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Leukemia Research
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omparison between multiparameter flow cytometry and WT1-RNAuantification in monitoring minimal residual disease in acute myeloid leukemiaithout specific molecular targets
iovanni Rossia,∗, Maria Marta Minervinia, Angelo Michele Carellaa, Chiara de Waureb,rancesco di Nardob, Lorella Melilloa, Giovanni D’Arenaa, Gina Zini c, Nicola Cascavillaa
Department of Hematology and Stem Cell Unit, IRCCS “Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo, ItalyDepartment of Hygiene, Catholic University of Sacred Heart, Rome, ItalyDepartment of Hematology and Laboratory, Center of Research ReCAMH, Catholic University of Sacred Heart, Rome, Italy
r t i c l e i n f o
rticle history:eceived 6 October 2011eceived in revised form0 November 2011ccepted 27 November 2011vailable online 21 December 2011
eywords:
a b s t r a c t
Despite a high remission rate, a significant number of patients with acute myeloid leukemia (AML) relapse.Thus, the evaluation of minimal residual disease (MRD) in AML is an important strategy to better identifyhigh risk patients. Most sensitive methodology to detect MRD is molecular polymerase chain reaction(PCR) but its applicability is restricted to AML with leukemia-specific molecular targets (e.g. AML1-ETO,CBFB-MYH11, MLL, FLT-3). In our study, MRD was monitored at different time points with both MFCand WT1-RNA quantification in 23 AML patients who did not present specific molecular targets. As pre-viously published, we considered values of 10−3 (0.1%) in MFC and 90 WT1-RNA ×104 ABL copies as
cute myeloid leukemiainimal residual disease
low-cytometryeukemia-associated immunophenotypes
T1-RNA
optimal thresholds. Receiver operating characteristics (ROC) analysis was used to confirm these data. Torealize the methodology that better identify high risk patients, an analysis of sensitivity, specificity, pre-dictive values (PV) and likelihood ratio (LR) was provided and similar results were showed. MRD levels≥10−3 in MFC as well MRD levels ≥90 WT1-RNA copies in RQ-PCR, identify risk groups of patients withpoor prognosis. Therefore, MFC and WT1-RNA quantification showed a comparable capacity in terms oftechnical performance and clinical significance to identify high risk patients who eventually relapsed.
. Introduction
Monitoring of the minimal residual disease (MRD) in acuteyeloid leukemia (AML) has become essential to predict progno-
is and to select the type of post-remission treatment [1]. Severaltudies have shown how multiparameter flow-cytometry (MFC)2,3] polymerase chain reaction (PCR) [4,5] and fluorescence in situybridization (FISH) [6] are useful for detection of MRD in AMLatients. PCR-based quantification of MRD has a high sensitiv-
ty but its applicability is restricted to subgroups of AML witheukemia-specific molecular targets (e.g. AML1-ETO, CBFb-MYH11,
LL, FLT3, NPM1) [7]. These cases comprise only 40–50% of all AMLases [8]. For this reason, several investigators studied WT1 expres-
ion as alternative marker of leukemic cells and tested it for MRDetection in AML patients [9–12]. However the results concern-
ng its use as follow-up marker during post-remission have been
∗ Corresponding author at: Department of Hematology and Stem Cell Unit, IRCCSCasa Sollievo della Sofferenza” Hospital, v.le Cappuccini 1, 71013 San Giovanniotondo, Italy. Tel.: +39 3922323949; fax: +39 3922323949.
