Comparison between multiparameter flow cytometry and WT1-RNA quantification in monitoring minimal residual disease in acute myeloid leukemia without specific molecular targets

Leukemia Research 36 (2012) 401–406

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Comparison between multiparameter flow cytometry and WT1-RNA quantification in monitoring minimal residual disease in acute myeloid leukemia without specific molecular targets Giovanni Rossi a,∗ , Maria Marta Minervini a , Angelo Michele Carella a , Chiara de Waure b , Francesco di Nardo b , Lorella Melillo a , Giovanni D’Arena a , Gina Zini c , Nicola Cascavilla a a

Department of Hematology and Stem Cell Unit, IRCCS “Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo, Italy Department of Hygiene, Catholic University of Sacred Heart, Rome, Italy c Department of Hematology and Laboratory, Center of Research ReCAMH, Catholic University of Sacred Heart, Rome, Italy b

a r t i c l e

i n f o

Article history: Received 6 October 2011 Received in revised form 20 November 2011 Accepted 27 November 2011 Available online 21 December 2011 Keywords: Acute myeloid leukemia Minimal residual disease Flow-cytometry Leukemia-associated immunophenotypes WT1-RNA

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 identify high 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 MFC and WT1-RNA quantification in 23 AML patients who did not present specific molecular targets. As previously published, we considered values of 10−3 (0.1%) in MFC and 90 WT1-RNA ×104 ABL copies as optimal thresholds. Receiver operating characteristics (ROC) analysis was used to confirm these data. To realize the methodology that better identify high risk patients, an analysis of sensitivity, specificity, predictive 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 with poor prognosis. Therefore, MFC and WT1-RNA quantification showed a comparable capacity in terms of technical performance and clinical significance to identify high risk patients who eventually relapsed. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Monitoring of the minimal residual disease (MRD) in acute myeloid leukemia (AML) has become essential to predict prognosis and to select the type of post-remission treatment [1]. Several studies have shown how multiparameter flow-cytometry (MFC) [2,3] polymerase chain reaction (PCR) [4,5] and fluorescence in situ hybridization (FISH) [6] are useful for detection of MRD in AML patients. PCR-based quantification of MRD has a high sensitivity but its applicability is restricted to subgroups of AML with leukemia-specific molecular targets (e.g. AML1-ETO, CBFb-MYH11, MLL, FLT3, NPM1) [7]. These cases comprise only 40–50% of all AML cases [8]. For this reason, several investigators studied WT1 expression as alternative marker of leukemic cells and tested it for MRD detection in AML patients [9–12]. However the results concerning its use as follow-up marker during post-remission have been

∗ Corresponding author at: Department of Hematology and Stem Cell Unit, IRCCS “Casa Sollievo della Sofferenza” Hospital, v.le Cappuccini 1, 71013 San Giovanni Rotondo, Italy. Tel.: +39 3922323949; fax: +39 3922323949. E-mail address: [email protected] (G. Rossi). 0145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2011.11.020

conflicting. Some groups reported a good association between the clinical course and WT1 levels [13,14] while others failed to show [15,16]. This discrepancy may be due to differences in methodology in measuring WT1 and in patients populations or in the intensity of the regimens used. On the other hand, using an accurate combination of monoclonal antibodies in the MFC allows specific detection of leukemic cells. This approach may be applicable to a percentage of AML patients that ranges from 80% to 94% [17–20] and shows a sensitivity better than 1 leukemic cell per 104 to 105 normal cells [8]. However, studies differ in the threshold use for MRD detection and in the timing of testing. A strong association with clinical outcome was found at the presence of both 10−4 [2,20] and 10−3 Leukemia-associated immunophenothypes (LAIPs) [3,21–24] positive cells and it is still unclear whether post-induction or postconsolidation have a stronger prognostic power. The majority of previous studies on MRD by immunophenotyping used three-four color MFC but some groups demonstrated that a six color approach improves the identification of LAIP positive cells respect to fourcolor examination [24]. We provided a six-color MFC analysis for a more sensitive and accurate quantification of MRD in AML. The purpose of the present study was to compare the performance of both MFC and WT1-RNA quantification in detecting MRD in order to

