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Outcome and Minimal Residual Disease Monitoring in Patients with t(16;21) Acute Myelogenous Leukemia Undergoing Allogeneic Hematopoietic Stem Cell Transplantation
Accepted Manuscript Title: Outcome and Minimal Residual Disease Monitoring in t(16;21) Acute Myeloid Leukemia Patients Receiving Allogeneic Hematologic Stem Cell Transplantation Author: Ya-Zhen Qin, Yao Chen, Lan-Ping Xu, Yu Wang, Xiao-Hui Zhang, Huan Chen, Xiao-Su Zhao, Kai-Yan Liu, Xiao-Jun Huang PII: DOI: Reference:
S1083-8791(17)30696-1 http://dx.doi.org/doi: 10.1016/j.bbmt.2017.09.002 YBBMT 54793
To appear in:
Biology of Blood and Marrow Transplantation
Received date: Accepted date:
5-8-2017 1-9-2017
Please cite this article as: Ya-Zhen Qin, Yao Chen, Lan-Ping Xu, Yu Wang, Xiao-Hui Zhang, Huan Chen, Xiao-Su Zhao, Kai-Yan Liu, Xiao-Jun Huang, Outcome and Minimal Residual Disease Monitoring in t(16;21) Acute Myeloid Leukemia Patients Receiving Allogeneic Hematologic Stem Cell Transplantation, Biology of Blood and Marrow Transplantation (2017), http://dx.doi.org/doi: 10.1016/j.bbmt.2017.09.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Outcome and Minimal Residual Disease Monitoring in t(16;21) Acute Myeloid Leukemia Patients Receiving Allogeneic Hematologic Stem Cell Transplantation Ya-Zhen Qin, MS1*; Yao Chen, MD1*; Lan-Ping Xu, MD 1; Yu Wang, MD 1; Xiao-Hui Zhang, MD 1; Huan Chen, MD 1; Xiao-Su Zhao, MD1; Kai-Yan Liu, MD1, Xiao-Jun Huang, MD 1,2** * Equal contributors ** Correspondence: No. 11 Xizhimen South Street, Beijing, China. 100044. Phone: 8610-88326006; Fax: 8610-88326006. [email protected]
1. Peking University People's Hospital, Peking University Institute of Hematology,
Beijing
Key
Laboratory
of
Hematopoietic
Stem
Cell
Transplantation, Beijing, China 2. Peking-Tsinghua Center for Life Sciences, Beijing (100871), China.
Running title Outcome and MRD monitoring in t(16;21)AML receiving allo-HSCT
1
Page 1 of 30
Highlights
t(16;21) AML is an indication for allo-HSCT.
The TLS-ERG transcript levels reflected MRD and may predict relapse after HSCT.
The TLS-ERG transcript levels might guide effective intervention.
ABSTRACT Patients
with
t(16;21)
acute
myeloid
leukemia
(AML)
who
receive
chemotherapy have poor outcomes. The treatment efficacy of allogeneic hematologic stem cell transplantation (allo-HSCT) must be identified, and the usefulness of minimal residual disease (MRD) monitoring requires evaluation. Fourteen t(16;21) AML patients consecutively receiving allo-HSCT at our institute were included in this study. The TLS-ERG transcript levels were serially monitored for a median of 15 (3–51) months after allo-HSCT. Eight patients relapsed, 7 patients died from relapse-related causes, and 1 patient died from a non-relapse-related cause. The 2-year cumulative incidence rates of relapse, disease-free survival and overall survival after HSCT were 66.2%, 30.8% and 46.2%, respectively. Of the 3 patients who received HLA-matched sibling transplantation, 2 relapsed, and 1 (33.3%) was in hematologic complete remission but die of non-relapse mortality, while 5 of 11 patients (45.5%) who received haploidentical transplantation were in complete remission (CR) and were alive. In total, 2 of 6 patients with undetectable TLS-ERG at the time of transplantation individually relapsed at 14 and 15 months, and 3 of 4 PCR-positive patients relapsed at a median of 10 months after HSCT. Four patients with continually low TLS-ERG levels (mostly <0.01%) after HSCT 2
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remained alive and in CR. The TLS-ERG levels of all 8 relapsed patients were significantly increased before the relapse, exceeding 1.0% in all 7 patients who experienced hematologic relapse. In total, 7 patients received modified donor lymphocyte infusions (DLIs), and 1 patient received interferon–a (IFN-a); all 7 patients with TLS-ERG levels >5.0% at the time of intervention experienced an increase or a brief decrease followed by an increase in the level of TLS-ERG, and 6 relapsed, whereas the TLS-ERG levels of 1 patient with TLS-ERG levels <1.0% at intervention were decreased to be undetectable. Therefore, t(16;21) AML is an indication for allo-HSCT. Of the patients who received HSCT, 30.8% responded to the treatment with a CR. The TLS-ERG transcript levels reflected MRD and might predict relapse and guide effective intervention. Key words:
t(16;21) acute myeloid leukemia; TLS-ERG transcript levels;
allogeneic hematologic stem cell transplantation; intervention
3
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INTRODUCTION Translocation t(16;21) (p11;q22) is a rare reciprocal chromosomal change that accounts for approximately 1% of acute myeloid leukemia (AML) cases [1,2]. Although t(16;21) AML is not considered an adverse genetic abnormality, patients with t(16;21) AML have an unfavorable prognosis and poor survival rates [2-5]. Therefore, allogeneic hematologic stem cell transplantation (allo-HSCT) is recommended as a post-remission treatment choice. Compared with chemotherapy, allo-HSCT has been recently shown to prolong survival in t(16;21) AML patients [5]. The potential capability of allo-HSCT to overcome the poor prognosis of patients with t(16;21) AML requires further evaluation. Furthermore, relapse after transplantation remains a challenge. Currently, monitoring minimal residual disease (MRD) has become a routine method for identifying patients with a high risk of relapse and directing preemptive treatment [6-8]; measurements of leukemic-specific fusion transcript levels obtained via the real-time quantitative polymerase chain reaction (RQ-PCR) technique could accurately and sensitively reflect MRD [9-11].