conflicting. Some groups reported a good association between theclinical course and WT1 levels [13,14] while others failed to show[15,16]. This discrepancy may be due to differences in methodologyin measuring WT1 and in patients populations or in the intensity ofthe regimens used. On the other hand, using an accurate combina-tion of monoclonal antibodies in the MFC allows specific detectionof leukemic cells. This approach may be applicable to a percent-age of AML patients that ranges from 80% to 94% [17–20] andshows a sensitivity better than 1 leukemic cell per 104 to 105 nor-mal cells [8]. However, studies differ in the threshold use for MRDdetection and in the timing of testing. A strong association withclinical outcome was found at the presence of both 10−4 [2,20] and10−3 Leukemia-associated immunophenothypes (LAIPs) [3,21–24]positive cells and it is still unclear whether post-induction or post-consolidation have a stronger prognostic power. The majority ofprevious studies on MRD by immunophenotyping used three-fourcolor MFC but some groups demonstrated that a six color approachimproves the identification of LAIP positive cells respect to four-
color examination [24]. We provided a six-color MFC analysis fora more sensitive and accurate quantification of MRD in AML. Thepurpose of the present study was to compare the performance ofboth MFC and WT1-RNA quantification in detecting MRD in order to
No. patients 23Male/female 13/10Age at diagnosis, median (range) 52.5 (19–76)WBC count at diagnosis × 109/L median (range) 7.56 (1.0–25.2)PLT count at diagnosis at diagnosis × 109/L median (range) 34 (10–35)Hb level (g/L) at diagnosis median (range) 96 (54–114)WHO classificationa
AML with minimal differentiation 4 (17%)AML without maturation 3 (13%)AML with maturation 5 (22%)Acute myelomonocytic leukemia 1 (4%)Acute monoblastic/monocytic leukemia 6 (27%)Acute erithroid leukemia 1 (4%)Acute Myeloid leukemia, with myelodysplasia-related
changes3 (13%)
Induction 23 (100%)Consolidation I 22 (96%)Consolidation II 8 (34%)AutoSCT/AlloSCT 0 (0%)/9 (39%)
02 G. Rossi et al. / Leukemia
valuate the best methodology to identify patients with MRD. Thus,ur primary goal was to study sensitivity, specificity, predictivealues (PV), likelihood ratio (LR), receiver operating characteristicurve (ROC) and the area under the curve (AUC) of two methodolo-ies at different time points. Secondary goals were to choose theeference time with the best performance and to analyze predic-ive factors for disease free survival (DFS) and overall survival (OS),ncluding MRD at the reference time.
. Patients and methods
Fresh bone marrow (BM) samples from 74 consecutive, unselected AML patientsere obtained at diagnosis between January 2008 and June 2011 from the Hematol-
gy Department of IRCCS “Casa Sollievo della Sofferenza” Hospital (Italy). Diagnosisf patients was based on morphology, immunophenotyping, cytogenetics andolecular biology. Of the 74 patients diagnosed with AML, 59 (79%) received
hemotherapy and of these 47 (80%) achieved a complete remission (CR). Twenty-our patients with leukemia-specific genetic alterations were excluded from thistudy. Thus, 23 patients without specific genetic alterations were analyzed atifferent time points for MRD detection: after induction therapy (T1) and afteronsolidation therapy (T2). Patients younger than 60 years old were uniformlyreated according to the protocols of EORTC/GIMEMA AML-12 with cytarabine3 g/m2/q12 h, days 1, 3, 5 and 7), daunorubicin (50 mg/m2, days 1, 3 and 5)nd etoposide (50 mg/m2 days 1–5). As consolidation, patients received cytara-ine (1.5 g/m2/q12 h, days 1, 3, 5, 7) and daunorubicin (50 mg/m2, days 1, 3 and). Patients with a HLA-compatible sibling were given allograft, whereas the oth-rs received autologous stem cell transplantation (auSCT). Patients older than0 years old were treated according to regimen FLAG-Myocet® with fludara-ine (20 mg/m2/day, days 1–5) and cytarabine (2 g/m2/day, days 1–5), G-CSF 5cg/kg/day until neutrophils >1 × 109/L and liposomial doxorubicin (Myocet®)
0 mg/m2 infused at first day. Patients who achieved morphologic CR o partialemission (PR) received an identical consolidation course. CR was defining wasefined according to criteria reported by Cheson [25] (<5% BM blast cells, absencef extramedullary leukemia and recovery of hematologic parameters) while PR wasonsidered when more than 5% and less of 15% BM blast cells were counted. Relapseas defined as reappearance of at least 5% blasts in the bone marrow or in peripheral
lood, or development of extramedullary leukemia.
