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Prognostic implication of gene mutations on overall survival in the adult acute myeloid leukemia patients receiving or not receiving allogeneic hematopoietic stem cell transplantations
- We evaluate prognostic impact of mutations in AML patients receiving allo-HSCT.
- FLT3-ITD and mutation ofNPM1do not affect the outcome.
- CEBPAdouble muthas a trend to be an independent good prognostic factor.
- Surprisingly,RUNX1mutation is an independent good prognostic factor.
Several gene mutations have been shown to provide clinical implications in patients with acute myeloid leukemia (AML). However, the prognostic impact of gene mutations in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains unclear. We retrospectively evaluated the clinical implications of 8 gene mutations in 325 adult AML patients; 100 of them received allo-HSCT and 225 did not. The genetic alterations analyzed includedNPM1, FLT3-ITD, FLT3-TKD, CEBPA, RUNX1, RAS, MLL-PTD, andWT1. In patients who did not receive allo-HSCT, older age, higher WBC count, higher lactate dehydrogenase level, unfavorable karyotype, andRUNX1mutation were significantly associated with poor overall survival (OS), whileCEBPAdouble mutation (CEBPAdouble-mut) andNPM1mut/FLT3-ITDnegwere associated with good outcome. However, in patients who received allo-HSCT, only refractory disease status at the time of HSCT and unfavorable karyotype were independent poor prognostic factors. Surprisingly,RUNX1mutation was an independent good prognostic factor for OS in multivariate analysis. The prognostic impact ofFLT3-ITD orNPM1mut/FLT3-ITDnegwas lost in this group of patients receiving allo-HSCT, whileCEBPAdouble-mutshowed a trend to be a good prognostic factor. In conclusion, allo-HSCT can ameliorate the unfavorable influence of some poor-risk gene mutations in AML patients. Unexpectedly, theRUNX1mutation showed a favorable prognostic impact in the context of allo-HSCT. These results need to be confirmed by further studies with more AML patients.
Keywords: Acute myeloid leukemia, Gene mutations, Allogeneic hematopoietic stem cell transplantation.
Acute myeloid leukemia (AML) is a heterogeneous neoplasm with great variability in the pathogenesis, response to therapy, and clinical outcome  . Thus, individualized treatment strategy according to patients’ risk and prognostic factors is the key to achieve better survival. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most powerful treatment modalities, but it also brings considerable treatment related risks to AML patients. Hence, careful selection of patients who may benefit from allo-HSCT is essential to get the best survival results  .
Several gene mutations were shown to have clinical implications in AML patients, , and , especially in those with normal karyotype, , and . Yet, the prognostic relevance of these gene mutations was derived without taking the effect of HSCT into account. Cytogenetic risk factors have been routinely used as guides to choose proper AML patients for allo-HSCT and . Recent reports suggested that gene mutations might also have similar prognostic implications in this aspect. Cytogenetically normal AML patients with mutantCEBPAor mutantNPM1but withoutFLT3-ITD (NPM1mut/FLT3-ITDneg) have more favorable prognosis  , but these patients do not benefit from allo-HSCT in the first complete remission (CR) when compared with traditional consolidation chemotherapy alone  . In contrast, allo-HSCT was shown to have a beneficial effect in AML patients with other genotypes includingFLT3-ITD orNPM1wild/FLT3-ITDneg. However, different result was reported in patients withFLT3-ITD.  Interestingly, a report showed thatRUNX1mutation, a poor-risk genotype  , was associated with better relapse-free survival in patients receiving allo-HSCT  . Nevertheless, the available literature concerning clinical implications of gene mutations in AML patients receiving all-HSCT is still very limited. Therefore, we investigated the prognostic implication of gene mutations on clinical outcomes in this clinical epidemiologic study by comparing survival between patients with and without gene mutations among 100 AML patients who received allo-HSCT and 225 others who did not.