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evaluate the best methodology to identify patients with MRD. Thus, our primary goal was to study sensitivity, specificity, predictive values (PV), likelihood ratio (LR), receiver operating characteristic curve (ROC) and the area under the curve (AUC) of two methodologies at different time points. Secondary goals were to choose the reference time with the best performance and to analyze predictive factors for disease free survival (DFS) and overall survival (OS), including MRD at the reference time. 2. Patients and methods Fresh bone marrow (BM) samples from 74 consecutive, unselected AML patients were obtained at diagnosis between January 2008 and June 2011 from the Hematology Department of IRCCS “Casa Sollievo della Sofferenza” Hospital (Italy). Diagnosis of patients was based on morphology, immunophenotyping, cytogenetics and molecular biology. Of the 74 patients diagnosed with AML, 59 (79%) received chemotherapy and of these 47 (80%) achieved a complete remission (CR). Twentyfour patients with leukemia-specific genetic alterations were excluded from this study. Thus, 23 patients without specific genetic alterations were analyzed at different time points for MRD detection: after induction therapy (T1) and after consolidation therapy (T2). Patients younger than 60 years old were uniformly treated according to the protocols of EORTC/GIMEMA AML-12 with cytarabine (3 g/m2 /q12 h, days 1, 3, 5 and 7), daunorubicin (50 mg/m2 , days 1, 3 and 5) and etoposide (50 mg/m2 days 1–5). As consolidation, patients received cytarabine (1.5 g/m2 /q12 h, days 1, 3, 5, 7) and daunorubicin (50 mg/m2 , days 1, 3 and 5). Patients with a HLA-compatible sibling were given allograft, whereas the others received autologous stem cell transplantation (auSCT). Patients older than 60 years old were treated according to regimen FLAG-Myocet® with fludarabine (20 mg/m2 /day, days 1–5) and cytarabine (2 g/m2 /day, days 1–5), G-CSF 5 mcg/kg/day until neutrophils >1 × 109 /L and liposomial doxorubicin (Myocet® ) 30 mg/m2 infused at first day. Patients who achieved morphologic CR o partial remission (PR) received an identical consolidation course. CR was defining was defined according to criteria reported by Cheson [25] (<5% BM blast cells, absence of extramedullary leukemia and recovery of hematologic parameters) while PR was considered when more than 5% and less of 15% BM blast cells were counted. Relapse was defined as reappearance of at least 5% blasts in the bone marrow or in peripheral blood, or development of extramedullary leukemia. 2.1. Flow cytometry Immunophenotypic analysis was performed on erythrocyte-lysed whole BM with direcly conjugated monoclonal antibodies (MoAbs). Antigen expression was analyzed using 6-color combinations of MoAbs with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), allophycocyanin-cyanin7 (APC-Cy7), peridin-chlorophyll proteins (PerCp), phycoerythrin-cyanin 7 (Pe-Cy7) (Becton Dickinson Biosciences, San Jose, CA, USA) at diagnosis and follow-up. Isotype-matched nonreactive Abs were used as controls. Blasts were identified by using a CD45/SSC log gating strategy. CD45/CD34 were used in combination with myeloid and lymphoid markers in a six-color combination to increase the sensitivity of the LAIPs detection. Monoclonal antibodies against 30 surface and cytoplasmatic antigens were used at diagnosis in the following combinations designed for the detection of LAIPs and for the study of maturation: CD117/CD15/CD13/HLACD64/CD14/CD33/56, CD13/CD11b/CD16/7, CD2/CD135/CD4/CD36, DR, MPO/TdT/cyCD3/CD19, CD22/CD20/CD10/CD19, CD61/GlyA/CD42a/CD36. During follow-up MoAbs CD45/CD34/CD117/CD15 and CD45/CD34/CD64/CD14 were respectively combined with different myeloid and lymphoid markers in a six-color combination to better identify the MRD (Supplementary Figure). For samples at the time of diagnosis, at least 20.000 events were acquired instead of 250.000 events acquired for MRD samples during follow-up. At the time of relapse, the same combinations of antibodies were applied and 20.000 events were collected. Data were acquired by FACS Canto ITM (Becton Dickinson) flow cytometer and analysis of list mode data was performed by FACS Diva (Becton Dickinson) software. Based on previous published data [3,17,22] a threshold of 10−3 (0.1%) was chosen to define the lower level of positive MRD (Fig. 4). 2.2. Quantification of WT1 The reverse transcription (RT) step was adapted from the BIOMED ITM protocol. Starting from 1 ␮g of total RNA, random hexamers were used at a concentration of 25 ␮M and 100 U of reverse transcriptase were added to the reaction mixture, obtaining a significant enhancement of the assay sensitivity. RQ-PCR reactions and fluorescence measurements were performed on the ABI PRISM 7000 Sequence detection SystemTM (Applied ByosystemsTM ). For quantitative analysis of WT1 we used wt1 Profile Quant kit ELNTM (Ipsogen, Marseille, France) in accordance with new method of European Leukemia Net previously described by Cilloni [12]. Primers and probe were localized on exons 1 and 2. Quantitative analysis of WT1 expression was performed by a calibration curve with plasmid containing the WT1 target sequence (Ipsogen). The WT1 transcripts values obtained by RQ-PCR were normalized with respect to the number of ABL transcripts and expressed as WT1 copy

