinA/CD45/CD34; CD65/7.1/ CD45/CD34;D71/CD56/CD45/CD34.

To investigate MRD in mCR BM samples obtained after induc-ion treatment, MoAb combinations aimed at identifying aberrant

henotypes, which had already been designed by the time ofiagnosis, were used in a two-step data acquisition procedure.

n the first step, 20,000 events corresponding to overall bonearrow cellularity were acquired; in the second, blast cells

), intermediate (n = 129) and high (n = 148) MRD levels at the time of attaining mCR.

were acquired through an SSC/marker live gate, defined accord-ing to the immunophenotype, and information was collectedfrom at least 106 BM nucleated cells. Data analysis was basedon identifying cells with the same or similar aberrant phe-notypic features to those identified in the leukemic cells atdiagnosis.

The LYSIS II and Cell Quest programs (Becton/Dickinson)were used for data acquisition. The PAINT-A-GATE PRO (Bec-

ton/Dickinson) and Infinicyt (Cytognos) program with the poly-nomial SSC transformation capability was used for further dataanalysis. In all cases the information was prospectively analysedand recorded for further examination.

4 M.-B. Vidriales et al. / Leukemia Research 40 (2016) 1–9

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ig. 2. Impact of MRD levels at the time of attaining mCR with respect to cytogenedverse (C) (n = 41) cytogenetic risk groups.

.3. Statistical analysis

The Chi square and Mann–Whitney U tests were used to esti-ate the statistical significance of differences between groups.

urvival curves were plotted according to the Kaplan–Meierethod and curves were compared using the log-rank and Bres-

ow tests. In the univariate analysis of relapse-free survival (RFS),he following variables were tested: age, WBC, platelet count, pro-ortion of blast cells in BM and absolute number of blasts in PB,umber of cycles to achieve CR (one vs. two courses), type of treat-ent, FAB classification, cytogenetics and MRD level at the end of

nduction therapy. Subsequently, a stepwise multivariate regres-ion was performed to explore the independent effect of variableshat showed a significant influence on disease-free survival in thenivariate analysis. Statistical analyses were performed using SPSSersion 18.0 (SPSS Inc., IL USA)

. Results

.1. Validation of previous cut-off MRD levels

We have previously reported in a series of 83 non-APL AMLatients that the levels of residual cells with leukaemia-associatedhenotype (LAP) at the time of attaining mCR provide prognostic

nformation (7). In the present paper, based on a series of 306 deovo non-APL AML patients, we confirmed the prognostic influencef the previously defined MRD threshold levels: 148 cases were inhe high-risk category (≥0.1% LAP + cells) and had a 5-year RFS of8%; 129 cases were in the intermediate-risk category (≥0.01–0.1%AP + cells), with a 5-year RFS of 50%; and the other 29 patients weren the low-risk group (<0.01% LAP + cells) with a RFS at 5-years of1% (p = 0.002) (Fig. 1). Moreover, the higher the level of MRD (pro-ressive thresholds of 0.01%, 0.03%, 0.1%, 0.3% and 0.5%), the shorterere the RFS observed.

.2. Impact of flow-MRD levels at the time of mCR attainment

ccording to cytogenetics

Cytogenetic information was available from 258 of the 306atients, and they were classified into the favourable (n = 30; 12%),

ve-year RFS in patients with favourable (A) (n = 30), intermediate (B) (n = 187), and

intermediate (n = 187; 72%), or adverse/poor (n = 41; 16%) cytoge-netic risk categories (37, 38).

Among patients (n = 187) classified as having intermediatecytogenetics, MRD levels discriminated three risk categories, witha 5-year RFS of 38%, 50% and 70% for patients with high (n = 86),intermediate (n = 83), and low MRD levels (n = 18) (p = 0.03)(Fig. 2B). Interestingly, upon analysing the outcome of patientswith intermediate and high MRD levels (n = 169) according to thetype of intensification received, we observed a clear advantage(p < 0.001) for allogeneic transplanted patients (n = 48; 5-yearRFS of 75%), followed by autologous transplantation (n = 69;5-year RFS of 43%), with poor results for chemotherapy (n = 52;5-year RFS of 20%). Moreover, when only patients with highMRD levels (n = 86) were considered, those receiving autologoustransplantation (n = 35) and chemotherapy (n = 35) had similarlyvery poor results (5-year RFS of 35% and 30%), and only allogeneictransplantation (n = 16) offered a favourable outcome for thesepatients, with a 5- year RFS of 66% (p = 0.06). These results sug-gest that the allogeneic transplantation would be the preferredoption for patients with intermediate risk cytogenetics but withpersistent high MRD levels after induction therapy. Moreover, inpatients with intermediate MRD levels (≥0.01–0.1% LAP + cells)intensification with conventional chemotherapy should bediscouraged.

