Wilms Tumor Gene 1 Expression as a Predictive Marker for Relapse and Survival after Hematopoietic Stem Cell Transplantation for Myelodysplastic Syndromes

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Wilms Tumor Gene 1 Expression as a Predictive Marker

for Relapse and Survival after Hematopoietic Stem Cell

Transplantation for Myelodysplastic Syndromes

Jae-Ho Yoon

1

, Young-Woo Jeon

1

, Seung-ah Yahng

1

, Seung-Hwan Shin

1

, Sung-Eun Lee

1

,

Byung-Sik Cho

1

, Dong-Gun Lee

1

, Ki-Seong Eom

1

, Hee-Je Kim

1

, Seok Lee

1

, Chang-Ki Min

1

,

Seok-Goo Cho

1

, Yonggoo Kim

2

, Dong-Wook Kim

1

, Jong-Wook Lee

1

, Kyungja Han

2

,

Woo-Sung Min

1

, Chong-Won Park

1

, Myungshin Kim

2

, Yoo-Jin Kim

1,*

1Catholic Blood and Marrow Transplantation Center, The Catholic University of Korea, Seoul, Korea

2Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea

Article history:

Received 20 September 2014 Accepted 11 November 2014 Key Words:

Myelodysplastic syndrome Hematopoietic stem cell transplantation

Wilms tumor gene 1 (WT1) Minimal residual disease Relapse

Survival

a b s t r a c t

Relapse after allogeneic hematopoietic stem cell transplantation (HSCT) is a major concern in myelodysplastic syndromes (MDS), but the role of Wilms tumor gene 1 (WT1) as a predictive marker for post-HSCT relapse remains to be validated. We measured WT1 transcript levels by real-time quantitative PCR from marrow samples of 82 MDS patients who underwent transplantation between 2009 and 2013. Pre-HSCT WT1 expression weakly correlated with marrow blast counts or International Prognostic Scoring System scores and failed to predict post-transplantation relapse. Regarding post-HSCT WT1, transcript levels of relapsed patients were significantly higher in comparison to those in remission. Further analysis using receiver operating characteristics curves showed that higher (>154 copies/104ABL) 1-month post-HSCT WT1 resulted in a higher 3-year relapse rate (47.2% versus 6.9%, P< .001) with poorer disease-free survival (DFS) and overall survival at 3 years (41.7% versus 79.0% and 54.3% versus 82.1%, P¼ .003 and P ¼ .033, respectively). Multivariate analysis after adjusting for pre-HSCT karyotype and chronic graft-versus-host disease (GVHD) also revealed that higher 1-month post-HSCT WT1 was an independent predictive marker for subsequent relapse (P¼ .002) and poorer DFS (P¼ .010). In the higher 1-month post-HSCT WT1 subgroup, patients with chronic GVHD showed lower relapse rate and favorable survival outcome. One month post-HSCT WT1 expression was a useful marker for minimal residual disease and relapse prediction in association with chronic GVHD in the context of HSCT for MDS.

Ó 2015 American Society for Blood and Marrow Transplantation.

INTRODUCTION

Several therapeutic options, including growth factors, lenalidomide, azanucleosides, intensive chemotherapy, and immunosuppressive therapy, have been used to treat mye-lodysplastic syndromes (MDS). However, allogeneic he-matopoietic stem cell transplantation (allo-HSCT) is still known to be the only curative treatment and the results show prolonged disease-free survival (DFS) rates of approx-imately 30% to 50%[1-3]. The recent introduction of high-resolution sequenced-based typing of human leukocyte

antigen, as well as reduced-intensity conditioning (RIC) regimens and improvement of supportive modalities, have reduced treatment-related mortality[4]. Therefore, relapse is now considered to be a major cause of treatment failure after allo-HSCT. Previous studies reported a relapse rate of 5% to 20% in early MDS and up to 60% in advanced MDS, and they suggested MDS stage, bone marrow (BM) blast counts, advanced age, and cytogenetics are risk factors for relapse

[1,5-7]. For patients at high risk, various transplantation strategies can be applied, including modification of condi-tioning intensity, early withdrawal of immunosuppressive agents, preemptive cellular therapy of donor lymphocyte infusion (DLI) or natural killer cells, and post-HSCT mainte-nance of hypomethylating agents (HMA) [8]. Therefore, identification of MDS patients with a high risk of relapse is the first and most important step for improving post-transplantation survival outcomes.

Financial disclosure: See Acknowledgments on page 466.

* Correspondence and reprint requests: Yoo-Jin Kim, MD, PhD, Depart-ment of Hematology, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Republic of Korea.

E-mail address:yoojink@catholic.ac.kr(Y.-J. Kim).

http://dx.doi.org/10.1016/j.bbmt.2014.11.008

1083-8791/Ó 2015 American Society for Blood and Marrow Transplantation.