.1. Flow cytometry
Immunophenotypic analysis was performed on erythrocyte-lysed whole BMith direcly conjugated monoclonal antibodies (MoAbs). Antigen expression was
nalyzed using 6-color combinations of MoAbs with fluorescein isothiocyanateFITC), phycoerythrin (PE), allophycocyanin (APC), allophycocyanin-cyanin7APC-Cy7), peridin-chlorophyll proteins (PerCp), phycoerythrin-cyanin 7 (Pe-Cy7)Becton Dickinson Biosciences, San Jose, CA, USA) at diagnosis and follow-up.sotype-matched nonreactive Abs were used as controls. Blasts were identified bysing a CD45/SSC log gating strategy. CD45/CD34 were used in combination withyeloid and lymphoid markers in a six-color combination to increase the sensitivity
f the LAIPs detection. Monoclonal antibodies against 30 surface and cytoplasmaticntigens were used at diagnosis in the following combinations designed for theetection of LAIPs and for the study of maturation: CD117/CD15/CD13/HLA-R, CD64/CD14/CD33/56, CD13/CD11b/CD16/7, CD2/CD135/CD4/CD36,PO/TdT/cyCD3/CD19, CD22/CD20/CD10/CD19, CD61/GlyA/CD42a/CD36. During
ollow-up MoAbs CD45/CD34/CD117/CD15 and CD45/CD34/CD64/CD14 wereespectively combined with different myeloid and lymphoid markers in a six-colorombination to better identify the MRD (Supplementary Figure). For samples at theime of diagnosis, at least 20.000 events were acquired instead of 250.000 eventscquired for MRD samples during follow-up. At the time of relapse, the sameombinations of antibodies were applied and 20.000 events were collected. Dataere acquired by FACS Canto ITM (Becton Dickinson) flow cytometer and analysis of
ist mode data was performed by FACS Diva (Becton Dickinson) software. Based onrevious published data [3,17,22] a threshold of 10−3 (0.1%) was chosen to definehe lower level of positive MRD (Fig. 4).
.2. Quantification of WT1
The reverse transcription (RT) step was adapted from the BIOMED ITM protocol.tarting from 1 �g of total RNA, random hexamers were used at a concentrationf 25 �M and 100 U of reverse transcriptase were added to the reaction mixture,btaining a significant enhancement of the assay sensitivity. RQ-PCR reactions anduorescence measurements were performed on the ABI PRISM 7000 Sequenceetection SystemTM (Applied ByosystemsTM). For quantitative analysis of WT1 wesed wt1 Profile Quant kit ELNTM (Ipsogen, Marseille, France) in accordance with
ew method of European Leukemia Net previously described by Cilloni [12]. Primersnd probe were localized on exons 1 and 2. Quantitative analysis of WT1 expres-ion was performed by a calibration curve with plasmid containing the WT1 targetequence (Ipsogen). The WT1 transcripts values obtained by RQ-PCR were normal-zed with respect to the number of ABL transcripts and expressed as WT1 copy
a The 2008 World Health Organization (WHO) classification of myeloid neoplasmsand acute leukemia [26].
number every 104 copies of ABL. The cut-off of normal value was 90 copies WT1/104
copies ABL, as was previously established [9,11]. All experiments were carried out induplicate with appropriate negative controls. The samples containing less of 1000copies of ABL were evaluated as degraded and inadequate for analysis.
