- Email: [email protected]
TP53 mutation in allogeneic hematopoietic cell transplantation for de novo myelodysplastic syndrome
Accepted Manuscript Title: TP53 mutation in allogeneic hematopoietic cell transplantation for de novo myelodysplastic syndrome Authors: Yoo-Jin Kim, Seung-Hyun Jung, Eun-Hye Hur, Eun-Ji Choi, Kyoo-Hyung Lee, Seon-Hee Yim, Hye-Jung Kim, Yong-Rim Kwon, Young-Woo Jeon, Sug Hyung Lee, Yeun-Jun Chung, Je-Hwan Lee PII: DOI: Reference:
S0145-2126(18)30429-6 https://doi.org/10.1016/j.leukres.2018.10.004 LR 6054
To appear in:
Leukemia Research
Received date: Revised date: Accepted date:
30-7-2018 5-10-2018 10-10-2018
Please cite this article as: Kim Y-Jin, Jung S-Hyun, Hur E-Hye, Choi EJi, Lee K-Hyung, Yim S-Hee, Kim H-Jung, Kwon Y-Rim, Jeon Y-Woo, Lee SH, Chung Y-Jun, Lee J-Hwan, TP53 mutation in allogeneic hematopoietic cell transplantation for de novo myelodysplastic syndrome, Leukemia Research (2018), https://doi.org/10.1016/j.leukres.2018.10.004 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.
TP53 mutation in allogeneic hematopoietic cell transplantation for de novo myelodysplastic syndrome
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Running short title: TP53 mutation in HCT for de novo MDS
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Yoo-Jin Kima,1, Seung-Hyun Jungb,1, Eun-Hye Hurc, Eun-Ji Choic, Kyoo-Hyung Leec, SeonHee Yimb, Hye-Jung Kima, Yong-Rim Kwona, Young-Woo Jeona, Sug Hyung Leed, Yeun-Jun
Seoul St. Mary's Hematology Hospital, College of Medicine, The Catholic University of
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Chungb,**, Je-Hwan Leec,*
Integrated Research Center for Genome Polymorphism, Precision Medicine Research Center,
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Korea, Seoul, Korea
Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul,
Department of Hematology, Asan Medical Center, University of Ulsan College of Medicine,
Seoul, Korea
Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul,
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Korea
Korea
These two authors contributed equally to this work.
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Correspondence: * Je-Hwan Lee, Department of Hematology, Asan Medical Center, University of Ulsan
College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Korea. Phone: +822-3010-3218, Fax: +82-2-3010-6885, Email: [email protected] ** Yeun-Jun Chung, Precision Medicine Research Center, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea. Phone:
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+82-2-2258-7343, Fax: +82-2-537-0572, Email: [email protected]
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Highlights
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Graphical abstract
76% of patients with de novo myelodysplastic syndrome had at least one mutation.
TP53 mutations were present in 11.4% of the patients.
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TP53 mutations were associated with poor outcomes after transplantation.
Only TP53 mutations were independently associated with shorter survival.
TP53 mutations were also associated with higher relapse.
ABSTRACT We investigated the prognostic role of somatic mutations in allogeneic hematopoietic cell transplantation (HCT) for de novo myelodysplastic syndrome (MDS). We performed targeted
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deep sequencing analysis of 26 genes on bone marrow samples obtained within 6 weeks before HCT from 202 patients with de novo MDS. Overall, 76% of patients carried one or
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more somatic mutations, and TP53 mutation was present in 23 patients (11.4%). Overall
survival (OS) at 5 years was 63.6%, cumulative incidence of relapse (CIR) was 18.6%, eventfree survival (EFS) was 58.5%, and non-relapse mortality (NRM) was 22.9%. TP53 mutation
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was an independent risk factor for lower OS (41% vs. 67%; P = 0.001), higher CIR (49% vs.
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15%; P = 0.001), and lower EFS (38% vs. 61%; P = 0.005), but not for NRM (13% vs. 24%).
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N-RAS mutation was an independent risk factor for higher CIR (HR, 5.91; P = 0.008). TP53
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mutation did not have significant interactions with conditioning intensity or the occurrence of
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graft-versus-host disease with regard to post-transplant outcomes. TP53 mutation was significantly associated with poor outcomes after HCT for patients with de novo MDS,
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mainly due to a higher incidence of disease relapse.
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Keywords: de novo MDS, allogeneic HCT, somatic mutation, TP53 mutation, survival
Introduction
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1.
Allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment modality available for patients with myelodysplastic syndrome (MDS). Clinical outcomes after HCT are highly variable and the selection of patients who will benefit from allogeneic HCT is essential. Allogeneic HCT is primarily recommended for higher-risk MDS patients based on
findings from previous studies using Markov models that suggested the importance of appropriate timing for HCT [1]. These statistical models showed that a decision to delay HCT would maximize survival in patients with low or intermediate-1 scores in the International Prognostic Scoring System (IPSS), whereas immediate HCT at the time of diagnosis would
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lead to longer survival in IPSS intermediate-2 or high risk patients [2, 3]. Other clinical variables usually taken into consideration when judging whether a patient should receive
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allogeneic HCT include disease risk determined with newer prognostic scoring systems or
cytogenetic features [4, 5], age [6], and the presence of comorbidities [7, 8]. Recently, a prognostic scoring system for patients undergoing allogeneic HCT for MDS was developed
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using data from the Center for International Blood and Marrow Transplant Research registry
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[9]. The scoring system includes several clinical variables such as blood blast percentage,
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platelets at HCT, Karnofsky performance status, comprehensive cytogenetic risk score, and
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age [9-11].
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Besides clinical variables, somatic mutations, which are involved in the pathogenesis of MDS,
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have become relevant in MDS prognostication. Several studies have investigated whether genetic mutations are associated with clinical outcomes after allogeneic HCT. Mutations in
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TP53, RAS pathway genes, JAK2, CBL, ASXL1, RUNX1, TET2, IDH2, and U2AF1 have each been reported to influence overall survival (OS) in the setting of HCT [12-17]. However, only mutations in the TP53 gene were consistently reported as poor prognostic factors in most
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studies. Previous studies included patients with therapy-related MDS and/or secondary acute myeloid leukemia (AML) from MDS as well as those with de novo MDS. TP53 gene mutations were found much more frequently in patients with therapy-related MDS than in those with de novo MDS [16], and the outgrowth or emergence of subclones harboring new mutations was found in secondary AML patients [18]. Thus, it is necessary to confirm
whether somatic mutations should be considered before a patient is determined to receive allogeneic HCT for de novo MDS.
In this study, we investigated the prognostic role of somatic mutations including TP53
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mutation in patients with de novo MDS who underwent allogeneic HCT.
2.
Methods
2.1. Patients This study included a total of 202 patients who underwent allogeneic HCT for de novo MDS at two Korean Institutes (Seoul St. Mary’s Hospital, Seoul, Korea and Asan Medical
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Center, Seoul, Korea). Patients who showed bone marrow blasts of 20% or more at any time prior to HCT, as well as those with therapy-related MDS or chronic myelomonocytic
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leukemia were excluded from this study. Only patients from whom bone marrow samples available for molecular analysis were obtained within 6 weeks before HCT were enrolled.