Biology of Blood and

Marrow Transplantation

j o u r n a l h o m e p a g e : w w w . b b m t . o r g

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The molecular basis of MDS has been recently elucidated by novel DNA sequencing technologies, and up to 80% of patients are known to have somatic genetic mutations[9-11]. Among the many genetic mutations, several genes such as TP53, RUNX1, ETV6, EZH2, ASXL1, U2AF1, and SRSF2 have been identified to have poor prognostic significance in MDS [12-15]. In addition to well-known clinical parameters, these genetic and molecular markers could be used as prognostic markers of transplantation outcomes in MDS. Unfortunately, none of the markers are currently available for detection of minimal residual disease (MRD) or prediction of relapse in a HSCT setting. Several studies investigating post-transplantation MRD in MDS have focused on Wilms tumor gene 1 (WT1) expression[16,17].

From the early 1990s, WT1 expression has been specif-ically studied in acute leukemia and the blast phase of chronic myeloid leukemia. Such studies showed a high level of WT1 expression in these diseases but not in indolent disease or chronic leukemia[18,19]. Therefore, WT1 is likely involved in the early stage of hematological cell differentia-tion and its expression correlates with BM or peripheral blood (PB) blast counts. Many studies have already investi-gated the role of aberrant expression of WT1 as a marker for residual disease in myeloid malignancy[20-22].

Several studies have reported that higher WT1 expression at diagnosis or after induction chemotherapy is a significant predictor for relapse and worse survival outcomes in acute myeloid leukemia (AML) [23-25], even after allo-HSCT

[26,27]. Also, in MDS patients, several studies reported the utility of WT1 expression. Higher WT1 expression was shown to be associated with higher International Prognostic Scoring System (IPSS) scores[28]and higher blast counts with poor survival outcomes[29]. Recently, Lange et al. reported that WT1 expression is also applicable after allo-HSCT, but further studies are necessary because of the small number of enrolled MDS patients[16].

In this study, we tried to identify the prognostic impact of WT1 expression in 82 MDS patients receiving allo-HSCT by analyzing the relationship between WT1 expression and pre-and post-transplantation disease status. We also tried to identify a clinically relevant cut-off level of WT1 expression that could predict subsequent relapse independently of well-known clinical predictors.

PATIENTS AND METHODS

Enrollment and Disease Risk Classification

Adult MDS patients receiving HSCT from May 2009 to May 2013 at our institution were screened. Subjects eligible for the study were those having WT1 transcript results available at the time of HSCT and not receiving any chemotherapeutic agents or cell therapy for the purpose of relapse pre-vention. This retrospective study was approved by the institutional review board of Seoul St. Mary’s Hospital, The Catholic University of Korea. MDS patients diagnosed by French-American-British classification [30] were included and reclassified by World Health Organization (WHO) 2008 clas-sification[31]. Risk stratification was assessed by IPSS and risk groups that were low and intermediate-1 were classified into lower risk MDS (LrMDS) and those that were intermediate-2 and high into higher risk MDS (HrMDS)

[32]. In addition to IPSS at diagnosis, IPSS score was also assessed just before the initial treatment, which consisted of HMA, intensive chemotherapy, or HSCT. For analytical purposes, chronic myelomonocytic leukemia and MDS/ myeloproliferative neoplasms were also assessed by IPSS. BM blasts and cytogenetic risk groups determined by IPSS, well-known predictors for transplantation outcomes in patients with MDS, were also evaluated within 1 month before the conditioning regimen and used for disease character-istics at the time of HSCT.

Pretransplantation Treatment Strategy

Pre-HSCT and HSCT treatment were performed according to the in-stitution’s guidelines for MDS as described elsewhere [33]. Briefly,

immediate HSCT was planned for HrMDS, and bridging therapy with HMA was planned for the preparation of HSCT when BM blast counts were 6% or more. LrMDS patients with profound cytopenias with or without prior HMA failure were considered candidates for HSCT. Standard protocols were used for azacitidine (75 mg/m2

for 7 days, every 28 days) or decitabine (20 mg/m2 for 5 days, every 28 days). Intensive chemotherapy, consisting of an anthracycline (idarubicin 12 mg/m2) for 3 days plus cytarabine (100 mg/m2

for 24 hours) for 7 days, was used to treat advanced diseases when patients were evaluated to tolerate the treatment.