All
t(16;21)
(p11;q22)
forms
of
the
translocation
liposarcoma-ETS-related gene (TLS-ERG) fusion gene, and are potential molecular MRD markers [12]. However, due to the fairly low incidence of t(16;21) AML, there were only case reports on TLS-ERG-based MRD monitoring [13,14]. In the present study, the treatment efficacy of allo-HSCT was evaluated in 14 4
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consecutive t(16;21) AML patients. Furthermore, the prognostic impact of serially monitoring the TLS-ERG transcript levels on relapse prediction and intervention guidance were also evaluated in a preliminary analysis.
PATIENTS AND METHODS Patients and samples In total, 14 t(16;21) AML patients were included in this study. The patients were in complete remission (CR, 1st or 2nd) at the time of HSCT and consecutively received allo-HSCT at our institute between August 2010 and April 2016. The patients were eligible for haploidentical HSCT without matched sibling donors or unrelated donors. The preconditioning consisted of cytarabine (4 g/m2/day, days −10 to −9), busulfan (3.2 mg/kg/day, days −8 to −6), cyclophosphamide (1.8 g/m2/day, days −5 to −4), simustine (250 mg/m 2, day −3) and rabbit anti-thymocyte globulin (ATG) (2.5 mg/kg, days −5 to −2) [15]. The prophylaxis for graft-versus-host disease (GVHD) was the same for all patients [16]. Recipients received cyclosporine (CsA), mycophenolate mofetil, and short-term methotrexate (MTX). A modified donor lymphocyte infusion (DLI) or interferon–a (IFN-a) were administered before hematological relapse as an intervention therapy, and a modified DLI was administered after relapse as a salvage therapy 3 months post-HSCT, followed by a trial of immunosuppressant withdrawal based on the donor availability and willingness [17,18]. Morphological evaluations and quantitative measurements 5
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of the TLS-ERG transcripts in bone marrow (BM) samples were performed at scheduled time points and when patients showed signs of relapse. The scheduled sampling time points were 0, 1, 2, 3, 4.5, 6, 9 and 12 months, followed by once every 6 months post-HSCT. The follow-up cutoff date was July 1, 2017. The study was approved by the Ethics Committee of Peking University People's Hospital, and all patients or their guardians provided written informed consent to participate in this study in accordance with the Declaration of Helsinki.
Measurements of TLS-ERG transcript levels TRIzol reagent (Invitrogen, CA, USA) was used to extract the total RNA. A High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used to synthesize complementary DNA (cDNA). TaqMan-based RQ-PCR was performed as we previously described [19]. The primers and probes for ABL were obtained from the Europe Against Cancer Program report [20], and the primers and probes for TLS-ERG will be provided upon request. Quality control samples were included in each PCR run. All amplifications were performed in at least duplicates. The TLS-ERG transcript level was calculated as the percentage of TLS-ERG transcript copies/ABL copies. The reproducible sensitivity of the RQ-PCR was five copies. All samples with an undetectable fusion transcript had ≥30000 copies of ABL to ensure that TLS-ERG transcript levels >0.002% could be detected. 6
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Definitions All outcomes were measured from the time of the stem cell infusion. Patients who were alive without relapse were censored at the time of the final contrast. The overall survival (OS) was based on death from any cause. The events for measuring the disease-free survival (DFS) included death or relapse. The standard definitions of relapse were used, including BM and/or extra-medullary sites. Non-relapse mortality (NRM) was defined as death without previous relapse. Neutrophil engraftment was defined as the first of 3 successive days with an absolute neutrophil count >0.5 ×109/L after post-transplantation nadir. A platelet engraftment was considered to have occurred in the first of three consecutive days with a platelet count of 20 × 109/L or higher in the absence of platelet transfusion for 7 seven consecutive days. The definitions of the acute GVHD (aGVHD) and chronic GVHD (cGVHD) diagnoses and staging were based on previously described definitions in the literature [21,22].
Statistical analysis The survival functions were estimated using the Kaplan-Meier method and compared using the log-rank test. Comparisons between two groups were performed using the Mann-Whitney U test for continuous variables and Fisher’s exact test for categorical variables. The statistical significance level was set at P ≤ 0.05. R version 2.6.1 (R Foundation for Statistical Computing, 7
Page 7 of 30
Vienna, Austria) and GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA) software were used.
RESULTS Patient characteristics and pre-HSCT treatment The patient characteristics at the time of diagnosis and HSCT are shown in Table 1. In total, 5 patients were children (≤14 years), and 9 patients were adults. Their French-American-British (FAB) subtypes included M2, M4, M5 and M7. In total, 30% of the patients had a complex karyotype. None of the 8 patients
who
were
screened
at
diagnosis
had
RUNX1-RUNX1T1,
CBFB-MYH11, PML-RARA, and MLL-translocations (i.e., MLL-AF4, MLL-AF6, MLL-AF9, MLL-AF10, MLL-ELL and MLL-ENL) or FLT3-ITD and NPM1 mutations. In total, 12 patients achieved CR after 1 course of induction therapy, and 2 patients achieved CR after 2 courses of induction therapy. The patients received 1-3 courses of consolidation therapy before receiving allo-HSCT, and the median interval between diagnosis and HSCT was 6 (4-12) months. In total, 79% of the patients received haploidentical HSCT.
Engraftment and GVHD Engraftment was successful in all patients (100%). A neutrophil count >0.5 ×109/L was reached at a median of 14 days after transplantation (range 11-21 8
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days), and the median time to reach a stable platelet count >20×10 9/L was 14 days (range 10-36 days). The cumulative incidence of grade II-IV aGVHD was 21.4% (3/14). The cumulative incidence of cGVHD was 72.5%.