Clinical and laboratory data at the time of HCT, and data for MDS treatment prior to
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HCT were collected. Karyotyping data just before HCT were available for all patients except
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one. IPSS and revised IPSS (R-IPSS) scores were recalculated at the time of HCT. This study
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was approved by the institutional review board at each institute. Written informed consent for
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molecular analysis was obtained from each patient.
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2.2. Targeted deep sequencing
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We performed targeted deep sequencing as previously described [19]. In brief, an MDS panel targeting 26 well-known MDS-related genes (DNMT3A, TET2, EZH2, RUNX1, ASXL1,
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STAG2, CBL, TP53, SRSF2, SF3B1, U2AF1, LAMB4, DNMT1, ETV6, KRAS, NF1, NPM1, NRAS, PRPF8, IDH1, IDH2, JAK2, FLT3, SETBP1, ATRX, and ZRSR2) was used to generate sequencing libraries. This MDS panel consisted of 1,088 amplicons covering 98.4% of all
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coding exons in 26 target genes. Sequencing libraries were generated using the AmpliSeq Library Kit 2.0 (Life Technologies, Carlsbad, CA) and the MDS panel was then sequenced using the Ion Torrent Proton system (Life Technologies) according to the manufacturer's instructions. Sequencing reads were aligned to UCSC hg19 and genomic variants were called using the Torrent Suite 5.2. To achieve reliable and robust mutation calling, stringent post-
filtering processes were conducted. First, we selected the functional variants (non-silent) in coding exons. The known polymorphic sites for East Asians (>1% of minor allele frequency) in public databases (dbSNP137, ESP6500, and the 1000 genomes project) were filtered out as germline polymorphisms. The variants with >1% of minor allele frequency in our in-house
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normal database (comparison with 38 whole genome and 2,283 whole exome sequencing data from Koreans) were also filtered out. Then, we filtered the variant with total read depth
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fewer than 20 or variant support reads depth fewer than 3. The variant allele frequency cut off value was 3%. The remaining variants were considered candidate somatic mutations.
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2.3. Statistical analysis
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The purpose of this study was to determine whether somatic mutations were
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significantly associated with clinical outcomes after allogeneic HCT for de novo MDS. The
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end points included OS, cumulative incidence of relapse (CIR), event-free survival (EFS), and non-relapse mortality (NRM). The time-to-event variables were defined as the duration
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from the date of HCT to the date of death from any cause (OS) or to the date of death or
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relapse (EFS). Cumulative incidences were calculated from the date of HCT to the date of relapse (CIR) or to the date of death not related to underlying disease (NRM).
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The Chi-square test was used to compare categorical variables, and the Mann–Whitney U test or t test was used to compare continuous variables. Survival was estimated using the Kaplan–Meier method, and differences in survival were compared using the log-rank test.
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Cumulative incidence was estimated using the method described by Gray, and differences between groups were analyzed using the test developed by Gray. Multivariate analysis was performed using the Cox proportional hazards model for survival and the Fine and Gray methods were used for cumulative incidence.
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Results
3.1. Patient characteristics The median age of the patients, 74 females and 128 males, was 51 years (range, 19-69) (Table 1). One hundred and thirty-two patients had received treatment for MDS prior to HCT:
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hypomethylating therapy (azacitidine or decitabine) for 125, intensive chemotherapy for 6, and immunosuppressive therapy for 6. Final responses to the treatment just before HCT were
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ongoing response in 54, stable disease in 44, primary progression in 11, and progression after
initial response in 23. At the time of HCT, 37.8% of patients had higher risk scores for IPSS
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3.2. Allogeneic hematopoietic cell transplantation
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and 48.8% had high or very high risk scores for R-IPSS (Table 1).
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The HCT donors were HLA matched siblings in 69 cases, unrelated volunteers in 74
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cases, and HLA haplo-identical family members in 59 cases (Table 1). The conditioning regimen was myeloablative for 62 patients (busulfan-cyclophosphamide for 1 patient and
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busulfan [4 days]-fludarabine ± antithymocyte globulin [ATG] for 61) and reduced-intensity
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for 140 patients (busulfan [2 days]-fludarabine-ATG ± total body irradiation [8 Gy] for 133, cyclophosphamide-fludarabine for 6, and other for 1 patient).
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Acute graft-versus-host disease (GVHD) occurred in 95 patients (47.0%) at a median of 33 days (range, 6–314) after HCT: grade I in 24, grade II in 51, grade III in 10, and grade IV in 10. Chronic GVHD (cGVHD) occurred in 89 (46.1%) of 193 assessable patients at a
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median of 139 days (range, 32-955) after HCT: mild in 40, moderate in 22, and severe in 27 patients. The median follow-up duration for the surviving patients was 3.4 years (range, 0.8–8.6). During this time, 68 deaths were recorded. Forty-three of the 68 deaths were non-relapse deaths. Thirty-three patients experienced MDS relapse, and 77 patients experienced death or
relapse. OS at 5 years was 63.6%, CIR was 18.6%, EFS was 58.5%, and NRM was 22.9%.
3.3. General features of somatic alterations in de novo MDS Targeted deep sequencing was performed using the MDS target panel consisting of 26
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genes [19] and the median depth of coverage for targeted deep sequencing was 2,355x (range, 1,082x–5,364x) across the target genome (Supplementary Table S1). A total of 379 somatic
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mutations (327 SNVs and 52 indels) were identified in the 202 de novo MDS patients
(Supplementary Table S2, Fig. 1). A majority of the MDS genomes (153/202, 75.7%) had mutations in at least one target gene. Consistent with previous reports [12,14,16,21],
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mutations in six genes were detected in more than 10% of the patients: U2AF1 (21.3%),
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TET2 (16.8%), ASXL1 (14.9%), SF3B1 (14.4), RUNX1 (11.9%), and TP53 (11.4%).
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3.4. Prognostic factor analysis
We assessed the prognostic values of clinical variables and genetic alterations.
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Univariate analysis showed that among the clinical variables, age (CIR and EFS), sex (OS,
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EFS, and NRM), hemoglobin (OS), platelet count (OS), IPSS cytogenetic risk (OS, CIR, and EFS), HCT comorbidity index (CI) (EFS), donor-recipient sex pair (OS), and donor age (EFS)
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were significantly associated with clinical outcomes after HCT (Supplementary Table S3). A significant association with clinical outcomes was identified for NRAS mutations (CIR and EFS) and TP53 mutations (OS, CIR, and EFS) among genetic alterations. Among the clinical
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variables, male sex (OS, NRM), thrombocytopenia (OS), poor cytogenetics (CIR), and older donor (CIR) were independent poor prognostic factors (Table 2). Among genetic alterations, mutation status of the TP53 gene was an independent prognostic factor for OS (wild type vs. mutant type, hazard ratio [HR] 2.897, P = 0.001), CIR (HR, 3.38; P = 0.001), and EFS (HR 2.400, P = 0.005) (Fig. 2), and NRAS mutations were associated with higher CIR (HR, 5.91;
P = 0.008) (Table 2). When we excluded non-hot-spot mutations, 209 mutations of 44 genes except NRAS were filtered out (Supplementary Table S2). Prognostic impacts of hot-spot mutations only were similar to those of total mutations: the TP53 mutations only were prognostic for overall
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survival (data not shown).