Transplantation Procedures

The intensity of the conditioning regimen for conventional donor HSCT was selected according to the IPSS risk group at peak during the disease course before HSCT. For HrMDS, a myeloablative conditioning (MAC) regimen consisting of fludarabine (150 mg/m2

) plus 3.2 mg/kg/d i.v. busulfan for 4 days (FB4) was used for 33 patients. For 28 patients with LrMDS or some HrMDS patients who were older or had comorbidities, flu-darabine (150 mg/m2) plus 3.2 mg/kg/d i.v. busulfan for 2 days (FB2) was

used, according to our institution’s transplantation protocol[34]. For hap-loidentical related donor transplantations (n¼ 21), 400 or 800 cGy of total body irradiation was added to FB2. For graft-versus-host disease (GVHD) prophylaxis, patients undergoing HSCT from matched sibling donors received cyclosporine and short-course methotrexate (10 mg/m2 i.v. on

daya þ1, þ3, þ6, and þ11), whereas patients with unrelated or hap-loidentical related donors received tacrolimus and short-course metho-trexate (5 mg/m2i.v. on dayaþ1, þ3, þ6, and þ11). GVHD prophylaxis with

rabbit antithymocyte globulin (Genzyme, Cambridge, MA) was added at a median dose of 8 mg/kg (range, 2.5 to 10.0 mg/kg) for all recipients. Assessment of WT1 Transcript Levels

WT1 transcript levels from BM samples were measured by real-time quantitative PCR (WT1 ProfileQuant kit; Ipsogen, France). The assays were performed in duplicate for greater accuracy. Real-time quantitative PCR levels represented the ratio of WT1 expression to that of normal ABL1 expression (copies/104ABL). In practice, serial monitoring of WT1 expression

levels was planned before HSCT (within 1 month of conditioning), 1, 2 to 6, and 7 to 12 months after the day of stem cell infusion, and whenever relapse was suspected. To determine the significant cut-off level for relapse pre-diction, we used receiver operating characteristic (ROC) curve analysis. Statistical Analyses

The definition of transplantation-related events followed those described in our previous report[34]and cause of death was defined ac-cording to the scheme by Copelan et al.[35]. GVHD was diagnosed and graded according to consensus criteria[36-38]. All categorical variables were compared by chi-square analysis and the Fisher’s exact test. Contin-uous variables were assessed with the Student’s t-test or Mann-Whitney U test for comparison between 2 groups, and Pearson analysis for calculating correlation coefficients. DFS rates were calculated using the Kaplan-Meier survival method, and log-rank analysis was used to evaluate differences between subgroups. Survival analyses were performed from the date of stem cell infusion to the last date of analysis on March 31, 2014. Cumulative incidence of relapse (CIR) and nonrelapse mortality were calculated by cu-mulative incidence estimation, treating nonrelapse deaths and relapse as competing risks, respectively. GVHD was also calculated by cumulative incidence estimates, treating deaths and relapse as competing risks. All results were compared using the Gray test[39]. Multivariate analyses by the Cox’s proportional regression model were used to calculate the survival hazard ratio. The significance of factors affecting the CIR was determined using a semi-parametric model known as the proportional hazard model for subdistribution of competing risks. All statistical analyses were performed using the Statistical Package for Social Sciences, version 14.0 (SPSS Inc., Chicago, IL). Cumulative incidence analyses were carried out with‘R’ soft-ware version 2.15.1 (R Foundation for Statistical Computing, 2012). Statis-tical significance was set at a P value <.05.

RESULTS

Patient Characteristics

Eighty-two patients with a median age of 49 (range, 20 to 66) years were enrolled in the study. Among the patients, 53 of 82 (64.6%) were male, and the median disease duration before transplantation was 8.9 (range 3.0 to 173.8) months. WHO-based diagnoses included 77 MDS, 3 chronic myelo-monocytic leukemia, and 2 MDS/myeloproliferative neoplasm. IPSS scores just before initial treatment showed HrMDS in 41 (50.0%) patients and LrMDS in 41 (50.0%)

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patients. Out of 55 patients (31 HrMDS and 24 LrMDS) who received treatment before HSCT, 1 patient was treated with intensive chemotherapy and 54 were treated with HMA, which led to marrow complete remission in 24 (43.6%) pa-tients, stable disease in 21 (38.2%) papa-tients, and treatment failure in 10 (18.2%) patients (6 [60.0%] HrMDS and 4 [40.0%] LrMDS) at the time of HSCT. Changes in WHO-based di-agnoses or IPSS risk during the clinical course until HSCT are summarized inTable 1. All patients received PB stem cells of which the graft source from donors was matched related (n ¼ 32), suitably matched unrelated (n ¼ 29), or hap-loidentical related (n¼ 21).