2. Subjects and methods
This study was approved by the Institutional Review Board of the National Taiwan University Hospital (NTUH) in advance. The written informed consents were obtained from all participants. A total of 542 patients with newly diagnosedde novoAML at the NTUH from 1995 to 2007 were screened for gene mutations. Pediatric patients less than 16 years of age and patients with acute promyelocytic leukemia were excluded from this study. And, 138 AML patients who received only palliative or supportive cares due to comorbidity or based on patient's own decision were not included in analysis. Finally, 325 AML patients who received conventional induction chemotherapy (idarubicin 12 mg/m2per day on days 1–3 and cytarabine 100 mg/m2per day on days 1–7) were recruited into this study. Treatment response was defined according to the criteria of Cheson et al.  . Among the 325 recruited AML patients, 100 received allo-HSCT (the allo-HSCT group) and 225 did not (the non allo-HSCT group). For patients who did not received allo-HSCT in first complete remission, 3 to 4 courses of consolidation chemotherapy with high dose cytarabine (2 g/m2q12 h for 4 days) with or without an anthracycline (idarubicin or mitoxatrone) were given after achievement of complete remission and . Patients who received allo-HSCT in first CR might receive 1 or 2 courses of consolidation chemotherapy before transplantation was performed. The median follow-up time of them was 64 months. Overall survival (OS) was defined as the time period from diagnosis to death or end of follow-up.
Detailed transplant related characteristics are summarized in Supplementary Table 1. Briefly, 75 patients received peripheral blood stem cell transplantation, 24 patients received bone marrow transplantation, and 1 patient received double-unit cord blood stem cell transplantation. The choices of allo-HSCT for AML patients were made by the responsible attending physicians based on patients’ demographic and clinical characteristics. Patients with age <60 years and unfavorable karyotype usually received allo-HSCT in the first CR if HLA-matched donors were available, whereas patients with favorable karyotype did not receive allo-HSCT until relapse. The HLA typing was performed by intermediate-resolution (1995–2007) or sequence-based genotyping methods (since 2007) for loci A, B, C, DR, and DQ. All unrelated donors, except that of the cord blood, came from the Buddhist Tzu-Chi Stem Cell Center in Taiwan. Unrelated donors were either fully matched in six HLA (A, B, and DR) loci (n = 15) or mismatched in one locus (5/6 loci,n = 3). Haploidentical donors (n = 9) have varied HLA matching at 5–9/10 loci.
The choices of conditioning regimens in the allo-HSCT group were also determined by physicians’ judgments. Standard busulfan plus cyclophosphamide (BuCy, 48 out of 77 patients) or total body irradiation plus cyclophosphamide (TBICy, 29 out of 77) was used for the myeloablative conditioning (n = 77), whereas reduced intensity conditioning (RIC) with fludarabine 150 mg/m2combined with half-dose BuCy was used for the 23 patients with comorbidity. Anti-thymocyte globulin (thymoglobulin, Sanofi, France) 5 mg/kg was given for matched unrelated donor and 6–7.5 mg/kg for mismatched and haploidentical transplantations. Additional fludarabine 90 to 150 mg/m2was used in haploidentical transplantations. Cyclosporin-A was used in all patients as the first line graft versus host disease prevention plus either methotrexate in patients receiving myeloablative conditioning (n = 77) or mycophenolate mofetil in patients receiving RIC (n = 23).
Bone marrow cells were harvested directly or after 1 to 3 days of unstimulated culture as previously described  . Metaphase chromosomes were banded by the trypsin-Giemsa method and karyotyped according to the International System for Human Cytogenetic Nomenclature. Cytogenetic risk was stratified according to the refined classification of the MRC trial  .
2.5. Statistical analysis
All statistical analyses were performed using the SPSS software, version 17 (SPSS Inc., Chicago, IL, U.S.A.) and the XLSTAT software for Microsoft Excel, version 2010.5.02 (Addinsoft, New York, NY, U.S.A.). In statistical testing, two-sidedPvalue ≤0.05 was considered statistically significant. The distributional properties of continuous variables were expressed by median and range, categorical variables were presented by frequency and percentage, and the survival curve of OS was estimated by the Kaplan–Meier method. In univariate analysis, the Mann–WhitneyUtest, chi-square test or Fisher's exact test (if an expected cell frequency of a contingency table <5), and log-rank test were used to examine the differences in the distributions of continuous, categorical, and OS variables between the allo-HSCT and non allo-HSCT groups, respectively. Next, multivariate analysis was conducted by fitting Cox's proportional hazards models to the AML patients in the allo-HSCT and non allo-HSCT groups for identifying important prognostic factors of OS in them.