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3.5. TP53 gene mutations
Twenty-three patients had TP53 mutations (21 missense mutations, 1 splicing mutation, and 1 frameshift mutation). The variant allele frequency was ≥5% for the majority of patients
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(20/23), with the exception of 3 patients with <5%. Mutations were located on one allele in
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exon 2, seven alleles in exon 5, five in exon 6, five in exon 7, three in exon 8, and one in
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exon 10 (Fig. 3).
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TP53 mutations were more frequently found in patients with poor IPSS cytogenetic risk and those with higher scores of IPSS or R-IPSS. The scores were recalculated at the time
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of HCT. Other transplant characteristics were not significantly different between patients with
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TP53 mutations and those without (Supplementary Table S4). Of the 76 patients with IPSS higher-risk scores, 18 patients (23.7%) had TP53 mutations. These patients showed
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significantly lower OS (25.0%. vs. 59.1%, P = 0.001), higher CIR (63.3% vs. 11.6%, P < 0.001), lower EFS (20.0% vs. 58.1%, P < 0.001), and similar NRM (16.7% vs. 30.3%, P =
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0.441).
We investigated the interaction between TP53 mutations and the conditioning regimen
or cGVHD to determine whether a high conditioning regimen intensity or induction of graftversus-leukemia (GVL) effects could improve the poor outcomes for patients with TP53 mutations. Disappointingly, there were no significant differences in the post-transplant outcomes according to the conditioning regimen intensity or occurrence of cGVHD (Table 3).
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Discussion From analyzing patients with de novo MDS receiving HCT, the results of this study
showed that TP53 mutation was the only molecular signature predictive of poor survival and disease relapse. NRAS mutation was also identified as a marker of post-transplant relapse, but
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its influence on survival did not reach statistical significance, probably due to the small number of mutated cases in this study. Although similar results were also reported by other
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investigators, it should be noted that our analyses were confined to patients with de novo MDS by excluding therapy-related MDS, secondary AML, and CMML. Secondly, our cohort
was relatively homogeneous as almost all of the patients received fludarabine-busulfan-based
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conditioning regimens. Thirdly, bone marrow samples obtained within 6 weeks of
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transplantation were used to uncover the real influences of the mutations present at the time
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of HCT, as the mutational profile varies during the disease course or pre-transplant treatment
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[21].
We chose to separate de novo MDS from other types of MDS and secondary AML
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when analyzing the influences of genetic mutations upon HCT outcomes due to their wildly
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different disease prognosis and genetic backgrounds. Patients with therapy-related MDS and secondary AML evolving from MDS exhibit suboptimal responses to conventional
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chemotherapy and an extremely low survival rate following allogeneic HCT [22-26]. In addition to an innate resistance to chemotherapy, the dismal prognosis for these conditions could also be associated with their distinct disease biology as evidenced by the acquisition of
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new mutations and their poor prognostic karyotypes [18, 27-29]. TP53 mutations are the most frequent genetic mutations in these patients. The incidence of TP53 mutations in therapyrelated MDS is quite high compared to that in de novo MDS [30, 31]. As a result, mutational genes, which are predictive of transplant outcomes, may differ between de novo MDS and therapy-related MDS or secondary AML. These observations justify the separate analysis of
genetic mutations only in de novo MDS to identify a specific molecular predictor for the disease. In the cohort comprised only of de novo MDS patients, our study results showed the poor prognostic influences of TP53 mutations, evidenced by short median survival and a high
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relapse rate. With these results, we raised the question of whether the negative influences attributed to TP53 mutation in other studies resulted from the inclusion of patients with de
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novo MDS, therapy-related MDS, or secondary AML, due to the heterogeneous nature of the
enrolled patients [12-17]. Two of the previous studies excluded secondary AML patients [16, 32]. One of them identified the prognostic implications of genetic mutations including TP53
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mutation. However, 21% of the enrolled patients had therapy-related MDS, which prevented
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the identification of de novo MDS-specific genetic mutations [16]. The other study failed to
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identify any genetic mutations associated with transplant outcomes, but the sample size was
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relatively small and the inclusion of therapy-related MDS was not clarified [32]. One recent study investigated the outcomes after allogeneic HCT for therapy-related MDS patients and
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the authors reported that there was no survival difference according to the presence or
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absence of TP53 mutation [33]. The study results suggest that the impacts of TP53 mutation may be different between de novo and therapy-related MDS. Further validation of the
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influence of TP53 mutations according to the type of MDS remains to be conducted and assessment of secondary AML should be performed first.
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Even with the poorer outcomes for patients with TP53 mutants compared to non-
mutant cases, one interesting observation in our study is that the estimated long-term survival rate for the former group reached around 40% at 5 years, which is a relatively high survival rate compared to the other study groups. Of course, data for the post-HCT survival rate in TP53 mutated MDS are still insufficient and the relatively small number of cases in each
study should be considered when comparing survival rate among the studies. Additionally, many factors other than genetic mutations may have affected the transplant outcomes among the patients with TP53 mutations. For example, although Lindsley et al. reported a 5-year survival rate approximately 20% lower than ours, their results could be associated with
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unfavorable influences from therapy-related MDS in their study [16]. Likewise, differences in conditioning intensity, disease risk and responses to pre-transplant treatment at HCT, age of
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the patients, and performance or comorbidities might have influenced the results. Apart from
these factors, we also speculate that MDS with TP53 mutation may not be a homogeneous entity and may harbor innate differences at the individual level. However, regardless of the
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reasons for different survival rates among patients with TP53 mutations, our study again
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novo MDS patients harboring TP53 mutations.
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showed that allogeneic HCT could be a curative option for this disease, especially for de
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Since allogeneic HCT is the most potent chemoimmunotherapy due to the potent
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cytotoxic preparation and subsequent allogeneic immune reaction, we sought to determine whether intense conditioning or GVL effects accompanying GVHD could control the
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reemergence of mutated clones. For patients with TP53 mutations, our results showed no association between the relapse rate and the conditioning intensity, a result also observed in
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the study by Lindsley et al [16]. This study also showed that there was no difference in relapse rate according to the presence of cGVHD. Most relapses in patients with TP53
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mutations in our study occurred within 6 months of HCT. This suggests that TP53 mutant clones are resistant to cytotoxic preparation and outpace the tumor control by GVL effects. In patients with RAS pathway mutations, intensification of the conditioning regimen has been proposed to be effective in lowering the relapse rate [16], but such an association was not observed in our study. We also could not observe any beneficial effects of cGVHD in
lowering the relapse rate in RAS pathway mutant cases (data not shown), but the number of patients with RAS pathway mutations in our study was too small. The role of conditioning intensity and allogeneic immune reaction upon disease relapse remains to be verified for the development of mutation-adapted therapeutic strategies.