Transplant outcomes The median follow-up time was 22 (9–56) months from diagnosis and 15 (3–51) months after allo-HSCT. In total, 8 (57.1%) patients experienced relapse at a median of 11 months (range 4–23 months) after HSCT; 2 patients experienced relapse during the first 6 months; 6 patients experienced relapse after 6 months following HSCT; 6 patients suffered hematologic-only relapse; 1 patient suffered extra-medullary-only relapse; and 1 patient suffered extra-medullary followed by hematologic relapse. At the final follow-up, 6 (42.9%) patients were alive, and their median follow-up times were 25 (12-56) months from diagnosis and 20 (9-51) months post- HSCT; 7 patients died from relapse, 1 patient died from NRM, and the NRM rate was 7.1%. The CIR rate was 40.8% (95% confidence interval (CI), 11.2–69.3%) at 1-year and 66.2% (95% CI, 42.4–82.0%) at 2-years, The 1-year DFS and OS rates were 53.8% (95% CI, 24.7–75.9%) and 69.2% (95% CI, 37.3–87.1%), and the 2-year DFS and OS rates were 30.8% (95% CI, 9.5–55.5%) and 46.2% (95% CI, 19.2–69.7%), respectively (Figure 1). In the subgroup analysis, 3 patients receiving human leukocyte antigen (HLA)-matched sibling transplantation were as follows: 1 patient experienced 9
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extra-medullary relapse, 1 patient experienced hematologic relapse and died and 1 patient died of NRM. The remaining 11 patients received haploidentical transplantation; of these patients, 5 patients were in continuous CR and alive, and 4 of them exhibited continuously low MRD levels. In addition, of the 6 patients who were PCR negative (transcript level 0) at the time of transplantation, 4 patients received haploidentical transplantation: 3 remained PCR negative (PTN1-3, Figure 1), and 1 patient was PCR negative for 1 year, followed by a significant increase in TLS-ERG (PTN5, Figure 1). The remaining 2 PCR-negative patients received matched sibling transplantation, and both became PCR-positive and ultimately relapsed (PTN7 and 8, Figure 1).
TLS-ERG transcript levels at diagnosis and pre-HSCT In total, 7 of the 14 patients were screened TLS-ERG transcript levels at diagnosis (Figure 2) with median level 451.1% (range 193.1%-967.6%), and all the other 7 patients had detectable TLS-ERG transcript levels post-HSCT. All 14 patients were tested for TLS-ERG at the time of HSCT, and their median transcript levels were 0.025% (range 0 to 32.7%, Figure 2). At the time of HSCT, 6 patients were PCR negative, and 8 patients were PCR positive with TLS-ERG levels ranging from 0.0095% to 32.7%. Two PCR-negative patients (2/6, 33.3%) relapsed at 14 and 15 months after HSCT, whereas 6 PCR-positive patients (6/8, 75.0%) relapsed at a median of 10 months (range 10
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4-23 months; 5 relapsed within the first 12 months) after HSCT. At the time of HSCT, all 3 patients who relapsed after 12 months had TLS-ERG levels <0.1%, and all 5 patients who relapsed within 12 months had TLS-ERG levels >0.1%. Therefore, the low TLS-ERG transcript levels at the time of HSCT seems to be related to the low relapse rate and late relapse time following HSCT.
TLS-ERG expression patterns post-HSCT Figure 2 shows the sequential TLS-ERG transcript levels pre- and post-HSCT for the entire cohort. The patients were divided into the following 3 groups according to their expression patterns: 1) PTN1 to PTN4 were in continuous CR, received no intervention and were alive at the final follow-up. These patients maintained low TLS-ERG transcript levels after receiving HSCT until the last follow-up (mostly PCR negative or <0.01%, occasionally 0.01%-0.1% in PTN4). 2) PTN5 and PTN6 had TLS-ERG transcript levels that increased over 10% after HSCT. PTN5 received preemptive DLI at a TLS-ERG level of 39.1%, and the TLS-ERG level decreased to 0.03% and then increased to 0.69% at the final follow-up (Figure 3). PTN6 had TLS-ERG levels of 19.3% at 6 months and died of severe GVHD. 3) PTN7 to PTN14 relapsed after HSCT. Before relapse, all patients experienced an increase in TLS-ERG. In total, 7 patients experienced hematologic
relapse
(PTN12
had
extra-medullary
relapse
before 11
Page 11 of 30
hematologic relapse), and the median transcript levels of TLS-ERG in 5 of these patients was 111.8% at the time of hematologic relapse (range 97.4% to 293.6%). The TLS-ERG levels in all 7 patients exceeded 1.0%, and 4 patients showed levels that exceeded 10.0% before hematologic relapse. The TLS-ERG levels in 2 patients were 0.24% and 8.8% at the time of extra-medullary relapse, and their TLS-ERG levels were <0.1% (0.030% and 0.031%) before relapse. Therefore, the TLS-ERG transcript levels accurately represented the MRD levels, and the TLS-ERG dynamics might be relevant to relapse.
Dynamics of TLS-ERG transcript levels after intervention therapy Five patients received preemptive DLI and 1 patient received IFN-a to prevent hematologic relapse, and 2 patients received modified DLI after extra-medullary relapse as a salvage therapy. Figure 3 shows the dynamics of the TLS-ERG transcript levels after the DLI and IFN-a treatments. In total, 7 patients had high levels of the TLS-ERG transcripts (range 5.0% to 170.3%) at the start of the DLI (IFN-a) treatment, and 6 patients experienced hematologic relapse 1-9 months (median 2.5 months) after the DLI or IFN-a treatments. Their TLS-ERG expression patterns were as follows: 4 patients (PTN10, 11, 12, and 14) had continually increased TLS-ERG levels, and the other 3 patients (PTN5, 8, and 13) had briefly decreased followed by increased TLS-ERG levels after the interventions. One patient (PTN7) received DLI when the 12
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TLS-ERG levels were low (0.14%) and remained PCR negative for 4 months after the DLI treatment. Therefore, modified DLI and IFN-a might be effective only when the TLS-ERG transcript levels are low.