Overall Transplantation Outcomes

A total of 28 patients developed acute GVHD grade II (grade II [n¼ 17], grade III [n ¼ 5], and grade IV [n ¼ 6]). The cumulative incidence of acute GVHD grade II at 3 years was 34.8%. Out of the 76 patients who survived at least 100 days with sustained engraftment after HSCT, chronic GVHD developed in 39 patients (mild [n¼ 10], moderate [n ¼ 24], and severe [n¼ 5]), which resulted in an overall cumulative incidence of 55.6%. After a median follow-up of 28.1 months (range, 10.7 to 58.2 months) for surviving transplant re-cipients, 55 patients (67.1%) remained alive. In all, 27 (32.9%) of the 82 patients succumbed; 12 died of relapse and the remaining 15 died of causes other than MDS. The primary causes were acute GVHD (n¼ 5), pneumonia (n ¼ 4), hem-orrhagic cystitis (n¼ 1), cytomegalovirus colitis (n ¼ 1), brain abscess (n¼ 1), viral meningitis (n ¼ 1), pulmonary hem-orrhage (n¼ 1), and organ failure followed by veno-occlusive disease (n¼ 1). Persistent disease at 1 month after HSCT was observed in 3 patients, and after which 12 patients relapsed at a median of 4.3 (range, 2.1 to 24.1) months after HSCT. Their WHO diagnoses at relapse were refractory cytopenia with multilineage dysplasia in 10 patients, refractory anemia with excess blasts (RAEB)-1 in 3 patients, and RAEB-2 in 2 patients; 3 of them remained alive after salvage treatment including DLI, second allo-HSCT, or azacytidine therapy, all of which induced additional GVHD. CIR and nonrelapse mor-tality at 3 years were 21.5% and 16.0%, respectively, and 3-year DFS and overall survival (OS) were 60.9% 2.8% and 65.0% 2.7%, respectively.

WT1 Transcript Levels According to Pre- and Post-HSCT Disease Status

A total of 245 samples were assayed for WT1 transcripts; 82 samples before transplantation, 74 at 1 month, 49 at 2 to 6 months, and 40 at 7 to 12 months. First, we analyzed serial changes of WT1 transcript levels. Median WT1 transcripts were 996.5 copies/104 ABL (range, 10.3 to 15680.0) before transplantation, 60.1 copies/104ABL (range, 8.3 to 65,770.0) at 1 month, 52.2 copies/104ABL (range, 3.5 to 15,830.0) at 2 to 6 months, and 128.5 copies/104ABL (range, 7.7 to 11,310.0) at 7 to 12 months. When we calculated the log reduction of WT1 expression at 1 month after HSCT compared with pre-HSCT levels, the median log reduction was 1.0 log (range,2.4 log to 2.8 log). Comparison of WT1 transcripts between MAC and RIC showed no significant differences in transcript levels at 1 month (median, 48.4 versus 87.7 copies/ 104ABL, P¼ .077). However, the MAC regimen showed more log reduction of WT1 expression (median, 1.3 log versus .8 log, P¼ .026). Next, we determined whether WT1 transcript levels differed according to disease status in pre- and post-HSCT samples. We analyzed WT1 expression levels accord-ing to pre-HSCT disease status from 82 samples, which showed weak correlation with BM blast counts (r¼ .157, P ¼ .025) and IPSS scores (r¼ .189, P ¼ .013). We obtained 163 samples of post-transplantation WT1 transcripts and iden-tified transcript levels according to the relapse status. WT1 transcript levels of 18 samples at various time points after HSCT from 15 relapsed patients were significantly higher in comparison to those from 145 samples in remission (median, 13,420.0 [range, 53.7 to 65,770.0] versus 59.3 [range, 3.5 to 12,510.0] copies/104ABL, P< .001). Further comparison of WT1 transcript levels between relapsed and nonrelapsed samples at given time points showed that median transcript levels were significantly different (Figure 1). The median levels of relapsed versus nonrelapsed patients at 1 month, 2 Table 1

Baseline Characteristics of Enrolled Patients

Characteristic Value

Total no. patients 82

Age, median (range), yr 49 (20-66)

Gender (male) 53 (64.6%)

WHO classification at diagnosis

RCUD or RARS 6 (7.3%) RCMD 30 (36.6%) RAEB-1 18 (22.0%) RAEB-2 21 (25.6%) MDS-u 2 (2.4%) MDS/MPN-u 2 (2.4%) CMML 3 (3.7%)

WHO classification at initial treatments

RCUD or RARS 3 (3.7%) RCMD 30 (36.6%) RAEB-1 11 (13.3%) RAEB-2 30 (36.6%) MDS-u 3 (3.7%) MDS/MPN-u 2 (2.4%) CMML 3 (3.7%)

Karyotype (IPSS risk) at diagnosis

Good 36 (43.9%)

Intermediate 27 (32.9%)

Poor 19 (23.2%)

IPSS score at diagnosis

Low 2 (2.4%)

Intermediate-1 44 (53.7%)

Intermediate-2 27 (32.9%)