The clinical characteristics and laboratory features for the patients receiving allo-HSCT (n = 100) and those without (n = 225) were shown in Table 1 . Except that patients receiving allo-HSCT were younger (35.4 yearsvs.49.5 years,P < 0.001), there was no difference in other clinical parameters between the two groups. The prevalence of most gene mutations including mutations ofCEBPA(double or single),RUNX1,NRAS,FLT3-ITD,FLT3-TKD, andMLL-PTD were similar between allo-HSCT group and non allo-HSCT group ( Table 2 ). However, the prevalence ofNPM1mutation was lower (13%vs.25%,P = 0.016) and that ofWT1mutation was higher (13% vs. 6%,P = 0.05) in allo-HSCT group. There was no difference in the distribution ofNPM1mut/FLT3-ITDnegbetween the two groups. In allo-HSCT group, the source of hematopoietic stem cells came from HLA-identical sibling donors in 73 patients, from unrelated donors in 18, and haploidentical family donors in nine patients. The median time from diagnosis to allo-HSCT was 6.3 months. At the time of allo-HSCT, 42 patients were in first CR, 17 in second or third CR and 41 in relapsed/refractory status. Disease status at the time of transplant stratified by gene alterations is summarized in Supplementary Table 2.
|Variable||Total (n = 325)||Allo-HSCT (n = 100)||Non allo-HSCT (n = 225)||P value|
|Age a||45 (16–85)||35 (17–60)||50 (16–85)||<0.001|
|WBC (×109 L−1) a||22.59 (0.12–627.8)||20.02 (0.98–420.3)||23.32 (0.12–627.8)||0.349|
|Hb (g/dl) a||8.0 (2.9–14.0)||7.8 (2.9–13.6)||8.1 (3.0–14.0)||0.515|
|Platelet (×109 L−1) a||46 (3–802)||51 (5–712)||45 (3–802)||0.414|
|LDH (U/L) a||857.5 (206–13130)||832 (206–8693)||877 (291–13,130)||0.390|
|M0||5 (1.5%)||3 (3%)||2 (9%)|
|M1||85 (26.2%)||30 (30%)||55 (24.4%)|
|M2||126 (38.8%)||31 (31%)||95 (42.2%)|
|M4||82 (25.2%)||27 (27%)||55 (24.4%)|
|M5||16 (4.9%)||5 (5%)||11 (4.9%)|
|M6||9 (2.8%)||4 (4%)||5 (2.2%)|
|M7||0 (0%)||0 (0%)||0 (0%)|
|Unclassified||2 (0.6%)||0 (0%)||2 (0.9%)|
|Karyotypeb and c||0.175|
|Favorable||51 (16.1%)||10 (10.4%)||41 (18.6%)|
|Intermediate||225 (71.2%)||72 (75.0%)||153 (69.5%)|
|Unfavorable||40 (12.7%)||14 (14.6%)||26 (11.8%)|
a Median (range).
b Number of patients (%).
c Chromosome data at diagnosis was available in 316 patients including 96 in the allo-HSCT group and 220 in the non allo-HSCT group. Classification was made according to the refined MRC 2010 criteria. (Grimwade et al., Blood 2010.) Abbreviation: WBC, white blood cell count; Hb, hemoglobin; LDH, lactate dehydrogenase; FAB, French–American–British classification; HSCT, hematopoietic stem cell transplantation.