DISCUSSION t(16;21) AML is a very rare subtype of AML. Although t(16;21) AML is not considered an adverse genetic abnormality, patients with t(16;21) AML have a poor prognosis [2-5]. According to a review of studies investigating t(16;21) AML that was published in 2009, of the 42 cases with reported information regarding prognosis, only 4 patients were in CR, and only 5 patients were alive at the time of the report [3]. Therefore, the efficacy of intensive treatments, such as allo-HSCT, must be evaluated. The current analysis shows that 30.8% of the patients responded to allogeneic HSCT and were in CR. Furthermore, our previous study showed that haploidentical allografts might have strong anti-leukemia effects and may be superior to matched sibling donor allografts in eradicating pre-transplantation MRD in AML patients [23]. Similarly in this study, of the 3 patients who received HLA-matched sibling transplantation, 2 patients relapsed, and 1 (33.3%) patient was in CR but died of NRM, whereas 5 of the 11 patients (45.5%) who received haploidentical transplantation were in CR and alive. Due to the relatively low incidence rate, transplantation relevant evaluations are scarce, and more clinical data are needed. According to Kong et al, 2 of 19 13
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cases received allo-HSCT, 1 died of NRM, and the remaining cases were alive 22 months following HSCT [2]. Okoshi et al reported that a patient who received HSCT relapsed after 9 months [12]. Kim et al reported a case who suffered from grade IV GVHD after HSCT [4]. Recently, Pan et al evaluated 20 patients with t(16;21) AML and demonstrated that 11 cases who received HSCT had a longer OS than 9 cases who received chemotherapy [5]; however, 8 of the 11 cases had a hematological relapse after transplantation. Similarly, in the current analysis, 8 of 14 patients experienced relapse at a median of 11 months after HSCT. In addition, all patients had successful engraftments, and only 1 patient died of NRM. Therefore, relapse remained the primary cause of treatment failure in t(16;21) AML after HSCT. MRD monitoring plays an increasingly vital role in predicting relapse [6-8]. The TLS-ERG chimeric transcript is the molecular entity of t(16;21) AML and has been implied to represent MRD [2-4]. Pre-transplantation MRD detected by both flow cytometry and RQ-PCR strongly predicts relapse in patients with AML [24-26]. The current study showed a similar tendency, although it is difficult to draw conclusions due to the small sample size. The TLS-ERG negative patients and patients with very low TLS-ERG levels experienced a lower and delayed incidence of relapse post-transplantation. Therefore, MRD might be needed to be decreased to as low as possible at the time of transplantation in t(16;21) AML. The potential role of TLS-ERG follow-up in relapse prediction has not been 14
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comprehensively assessed to date. All 14 patients in the current study were serially monitored for the TLS-ERG transcript levels from the time of transplantation at scheduled time points. The TLS-ERG dynamics were significantly different among patients with different outcomes as follows: patients with good outcomes maintained low or undetectable TLS-ERG levels (<0.1%), whereas a continually high or significant increase in the TLS-ERG levels always occurred prior to relapse, that is, all patients experienced >1.0% of TLS-ERG before hematologic relapse. This preliminary data implied that the threshold for predicting impending hematologic relapse might be between 0.1% and 1.0%. However, the optimal cutoff value for relapse prediction can be identified only after a large sample size is acquired. DLI can be used to effectively prevent impending relapse or as salvage therapy after transplantation [17,27,28]. IFN-a was also effective in preventing relapse [18]. We previously demonstrated that low RUNX1-RUNX1T1 transcript levels in patients with t(8;21) AML at the time of intervention were related to low relapse rates after intervention [29]. This finding is consistent with that observed in patients with t(16;21) AML. After the DLI treatment, the TLS-ERG levels in all 7 patients with high TLS-ERG levels (>5.0%) at the time of the intervention increased, and 6 of these patients relapsed shortly after this increase, whereas 1 patient with low TLS-ERG levels (0.14%) achieved a stable PCR-negative result. Considering that all 5 patients with good outcomes had TLS-ERG levels <0.1%, TLS-ERG transcript levels in the range of 15
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0.1%-1.0% might be indicative of effective intervention; however, this hypothesis must be confirmed in more patients. The limitation of the current study is the small sample size and that it was retrospective. However, although only 14 patients were included, this is the largest study to evaluate the efficacy of allo-HSCT and the first study to comprehensively evaluate the usefulness of molecular MRD monitoring in patients with t(16;21) AML to date. In conclusion, this high-risk rare AML type is an indication for allo-HSCT. Haploidentical donors might be the preferred donor source over HLA-matched sibling donors. In order to reduce relapse, a cohort intervention, including modified preconditioning and new innovative donor cell strategies, might be added and given as an early prophylactic or maintenance strategy. In addition, the serial monitoring of the TLS-ERG transcript levels reflected the MRD and might predict relapse and guide effective interventions. The multi-center prospective study is warranted.
ACKNOWLEDGEMENTS This work was supported by the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (81621001) and the National Natural Science Foundation of China (81530046 and 81570130).
CONFLICTS OF INTEREST The authors have no conflict of interest. 16
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21
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Figure 1. CIR (A), DFS (B) and OS (C) rates of the whole cohort. Figure 2. Sequential TLS-ERG transcript levels at diagnosis, pre- and post-HSCT in 14 patients. Number in the box represents TLS-ERG transcript levels at diagnosis; The arrow represents the intervention; 8 patients received preemptive DLI, and 1 patient received IFN-a. Dx: at diagnosis; UKN: unknown; HR: hematologic relapse; EMR: extra-medullary relapse; NRM: non-relapse mortality. Figure 3. Dynamics of TLS-ERG transcript levels after modified DLI (7 patients, solid lines) and IFN-a (1 patient, broken line) treatments.
22
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Table 1. Patient characteristics Parameters
n=14
At diagnosis Median age, y (range)
24 (5-56)
Sex Male
9 (64%)
Female
5 (36%) 9
Median WBC count (range), ×10 /L
16.3 (1.1-117)
Median hemoglobin level (range), g/dL
98 (40-121)
9
Median PLT count (range), ×10 /L
40 (2-156)
FAB subtype M2
7 (50%)
M4
1 (7%)
M5
5 (36%)
M7
1 (7%)
Cytogenetics Complex karyotype
4 (29%)
Non-complex karyotype
9 (64%)
Unknown
1 (7%)
FLT3-ITD mutations Yes
0 (0%)
No
8 (67%)
Unknown
4 (33%)
During treatment 1st CR induction courses 1
12 (86%)
2
2 (14%)
Disease status when receiving HSCT 1st CR
13 (93%)
2nd CR
1 (7%) 23
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Donor source HLA-matched sibling
3 (21%)
Haploidentical
11 (79%)
24
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Fig 1-survival.tif
25
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Fig 1-survival_bestsetConverted.png
26
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Fig 2-expression patterns.tif
27
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Fig 2-expression patterns_bestsetConverted.png
28
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Fig 3-intervention.tif
29
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Fig 3-intervention_bestsetConverted.png
30
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S1083-8791(17)30696-1 http://dx.doi.org/doi: 10.1016/j.bbmt.2017.09.002 YBBMT 54793
To appear in:
Biology of Blood and Marrow Transplantation
Received date: Accepted date:
5-8-2017 1-9-2017
Please cite this article as: Ya-Zhen Qin, Yao Chen, Lan-Ping Xu, Yu Wang, Xiao-Hui Zhang, Huan Chen, Xiao-Su Zhao, Kai-Yan Liu, Xiao-Jun Huang, Outcome and Minimal Residual Disease Monitoring in t(16;21) Acute Myeloid Leukemia Patients Receiving Allogeneic Hematologic Stem Cell Transplantation, Biology of Blood and Marrow Transplantation (2017), http://dx.doi.org/doi: 10.1016/j.bbmt.2017.09.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Outcome and Minimal Residual Disease Monitoring in t(16;21) Acute Myeloid Leukemia Patients Receiving Allogeneic Hematologic Stem Cell Transplantation Ya-Zhen Qin, MS1*; Yao Chen, MD1*; Lan-Ping Xu, MD 1; Yu Wang, MD 1; Xiao-Hui Zhang, MD 1; Huan Chen, MD 1; Xiao-Su Zhao, MD1; Kai-Yan Liu, MD1, Xiao-Jun Huang, MD 1,2** * Equal contributors ** Correspondence: No. 11 Xizhimen South Street, Beijing, China. 100044. Phone: 8610-88326006; Fax: 8610-88326006. [email protected]
1. Peking University People's Hospital, Peking University Institute of Hematology,
Beijing
Key
Laboratory
of
Hematopoietic
Stem
Cell
Transplantation, Beijing, China 2. Peking-Tsinghua Center for Life Sciences, Beijing (100871), China.