Table 1 Study population characteristics. Patients characteristics No. patients Male/female Age at diagnosis, median (range) WBC count at diagnosis × 109 /L median (range) PLT count at diagnosis at diagnosis × 109 /L median (range) Hb level (g/L) at diagnosis median (range) WHO classificationa AML with minimal differentiation AML without maturation AML with maturation Acute myelomonocytic leukemia Acute monoblastic/monocytic leukemia Acute erithroid leukemia Acute Myeloid leukemia, with myelodysplasia-related changes Induction Consolidation I Consolidation II AutoSCT/AlloSCT

23 13/10 52.5 (19–76) 7.56 (1.0–25.2) 34 (10–35) 96 (54–114) 4 (17%) 3 (13%) 5 (22%) 1 (4%) 6 (27%) 1 (4%) 3 (13%) 23 (100%) 22 (96%) 8 (34%) 0 (0%)/9 (39%)

a The 2008 World Health Organization (WHO) classification of myeloid neoplasms and 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 in duplicate with appropriate negative controls. The samples containing less of 1000 copies 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 frequencies for qualitative ones. An analysis of sensitivity, specificity, positive and negative predictive values (PPV (true positive/(true positive + false positive)) and NPV (true negative/(true negative + false negative)) and positive and negative likelihood ratio (LR + (sensitivity/(1 − specificity)) and LR − ((1 − sensitivity)/specificity)) was carried out in order to compare the performance of MFC and WT1 quantification at different time points. Furthermore, the (ROC) and (AUC) for both techniques were used in order to compare their performance at each point in time. In particular, the choice of the best technique was relied on AUC, LR+ and LR−. An analysis of sensitivity, specificity, PPV, NPV, LR+ and LR− was performed also for the combination of the two techniques. Furthermore, in order to validate the standard cut-off used for MCF and WT1 quantification in RQ-PCR, a ROC analysis was carried out in the subgroup of population who did not undergo a bone marrow transplant. To evaluate the prognosis of patients in terms of OS and DFS a univariable analysis was performed with respect to: age, gender, allogeneic bone marrow transplant, results of MCF and WT1-RNA (in accordance to standard cut-off), the presence of comorbidities, hemoglobin level, white blood cell and platelet counts. The Kaplan Meier methods with Log-Rank test and univariable Cox regression model were used in order to do it. Results were expressed as hazard ratio (HR) and 95% confidence interval (95% CI). Only variables for which the 80% of data were available were used in the analysis. The analysis was performed using SPSS software version 12.0 for Windows and statistical significance was set at p = 0.05.