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Another important factor associated with post-transplant relapse is responsiveness to pre-transplant debulking treatment, especially for higher risk disease [34]. A recent study
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showed that decitabine induces a high promising response rate in TP53-mutant AML or MDS
[35, 36], suggesting that bridging treatment with decitabine and subsequent allogeneic HSCT could be an effective treatment strategy for patients with this dismal prognosis. Other
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possible candidate treatments include novel agents being developed to effectively target
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mutated genes such as IDH1 or IDH2 mutation [37]. Of course, one more basic but important
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way to increase HCT efficiency is the selection of optimal transplantation candidates.
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Incorporation of specific genetic mutations to transplantation-specific prognostic scoring
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systems in MDS [10, 11] may enhance the predictability of the systems and may be useful to minimize unwanted toxicity due to overtreatment.
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Our study has some limitations. First, we did not use matched normal samples. We
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tried to filter the germline variants out using the public and in-house database, but it might not be sufficient to exclude the germline variants completely. Second, the number of patients analyzed in our study seems to be relatively small considering the heterogeneous nature of
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MDS. Third, the number of genes for targeted sequencing was small and specific mutations having critical prognostic implications might be missed. Lastly, the retrospective nature of our study might cause bias in interpretation of the data. In conclusion, for de novo MDS patients receiving HCT, this study showed that TP53 mutation was the only molecular signature predictive of poor survival and disease relapse.
Our study also showed that allogeneic HCT could be a curative option for patients with this dismal prognostic molecular feature.
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Acknowledgments This study was supported by a grant from the Korean Health Technology R&D project, Ministry of Health & Welfare, Republic of Korea (HI12C0129) and a grant from National
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Research Foundation of Korea (2017R1E1A1A01074913). Part of the biospecimen and data
used in this study was provided by Asan Bio-Resource Center, Korea Biobank Network
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(2012-14(57)).
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Figure legends Fig. 1. Mutational features of the 26 target genes in 202 patients. Each row represents the mutated gene and each column represents an individual patient. Fig. 2. Post-transplant clinical outcomes according to TP53 mutation status. (A) Overall
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survival; (B) Cumulative incidence of relapse; (C) Event-free survival; and (D) Non-relapse
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mortality
Fig. 3. Schematic diagram of TP53 mutations in de novo MDS cases. The x axis represents
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ED
M
A
N
U
amino acid position. The y axis represents the number of mutations.
Table 1 Patient and transplant characteristics at the time of hematopoietic cell transplantation Characteristic
No. patients (%)
Female
74 (36.6%)
Male
128 (63.4%)
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Sex
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Age, year Median (range)
51 (19-69)
13 (6.4%)
RCMD
51 (25.2%)
RAEB-1
54 (26.7%)
RAEB-2
84 (41.6%)
M
A
N
RCUD/RARS/5q-
IPSS
ED
Lower risk
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R-IPSS
PT
Higher risk Unknown
U
Subtype
125 (62.2%) 76 (37.8%) 1
42 (20.9%)
Intermediate
61 (30.3%)
High/Very high
98 (48.8%)
Unknown
1
A
Very low/Low
HCT donor Matched sibling
69 (34.2%)
Matched unrelated
66 (32.7%)
Mismatched unrelated
8 (4.0%)
Familial haplo-identical
59 (29.2%)
Myeloablative
62 (30.7%)
Reduced-intensity
140 (69.3%)
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Conditioning regimen
4 (2.0%)
>0-5
53 (26.2%)
>5
145 (71.8%)
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0
N
GVHD prophylaxis 114 (56.4%)
FK506/MTX
88 (43.6%)
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A
CSA/MTX
HCT-CI
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0-1
129 (63.9%) 73 (36.1%)
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>1 Donor age, year
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ATG, mg/kg
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Median (range)
35.5 (15-70)
Donor-recipient sex pair 46 (22.8%)
Others
156 (77.2%)
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Female-to-male
RCUD, refractory cytopenia with unilineage dysplasia; RARS, refractory anemia with ringed sideroblasts; 5q-, myelodysplastic syndrome associated with isolated del(5q); RCMD, refractory cytopenia with multilineage dysplasia; RAEB, refractory anemia with excess of blasts; IPSS, International Prognostic Scoring System; RIPSS, revised IPSS; HCT, hematopoietic cell transplantation; ATG, antithymocyte globulin; GVHD, graftversus-host disease; CSA, cyclosporine; MTX, methotrexate; HCT-CI, HCT comorbidity index
Table 2 Multivariate analysis of prognostic factors for clinical outcomes after HCT HR
95% CI
P-value
Overall survival
1
Male
3.126
1.691-5.781
Platelets, x103/μL 1
<100
2.193
1.209-3.977
1
Mutant type
2.897
A
Wild type
IPSS cytogenetic risk
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PT
Good
1.552-5.409
0.001
0.254
0.055-1.17
0.079
2.47
1.20-5.10
0.015
1.02-4.13
0.043
1.59-21.9
0.008
M
ED
Cumulative incidence of relapse
Poor
N
TP53
Intermediate
0.010
U
≥100
< 0.001
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Female
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Sex
1
Donor age
1
≥40
2.06
A
<40
NRAS Wild type
1
Mutant type
5.91
TP53 Wild type
1
Mutant type
3.38
1.62-7.06
0.001
Event-free survival
1
Male
2.689
1.537-4.705
0.001
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Female
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Sex
IPSS cytogenetic risk
0.033
1
Intermediate
1.305
0.565-1.897
0.911
Poor
1.990
0.013
1.040-2.603
0.033
1.311-4.394
0.005
1.022-5.11
0.044
N
1.159-3.419
A
HCT-CI 1
M
0-1 >1
1.645
ED
TP53
PT
Wild type Mutant type
U
Good
1 2.400
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Non-relapse mortality Sex
1
Male
2.285
A
Female
HR, hazard ratio; CI, confidence interval; IPSS, International Prognostic Scoring System; HCT-CI, hematopoietic cell transplantation-comorbidity index
Table 3 Clinical outcomes for patients with TP53 mutations according to conditioning regimen intensity or occurrence of chronic graft-versus-host disease
N
OS
P
CIR
P
EFS
Myeloablative
9
29.6%
0.655
44.4%
0.893
33.3%
0.726 22.2% 0.338
Reduced-intensity
14
50.0%
41.7%
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Survival probabilities or cumulative incidences at 5 years
7.1%
No
11
34.1%
66.7%
0.689 21.4% 0.173
Yes
8
75.0%
60.0%
0.0%
51.2%
Chronic GVHD* 50.0% 40.0%
0.454
U
0.124
NRM
P
SC R
Conditioning regimen
P
N
N, number of patients; OS, overall survival; P, P-value; CIR, cumulative incidence of relapse; EFS, event-free
A
survival; NRM, non-relapse mortality; GVHD, graft-versus-host disease
A
CC E
PT
ED
chronic GVHD in our study population.
M
*Analysis included only patients who survived more than 138 days after HCT, which was the earliest onset of
Fig. 1. Mutational features of the 26 target genes in 202 patients. Each row represents the
A
CC E
PT
ED
M
A
N
U
SC R
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mutated gene and each column represents an individual patient.
Fig. 2. Post-transplant clinical outcomes according to TP53 mutation status. (A) Overall survival; (B) Cumulative incidence of relapse; (C) Event-free survival; and (D) Non-relapse
A
CC E
PT
ED
M
A
N
U
SC R
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mortality
Fig. 3. Schematic diagram of TP53 mutations in de novo MDS cases. The x axis represents
A
CC E
PT
ED
M
A
N
U
SC R
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amino acid position. The y axis represents the number of mutations.