Running title Outcome and MRD monitoring in t(16;21)AML receiving allo-HSCT
1
Page 1 of 30
Highlights
t(16;21) AML is an indication for allo-HSCT.
The TLS-ERG transcript levels reflected MRD and may predict relapse after HSCT.
The TLS-ERG transcript levels might guide effective intervention.
ABSTRACT Patients
with
t(16;21)
acute
myeloid
leukemia
(AML)
who
receive
chemotherapy have poor outcomes. The treatment efficacy of allogeneic hematologic stem cell transplantation (allo-HSCT) must be identified, and the usefulness of minimal residual disease (MRD) monitoring requires evaluation. Fourteen t(16;21) AML patients consecutively receiving allo-HSCT at our institute were included in this study. The TLS-ERG transcript levels were serially monitored for a median of 15 (3–51) months after allo-HSCT. Eight patients relapsed, 7 patients died from relapse-related causes, and 1 patient died from a non-relapse-related cause. The 2-year cumulative incidence rates of relapse, disease-free survival and overall survival after HSCT were 66.2%, 30.8% and 46.2%, respectively. Of the 3 patients who received HLA-matched sibling transplantation, 2 relapsed, and 1 (33.3%) was in hematologic complete remission but die of non-relapse mortality, while 5 of 11 patients (45.5%) who received haploidentical transplantation were in complete remission (CR) and were alive. In total, 2 of 6 patients with undetectable TLS-ERG at the time of transplantation individually relapsed at 14 and 15 months, and 3 of 4 PCR-positive patients relapsed at a median of 10 months after HSCT. Four patients with continually low TLS-ERG levels (mostly <0.01%) after HSCT 2
Page 2 of 30
remained alive and in CR. The TLS-ERG levels of all 8 relapsed patients were significantly increased before the relapse, exceeding 1.0% in all 7 patients who experienced hematologic relapse. In total, 7 patients received modified donor lymphocyte infusions (DLIs), and 1 patient received interferon–a (IFN-a); all 7 patients with TLS-ERG levels >5.0% at the time of intervention experienced an increase or a brief decrease followed by an increase in the level of TLS-ERG, and 6 relapsed, whereas the TLS-ERG levels of 1 patient with TLS-ERG levels <1.0% at intervention were decreased to be undetectable. Therefore, t(16;21) AML is an indication for allo-HSCT. Of the patients who received HSCT, 30.8% responded to the treatment with a CR. The TLS-ERG transcript levels reflected MRD and might predict relapse and guide effective intervention. Key words:
t(16;21) acute myeloid leukemia; TLS-ERG transcript levels;
allogeneic hematologic stem cell transplantation; intervention
3
Page 3 of 30
INTRODUCTION Translocation t(16;21) (p11;q22) is a rare reciprocal chromosomal change that accounts for approximately 1% of acute myeloid leukemia (AML) cases [1,2]. Although t(16;21) AML is not considered an adverse genetic abnormality, patients with t(16;21) AML have an unfavorable prognosis and poor survival rates [2-5]. Therefore, allogeneic hematologic stem cell transplantation (allo-HSCT) is recommended as a post-remission treatment choice. Compared with chemotherapy, allo-HSCT has been recently shown to prolong survival in t(16;21) AML patients [5]. The potential capability of allo-HSCT to overcome the poor prognosis of patients with t(16;21) AML requires further evaluation. Furthermore, relapse after transplantation remains a challenge. Currently, monitoring minimal residual disease (MRD) has become a routine method for identifying patients with a high risk of relapse and directing preemptive treatment [6-8]; measurements of leukemic-specific fusion transcript levels obtained via the real-time quantitative polymerase chain reaction (RQ-PCR) technique could accurately and sensitively reflect MRD [9-11].
All
t(16;21)
(p11;q22)
forms
of
the
translocation
liposarcoma-ETS-related gene (TLS-ERG) fusion gene, and are potential molecular MRD markers [12]. However, due to the fairly low incidence of t(16;21) AML, there were only case reports on TLS-ERG-based MRD monitoring [13,14]. In the present study, the treatment efficacy of allo-HSCT was evaluated in 14 4
Page 4 of 30
consecutive t(16;21) AML patients. Furthermore, the prognostic impact of serially monitoring the TLS-ERG transcript levels on relapse prediction and intervention guidance were also evaluated in a preliminary analysis.
PATIENTS AND METHODS Patients and samples In total, 14 t(16;21) AML patients were included in this study. The patients were in complete remission (CR, 1st or 2nd) at the time of HSCT and consecutively received allo-HSCT at our institute between August 2010 and April 2016. The patients were eligible for haploidentical HSCT without matched sibling donors or unrelated donors. The preconditioning consisted of cytarabine (4 g/m2/day, days −10 to −9), busulfan (3.2 mg/kg/day, days −8 to −6), cyclophosphamide (1.8 g/m2/day, days −5 to −4), simustine (250 mg/m 2, day −3) and rabbit anti-thymocyte globulin (ATG) (2.5 mg/kg, days −5 to −2) [15]. The prophylaxis for graft-versus-host disease (GVHD) was the same for all patients [16]. Recipients received cyclosporine (CsA), mycophenolate mofetil, and short-term methotrexate (MTX). A modified donor lymphocyte infusion (DLI) or interferon–a (IFN-a) were administered before hematological relapse as an intervention therapy, and a modified DLI was administered after relapse as a salvage therapy 3 months post-HSCT, followed by a trial of immunosuppressant withdrawal based on the donor availability and willingness [17,18]. Morphological evaluations and quantitative measurements 5
Page 5 of 30
of the TLS-ERG transcripts in bone marrow (BM) samples were performed at scheduled time points and when patients showed signs of relapse. The scheduled sampling time points were 0, 1, 2, 3, 4.5, 6, 9 and 12 months, followed by once every 6 months post-HSCT. The follow-up cutoff date was July 1, 2017. The study was approved by the Ethics Committee of Peking University People's Hospital, and all patients or their guardians provided written informed consent to participate in this study in accordance with the Declaration of Helsinki.