High 9 (11.0%)

IPSS score at initial treatments

Low 1 (1.2%)

Intermediate-1 40 (48.8%)

Intermediate-2 33 (40.2%)

High 8 (9.8%)

Time from diagnosis to HSCT, median (range), mo 8.9 (3.0-173.8) Treatment before HSCT

Best supportive care 27 (32.9%)

Azacitidine alone 28 (34.1%)

Decitabine alone 26 (31.7%)

Intensive chemotherapy alone 1 (1.2%) Karyotype (IPSS risk) before HSCT

Good 40 (48.8%)

Intermediate 27 (32.9%)

Poor 15 (18.3%)

Marrow blast before HSCT, % (range) 3.0 (1-17) HSCT donor

Matched sibling donor 32 (39.0%)

Unrelated donor 29 (35.4%)

Haploidentical related donor 21 (25.6%) Conditioning intensity

MAC 33 (40.2%)

RIC 49 (59.8%)

CD34þcells, median (range), 106/kg 5.1 (.4-21.5)

CD3þcells, median (range), 107/kg 41.9 (1.4-118.0)

RCUD indicates refractory cytopenia with unilineage dysplasia; RARS, re-fractory anemia with ringed sideroblast; RCMD, rere-fractory cytopenia with multilineage dysplasia; MDS-u, myelodysplastic syndrome unclassifiable; MPN, myeloproliferative neoplasm; CMML, chronic myelomonocytic leu-kemia.

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to 6 months, and 7 to 12 months were 189.0 versus 57.6, 3730.0 versus 46.1, and 2440.0 versus 109.0 copies/104ABL, respectively (P< .001). In patients with persistent disease or relapse, the median WT1 transcript levels at the time of relapse was 1640 copies/104 ABL (range, 210.0 to 9170.0) in refractory cytopenia with multilineage dysplasia; 10,106 copies/104 ABL (range, 924.0 to 15,830.0) in RAEB-1; and 34,490 copies/104ABL (range, 3210.0 to 65,770.0) in RAEB-2, respectively. As a result, relapsed patients also showed increased WT1 expression at the time of relapse compared with the level at 1 month after HSCT (from 240.3 copies/104 ABL to 1760.0 copies/104ABL, P¼ .093).

Prediction of Subsequent Relapse by WT1 Transcript Levels

Next, we tried to identify the cut-off of WT1 expression levels or its kinetics, which could predict subsequent relapse by using ROC curve analysis on the results before HSCT and those at 1 month. Pre- or post-HSCT WT1 expression results from all 82 patients were analyzed for prediction of subse-quent relapse after WT1 assays. For WT1 expression levels at 1 month after HSCT, results from 71 patients were used to predict relapse after 1 month by using WT1 expression levels or WT1 log reduction, excluding cases with unavailable WT1 results (n¼ 8) or early relapse (n ¼ 3). Neither pre-HSCT WT1 expression nor WT1 log reduction showed a significant cut-off for relapse prediction by ROC curve analysis. On the contrary, 1-month post-HSCT WT1 expression revealed a significant cut-off for subsequent relapse prediction at 154 copies/104 ABL with 71.4% sensitivity and 83.3% specificity (Figure 2A, area under curve¼ .840; P < .001). The 3-year CIR rate was 47.2% (8 of 18 patients) in the higher WT1 group 1 month after HSCT and 6.9% (3 of 53 patients) in the lower WT1 group 1 month after HSCT, which was significantly different (Figure 2B, P < .001). As a result, patients with higher WT1 expression 1 month after HSCT also showed poorer survival outcome. The 3-year DFS and OS rates were 41.7% and 54.3% in the higher WT1 group at 1 month after HSCT, respectively, and 79.0% and 82.1% in the lower WT1 group at 1 month after HSCT, respectively (Figure 2C,D) (P¼ .003 for DFS and P ¼ .033 for OS). Multivariate analyses including clinical predictors showed that higher WT1 expression at 1 month (hazard ratio, 9.94; 95% confidence Figure 1. Median WT1 transcript levels of relapsed and nonrelapsed samples

over the post-HSCT time period.

Figure 2. (A) ROC curve analysis revealed a significant WT1 cut-off level at 1 month after HSCT that could predict subsequent relapse after allo-HSCT. (B-D), Higher (>154 copies/104ABL) WT1 expression at 1 month after HSCT showed significantly higher incidence of subsequent relapse and poorer DFS and OS.