|Gene mutation||Number of patients with mutation (%)||P value|
|Total population (n = 325)||Allo-HSCT (n = 100)||Non allo-HSCT (n = 225)|
|FLT3-ITD||81 (25%)||25 (25%)||56 (25%)||0.983|
|NPM1||69 (21%)||13 (13%)||56 (25%)||0.016|
|NPMmut/FLT3ITDneg||33 (10%)||7 (7%)||26 (12%)||0.209|
|CEBPA||54 (17%)||15 (15%)||39 (17%)||0.632|
|CEBPA double mut||42 (13%)||9 (9%)||33 (15%)||0.210|
|NRAS||41 (13%)||15 (15%)||26 (12%)||0.388|
|RUNX1||36 (11%)||12 (12%)||24 (11%)||0.724|
|WT1||27 (8%)||13 (13%)||14 (6%)||0.050|
|FLT3-TKD||25 (8%)||9 (9%)||16 (7%)||0.555|
|MLL-PTD||18 (6%)||8 (8%)||10 (4%)||0.199|
In non allo-HSCT patients, older age (≥45 years), higher initial white blood cell (WBC) counts (≥50 × 109 L−1), elevated lactate dehydrogenase (LDH) levels (>460 U/L), unfavorable karyotypes (by refined MRC classification  : abn(3q) [excluding t(3;5)], inv(3)/t(3;3), −5, add(5q)/del(5q), −7, add(7q)/del(7q), t(6;11), t(10;11), t(11q23) [excluding t(9;11) and t(11;19)], t(9;22), −17/abn(17p), and complex changes with ≥4 unrelated abnormalities),FLT3-ITD andRUNX1mutations were all significantly associated with poorer OS in univariate analysis. On the contrary,NPM1mut/FLT3-ITDnegandCEBPAdouble mutations (CEBPAdouble-mut) were significant good prognostic factors. The Kaplan–Meier survival curves stratified byCEBPAdouble-mut,RUNX1mutation andFLT3-ITD are shown in Fig. 1 a,c, and e, respectively.MLL-PTD showed a trend to be a poor prognostic factor (P = 0.053). Other parameters, such as gender, platelet counts, hemoglobin levels, and mutations ofNRAS, WT1andFLT3-TKD had no significant impact on OS. In multivariate analysis, older age, higher WBC count, unfavorable karyotypes, mutations ofRUNX1,CEBPAdouble mutandNPM1mut/FLT3-ITDnegremained to be independent prognostic factors in non allo-HSCT group, while elevated LDH levels had borderline significance ( Table 3 ). In the allo-HSCT group, univariate analyses showed that only three variables were significant prognostic factors for OS: disease status before HSCT, use of HLA-identical sibling donor and unfavorable karyotype.RUNX1mutation andCEBPAdouble-muthad a trend to be good prognostic factors (P = 0.073 and 0.057, respectively). All other gene mutations failed to have significant impact on OS in allo-HSCT group. Conditioning regimen (BuCyvs. TBICyvs. RIC) also had no impact on survival. Mutation ofNPM1andFLT3-ITD, either alone or in combination as a factor, had no prognostic effect. The Kaplan–Meier survival curves for OS stratified byCEBPAdouble-mut,RUNX1mutation andFLT3-ITD are shown in Fig. 1 b, d, and f, respectively. Multivariate analysis ( Table 3 ) showed that only three factors significantly affected OS in the allo-HSCT group: unfavorable karyotype and refractory disease status at the time of HSCT as independent poor prognostic factors while mutation ofRUNX1as an independent favorable prognostic factor. Besides,CEBPAdouble-muttended to be a favorable prognostic factor (P = 0.085). In univariate analysis, the prognostic factors for relapse-free survival (RFS) after transplant in allo-HSCT group were similar to those for OS except that unfavorable karyotypes had only borderline significance (P = 0.09). In the multivariate analysis of RFS, (Supplementary Table 3) disease status before HSCT, transplantation with HLA-identical sibling donor,RUNX1mutation andCEBPAdouble-mutwere significantly independent prognostic factors. The mutations of other genes did not influence the RFS.
|Covariate||Hazard ratio||95% C.I.||P value|
|The non allo-HSCT patients (n = 225)|
|Age ≥45 years a||2.027||1.393–2.947||<0.001|
|Unfavorable karyotype b||2.333||1.442–3.776||0.001|
|WBC ≥50 × 109 L−1 c||1.966||1.368–2.824||0.001|
|LDH > 460 U/L d||1.673||0.929–3.011||0.086|
|CEBPA double mut||0.468||0.265–0.828||0.009|
|The allo-HSCT patients (n = 100)|
|Relapse/refractory before HSCT e||3.903||2.197–6.933||<0.001|
|Unfavorable karyotype b||5.661||2.896–11.067||<0.001|
|CEBPA double mut||0.286||0.069–1.188||0.085|
a Age ≥ 45 years vs. Age < 45 years.
b Unfavorable karyotype vs. favorable or intermediate karyotype according to the refined MRC 2010 classification.
c WBC ≥ 50 × 109 L−1vs. WBC < 50 × 109 L−1.
d LDH > 460 U/L (normal upper limit) vs. LDH ≤ 460 U/L.
e Relapse/refractory vs. complete remission.