3. Results Twenty-three patients with AML and in morphological complete remission were studied for MRD monitoring. Patients characteristics were summarized in Table 1. Ten patients (43.5%) had an AML relapse at the end of the study. Sixteen patients (69.6% of all patients) were still alive at the end of the study: median survival time was 513 days (IQR: 349). One patient died after induction, hence 22 patients were evaluable for MRD post consolidation. LAIPs were identified in 91% patients (Table 2). Patients studied showed WT1-RNA elevated at diagnosis with a mean value of 3320.457 copies WT1/104 ABL. 3.1. Analysis of technical performance The best performance of both techniques was observed at the time of induction therapy: T1 was thus chosen as reference time.

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Table 2 Leukemia-associated immunophenotypes (LAIPs) and their distribution in 21 patients at diagnosis. LAIPs

No. of cases (%)

Lineage infidelity CD34/CD7 CD34/CD19 CD33/CD4 Asynchronous antigens CD34/CD11b CD34/CD14 CD34/CD15 CD34/CD64 CD34/CD56 CD117/CD11b CD117/CD14 CD117/CD15 CD117/CD64 CD117/56 CD33/CD56 Lack of lineage specific CD33+ /CD13− CD33− /CD13+ CD33+ /HLADR− Overexpression antigens CD34++ CD117++ CD33++ HLA-DR++

1 (4) 2 (9) 5 (24) 4 (19) 1 (4) 6 (29) 1 (4) 4 (19) 4 (19) 1 (4) 5 (24) 1 (4) 4 (19) 5 (24) 5 (24) 6 (29) 3 (14)

Fig. 1. ROC curve for flow-cytometry (MFC) and WT1 tests (patients who underwent 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).

2 (9) 2 (9) 3 (14) 5 (24)

MFC showed higher sensitivity than WT1 in RQ-PCR (80% vs 70% at T1) but a less value of specificity than WT1 quantification (Table 3). Although both methodologies showed comparable LR+ at each time point, a better LR− was found (LR+: 1.73 vs 1.82; LR−:0.37 vs 0.48 at T1) for MFC analysis. However both techniques showed low PPV (57.1 vs 58.3 at T1) and NPV (77.8% vs 72.7% at T1) (Table 3). AUC and 95% confidence intervals demonstrated a moderate accuracy for

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 value of sensitivity, specificity, PPV and NPV were obtained for both the techniques as well as better values of LR (MFC LR+/LR−: 5.25/0.15 vs RQ-PCR LR+/LR−: +∞/0.25 at T1) were obtained (Table 4). In order to evaluate if the techniques together ameliorated the detection of MRD, the performance of combined results was also investigated but any advantage was shown (Table 5). The ROC allowed identifying the optimal threshold for both MFC and WT1 test (Fig. 1). Optimal cut-off values were identified at 0.15% for MFC

Table 3 MFC and WT1 RNA levels performance at different time points.

Specificity Sensitivity PPV NPV LR+ LR−

T1 post-induction

T2 post-consolidation

T1 post-induction

T2 post-consolidation

53.8% 80.0% 57.1% 77.8% 1.73 0.37

53.8% 77.8% 53.8% 77.8% 1.68 0.41

61.5% 70.0% 58.3% 72.7% 1.82 0.48

76.9% 44.4% 57.1% 66.7% 1.92 0.72

Table 4 MFC and WT1 RNA performance at different time points in patients who did not undergo allotransplantation.

Specificity Sensitivity PPV NPV LR+ LR−

T1 post-induction

T2 post-consolidation

T1 post-induction

T2 post-consolidation

83.3% 87.5% 87.5% 83.3% 5.25 0.15

66.7% 85.7% 75.0% 80.0% 2.57 0.21

100.0% 75.5% 100.0% 75.0% +∞ 0.25

83.3% 57.1% 80.0% 62.5% 3.4 0.51

Table 5 Combination of both MFC and WT1 RNA performance at different time points in all patients studied and excluding patients who underwent allotransplantation.