Upon considering only patients with favourable cytogenetics(n = 30), MRD levels showed a tendency to discriminate three riskcategories (5-year RFS of 57%, 78% and 100% for patients withhigh (n = 14), intermediate (n = 19), and low MRD levels (n = 4),respectively), although the differences did not reach statistical sig-nificance probably due to the small number of cases (Fig. 2A).A more detailed analysis of the critical group of the 14 patientswith a favourable karyotype and high MRD levels showed thatsix of them had a high WBC count (>50 × 109/l) at diagnosis andone required a second induction cycle to achieve mCR. These14 patients were consolidated with chemotherapy (n = 7), autol-ogous (n = 6) and allogeneic (n = 1) transplantation, the outcomebeing better for patients who underwent transplantation (the allo-transplanted patient was relapse-free, and the 5-year RFS for the

autologous transplantation was 67%) compared with that of thepatients who were consolidated with chemotherapy (5-year RFS of43%). Although the number of cases is small, these results argue that

M.-B. Vidriales et al. / Leukemia Research 40 (2016) 1–9 5

F sificato ct of tw the ti

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ig. 3. First line: Impact of MRD levels at the time of attaining mCR by type of intenr allogeneic (B) (n = 83) transplant or chemotherapy (C) (n = 105). Second line: Impaith low (D) (n = 29), intermediate (E) (n = 129) and high (F) (n = 148) MRD levels at

atients with favourable cytogenetics but high MRD levels shouldndergo stem cell transplantation.

Finally, of the patients with adverse cytogenetics (n = 41), onlyases with no detectable level of MRD (n = 5) displayed a good out-ome (no relapses to date), while results were very poor for patientsith intermediate (n = 17) or high (n = 19) MRD levels, with a 5-yearFS of 20% in both groups (p = 0.02) (Fig. 2C). Most patients withdverse cytogenetics received an allogeneic (49%) or autologous

29%) transplant; we would like to note that there were not sig-ificant differences in the distribution of intensification receivedhrough the different MRD levels groups, with 49% of patientseceiving allogeneic and 29% autologous transplant.

ion therapy (A–C): 5-year RFS in patients who underwent autologous (A) (n = 118),ype of intensification therapy in each MRD level group (D–F): 5-year RFS in patientsme of attaining mCR.

3.3. Impact of flow-MRD levels at the time of mCR attainment byintensification treatment type

Among patients who underwent intensification with autologoustransplantation (n = 118) the MRD levels evaluated at the time ofmCR allowed the identification of three risk groups, with 5-yearRFS of 40%, 57% and 83% for patients with high (n = 10), intermedi-ate (n = 53) and low (n = 55) MRD, respectively (p = 0.02) (Fig. 3A–C).

Upon analysing patients intensified with allogeneic transplantation(n = 83), MRD levels also discriminated three subgroups of patientswith 5-year RFS of 53%, 62%, and 75% for cases with high (n = 27),intermediate (n = 49) and low (n = 7) MRD levels, although the dif-

6 M.-B. Vidriales et al. / Leukemia Research 40 (2016) 1–9

Fig. 4. Impact of MRD levels at the time of mCR plus cytogenetic risk groups on 5-year RFS, defining a new scoring system. Scores of 0, 1 and 2 were respectively assigned tothe low-, intermediate- and high-risk groups for each variable (MRD level and cytogenetics), generating 5 risk groups, with scores between 0 and 4.

Table 2Variables with significant impact on RFS (Relapse Free Survival) in the univariate and multivariate analyses.

Univariate analysis Multivariate analysis

5-year RFS p p HR 95% CI

MRD level at mCR 0.002 <0.001 1.8 1.3–2.5≥0.1% LAP + cells (n = 148) 38%≥0.01–0.1% LAP + cells (n = 129) 50%<0.01 LAP + cells (n = 29) 71%

Cytogenetics 0.001 0.001 1.98 1.34–2.92Adverse (n = 41) 30%Intermediate (n = 187) 46%Favourable (n = 30) 68%

Age 0.004 0.010 1.02 1–1.03>60 years (n = 84) 31%≤60 years (n = 222) 52%

WBC count 0.009 NS≥100 × 109/L (n = 37) 30%<100 × 109/L (n = 269) 49%

mCR 0.030 NSAfter 1 cycle (n = 247) 49%After ≥ 2 cycles (n = 59) 35%

Intensification therapy NSChemotherapy (n = 105) 30% 0.001Autologous transplantation (n = 118) 50%