Among the 12RUNX1-mutated patients in allo-HSCT group, 10 achieved sustained CR with a median remission duration of 59 months (range 8 to 121+ months), including three out of four patients with refractory AML at the time of allo-HSCT (remission duration 78+, 60+ and 8+ months, respectively). In the patients (n = 2) who did not achieve sustained CR, one died of infection while in remission 3 months after allo-HSCT; the other had primary refractory AML and died in 2 months after allo-HSCT without achieving remission.
In this study, we showed that several gene mutations, such asCEBPAdouble-mut,NPM1mut/FLT3-ITDneg, andRUNX1, were independent prognostic factors in the non allo-HSCT group. However, the impact ofNPM1mut/FLT3-ITDnegon clinical outcome in the allo-HSCT group was lost andCEBPAdouble-mutshowed only a trend to be a good prognostic factor. Surprisingly, mutation ofRUNX1was an independent good prognostic factor in the allo-HSCT group while it was a significantly poor prognostic factor in the non allo-HSCT group.
Unfavorable karyotype had a negative impact on OS in both the allo-HSCT and non allo-HSCT groups. Patients with unfavorable karyotype had poorer outcome after allo-HSCT than those with other cytogenetic changes, and the effect was independent of the disease status at the time of allo-HSCT in multivariate analysis ( Table 3 ). Furthermore, subgroup analysis showed that the negative prognostic effect of unfavorable karyotypes remained significant in the subgroup of patients who were in remission status at the time of allo-HSCT (P = 0.003). Our finding was compatible with that reported by Ferrant et al.  who showed significantly poorer leukemia-free survival and OS in AML patients with unfavorable karyotype even though allo-HSCT was performed. However, in the study of Tallman et al.  , patients with unfavorable karyotype had similar OS and event-free survival to those with other cytogenetic changes after receiving allo-HSCT from unrelated donors. More studies are needed to clarify this point.
In this study,FLT3-ITD, a poor-risk genotype, no longer had prognostic impact in allo-HSCT group, a result similar to that reported by Gale et al.  and Bornhauser et al.  , but different from that of Brunet et al.  . Prospective studies on more patients are needed to clarify this point. Patients withNPM1mut/FLT3-ITDneg, a favorable genotype, did not fare better than patients with genotypes other thanNPM1mut/FLT3-ITDnegafter allo-HSCT in this study, a finding compatible to the report of Schlenk et al.  . From the findings of this study and others, it is suggested that allo-HSCT can somehow improve the survival of patients with unfavorable genotype, such asFLT3-ITD or genotypes other thanNPM1mut/FLT3-ITDneg. On the other hand, allo-HSCT seems to be less effective to ameliorate the poor prognosis of patients with unfavorable karyotypes.
Although Schlenk et al. showed patients with mutation ofCEBPAmight not benefit from allo-HSCT in the first CR  , we found a trend of good prognostic effect ofCEBPAdouble mutin allo-HSCT group in this study. We also found all six patients withCEBPAdouble mutreceiving allo-HSCT in a remission status survived without relapse. It was found thatCEBPAdouble-mutcould better predict outcome than combined single and double mutations ofCEBPAin AML patients and . It will be interesting if the prognostic impact ofCEBPAdouble-mutin patients receiving allo-HSCT can be evaluated prospectively or in a larger cohort. Such kind of studies may get different results from that usingCEBPAmutation, both single and double, in the analysis as previously published.