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interval, 2.3 to 43.2; P¼ .002) and poor pre-HSCT karyotype (hazard ratio, 3.52; 95% confidence interval, 1.01 to 12.3, P ¼ .049) were significantly predictive for subsequent relapse, when adjusted with chronic GVHD as a time-dependent covariate. Our data showed there was no definite associa-tion between WT1 expression 1 month after HSCT and development of chronic GVHD. Also, median WT1 expression level 1 month after HSCT of patients with chronic GVHD (71.9 copies/104ABL) and without chronic GVHD (49.8 copies/104 ABL) was not statistically different (P ¼ .578). Higher WT1 expression 1 month also showed poorer DFS but OS was not statistically significant, whereas poor pre-HSCT karyo-type and absence of chronic GVHD were significantly pre-dictive for poorer DFS and OS (Table 2). On the contrary, age, disease duration, graft source, and conditioning intensity were not significantly associated with relapse, DFS, and OS. Subgroup Analysis

As expected, MRD levels at 1 month induced by the conditioning regimen could be further reduced by graft-versus-leukemia (GVL) effects; therefore, we assessed the association of GVHD and relapse among 18 patients with higher WT1 expression 1 month after HSCT. Although the statistical value was not significant, chronic GVHD was associated with a relatively lower incidence of relapse (25.0% versus 60.0%, P¼ .210) and superior DFS (75.0% versus 20.0%, P ¼ .079) among patients with higher WT1 expression 1 month after HSCT, suggesting a preventive role of chronic GVHD in patients with a higher risk of relapse. On the con-trary, chronic GVHD was associated with superior OS (87.5% versus 30.0%, P ¼ .019) in the group with higher WT1 expression 1 month after HSCT (Figure 3).

DISCUSSION

Our current study showed that higher WT1 expression at any time point after HSCT was associated with hematological

relapse of MDS, suggesting that WT1 expression could be a useful marker for disease status of MDS after transplantation. Furthermore, we found that higher WT1 expression 1 month after HSCT could predict subsequent relapse at a significant cut-off level of 154 copies/104ABL in our cohort, thus indi-cating a role of WT1 expression as a relapse predictor. In this study, we evaluated WT1 expression as a marker of MRD and predictor of relapse in a transplantation setting from a rela-tively large cohort consisting of patients with MDS who were treated with the same strategy at a single institution. As our final cut-off of 154 copies/104ABL revealed 8 (44.4%) relapsed

patients out of 18 higher WT1 group with a relatively low sensitivity (71%), we also regarded lower levels of the cut-off associated with a higher sensitivity may be useful to identify more patients at high risk of relapse earlier. However, when we established the cut-off at the lower levels with higher sensitivity (81.8% for 66 copies/104 ABL or 90.9% for 55 copies/104ABL), 9 (28.1%) out of 32 patients and 9 (24.3%) out of 37 patients showed subsequent relapse, respectively. Lower cut-off showed only marginal gain of screening effect of relapse prediction with lower specificity in our cohort.

Although many investigators have evaluated the utility of WT1 expression in MDS, its role in an allo-HSCT setting has not been fully elucidated. In our analyses, wefirst evaluated the role of pre-HSCT WT1 expression, which showed weak correlations with BM blast counts and IPSS scores at the time of HSCT. There was no significant pre-HSCT WT1 cut-off level for prediction of relapse. We reasoned that heterogeneity of pretransplantation treatment modalities in the study cohort might have affected the results. Therefore, the predictive role of pre-HSCT WT1 expression and its kinetics in a trans-plantation setting for MDS should be reassessed in cohorts having homogeneous pre-HSCT treatment, as was previously assessed in AML[26].

Our next analyses on post-HSCT WT1 expression revealed that it could be used as a relapse predictor, similar to Table 2

One-Month Post-HSCT WT1 Additionally Adjusted with Other Parameters for Prediction of Relapse, DFS, and OS

Variables Relapse (CIR) DFS OS

Univariate Multivariate Univariate Multivariate Univariate Multivariate 3-Year CIR P HR (95% CI) P 3-Year DFS P HR (95% CI) P 3-Year OS P HR (95% CI) P Age at diagnosis >50 years 22.9% .574 59.8% .952 65.6% .790 50 years 19.8% 62.2% 67.8% Karyotype at HSCT Poor 41.7% .028* 3.52 (1.0-12.3) .049* 25.0% <.001* 4.55 (1.6-12.4) <.001* 33.3% <.001* 6.58 (2.0-21.3) .001* Not poor 16.6% 69.5% 74.2% BM blast at HSCT >5% 29.7% .139 56.0% .292 59.3% .239 5% 17.3% 63.2% 70.9%

IPSS before treatments

Higher 26.9% .312 52.4% .212 58.8% .163 Lower 14.6% 70.6% 75.1% Acute GVHD (Grade 2) Present 23.1% .796 51.9% .167 60.5% .256 Absent 20.7% 65.2% 69.4% Chronic GVHD Present 13.5% .088 .43 (.1-1.7) .240 78.0% <.001* .29 (.1-.8) .027* 85.8% <.001* .11 (.1-.4) .001* Absent 30.6% 43.6% 47.9% HSCT regimen MAC (FB4) 15.2% .432 62.4% .877 68.9% .390 RIC (FB2 TBI) 26.9% 58.7% 62.1% Post-HSCT 1 month WT1 >154 copies/104ABL 52.5% <.001* 9.94 (2.3-43.2) .002* 37.5% .001* 3.19 (1.2-8.3) .010* 53.0% .028* 2.72 (.9-7.5) .053 154 copies/104ABL 8.7% 77.6% 80.6%

HR indicates hazard ratio; CI, confidential interval; TBI, total body irradiation.