S0145-2126(18)30429-6 https://doi.org/10.1016/j.leukres.2018.10.004 LR 6054
To appear in:
Leukemia Research
Received date: Revised date: Accepted date:
30-7-2018 5-10-2018 10-10-2018
Please cite this article as: Kim Y-Jin, Jung S-Hyun, Hur E-Hye, Choi EJi, Lee K-Hyung, Yim S-Hee, Kim H-Jung, Kwon Y-Rim, Jeon Y-Woo, Lee SH, Chung Y-Jun, Lee J-Hwan, TP53 mutation in allogeneic hematopoietic cell transplantation for de novo myelodysplastic syndrome, Leukemia Research (2018), https://doi.org/10.1016/j.leukres.2018.10.004 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.
TP53 mutation in allogeneic hematopoietic cell transplantation for de novo myelodysplastic syndrome
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Running short title: TP53 mutation in HCT for de novo MDS
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Yoo-Jin Kima,1, Seung-Hyun Jungb,1, Eun-Hye Hurc, Eun-Ji Choic, Kyoo-Hyung Leec, SeonHee Yimb, Hye-Jung Kima, Yong-Rim Kwona, Young-Woo Jeona, Sug Hyung Leed, Yeun-Jun
Seoul St. Mary's Hematology Hospital, College of Medicine, The Catholic University of
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a
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Chungb,**, Je-Hwan Leec,*
Integrated Research Center for Genome Polymorphism, Precision Medicine Research Center,
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b
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Korea, Seoul, Korea
Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul,
Department of Hematology, Asan Medical Center, University of Ulsan College of Medicine,
Seoul, Korea
Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul,
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d
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c
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Korea
Korea
These two authors contributed equally to this work.
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1
Correspondence: * Je-Hwan Lee, Department of Hematology, Asan Medical Center, University of Ulsan
College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Korea. Phone: +822-3010-3218, Fax: +82-2-3010-6885, Email: [email protected] ** Yeun-Jun Chung, Precision Medicine Research Center, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea. Phone:
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+82-2-2258-7343, Fax: +82-2-537-0572, Email: [email protected]
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Highlights
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Graphical abstract
76% of patients with de novo myelodysplastic syndrome had at least one mutation.
TP53 mutations were present in 11.4% of the patients.
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TP53 mutations were associated with poor outcomes after transplantation.
Only TP53 mutations were independently associated with shorter survival.
TP53 mutations were also associated with higher relapse.
ABSTRACT We investigated the prognostic role of somatic mutations in allogeneic hematopoietic cell transplantation (HCT) for de novo myelodysplastic syndrome (MDS). We performed targeted
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deep sequencing analysis of 26 genes on bone marrow samples obtained within 6 weeks before HCT from 202 patients with de novo MDS. Overall, 76% of patients carried one or
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more somatic mutations, and TP53 mutation was present in 23 patients (11.4%). Overall
survival (OS) at 5 years was 63.6%, cumulative incidence of relapse (CIR) was 18.6%, eventfree survival (EFS) was 58.5%, and non-relapse mortality (NRM) was 22.9%. TP53 mutation
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was an independent risk factor for lower OS (41% vs. 67%; P = 0.001), higher CIR (49% vs.
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15%; P = 0.001), and lower EFS (38% vs. 61%; P = 0.005), but not for NRM (13% vs. 24%).
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N-RAS mutation was an independent risk factor for higher CIR (HR, 5.91; P = 0.008). TP53
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mutation did not have significant interactions with conditioning intensity or the occurrence of
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graft-versus-host disease with regard to post-transplant outcomes. TP53 mutation was significantly associated with poor outcomes after HCT for patients with de novo MDS,
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mainly due to a higher incidence of disease relapse.
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Keywords: de novo MDS, allogeneic HCT, somatic mutation, TP53 mutation, survival
Introduction
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1.
Allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment modality available for patients with myelodysplastic syndrome (MDS). Clinical outcomes after HCT are highly variable and the selection of patients who will benefit from allogeneic HCT is essential. Allogeneic HCT is primarily recommended for higher-risk MDS patients based on
findings from previous studies using Markov models that suggested the importance of appropriate timing for HCT [1]. These statistical models showed that a decision to delay HCT would maximize survival in patients with low or intermediate-1 scores in the International Prognostic Scoring System (IPSS), whereas immediate HCT at the time of diagnosis would
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lead to longer survival in IPSS intermediate-2 or high risk patients [2, 3]. Other clinical variables usually taken into consideration when judging whether a patient should receive
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allogeneic HCT include disease risk determined with newer prognostic scoring systems or
cytogenetic features [4, 5], age [6], and the presence of comorbidities [7, 8]. Recently, a prognostic scoring system for patients undergoing allogeneic HCT for MDS was developed
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using data from the Center for International Blood and Marrow Transplant Research registry
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[9]. The scoring system includes several clinical variables such as blood blast percentage,
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platelets at HCT, Karnofsky performance status, comprehensive cytogenetic risk score, and
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age [9-11].
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Besides clinical variables, somatic mutations, which are involved in the pathogenesis of MDS,
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have become relevant in MDS prognostication. Several studies have investigated whether genetic mutations are associated with clinical outcomes after allogeneic HCT. Mutations in
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TP53, RAS pathway genes, JAK2, CBL, ASXL1, RUNX1, TET2, IDH2, and U2AF1 have each been reported to influence overall survival (OS) in the setting of HCT [12-17]. However, only mutations in the TP53 gene were consistently reported as poor prognostic factors in most
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studies. Previous studies included patients with therapy-related MDS and/or secondary acute myeloid leukemia (AML) from MDS as well as those with de novo MDS. TP53 gene mutations were found much more frequently in patients with therapy-related MDS than in those with de novo MDS [16], and the outgrowth or emergence of subclones harboring new mutations was found in secondary AML patients [18]. Thus, it is necessary to confirm
whether somatic mutations should be considered before a patient is determined to receive allogeneic HCT for de novo MDS.
In this study, we investigated the prognostic role of somatic mutations including TP53
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mutation in patients with de novo MDS who underwent allogeneic HCT.
2.
Methods
2.1. Patients This study included a total of 202 patients who underwent allogeneic HCT for de novo MDS at two Korean Institutes (Seoul St. Mary’s Hospital, Seoul, Korea and Asan Medical
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Center, Seoul, Korea). Patients who showed bone marrow blasts of 20% or more at any time prior to HCT, as well as those with therapy-related MDS or chronic myelomonocytic
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leukemia were excluded from this study. Only patients from whom bone marrow samples available for molecular analysis were obtained within 6 weeks before HCT were enrolled.
Clinical and laboratory data at the time of HCT, and data for MDS treatment prior to
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HCT were collected. Karyotyping data just before HCT were available for all patients except
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one. IPSS and revised IPSS (R-IPSS) scores were recalculated at the time of HCT. This study
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was approved by the institutional review board at each institute. Written informed consent for
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molecular analysis was obtained from each patient.