RUNX1mutation was found to be an independent poor prognostic factor in patients withde novoAML  . However, the prognostic implication ofRUNX1mutation in AML patients receiving allo-HSCT was quite the opposite as shown in the report of the AML study group  and this study. The AML study group foundRUNX1-mutated patients had better RFS than others after receiving allo-HSCT. In our study, this particular gene mutation was also an independent good prognostic factor in the allo-HSCT group while it had significant poor prognostic impact in the non allo-HSCT group ( Table 3 ). In the allo-HSCT group, 10 of the 12 patients withRUNX1mutation, including 3 out of 4 patients with refractory disease, remained in sustained remission after allo-HSCT. As a comparison, among the 15 patients with refractory AML andFLT3-ITD, only 4 achieved sustained remission after receiving allo-HSCT, and 2 of these 4 patients remaining in remission also hadRUNX1mutation simultaneously. According to a report from the M. D. Anderson Cancer Center  , less than 20% of patients with active or refractory AML (failure of salvage therapy) at the time of allo-HSCT could achieve sustained remission and survive more than 1 year. In our cohort, although the number of patients was small, the finding that 3 out of 4RUNX1-mutated patients with refractory AML remained in sustained remission after allo-HSCT was surprising. In the report of AML study group, they found that all patients with mutantRUNX1relapsed after conventional consolidation therapy including repetitive high dose cytarabine and autologous HSCT. Clearly, mutantRUNX1indicates resistance to conventional treatments or even autologous HSCT in AML patients. The findings that in patients receiving allo-HSCT, mutantRUNX1correlated with better prognosis are interesting. It may suggest that patients withRUNX1mutation should receive allo-HSCT if patients’ condition is feasible. It is possible that the graft versus leukemia effect plays a role in improving the prognosis ofRUNX1-mutanted AML patients after allo-HSCT, but further studies are needed to explore the mechanisms. Currently, no published literature addresses this issue.
The limitations of this study included single center, retrospective design, and the heterogeneous disease status at the time of allo-HSCT. Many of our patients were transplanted at relapse/refractory disease status which led to poorer outcomes than those reported in literatures. However, the distribution of disease status at the time of allo-HSCT was similar between patients with individual gene mutations and those without (Supplementary Table 2), except that more patients withFLT3-ITD were transplanted at relapsed/refractory status than those without the gene mutation (60%vs. 35%, respectively,P = 0.035). Yet, the patients withFLT3-ITD showed no inferior OS than those without this gene mutation in the allo-HSCT group. Hence, inclusion of relapsed/refractory patients might not significantly affect the results of prognostic impacts of gene alterations in this group of patients. The presence of minimal residual disease (MRD) detected by flow cytometry before allo-HSCT was also found to be related to survival  , However, MRD monitoring was not available in our center at the time of this study, so we couldn’t add this data into our analyses. The transplants were performed over 12 years period in single center and there might be other unknown confounding factors influencing results. However, this cohort represented consecutive adult patients withde novoAML who received curative-intent chemotherapy and allo-HSCT in our center and had adequate cryopreserved samples for mutation analyses. Both groups of patients with and without individual mutations distributed similarly in the year recruited (data not shown), so the influences from the changes in this period might not be much between groups with and without the specific mutations. Prospective studies on more patients are needed to clarify the findings in this study.
In summary,FLT3-ITD and mutations ofNPM1do not affect the outcome of AML patients receiving allo-HSCT. However,CEBPAdouble muthas a trend of good prognostic impact in patients receiving allo-HSCT. Furthermore, mutation ofRUNX1is an independent good prognostic factor in allo-HSCT group, in contrast to its poor impact in patients who did not underwent allo-HSCT. Unfavorable karyotype remains to be an unfavorable prognostic factor in the allo-HSCT group, as in the non allo-HSCT group. These findings need to be confirmed by further prospective large cohort studies before they are applied to clinical decision making.
S-C Chou, J-L Tang, W-C Chou, and H-F Tien were responsible for literature collection, data management and interpretation, and manuscript writing; H-A Hou were responsible for manuscript writing and statistical analyses; and F-C Hu helped with some of statistical analyses and interpretation of result. C-Y Chen, M Yao, S-Y Huang, B-S Ko, W Tsay, and Y-C Chen contributed patient samples and clinical data; and J-L Tang and H-F Tien planned, designed, and coordinated the study over the entire period.
Conflict of interest statement
There was no conflict of interest in any of the authors who were involved in this study.
This work was partially sponsored by grants NSC 100-2314-B002-057-MY3, NSC 100-2314-B-002 -112 -MY3 and NSC 100-2628 -B-002-003-MY3 from the National Science Council (Taiwan), MOHW103-TD-B-111-04 from the Ministry of Health and Welfare (Taiwan) and NTUH 102P06 and UN 102-015 from the Department of Medical Research, National Taiwan University Hospital.
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a Division of Hematology, Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
b Department of Laboratory Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
c National Taiwan University Hospital, Taipei, Taiwan, Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
1 H.-F. Tien and S-C. Chou contributed equally to this research work.
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