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observations by Lange et al.[16]. They analyzed WT1 tran-script levels from 20 MDS patients in addition to 68 de novo AML patients receiving RIC HSCT for prediction of hemato-logic relapse by 4 months. They showed that PB WT1 expression, BM CD34þdonor chimerism kinetics, and their

combination significantly predict impending hematological relapse within 28 days. Their assays, however, did not work for later relapse, even within the following 84 days. Compared with their study in which WT1 expression levels at 1 month were not shown to be significantly different be-tween the relapsed and nonrelapsed groups, our data showed that WT1 expression levels at 1 month could predict subsequent relapse with 71.4% sensitivity and 83.3% speci-ficity. The discrepancy between the study by Lange et al. and our study could be associated with differences in the number of samples (12 versus 74) evaluated or the source of the WT1 assay (PB versus BM). The higher intensity of our condi-tioning regimen could have also separated those cases with a different risk of relapse at an early time point after HSCT.

Even though we observed a lower specificity of WT1 expression at 1 month compared with the prediction assays proposed by Lange et al., our approach has practical impli-cations for preventing overt hematological relapse in a clin-ical setting. Early identification 1 month after HSCT and preemptive treatment to lessen tumor burden may result in more effective treatment results and allow clinicians to do more careful and step-by-step approaches to minimize treatment toxicities, such as severe GVHD. Slow tapering of immunosuppressive agents or preemptive DLI by escalating doses rather than using bulk doses is an approach that has been used[40]. Also, early identification may optimize the frequency of testing for WT1 expression according to the risk of relapse. More frequent serial monitoring of WT1 expres-sion after 1 month in patients with a high risk of relapse may identify other cases at risk for later relapse. In addition to immune modulation previously reported, another course of immunotherapy or other novel therapies may be used in patients still having higher WT1 expression. Induction of an immune reaction against remnant MDS clones by WT1 cytotoxic T cell therapy, vaccines, or cytokine therapy can be considered[41,42]. Novel agents including HMA, lenalido-mide, or new targeted drugs can also be feasible treatment options, which should be validated in the near future

[8,43,44]. For measurement of WT1 expression, using PB samples might be more convenient for patients compared to using BM samples, although the diagnosis of MDS mainly depends on the morphologicalfindings of BM. To identify the possible utility of PB samples, several studies have already showed that WT1 expression levels in PB and BM are well correlated. Moreover, both of them tend to increase with disease progression, including patients with a higher risk of leukemic transformation in myeloid malignancy[22,25,29]. If a relevant cut-off level of PB WT1 expression for relapse prediction and the range of PB WT1 expression in healthy people are well validated, the results could be sufficient without performing repeated BM aspiration, especially for serial monitoring after 1 month.

MRD at 1 month by quantification of WT1 might not only reflect the amount of MDS clones but could also be a reflection of the sensitivity of MDS clones to chemothera-peutic agents and, therefore, should be strongly associated with the intensity of the conditioning regimen. Actually, our MAC regimen showed a bigger WT1 log reduction and rela-tively lower 1-month post-HSCT WT1 expression compared with the RIC regimen. Of course, the intensity of conditioning was primarily selected by disease risk and its influence on relapse was, therefore, of no importance in our analysis. However, the role of WT1 expression at 1 month remained significant when statistical analyses were performed sepa-rately for MAC and RIC transplantations (data not shown). Figure 3. Clinical outcomes according to the development of chronic GVHD

among those patients with higher WT1 expression at 1 month after HSCT (n¼ 18).