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2.2. Targeted deep sequencing
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We performed targeted deep sequencing as previously described [19]. In brief, an MDS panel targeting 26 well-known MDS-related genes (DNMT3A, TET2, EZH2, RUNX1, ASXL1,
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STAG2, CBL, TP53, SRSF2, SF3B1, U2AF1, LAMB4, DNMT1, ETV6, KRAS, NF1, NPM1, NRAS, PRPF8, IDH1, IDH2, JAK2, FLT3, SETBP1, ATRX, and ZRSR2) was used to generate sequencing libraries. This MDS panel consisted of 1,088 amplicons covering 98.4% of all
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coding exons in 26 target genes. Sequencing libraries were generated using the AmpliSeq Library Kit 2.0 (Life Technologies, Carlsbad, CA) and the MDS panel was then sequenced using the Ion Torrent Proton system (Life Technologies) according to the manufacturer's instructions. Sequencing reads were aligned to UCSC hg19 and genomic variants were called using the Torrent Suite 5.2. To achieve reliable and robust mutation calling, stringent post-
filtering processes were conducted. First, we selected the functional variants (non-silent) in coding exons. The known polymorphic sites for East Asians (>1% of minor allele frequency) in public databases (dbSNP137, ESP6500, and the 1000 genomes project) were filtered out as germline polymorphisms. The variants with >1% of minor allele frequency in our in-house
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normal database (comparison with 38 whole genome and 2,283 whole exome sequencing data from Koreans) were also filtered out. Then, we filtered the variant with total read depth
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fewer than 20 or variant support reads depth fewer than 3. The variant allele frequency cut off value was 3%. The remaining variants were considered candidate somatic mutations.
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2.3. Statistical analysis
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The purpose of this study was to determine whether somatic mutations were
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significantly associated with clinical outcomes after allogeneic HCT for de novo MDS. The
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end points included OS, cumulative incidence of relapse (CIR), event-free survival (EFS), and non-relapse mortality (NRM). The time-to-event variables were defined as the duration
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from the date of HCT to the date of death from any cause (OS) or to the date of death or
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relapse (EFS). Cumulative incidences were calculated from the date of HCT to the date of relapse (CIR) or to the date of death not related to underlying disease (NRM).
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The Chi-square test was used to compare categorical variables, and the Mann–Whitney U test or t test was used to compare continuous variables. Survival was estimated using the Kaplan–Meier method, and differences in survival were compared using the log-rank test.
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Cumulative incidence was estimated using the method described by Gray, and differences between groups were analyzed using the test developed by Gray. Multivariate analysis was performed using the Cox proportional hazards model for survival and the Fine and Gray methods were used for cumulative incidence.
3.
Results
3.1. Patient characteristics The median age of the patients, 74 females and 128 males, was 51 years (range, 19-69) (Table 1). One hundred and thirty-two patients had received treatment for MDS prior to HCT:
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hypomethylating therapy (azacitidine or decitabine) for 125, intensive chemotherapy for 6, and immunosuppressive therapy for 6. Final responses to the treatment just before HCT were
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ongoing response in 54, stable disease in 44, primary progression in 11, and progression after
initial response in 23. At the time of HCT, 37.8% of patients had higher risk scores for IPSS
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3.2. Allogeneic hematopoietic cell transplantation
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and 48.8% had high or very high risk scores for R-IPSS (Table 1).
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The HCT donors were HLA matched siblings in 69 cases, unrelated volunteers in 74
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cases, and HLA haplo-identical family members in 59 cases (Table 1). The conditioning regimen was myeloablative for 62 patients (busulfan-cyclophosphamide for 1 patient and
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busulfan [4 days]-fludarabine ± antithymocyte globulin [ATG] for 61) and reduced-intensity
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for 140 patients (busulfan [2 days]-fludarabine-ATG ± total body irradiation [8 Gy] for 133, cyclophosphamide-fludarabine for 6, and other for 1 patient).
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Acute graft-versus-host disease (GVHD) occurred in 95 patients (47.0%) at a median of 33 days (range, 6–314) after HCT: grade I in 24, grade II in 51, grade III in 10, and grade IV in 10. Chronic GVHD (cGVHD) occurred in 89 (46.1%) of 193 assessable patients at a
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median of 139 days (range, 32-955) after HCT: mild in 40, moderate in 22, and severe in 27 patients. The median follow-up duration for the surviving patients was 3.4 years (range, 0.8–8.6). During this time, 68 deaths were recorded. Forty-three of the 68 deaths were non-relapse deaths. Thirty-three patients experienced MDS relapse, and 77 patients experienced death or
relapse. OS at 5 years was 63.6%, CIR was 18.6%, EFS was 58.5%, and NRM was 22.9%.
3.3. General features of somatic alterations in de novo MDS Targeted deep sequencing was performed using the MDS target panel consisting of 26
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genes [19] and the median depth of coverage for targeted deep sequencing was 2,355x (range, 1,082x–5,364x) across the target genome (Supplementary Table S1). A total of 379 somatic
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mutations (327 SNVs and 52 indels) were identified in the 202 de novo MDS patients
(Supplementary Table S2, Fig. 1). A majority of the MDS genomes (153/202, 75.7%) had mutations in at least one target gene. Consistent with previous reports [12,14,16,21],
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mutations in six genes were detected in more than 10% of the patients: U2AF1 (21.3%),
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TET2 (16.8%), ASXL1 (14.9%), SF3B1 (14.4), RUNX1 (11.9%), and TP53 (11.4%).
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3.4. Prognostic factor analysis
We assessed the prognostic values of clinical variables and genetic alterations.
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Univariate analysis showed that among the clinical variables, age (CIR and EFS), sex (OS,
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EFS, and NRM), hemoglobin (OS), platelet count (OS), IPSS cytogenetic risk (OS, CIR, and EFS), HCT comorbidity index (CI) (EFS), donor-recipient sex pair (OS), and donor age (EFS)
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were significantly associated with clinical outcomes after HCT (Supplementary Table S3). A significant association with clinical outcomes was identified for NRAS mutations (CIR and EFS) and TP53 mutations (OS, CIR, and EFS) among genetic alterations. Among the clinical
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variables, male sex (OS, NRM), thrombocytopenia (OS), poor cytogenetics (CIR), and older donor (CIR) were independent poor prognostic factors (Table 2). Among genetic alterations, mutation status of the TP53 gene was an independent prognostic factor for OS (wild type vs. mutant type, hazard ratio [HR] 2.897, P = 0.001), CIR (HR, 3.38; P = 0.001), and EFS (HR 2.400, P = 0.005) (Fig. 2), and NRAS mutations were associated with higher CIR (HR, 5.91;
P = 0.008) (Table 2). When we excluded non-hot-spot mutations, 209 mutations of 44 genes except NRAS were filtered out (Supplementary Table S2). Prognostic impacts of hot-spot mutations only were similar to those of total mutations: the TP53 mutations only were prognostic for overall
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survival (data not shown).