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Allogeneic transplantation (n = 83) 61%

RD—minimal residual disease, LAP—leukaemia-associated phenotype, mCR—mor

erences did not reach statistical significance. Finally, among thoseatients who received chemotherapy alone as intensification treat-ent (n = 105), only cases that achieved low MRD (n = 12) had an

cceptable outcome, with a 5-year RFS of 65%, whereas those withntermediate (n = 27) or high (n = 66) MRD levels had a very poor

utcome, with 5-year RFS rates of only 11% and 28% (p = 0.020or the comparison of low vs. intermediate and high MRD levels).ig. 3D–F shows that, in spite of the type of intensification therapysed, the prognosis is poorer when the MRD level is higher; more-

gical complete remission, NS—non- significant.

over, allogeneic transplantation is the best option for patients withintermediate (Fig. 3E) and high (Fig. 3F) MRD levels at the time ofattaining morphological CR.

3.4. Other factors associated with RFS in univariate and

multivariate analyses

In addition to MRD levels, five other disease features had a sig-nificant prognostic association with the RFS (Table 2): cytogenetic

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lassification; age; WBC count; number of cycles of chemother-py required to achieve mCR; and intensification treatment typechemotherapy, and allogeneic or autologous transplantation).

The Cox regression analysis, performed in a cohort of 246atients, revealed an independent prognostic value for MRD levelst the time of mCR, whether considered as a continuous (p = 0.03)r categorical variable (3 risk groups: p < 0.001; HR = 1.8; 95%I = 1.3–2.5), together with the cytogenetic classification (p = 0.001;R = 1.98; 95% CI = 1.34–2.92), and patient age (continuous vari-ble: p = 0.002; HR = 1.02; 95% CI = 1.00–1.03). In addition, whennly patients younger than 65 years were included in the multivari-te analysis (n = 214), flow-MRD and cytogenetics again emerged asndependently significant variables.

Finally, using the two age-independent categorical variableselected in the multivariate model (MRD and cytogenetics), a scor-ng system was created that assigned 0 points if the variable fell inhe low-risk category (low MRD levels or favourable cytogenetics),

point for intermediate levels of MRD or medium-risk cytogenet-cs, and 2 points for high MRD levels or high-risk cytogenetics. Basedn these scores, five significantly different AML risk groups coulde defined for scores of 0 to 4. These groups had 5-year RFS of 100%n = 4), 70% (n = 30), 54% (n = 102), 35% (n = 103) and 19% (n = 19),espectively (p < 0.001) (Fig. 4).

. Discussion

At present both molecular and cytogenetic information guidehe treatment decision process in AML [37–39]. In addition, MRDssessment by MFC at the time of mCR could contribute to guidentensification therapy [7,10,27,29–31,35,40,41]. Our study basedn a large series of AML patients illustrates the independent valuef cytogenetics and MRD as the two most relevant prognosis factorsn AML. Unfortunately molecular information was only available in

small fraction of patients that precluded further analysis. Of noteur study highlighted the value of MRD investigations in patientsith intermediate risk cytogenetic, a grey zone in which other risk

tratification tools are needed.Several groups, including our own, have shown the

rognostic value of MFC for MRD investigation in AML4,6–11,15,20,23,25–28,30–33,40,41]. However, the informa-ion provide by flow MRD analysis has not been incorporated inoutine clinical practice to decide consolidation therapy, with onlyne study already published, in children population, in which thisnformation guides treatment (Rubnitz et al. [27]). This could beue, almost in part, to the facts that flow MRD in AML is technicallyhallenging, different threshold levels have been proposed forisk stratification, it is not well established if MRD is independentrom cytogenetics, and it has not yet been defined whether the

odality of intensification therapy (chemotherapy or transplant)odifies the influence of the MRD level assessed after induction

herapy. For this reason, our primary goal was to validate the MRDhreshold values used in our previous report [7], in a large seriesf patients, and we also decided to focus on the BM obtained inCR after induction because we considered this to be the criticaloment in AML for the treatment decision-making process, in lineith reports from other groups that also focus in the BM obtained

fter induction therapy [6,7,9,11,20,22–24,27,29–31,35,40,41].The present study has enabled us to confirm our previous MRD

hreshold values (0.1%; 0.01%) in a large series of 306 AML patients,nd indicate that these cut-off levels can be used for routine risktratification in AML.