Specificity Sensitivity PPV NPV LR+ LR−

T1 post-induction

T2 post-consolidation

T1 post-induction

T2 post-consolidation

61.5% 70.0% 58.3% 72.7% 1.82 0.48

76.9% 44.4% 57.1% 66.7% 1.92 0.72

75.0% 75.0% 75.0% 75.0% 3 0.33

83.3% 57.1% 80.0% 62.5% 3.42 0.51

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Table 6 Results of the univariable analysis for OS and DFS. HR: hazard ratio; 95% C.I.: 95% confidence interval. Variables

OS Crude HR (95% C.I.)

DSF Crude HR (95% C.I.)

MFC+ (positive: >0.1) WT1+ (positive: >90.0) Gender (male) Age Transplantation Leucocytes Hemoglobin Platelets Comorbidities

6.53 (0.77–55.61) 2.86 (0.54–15.05) 1.26 (0.28–5.79) 1.07 (0.99–1.16) 0.03 (0.01–27.40) 1.00 (0.99–1.01) 0.857 (0.59–1.24) 1.00 (0.99–1.01) 9.20 (1.06–79.73)

9.52 (1.18–77.04) 4.96 (1.02–24.05) 1.33 (0.34–5.11) 1.02 (0.98–1.07) 0.25 (0.03–1.99) 1.00 (0.99–1.01) 0.678 (0.47–0.97) 1.00 (0.99–1.01) 3.36 (0.83–13.62)

(sensitivity 87.0%; specificity: 83%) and 83.7 copies /104 ABL for WT1-RNA quantification (sensitivity: 87%; specificity 100%). Using 0.1% and 90 copies/104 ABL as classical thresholds for MFC and WT1 RQ-PCR, respectively the following results were obtained: sensitivity was 83.3% and specificity was 87.5% for MCF (LR+: 6.38 and LR−: 0.20) whereas for WT1 the sensitivity was 75% and specificity equal to 100% (LR+: +∞ and LR−: 0.25). Therefore, classical thresholds were considered comparable to optimal values obtained from our ROC analysis.

3.2. Overall survival Median overall survival was 607 days in the group of patients who had negative MFC test at T1 (IQR: 465 days) and 464 days in the positive group (IQR: 333 days). In the group of patients who had negative WT1 test the median survival was 421 days (IQR: 417) while median OS in those who had positive test was 518 (IQR: 323). Results of univariable analysis are shown in Table 6: the only variable demonstrated to be associated to OS was represented by the presence of comorbidities (HR 9.20; 95% C.I.: 1.06–79.73).

3.3. Disease free survival The results of the univariable analysis are shown in Table 6: hemoglobin level was significantly associated to disease free survival (HR: 0.678; 95% C.I.: 0.47–0.97) as well as the positivity to MFC (HR: 9.52; 95% C.I.: 1.18–77.04) and WT1 test (HR: 4.96; 95% C.I.: 1.02–24.05) (Figs. 2 and 3).

Fig. 2. DFS for patients who had a positive MRD for flow-cytometry (cut-off 0.1%) vs patients who had a negative MRD (p = 0.010).

Fig. 3. DFS for patients who presented a positive MRD for WT1 test (cut-off WT1RNA 90 × 104 ABL copies) vs patients who did not have MRD (p = 0.026).