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3.5. TP53 gene mutations
Twenty-three patients had TP53 mutations (21 missense mutations, 1 splicing mutation, and 1 frameshift mutation). The variant allele frequency was ≥5% for the majority of patients
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(20/23), with the exception of 3 patients with <5%. Mutations were located on one allele in
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exon 2, seven alleles in exon 5, five in exon 6, five in exon 7, three in exon 8, and one in
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exon 10 (Fig. 3).
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TP53 mutations were more frequently found in patients with poor IPSS cytogenetic risk and those with higher scores of IPSS or R-IPSS. The scores were recalculated at the time
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of HCT. Other transplant characteristics were not significantly different between patients with
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TP53 mutations and those without (Supplementary Table S4). Of the 76 patients with IPSS higher-risk scores, 18 patients (23.7%) had TP53 mutations. These patients showed
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significantly lower OS (25.0%. vs. 59.1%, P = 0.001), higher CIR (63.3% vs. 11.6%, P < 0.001), lower EFS (20.0% vs. 58.1%, P < 0.001), and similar NRM (16.7% vs. 30.3%, P =
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0.441).
We investigated the interaction between TP53 mutations and the conditioning regimen
or cGVHD to determine whether a high conditioning regimen intensity or induction of graftversus-leukemia (GVL) effects could improve the poor outcomes for patients with TP53 mutations. Disappointingly, there were no significant differences in the post-transplant outcomes according to the conditioning regimen intensity or occurrence of cGVHD (Table 3).
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Discussion From analyzing patients with de novo MDS receiving HCT, the results of this study
showed that TP53 mutation was the only molecular signature predictive of poor survival and disease relapse. NRAS mutation was also identified as a marker of post-transplant relapse, but
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its influence on survival did not reach statistical significance, probably due to the small number of mutated cases in this study. Although similar results were also reported by other
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investigators, it should be noted that our analyses were confined to patients with de novo MDS by excluding therapy-related MDS, secondary AML, and CMML. Secondly, our cohort
was relatively homogeneous as almost all of the patients received fludarabine-busulfan-based
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conditioning regimens. Thirdly, bone marrow samples obtained within 6 weeks of
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transplantation were used to uncover the real influences of the mutations present at the time
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of HCT, as the mutational profile varies during the disease course or pre-transplant treatment
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[21].
We chose to separate de novo MDS from other types of MDS and secondary AML
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when analyzing the influences of genetic mutations upon HCT outcomes due to their wildly
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different disease prognosis and genetic backgrounds. Patients with therapy-related MDS and secondary AML evolving from MDS exhibit suboptimal responses to conventional
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chemotherapy and an extremely low survival rate following allogeneic HCT [22-26]. In addition to an innate resistance to chemotherapy, the dismal prognosis for these conditions could also be associated with their distinct disease biology as evidenced by the acquisition of
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new mutations and their poor prognostic karyotypes [18, 27-29]. TP53 mutations are the most frequent genetic mutations in these patients. The incidence of TP53 mutations in therapyrelated MDS is quite high compared to that in de novo MDS [30, 31]. As a result, mutational genes, which are predictive of transplant outcomes, may differ between de novo MDS and therapy-related MDS or secondary AML. These observations justify the separate analysis of
genetic mutations only in de novo MDS to identify a specific molecular predictor for the disease. In the cohort comprised only of de novo MDS patients, our study results showed the poor prognostic influences of TP53 mutations, evidenced by short median survival and a high
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relapse rate. With these results, we raised the question of whether the negative influences attributed to TP53 mutation in other studies resulted from the inclusion of patients with de
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novo MDS, therapy-related MDS, or secondary AML, due to the heterogeneous nature of the
enrolled patients [12-17]. Two of the previous studies excluded secondary AML patients [16, 32]. One of them identified the prognostic implications of genetic mutations including TP53
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mutation. However, 21% of the enrolled patients had therapy-related MDS, which prevented
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the identification of de novo MDS-specific genetic mutations [16]. The other study failed to
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identify any genetic mutations associated with transplant outcomes, but the sample size was
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relatively small and the inclusion of therapy-related MDS was not clarified [32]. One recent study investigated the outcomes after allogeneic HCT for therapy-related MDS patients and
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the authors reported that there was no survival difference according to the presence or
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absence of TP53 mutation [33]. The study results suggest that the impacts of TP53 mutation may be different between de novo and therapy-related MDS. Further validation of the
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influence of TP53 mutations according to the type of MDS remains to be conducted and assessment of secondary AML should be performed first.
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Even with the poorer outcomes for patients with TP53 mutants compared to non-
mutant cases, one interesting observation in our study is that the estimated long-term survival rate for the former group reached around 40% at 5 years, which is a relatively high survival rate compared to the other study groups. Of course, data for the post-HCT survival rate in TP53 mutated MDS are still insufficient and the relatively small number of cases in each
study should be considered when comparing survival rate among the studies. Additionally, many factors other than genetic mutations may have affected the transplant outcomes among the patients with TP53 mutations. For example, although Lindsley et al. reported a 5-year survival rate approximately 20% lower than ours, their results could be associated with
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unfavorable influences from therapy-related MDS in their study [16]. Likewise, differences in conditioning intensity, disease risk and responses to pre-transplant treatment at HCT, age of
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the patients, and performance or comorbidities might have influenced the results. Apart from
these factors, we also speculate that MDS with TP53 mutation may not be a homogeneous entity and may harbor innate differences at the individual level. However, regardless of the
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reasons for different survival rates among patients with TP53 mutations, our study again
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novo MDS patients harboring TP53 mutations.
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showed that allogeneic HCT could be a curative option for this disease, especially for de
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Since allogeneic HCT is the most potent chemoimmunotherapy due to the potent
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cytotoxic preparation and subsequent allogeneic immune reaction, we sought to determine whether intense conditioning or GVL effects accompanying GVHD could control the
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reemergence of mutated clones. For patients with TP53 mutations, our results showed no association between the relapse rate and the conditioning intensity, a result also observed in
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the study by Lindsley et al [16]. This study also showed that there was no difference in relapse rate according to the presence of cGVHD. Most relapses in patients with TP53
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mutations in our study occurred within 6 months of HCT. This suggests that TP53 mutant clones are resistant to cytotoxic preparation and outpace the tumor control by GVL effects. In patients with RAS pathway mutations, intensification of the conditioning regimen has been proposed to be effective in lowering the relapse rate [16], but such an association was not observed in our study. We also could not observe any beneficial effects of cGVHD in
lowering the relapse rate in RAS pathway mutant cases (data not shown), but the number of patients with RAS pathway mutations in our study was too small. The role of conditioning intensity and allogeneic immune reaction upon disease relapse remains to be verified for the development of mutation-adapted therapeutic strategies.
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Another important factor associated with post-transplant relapse is responsiveness to pre-transplant debulking treatment, especially for higher risk disease [34]. A recent study
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showed that decitabine induces a high promising response rate in TP53-mutant AML or MDS
[35, 36], suggesting that bridging treatment with decitabine and subsequent allogeneic HSCT could be an effective treatment strategy for patients with this dismal prognosis. Other
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possible candidate treatments include novel agents being developed to effectively target
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mutated genes such as IDH1 or IDH2 mutation [37]. Of course, one more basic but important
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way to increase HCT efficiency is the selection of optimal transplantation candidates.