Measurements of TLS-ERG transcript levels TRIzol reagent (Invitrogen, CA, USA) was used to extract the total RNA. A High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used to synthesize complementary DNA (cDNA). TaqMan-based RQ-PCR was performed as we previously described [19]. The primers and probes for ABL were obtained from the Europe Against Cancer Program report [20], and the primers and probes for TLS-ERG will be provided upon request. Quality control samples were included in each PCR run. All amplifications were performed in at least duplicates. The TLS-ERG transcript level was calculated as the percentage of TLS-ERG transcript copies/ABL copies. The reproducible sensitivity of the RQ-PCR was five copies. All samples with an undetectable fusion transcript had ≥30000 copies of ABL to ensure that TLS-ERG transcript levels >0.002% could be detected. 6
Page 6 of 30
Definitions All outcomes were measured from the time of the stem cell infusion. Patients who were alive without relapse were censored at the time of the final contrast. The overall survival (OS) was based on death from any cause. The events for measuring the disease-free survival (DFS) included death or relapse. The standard definitions of relapse were used, including BM and/or extra-medullary sites. Non-relapse mortality (NRM) was defined as death without previous relapse. Neutrophil engraftment was defined as the first of 3 successive days with an absolute neutrophil count >0.5 ×109/L after post-transplantation nadir. A platelet engraftment was considered to have occurred in the first of three consecutive days with a platelet count of 20 × 109/L or higher in the absence of platelet transfusion for 7 seven consecutive days. The definitions of the acute GVHD (aGVHD) and chronic GVHD (cGVHD) diagnoses and staging were based on previously described definitions in the literature [21,22].
Statistical analysis The survival functions were estimated using the Kaplan-Meier method and compared using the log-rank test. Comparisons between two groups were performed using the Mann-Whitney U test for continuous variables and Fisher’s exact test for categorical variables. The statistical significance level was set at P ≤ 0.05. R version 2.6.1 (R Foundation for Statistical Computing, 7
Page 7 of 30
Vienna, Austria) and GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA) software were used.
RESULTS Patient characteristics and pre-HSCT treatment The patient characteristics at the time of diagnosis and HSCT are shown in Table 1. In total, 5 patients were children (≤14 years), and 9 patients were adults. Their French-American-British (FAB) subtypes included M2, M4, M5 and M7. In total, 30% of the patients had a complex karyotype. None of the 8 patients
who
were
screened
at
diagnosis
had
RUNX1-RUNX1T1,
CBFB-MYH11, PML-RARA, and MLL-translocations (i.e., MLL-AF4, MLL-AF6, MLL-AF9, MLL-AF10, MLL-ELL and MLL-ENL) or FLT3-ITD and NPM1 mutations. In total, 12 patients achieved CR after 1 course of induction therapy, and 2 patients achieved CR after 2 courses of induction therapy. The patients received 1-3 courses of consolidation therapy before receiving allo-HSCT, and the median interval between diagnosis and HSCT was 6 (4-12) months. In total, 79% of the patients received haploidentical HSCT.
Engraftment and GVHD Engraftment was successful in all patients (100%). A neutrophil count >0.5 ×109/L was reached at a median of 14 days after transplantation (range 11-21 8
Page 8 of 30
days), and the median time to reach a stable platelet count >20×10 9/L was 14 days (range 10-36 days). The cumulative incidence of grade II-IV aGVHD was 21.4% (3/14). The cumulative incidence of cGVHD was 72.5%.
Transplant outcomes The median follow-up time was 22 (9–56) months from diagnosis and 15 (3–51) months after allo-HSCT. In total, 8 (57.1%) patients experienced relapse at a median of 11 months (range 4–23 months) after HSCT; 2 patients experienced relapse during the first 6 months; 6 patients experienced relapse after 6 months following HSCT; 6 patients suffered hematologic-only relapse; 1 patient suffered extra-medullary-only relapse; and 1 patient suffered extra-medullary followed by hematologic relapse. At the final follow-up, 6 (42.9%) patients were alive, and their median follow-up times were 25 (12-56) months from diagnosis and 20 (9-51) months post- HSCT; 7 patients died from relapse, 1 patient died from NRM, and the NRM rate was 7.1%. The CIR rate was 40.8% (95% confidence interval (CI), 11.2–69.3%) at 1-year and 66.2% (95% CI, 42.4–82.0%) at 2-years, The 1-year DFS and OS rates were 53.8% (95% CI, 24.7–75.9%) and 69.2% (95% CI, 37.3–87.1%), and the 2-year DFS and OS rates were 30.8% (95% CI, 9.5–55.5%) and 46.2% (95% CI, 19.2–69.7%), respectively (Figure 1). In the subgroup analysis, 3 patients receiving human leukocyte antigen (HLA)-matched sibling transplantation were as follows: 1 patient experienced 9
Page 9 of 30
extra-medullary relapse, 1 patient experienced hematologic relapse and died and 1 patient died of NRM. The remaining 11 patients received haploidentical transplantation; of these patients, 5 patients were in continuous CR and alive, and 4 of them exhibited continuously low MRD levels. In addition, of the 6 patients who were PCR negative (transcript level 0) at the time of transplantation, 4 patients received haploidentical transplantation: 3 remained PCR negative (PTN1-3, Figure 1), and 1 patient was PCR negative for 1 year, followed by a significant increase in TLS-ERG (PTN5, Figure 1). The remaining 2 PCR-negative patients received matched sibling transplantation, and both became PCR-positive and ultimately relapsed (PTN7 and 8, Figure 1).