4. Discussion Despite high remission rate, a significant number of patients with acute myeloid leukemia (AML) incur in a relapse [27]: the monitoring of MRD has become essential to optimize the clinical management of postremission phase. Although molecular detection showed higher sensitivity (10−5 to 10−6 ) than other methodologies (FISH, flow-cytometry), its applicability is restricted to common targets, including specific fusion transcripts as AML1ETO, CBFB-MYH11, PML-RARA, NPM1, MLL gene fusions. More than 50% of all AML samples lack one of these specific genes, so it is crucial to identify molecular targets applicable for the majority of patients. Several studies on WT1 as molecular marker for MRD reported a good association between WT1 levels and clinical course [10,11,13,14] but the combination of markers from flow-cytometry allow to reach a high sensitivity in detecting MRD (10−4 to 10−5 ) [8]. Previous studies identified LAIPs in 80% of AML patients 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 MFC and a comprehensive panel of MoAbs were applied. Olaru [24] also demonstrated an improvement in detection of LAIPs with a six color combination. In our study MRD analysis was performed with a six color combination at different points in time and was compared to WT1 levels. MFC showed higher sensitivity and a smaller specificity than WT1 test at each time point. Although both methodologies showed comparable LR+ values at each time point, a better LR− for MFC analysis was found. Furthermore, the AUC and 95% confidence intervals demonstrated a moderate accuracy for both techniques, higher for MFC. Thinking to allotransplantation as a factor that could affect the accuracy of analysis we excluded allograft patients and repeated the analysis in the remaining patients. Better values of sensitivity, specificity, PV, LR were obtained. AUC was higher but did not reach optimal values because of the small number of patients involved in the study. Then, we combined both methodologies in order to evaluate the global performance in predicting relapse but any advantage was achieved. From this study emerged that post induction time (T1) is the time point with best performance observed. Thus, we chose T1 as reference time. These results agree with previous published data [3,12,23,28] that reported the post-induction phase as the most significant time to provide MRD. In the matter of clinical significance on MRD detection many controversies on WT1 test and immunophenotipic analysis were found in the literature. Some groups showed a good association between WT1 quantification for monitoring patients with acute leukemia and the outcome [11–14] while others was not able to demonstrate the same [15,16]. Different levels of

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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+ CD56+ ; the MRD was 0.087% after induction and completely absent after consolidation I and consolidation II chemotherapy. (B) Leukemic cells CD117+ CD13+ displayed the aberrant phenotype CD117+ CD33− ; the MRD was 0.456%, 0.786% and 6.7% after induction, consolidation chemotherapy and at relapse.

sensitivity of the RT-PCR procedures used, different treatment strategies and clinical settings may justify the observed discrepancies. On the other hand, using immunophenotipic method to detect MRD, differences among thresholds levels, time points, combination of markers and clinical outcomes were noticed [2,3,20,23]. Data presented in this paper showed a concordance between high levels of WT1 copy-number and MFC in predicting the possibility of disease recurrence: the positivity to MFC at post induction phase was associated with worse DFS (p = 0.010) as well as the positivity to WT1-RNA test (p = 0.026). Because of the limitedness of the sample size, a multivariable analysis was not performed. Further research should be indeed promoted in order to evaluate the adjusted value of the positivity to MCF and WT1. As threshold, we used a level of 0.1% in MFC and 90.0 copies/104 ABL in WT1 test, as already established in previous studies [3,9,23]. To confirm these cut-off values a ROC analysis was provided. Results of this analysis report optimal values of cut-off at 0.15% for MFC and 83.7 copies/104 ABL for WT1 RNA expression. Our threshold values (0.1% and 90.0, respectively) do not differ from the optimal values in terms of sensitivity, specificity and LR. Therefore, our data confirm the clinical significance of thresholds already established. In conclusion, according to our study, in AML patients without specific molecular targets MFC and WT1-RNA quantification showed a comparable capacity to identify high risk patients who eventually relapsed. We showed these results in terms of technical performance and clinical significance. The combination of both MFC and WT1-RNA quantification did not add any improvement to the performance of each technique. Therefore, to the best of our knowledge, in the absence of any specific molecular target each diagnostic laboratory could choice one of these methods for MRD detection in patients with AML. The allotransplatation seems to influence the performance of both techniques but more studies need to demonstrate it. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: 10.1016/j.leukres.2011.11.020.

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