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Incorporation of specific genetic mutations to transplantation-specific prognostic scoring
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systems in MDS [10, 11] may enhance the predictability of the systems and may be useful to minimize unwanted toxicity due to overtreatment.
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Our study has some limitations. First, we did not use matched normal samples. We
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tried to filter the germline variants out using the public and in-house database, but it might not be sufficient to exclude the germline variants completely. Second, the number of patients analyzed in our study seems to be relatively small considering the heterogeneous nature of
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MDS. Third, the number of genes for targeted sequencing was small and specific mutations having critical prognostic implications might be missed. Lastly, the retrospective nature of our study might cause bias in interpretation of the data. In conclusion, for de novo MDS patients receiving HCT, this study showed that TP53 mutation was the only molecular signature predictive of poor survival and disease relapse.
Our study also showed that allogeneic HCT could be a curative option for patients with this dismal prognostic molecular feature.
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Acknowledgments This study was supported by a grant from the Korean Health Technology R&D project, Ministry of Health & Welfare, Republic of Korea (HI12C0129) and a grant from National
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Research Foundation of Korea (2017R1E1A1A01074913). Part of the biospecimen and data
used in this study was provided by Asan Bio-Resource Center, Korea Biobank Network
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(2012-14(57)).
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ED
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Figure legends Fig. 1. Mutational features of the 26 target genes in 202 patients. Each row represents the mutated gene and each column represents an individual patient. Fig. 2. Post-transplant clinical outcomes according to TP53 mutation status. (A) Overall
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survival; (B) Cumulative incidence of relapse; (C) Event-free survival; and (D) Non-relapse
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mortality
Fig. 3. Schematic diagram of TP53 mutations in de novo MDS cases. The x axis represents
A
CC E
PT
ED
M
A
N
U
amino acid position. The y axis represents the number of mutations.
Table 1 Patient and transplant characteristics at the time of hematopoietic cell transplantation Characteristic
No. patients (%)
Female
74 (36.6%)
Male
128 (63.4%)
IP T
Sex
SC R
Age, year Median (range)
51 (19-69)
13 (6.4%)
RCMD
51 (25.2%)
RAEB-1
54 (26.7%)
RAEB-2
84 (41.6%)
M
A
N
RCUD/RARS/5q-
IPSS
ED
Lower risk
CC E
R-IPSS
PT
Higher risk Unknown
U
Subtype
125 (62.2%) 76 (37.8%) 1
42 (20.9%)
Intermediate
61 (30.3%)
High/Very high
98 (48.8%)
Unknown
1
A
Very low/Low
HCT donor Matched sibling
69 (34.2%)
Matched unrelated
66 (32.7%)
Mismatched unrelated
8 (4.0%)
Familial haplo-identical
59 (29.2%)
Myeloablative
62 (30.7%)
Reduced-intensity
140 (69.3%)
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Conditioning regimen
4 (2.0%)
>0-5
53 (26.2%)
>5
145 (71.8%)
U
0
N
GVHD prophylaxis 114 (56.4%)
FK506/MTX
88 (43.6%)
M
A
CSA/MTX
HCT-CI
ED
0-1
129 (63.9%) 73 (36.1%)
PT
>1 Donor age, year
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ATG, mg/kg
CC E
Median (range)
35.5 (15-70)
Donor-recipient sex pair 46 (22.8%)
Others
156 (77.2%)
A
Female-to-male
RCUD, refractory cytopenia with unilineage dysplasia; RARS, refractory anemia with ringed sideroblasts; 5q-, myelodysplastic syndrome associated with isolated del(5q); RCMD, refractory cytopenia with multilineage dysplasia; RAEB, refractory anemia with excess of blasts; IPSS, International Prognostic Scoring System; RIPSS, revised IPSS; HCT, hematopoietic cell transplantation; ATG, antithymocyte globulin; GVHD, graftversus-host disease; CSA, cyclosporine; MTX, methotrexate; HCT-CI, HCT comorbidity index
Table 2 Multivariate analysis of prognostic factors for clinical outcomes after HCT HR
95% CI
P-value
Overall survival
1
Male
3.126
1.691-5.781
Platelets, x103/μL 1
<100
2.193
1.209-3.977
1
Mutant type
2.897
A
Wild type
IPSS cytogenetic risk
CC E
PT
Good
1.552-5.409
0.001
0.254
0.055-1.17
0.079
2.47
1.20-5.10
0.015
1.02-4.13
0.043
1.59-21.9
0.008
M
ED
Cumulative incidence of relapse
Poor
N
TP53
Intermediate
0.010
U
≥100
< 0.001
SC R
Female
IP T
Sex
1
Donor age
1
≥40
2.06
A
<40
NRAS Wild type
1
Mutant type
5.91
TP53 Wild type
1
Mutant type
3.38
1.62-7.06
0.001
Event-free survival
1
Male
2.689
1.537-4.705
0.001
SC R
Female
IP T
Sex
IPSS cytogenetic risk
0.033
1
Intermediate
1.305
0.565-1.897
0.911
Poor
1.990
0.013
1.040-2.603
0.033
1.311-4.394
0.005
1.022-5.11
0.044
N
1.159-3.419
A
HCT-CI 1
M
0-1 >1
1.645
ED
TP53
PT
Wild type Mutant type
U
Good
1 2.400
CC E
Non-relapse mortality Sex
1
Male
2.285
A
Female
HR, hazard ratio; CI, confidence interval; IPSS, International Prognostic Scoring System; HCT-CI, hematopoietic cell transplantation-comorbidity index
Table 3 Clinical outcomes for patients with TP53 mutations according to conditioning regimen intensity or occurrence of chronic graft-versus-host disease
N
OS
P
CIR
P
EFS
Myeloablative
9
29.6%
0.655
44.4%
0.893
33.3%
0.726 22.2% 0.338
Reduced-intensity
14
50.0%
41.7%
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Survival probabilities or cumulative incidences at 5 years
7.1%
No
11
34.1%
66.7%
0.689 21.4% 0.173
Yes
8
75.0%
60.0%
0.0%
51.2%
Chronic GVHD* 50.0% 40.0%
0.454
U
0.124
NRM
P
SC R
Conditioning regimen
P
N
N, number of patients; OS, overall survival; P, P-value; CIR, cumulative incidence of relapse; EFS, event-free
A
survival; NRM, non-relapse mortality; GVHD, graft-versus-host disease
A
CC E
PT
ED
chronic GVHD in our study population.
M
*Analysis included only patients who survived more than 138 days after HCT, which was the earliest onset of
Fig. 1. Mutational features of the 26 target genes in 202 patients. Each row represents the
A
CC E
PT
ED
M
A
N
U
SC R
IP T
mutated gene and each column represents an individual patient.
Fig. 2. Post-transplant clinical outcomes according to TP53 mutation status. (A) Overall survival; (B) Cumulative incidence of relapse; (C) Event-free survival; and (D) Non-relapse
A
CC E
PT
ED
M
A
N
U
SC R
IP T
mortality
Fig. 3. Schematic diagram of TP53 mutations in de novo MDS cases. The x axis represents
A
CC E
PT
ED
M
A
N
U
SC R
IP T
amino acid position. The y axis represents the number of mutations.