2.3. Statistical analysis
A descriptive analysis of the sample was carried out by means of median,interquartile range (IQR) for continuous variables and absolute and relative fre-quencies for qualitative ones. An analysis of sensitivity, specificity, positive andnegative predictive values (PPV (true positive/(true positive + false positive)) andNPV (true negative/(true negative + false negative)) and positive and negative likeli-hood ratio (LR + (sensitivity/(1 − specificity)) and LR − ((1 − sensitivity)/specificity))was carried out in order to compare the performance of MFC and WT1 quantifica-tion at different time points. Furthermore, the (ROC) and (AUC) for both techniqueswere used in order to compare their performance at each point in time. In partic-ular, the choice of the best technique was relied on AUC, LR+ and LR−. An analysisof sensitivity, specificity, PPV, NPV, LR+ and LR− was performed also for the combi-nation of the two techniques. Furthermore, in order to validate the standard cut-offused for MCF and WT1 quantification in RQ-PCR, a ROC analysis was carried outin the subgroup of population who did not undergo a bone marrow transplant. Toevaluate the prognosis of patients in terms of OS and DFS a univariable analysiswas performed with respect to: age, gender, allogeneic bone marrow transplant,results of MCF and WT1-RNA (in accordance to standard cut-off), the presence ofcomorbidities, hemoglobin level, white blood cell and platelet counts. The KaplanMeier methods with Log-Rank test and univariable Cox regression model were usedin order to do it. Results were expressed as hazard ratio (HR) and 95% confidenceinterval (95% CI). Only variables for which the 80% of data were available were usedin the analysis. The analysis was performed using SPSS software version 12.0 forWindows and statistical significance was set at p = 0.05.
3. Results
Twenty-three patients with AML and in morphological completeremission were studied for MRD monitoring. Patients character-istics were summarized in Table 1. Ten patients (43.5%) had anAML relapse at the end of the study. Sixteen patients (69.6% of allpatients) were still alive at the end of the study: median survivaltime was 513 days (IQR: 349). One patient died after induction,hence 22 patients were evaluable for MRD post consolidation. LAIPswere identified in 91% patients (Table 2). Patients studied showedWT1-RNA elevated at diagnosis with a mean value of 3320.457copies WT1/104 ABL.
3.1. Analysis of technical performance
The best performance of both techniques was observed at thetime of induction therapy: T1 was thus chosen as reference time.
G. Rossi et al. / Leukemia Research 36 (2012) 401– 406 403
Table 2Leukemia-associated immunophenotypes (LAIPs) and their distribution in 21patients at diagnosis.
Fig. 1. ROC curve for flow-cytometry (MFC) and WT1 tests (patients who underwent
TM
TM
TC
CD33++ 3 (14)HLA-DR++ 5 (24)
FC showed higher sensitivity than WT1 in RQ-PCR (80% vs 70% at1) but a less value of specificity than WT1 quantification (Table 3).lthough both methodologies showed comparable LR+ at each time
oint, a better LR− was found (LR+: 1.73 vs 1.82; LR−:0.37 vs 0.48t T1) for MFC analysis. However both techniques showed low PPV57.1 vs 58.3 at T1) and NPV (77.8% vs 72.7% at T1) (Table 3). AUC and5% confidence intervals demonstrated a moderate accuracy for
able 3FC and WT1 RNA levels performance at different time points.
T1post-induction
T2post-consolidation
Specificity 53.8% 53.8%
Sensitivity 80.0% 77.8%
PPV 57.1% 53.8%
NPV 77.8% 77.8%
LR+ 1.73 1.68
LR− 0.37 0.41
able 4FC and WT1 RNA performance at different time points in patients who did not undergo
T1post-induction
T2post-consolidatio
Specificity 83.3% 66.7%
Sensitivity 87.5% 85.7%
PPV 87.5% 75.0%
NPV 83.3% 80.0%
LR+ 5.25 2.57
LR− 0.15 0.21
able 5ombination of both MFC and WT1 RNA performance at different time points in all patien
T1post-induction
T2post-consolidatio
Specificity 61.5% 76.9%
Sensitivity 70.0% 44.4%
PPV 58.3% 57.1%
NPV 72.7% 66.7%
LR+ 1.82 1.92
LR− 0.48 0.72
bone marrow transplantation excluded). Area under curve: MFC: 0.813 (95% C.I.:0.559–1.066); WT1: 0.896 (95% C.I.: 0.698–1.094).