TLS-ERG transcript levels at diagnosis and pre-HSCT In total, 7 of the 14 patients were screened TLS-ERG transcript levels at diagnosis (Figure 2) with median level 451.1% (range 193.1%-967.6%), and all the other 7 patients had detectable TLS-ERG transcript levels post-HSCT. All 14 patients were tested for TLS-ERG at the time of HSCT, and their median transcript levels were 0.025% (range 0 to 32.7%, Figure 2). At the time of HSCT, 6 patients were PCR negative, and 8 patients were PCR positive with TLS-ERG levels ranging from 0.0095% to 32.7%. Two PCR-negative patients (2/6, 33.3%) relapsed at 14 and 15 months after HSCT, whereas 6 PCR-positive patients (6/8, 75.0%) relapsed at a median of 10 months (range 10
Page 10 of 30
4-23 months; 5 relapsed within the first 12 months) after HSCT. At the time of HSCT, all 3 patients who relapsed after 12 months had TLS-ERG levels <0.1%, and all 5 patients who relapsed within 12 months had TLS-ERG levels >0.1%. Therefore, the low TLS-ERG transcript levels at the time of HSCT seems to be related to the low relapse rate and late relapse time following HSCT.
TLS-ERG expression patterns post-HSCT Figure 2 shows the sequential TLS-ERG transcript levels pre- and post-HSCT for the entire cohort. The patients were divided into the following 3 groups according to their expression patterns: 1) PTN1 to PTN4 were in continuous CR, received no intervention and were alive at the final follow-up. These patients maintained low TLS-ERG transcript levels after receiving HSCT until the last follow-up (mostly PCR negative or <0.01%, occasionally 0.01%-0.1% in PTN4). 2) PTN5 and PTN6 had TLS-ERG transcript levels that increased over 10% after HSCT. PTN5 received preemptive DLI at a TLS-ERG level of 39.1%, and the TLS-ERG level decreased to 0.03% and then increased to 0.69% at the final follow-up (Figure 3). PTN6 had TLS-ERG levels of 19.3% at 6 months and died of severe GVHD. 3) PTN7 to PTN14 relapsed after HSCT. Before relapse, all patients experienced an increase in TLS-ERG. In total, 7 patients experienced hematologic
relapse
(PTN12
had
extra-medullary
relapse
before 11
Page 11 of 30
hematologic relapse), and the median transcript levels of TLS-ERG in 5 of these patients was 111.8% at the time of hematologic relapse (range 97.4% to 293.6%). The TLS-ERG levels in all 7 patients exceeded 1.0%, and 4 patients showed levels that exceeded 10.0% before hematologic relapse. The TLS-ERG levels in 2 patients were 0.24% and 8.8% at the time of extra-medullary relapse, and their TLS-ERG levels were <0.1% (0.030% and 0.031%) before relapse. Therefore, the TLS-ERG transcript levels accurately represented the MRD levels, and the TLS-ERG dynamics might be relevant to relapse.
Dynamics of TLS-ERG transcript levels after intervention therapy Five patients received preemptive DLI and 1 patient received IFN-a to prevent hematologic relapse, and 2 patients received modified DLI after extra-medullary relapse as a salvage therapy. Figure 3 shows the dynamics of the TLS-ERG transcript levels after the DLI and IFN-a treatments. In total, 7 patients had high levels of the TLS-ERG transcripts (range 5.0% to 170.3%) at the start of the DLI (IFN-a) treatment, and 6 patients experienced hematologic relapse 1-9 months (median 2.5 months) after the DLI or IFN-a treatments. Their TLS-ERG expression patterns were as follows: 4 patients (PTN10, 11, 12, and 14) had continually increased TLS-ERG levels, and the other 3 patients (PTN5, 8, and 13) had briefly decreased followed by increased TLS-ERG levels after the interventions. One patient (PTN7) received DLI when the 12
Page 12 of 30
TLS-ERG levels were low (0.14%) and remained PCR negative for 4 months after the DLI treatment. Therefore, modified DLI and IFN-a might be effective only when the TLS-ERG transcript levels are low.
DISCUSSION t(16;21) AML is a very rare subtype of AML. Although t(16;21) AML is not considered an adverse genetic abnormality, patients with t(16;21) AML have a poor prognosis [2-5]. According to a review of studies investigating t(16;21) AML that was published in 2009, of the 42 cases with reported information regarding prognosis, only 4 patients were in CR, and only 5 patients were alive at the time of the report [3]. Therefore, the efficacy of intensive treatments, such as allo-HSCT, must be evaluated. The current analysis shows that 30.8% of the patients responded to allogeneic HSCT and were in CR. Furthermore, our previous study showed that haploidentical allografts might have strong anti-leukemia effects and may be superior to matched sibling donor allografts in eradicating pre-transplantation MRD in AML patients [23]. Similarly in this study, of the 3 patients who received HLA-matched sibling transplantation, 2 patients relapsed, and 1 (33.3%) patient was in CR but died of NRM, whereas 5 of the 11 patients (45.5%) who received haploidentical transplantation were in CR and alive. Due to the relatively low incidence rate, transplantation relevant evaluations are scarce, and more clinical data are needed. According to Kong et al, 2 of 19 13
Page 13 of 30
cases received allo-HSCT, 1 died of NRM, and the remaining cases were alive 22 months following HSCT [2]. Okoshi et al reported that a patient who received HSCT relapsed after 9 months [12]. Kim et al reported a case who suffered from grade IV GVHD after HSCT [4]. Recently, Pan et al evaluated 20 patients with t(16;21) AML and demonstrated that 11 cases who received HSCT had a longer OS than 9 cases who received chemotherapy [5]; however, 8 of the 11 cases had a hematological relapse after transplantation. Similarly, in the current analysis, 8 of 14 patients experienced relapse at a median of 11 months after HSCT. In addition, all patients had successful engraftments, and only 1 patient died of NRM. Therefore, relapse remained the primary cause of treatment failure in t(16;21) AML after HSCT. MRD monitoring plays an increasingly vital role in predicting relapse [6-8]. The TLS-ERG chimeric transcript is the molecular entity of t(16;21) AML and has been implied to represent MRD [2-4]. Pre-transplantation MRD detected by both flow cytometry and RQ-PCR strongly predicts relapse in patients with AML [24-26]. The current study showed a similar tendency, although it is difficult to draw conclusions due to the small sample size. The TLS-ERG negative patients and patients with very low TLS-ERG levels experienced a lower and delayed incidence of relapse post-transplantation. Therefore, MRD might be needed to be decreased to as low as possible at the time of transplantation in t(16;21) AML. The potential role of TLS-ERG follow-up in relapse prediction has not been 14