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Early MRD after transplantation can be further changed by GVL effects accompanying acute and chronic GVHD[45,46]. Contrary to acute GVHD, chronic GVHD showed a lower CIR rate and favorable DFS and OS, although its influence on relapse did not reach statistical significance in multivariate analyses combined with WT1 expression at 1 month. How-ever, especially in the 18 patients with higher post-HSCT WT1 expression, 60.0% of patients without chronic GVHD and 25.0% with chronic GVHD relapsed. Although this dif-ference of relapse rate and DFS did not reach statistical po-wer, OS was significantly superior in the chronic GVHD subgroup. We consider the lack of statistical power was caused by a small patient number, and there might be a possible GVL effect accompanied by chronic GVHD. There-fore, induction of chronic GVHD should be considered with caution to minimize its toxicity, especially in high-risk pa-tients with elevated WT1 expression at 1 month after HSCT. Although the results of this study are based on a retro-spective analysis of a limited number of patients, our ob-servations were from a cohort in which a consistent treatment strategy, including immunosuppressive agents and supportive management, were applied. We suggest that WT1 expression 1 month after HSCT is useful for relapse prediction in MDS patients receiving allo-HSCT. Further analysis to identify a certain threshold level of WT1 expres-sion in different conditioning regimens and its association with other molecular markers or gene mutations should be analyzed. Then, those results should be validated in large prospective studies in the near future.

ACKNOWLEDGMENTS

This study was supported by a grant of the Korea Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (A120175).

Financial disclosure: The authors have nothing to disclose. Conflict of interest statement: There are no conflicts of in-terest to report.

Authorship statement: M.K. and Y.-J.K. contributed equally to this work.

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Figure

Updating...

References

  1. http://dx.doi.org/10.1016/j.bbmt.2014.11.008
  2. w w w . b b m t . o r g
  3. Chang C, Storer BE, Scott BL, et al. Hematopoietic cell transplantation inpatients with myelodysplastic syndrome or acute myeloid leukemia
  4. Zeidan AM, Linhares Y, Gore SD. Current therapy of myelodysplasticsyndromes. Blood Rev. 2013;27:243-259
  5. Ades L, Itzykson R, Fenaux P. Myelodysplastic syndromes. Lancet. 2014;383:2239-2252
  6. Hahn T, McCarthy PL Jr, Hassebroek A, et al. Significant improvement insurvival after allogeneic hematopoietic cell transplantation during a
  7. Pavletic SZ, Kumar S, Mohty M, et al. NCI First International Workshopon the Biology, Prevention, and Treatment of Relapse after Allogeneic
  8. 2010;2010:325-329
  9. Parmar S, de Lima M. Hematopoietic stem cell transplantation formyelodysplastic syndrome. Biol Blood Marrow Transplant. 2010;16:
  10. de Lima M, Giralt S, Thall PF, et al. Maintenance therapy with low-doseazacitidine after allogeneic hematopoietic stem cell transplantation for
  11. Bejar R, Levine R, Ebert BL. Unraveling the molecular pathophysiologyof myelodysplastic syndromes. J Clin Oncol. 2011;29:504-515
  12. Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway muta-tions of splicing machinery in myelodysplasia. Nature. 2011;478:
  13. Kulasekararaj AG, Mohamedali AM, Mufti GJ. Recent advances in un-derstanding the molecular pathogenesis of myelodysplastic
  14. 2496-2506
  15. Wu SJ, Kuo YY, Hou HA, et al. The clinical implication of SRSF2 muta-tion in patients with myelodysplastic syndrome and its stability during
  16. Thol F, Kade S, Schlarmann C, et al. Frequency and prognostic impact ofmutations in SRSF2, U2AF1, and ZRSR2 in patients with
  17. 241-247.
  18. Lange T, Hubmann M, Burkhardt R, et al. Monitoring of WT1 expressionin PB and CD34() donor chimerism of BM predicts early relapse in
  19. Bacher U, Talano JA, Bishop MR. Monitoring and prevention of relapseafter allogeneic hematopoietic cell transplantation for myeloid
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  21. 1405-1412
  22. Inoue K, Sugiyama H, Ogawa H, et al. WT1 as a new prognostic factorand a new marker for the detection of minimal residual disease in
  23. Tamaki H, Ogawa H, Ohyashiki K, et al. The Wilms’ tumor gene WT1 isa good marker for diagnosis of disease progression of myelodysplastic
  24. Cilloni D, Gottardi E, De Micheli D, et al. Quantitative assessment ofWT1 expression by real time quantitative PCR may be a useful tool for
  25. Weisser M, Kern W, Rauhut S, et al. Prognostic impact of RT-PCR-basedquantification of WT1 gene expression during MRD monitoring of
  26. Lapillonne H, Renneville A, Auvrignon A, et al. High WT1 expres-sion after induction therapy predicts high risk of relapse and death
  27. Cilloni D, Messa F, Arruga F, et al. Early prediction of treatmentoutcome in acute myeloid leukemia by measurement of WT1 transcript
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  29. Yoon JH, Kim HJ, Shin SH, et al. BAALC and WT1 expressions fromdiagnosis to hematopoietic stem cell transplantation: consecutive
  30. Cilloni D, Gottardi E, Messa F, et al. Significant correlation between thedegree of WT1 expression and the International Prognostic Scoring
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