both MFC and WT1-RNA in RQ-PCR (0.715 (0.499–0.932) vs 0.713(0.506–0.940) at T1). Excluding allograft patients a higher valueof sensitivity, specificity, PPV and NPV were obtained for both thetechniques as well as better values of LR (MFC LR+/LR−: 5.25/0.15 vsRQ-PCR LR+/LR−: +∞/0.25 at T1) were obtained (Table 4). In orderto evaluate if the techniques together ameliorated the detection
of MRD, the performance of combined results was also investi-gated but any advantage was shown (Table 5). The ROC allowedidentifying the optimal threshold for both MFC and WT1 test(Fig. 1). Optimal cut-off values were identified at 0.15% for MFC
sensitivity 87.0%; specificity: 83%) and 83.7 copies /104 ABL forT1-RNA quantification (sensitivity: 87%; specificity 100%). Using
.1% and 90 copies/104 ABL as classical thresholds for MFC and WT1Q-PCR, respectively the following results were obtained: sensitiv-
ty was 83.3% and specificity was 87.5% for MCF (LR+: 6.38 and LR−:.20) whereas for WT1 the sensitivity was 75% and specificity equalo 100% (LR+: +∞ and LR−: 0.25). Therefore, classical thresholdsere considered comparable to optimal values obtained from ourOC analysis.
.2. Overall survival
Median overall survival was 607 days in the group of patientsho had negative MFC test at T1 (IQR: 465 days) and 464 days
n the positive group (IQR: 333 days). In the group of patients whoad negative WT1 test the median survival was 421 days (IQR: 417)hile median OS in those who had positive test was 518 (IQR: 323).esults of univariable analysis are shown in Table 6: the only vari-ble demonstrated to be associated to OS was represented by theresence of comorbidities (HR 9.20; 95% C.I.: 1.06–79.73).
.3. Disease free survival
The results of the univariable analysis are shown in Table 6:emoglobin level was significantly associated to disease free sur-ival (HR: 0.678; 95% C.I.: 0.47–0.97) as well as the positivity to
FC (HR: 9.52; 95% C.I.: 1.18–77.04) and WT1 test (HR: 4.96; 95%
.I.: 1.02–24.05) (Figs. 2 and 3).
ig. 2. DFS for patients who had a positive MRD for flow-cytometry (cut-off 0.1%)s patients who had a negative MRD (p = 0.010).
Fig. 3. DFS for patients who presented a positive MRD for WT1 test (cut-off WT1-RNA 90 × 104 ABL copies) vs patients who did not have MRD (p = 0.026).
4. Discussion
Despite high remission rate, a significant number of patientswith acute myeloid leukemia (AML) incur in a relapse [27]: themonitoring of MRD has become essential to optimize the clin-ical management of postremission phase. Although moleculardetection showed higher sensitivity (10−5 to 10−6) than othermethodologies (FISH, flow-cytometry), its applicability is restrictedto common targets, including specific fusion transcripts as AML1-ETO, CBFB-MYH11, PML-RARA, NPM1, MLL gene fusions. More than50% of all AML samples lack one of these specific genes, so itis crucial to identify molecular targets applicable for the major-ity of patients. Several studies on WT1 as molecular marker forMRD reported a good association between WT1 levels and clin-ical course [10,11,13,14] but the combination of markers fromflow-cytometry allow to reach a high sensitivity in detecting MRD(10−4 to 10−5) [8]. Previous studies identified LAIPs in 80% of AMLpatients using a three-four color MFC [2,3,17,20]. Al Mawali [23]was able to detect LAIPs in 94% of patients when five-color MFCand a comprehensive panel of MoAbs were applied. Olaru [24] alsodemonstrated an improvement in detection of LAIPs with a sixcolor combination. In our study MRD analysis was performed witha six color combination at different points in time and was com-pared to WT1 levels. MFC showed higher sensitivity and a smallerspecificity than WT1 test at each time point. Although both method-ologies showed comparable LR+ values at each time point, a betterLR− for MFC analysis was found. Furthermore, the AUC and 95%confidence intervals demonstrated a moderate accuracy for bothtechniques, higher