As far as the independent value of MRD information fromytogenetics is concerned, Buccisano et al. [10] showed that thessessment of BM MRD levels by MFC after consolidation therapyogether with cytogenetic information at diagnosis provided better

Research 40 (2016) 1–9 7

discrimination between patients with different risk of relapse thanwhen cytogenetics alone was used. These results are concordantwith Freeman et al. [30], that showed early assessment of MDR asan independent prognostic factor in a series of older AML patients,and with Walter et al. [28] that shows the impact of flow MRDanalysis in the intermediate-risk cytogenetic group in the trans-plantation settings. Recently, Köhnke et al. [40] also showed thevalue of flow MRD during aplasia after induction therapy. How-ever, others Langebrake et al. [24] have suggested that althoughflow-MRD in AML enables a more precise evaluation of the qualityof CR, it does not provide additional prognostic information to thatobtained from cytogenetics. In the present study, both variableswere independent predictors of RFS, and although MRD assessmentwas particularly informative in the intermediate cytogenetic group,it also yielded valuable information in the other cytogenetic riskgroups. Although the favourable and adverse cytogenetics groupsare smaller in the present series, our results support that patientswith favourable cytogenetics who maintain high MRD levels at mCRhave a poor outcome and should be considered candidates for moreintensive post- consolidation approaches such as transplantation.Conversely, the small group of patients with adverse cytogenet-ics who achieve a negative MRD appear to do as well as patientswith standard risk cytogenetics, suggesting that the achievementof undetectable MRD levels may overcome the adverse impact ofcytogenetics.

It is nor well established whether the MRD level has the samevalue for different types of intensification therapy (chemother-apy or transplantation). Maurillo et al. [26] have shown, in a shortseries of patients, that MRD investigation yields prognostic infor-mation for both patients intensified with allogeneic or autologoustransplantation. In the present series, the prognostic value of MRDwas maintained for each of the three different strategies used asintensification treatment (chemotherapy, autologous or allogeneictransplantation). It is important to emphasize those patients withhigh or intermediate MRD levels only had an acceptable outcomewhen they received an allogeneic transplant, while the RFS wasvery short when solely chemotherapy was used. Based on theseobservations it appears that allogeneic transplantation could atleast partially overcome the adverse impact of a high MRD levelat the time mCR is achieved, and should be the primary treatmentoption for this group of patients.

Finally, it should be emphasize that MRD assessment by mul-tiparametric flow will significantly improve in the coming yearsboth through new strategies for data analysis and interpretation[42–45].

In summary the combined use of MRD at the time of mCRand baseline cytogenetics information allowed to define a simplescoring system that could help not only to decide on the intensifica-tion strategies but also in the design of experimental therapeuticsoptions.

5. Conclusions

Our results show that MFC performed in the BM at the achieve-ment of mCR is a valuable tool in the prognostic evaluation of AMLpatients. Interestingly, MFC keeps its value through the differentcytogenetic prognostic groups, as well as in different consolidationapproaches, being of special interest in the intermediate cytoge-netic group that represents a grey zone of prognostic in AML. Inaddition, allogeneic transplantation could overcome the adverse

impact of having a high MRD level at the mCR achievement,whereas autologous transplantation and chemotherapy should beconsidered as an under-treatment approach for patients with highMRD levels. Taken together, these results suggest that MRD inves-

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igation by MFC should be used together with cytogenetics for therognostic stratification and therapeutic decisions in AML patients.

ontributors

María-Belén Vidriales and Estefanía Pérez-López contributedqually to this work.

JFSM and AO conceived the idea, and together with MBV andDM, designed the study protocol. MBV, EPL and JJP analyze the flowytometry data. EPL and MBV analysed and interpreted data ando-wrote the paper, together with JFSM. The paper was reviewednd corrected by JFSM and AO. EPL, CP, MC and MC were respons-ble of data bases clinical cases and follow-up of patients. JDM, CR,H, PFA, MC, AGC, JMA, CO, JMHR, MG, GM, RF, JGS, MG, and MJS,ere responsible of clinical cases and follow-up of patients, and

ontributed to the clinical data entry.

cknowledgements

This work was supported in part by Spanish grants from Fondoe Investigación Sanitaria-ISCIII (FIS 00/0023-03, PI12/02321),GCYT (SAF 94- 0308, SAF2001-1687), Conserjería de Educacióne Castilla y León (HUS416A12), and Red Temática de Investigaciónooperativa en Cáncer (RTICC-ISCIII) (RD12/0036/0069).

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