Page 14 of 30
comprehensively assessed to date. All 14 patients in the current study were serially monitored for the TLS-ERG transcript levels from the time of transplantation at scheduled time points. The TLS-ERG dynamics were significantly different among patients with different outcomes as follows: patients with good outcomes maintained low or undetectable TLS-ERG levels (<0.1%), whereas a continually high or significant increase in the TLS-ERG levels always occurred prior to relapse, that is, all patients experienced >1.0% of TLS-ERG before hematologic relapse. This preliminary data implied that the threshold for predicting impending hematologic relapse might be between 0.1% and 1.0%. However, the optimal cutoff value for relapse prediction can be identified only after a large sample size is acquired. DLI can be used to effectively prevent impending relapse or as salvage therapy after transplantation [17,27,28]. IFN-a was also effective in preventing relapse [18]. We previously demonstrated that low RUNX1-RUNX1T1 transcript levels in patients with t(8;21) AML at the time of intervention were related to low relapse rates after intervention [29]. This finding is consistent with that observed in patients with t(16;21) AML. After the DLI treatment, the TLS-ERG levels in all 7 patients with high TLS-ERG levels (>5.0%) at the time of the intervention increased, and 6 of these patients relapsed shortly after this increase, whereas 1 patient with low TLS-ERG levels (0.14%) achieved a stable PCR-negative result. Considering that all 5 patients with good outcomes had TLS-ERG levels <0.1%, TLS-ERG transcript levels in the range of 15
Page 15 of 30
0.1%-1.0% might be indicative of effective intervention; however, this hypothesis must be confirmed in more patients. The limitation of the current study is the small sample size and that it was retrospective. However, although only 14 patients were included, this is the largest study to evaluate the efficacy of allo-HSCT and the first study to comprehensively evaluate the usefulness of molecular MRD monitoring in patients with t(16;21) AML to date. In conclusion, this high-risk rare AML type is an indication for allo-HSCT. Haploidentical donors might be the preferred donor source over HLA-matched sibling donors. In order to reduce relapse, a cohort intervention, including modified preconditioning and new innovative donor cell strategies, might be added and given as an early prophylactic or maintenance strategy. In addition, the serial monitoring of the TLS-ERG transcript levels reflected the MRD and might predict relapse and guide effective interventions. The multi-center prospective study is warranted.
ACKNOWLEDGEMENTS This work was supported by the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (81621001) and the National Natural Science Foundation of China (81530046 and 81570130).
CONFLICTS OF INTEREST The authors have no conflict of interest. 16
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measurable ('minimal') residual disease via multiparameter flow cytometry in adult acute myeloid leukemia. Leukemia. 2016;30:1456-1464. 25. Buckley SA, Wood BL, Othus M, et al. Minimal residual disease prior to
allogeneic hematopoietic cell transplantation in acute myeloid leukemia: a meta-analysis. Haematologica. 2017;102:865-873. 26. Ino K, Fuji S, Tajima K, et al. Clinical Utility of Wilms' Tumor 1 Monitoring in
Patients with Myeloid Malignancy and Prior Allogeneic Hematopoietic Stem Cell Transplantation. Biol Blood Marrow Transplant. 2017 Jun 30. [Epub ahead of print] 27. Takami A, Yano S, Yokoyama H, et al. Donor lymphocyte infusion for the
treatment
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hematopoietic stem cell transplantation: a retrospective analysis by the Adult Acute Myeloid Leukemia Working Group of the Japan Society for Hematopoietic Cell Transplantation. Biol Blood Marrow Transplant. 20
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2014;20:1785-1790. 28. Tsirigotis P, Byrne M, Schmid C, Baron F, Ciceri F, Esteve J, et al.
Relapse of AML after hematopoietic stem cell transplantation: methods of monitoring and preventive strategies. A review from the ALWP of the EBMT. Bone Marrow Transplant. 2016;51:1431-1438. 29. Qin YZ, Wang Y, Xu LP, et al. The dynamics of RUNX1-RUNX1T1
transcript levels after allogeneic hematopoietic stem cell transplantation predict relapse in patients with t(8;21) acute myeloid leukemia. J Hematol Oncol. 2017;10:44.
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Figure 1. CIR (A), DFS (B) and OS (C) rates of the whole cohort. Figure 2. Sequential TLS-ERG transcript levels at diagnosis, pre- and post-HSCT in 14 patients. Number in the box represents TLS-ERG transcript levels at diagnosis; The arrow represents the intervention; 8 patients received preemptive DLI, and 1 patient received IFN-a. Dx: at diagnosis; UKN: unknown; HR: hematologic relapse; EMR: extra-medullary relapse; NRM: non-relapse mortality. Figure 3. Dynamics of TLS-ERG transcript levels after modified DLI (7 patients, solid lines) and IFN-a (1 patient, broken line) treatments.
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Table 1. Patient characteristics Parameters
n=14
At diagnosis Median age, y (range)
24 (5-56)
Sex Male
9 (64%)
Female
5 (36%) 9
Median WBC count (range), ×10 /L
16.3 (1.1-117)
Median hemoglobin level (range), g/dL
98 (40-121)
9
Median PLT count (range), ×10 /L
40 (2-156)
FAB subtype M2
7 (50%)
M4
1 (7%)
M5
5 (36%)
M7
1 (7%)
Cytogenetics Complex karyotype
4 (29%)
Non-complex karyotype
9 (64%)
Unknown
1 (7%)
FLT3-ITD mutations Yes
0 (0%)
No
8 (67%)
Unknown
4 (33%)
During treatment 1st CR induction courses 1
12 (86%)
2
2 (14%)
Disease status when receiving HSCT 1st CR
13 (93%)
2nd CR
1 (7%) 23
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Donor source HLA-matched sibling
3 (21%)
Haploidentical
11 (79%)
24
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Fig 1-survival.tif
25
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Fig 1-survival_bestsetConverted.png
26
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Fig 2-expression patterns.tif
27
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Fig 2-expression patterns_bestsetConverted.png
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Fig 3-intervention.tif
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Fig 3-intervention_bestsetConverted.png
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