for MFC. Thinking to allotransplantation as afactor that could affect the accuracy of analysis we excluded allo-graft patients and repeated the analysis in the remaining patients.Better values of sensitivity, specificity, PV, LR were obtained. AUCwas higher but did not reach optimal values because of the smallnumber of patients involved in the study. Then, we combinedboth methodologies in order to evaluate the global performancein predicting relapse but any advantage was achieved. From thisstudy emerged that post induction time (T1) is the time pointwith best performance observed. Thus, we chose T1 as referencetime. These results agree with previous published data [3,12,23,28]that reported the post-induction phase as the most significanttime to provide MRD. In the matter of clinical significance onMRD detection many controversies on WT1 test and immunophe-notipic analysis were found in the literature. Some groups showed
a good association between WT1 quantification for monitoringpatients with acute leukemia and the outcome [11–14] while oth-ers was not able to demonstrate the same [15,16]. Different levels of
G. Rossi et al. / Leukemia Research 36 (2012) 401– 406 405
Fig. 4. MRD detection in consecutive BM samples of a patient still in remission (A) and of a relapsing patient (B). (A) Leukemic cells CD34+CD117+ showed the LAIP CD34+
C tion I aa uction
sscMtpoeaWssooevoArsiatcWcqoacwmi
A
t
[
[
[
D56+; the MRD was 0.087% after induction and completely absent after consolidaberrant phenotype CD117+CD33−; the MRD was 0.456%, 0.786% and 6.7% after ind
ensitivity of the RT-PCR procedures used, different treatmenttrategies and clinical settings may justify the observed discrepan-ies. On the other hand, using immunophenotipic method to detectRD, differences among thresholds levels, time points, combina-
ion of markers and clinical outcomes were noticed [2,3,20,23]. Dataresented in this paper showed a concordance between high levelsf WT1 copy-number and MFC in predicting the possibility of dis-ase recurrence: the positivity to MFC at post induction phase wasssociated with worse DFS (p = 0.010) as well as the positivity toT1-RNA test (p = 0.026). Because of the limitedness of the sample
ize, a multivariable analysis was not performed. Further researchhould be indeed promoted in order to evaluate the adjusted valuef the positivity to MCF and WT1. As threshold, we used a levelf 0.1% in MFC and 90.0 copies/104 ABL in WT1 test, as alreadystablished in previous studies [3,9,23]. To confirm these cut-offalues a ROC analysis was provided. Results of this analysis reportptimal values of cut-off at 0.15% for MFC and 83.7 copies/104
BL for WT1 RNA expression. Our threshold values (0.1% and 90.0,espectively) do not differ from the optimal values in terms ofensitivity, specificity and LR. Therefore, our data confirm the clin-cal significance of thresholds already established. In conclusion,ccording to our study, in AML patients without specific molecularargets MFC and WT1-RNA quantification showed a comparableapacity to identify high risk patients who eventually relapsed.
e showed these results in terms of technical performance andlinical significance. The combination of both MFC and WT1-RNAuantification did not add any improvement to the performancef each technique. Therefore, to the best of our knowledge, in thebsence of any specific molecular target each diagnostic laboratoryould choice one of these methods for MRD detection in patientsith AML. The allotransplatation seems to influence the perfor-ance of both techniques but more studies need to demonstrate
t.
ppendix A. Supplementary data
Supplementary data associated with this article can be found, inhe online version, at doi: 10.1016/j.leukres.2011.11.020.
[
nd consolidation II chemotherapy. (B) Leukemic cells CD117+CD13+ displayed the, consolidation chemotherapy and at relapse.
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