Clinical outcomes and characteristics of patients with TP53-mutated myelodysplastic syndromes

ABSTRACT Objective To explore the clinical outcomes and characteristics of TP53-mutated primary myelodysplastic syndromes (MDS). Methods A total of 74 de novo primary MDS patients who were diagnosed and treated in the Department of Hematology of our hospital from January 2018 and September 2021 were analyzed retrospectively. All patients had evaluable blood cell counts, mean corpuscular volume (MCV), lactate dehydrogenase (LDH), bone marrow (BM) morphology, biopsy, and MDS-related 20-gene mutations sequencing. In addition, 69 of 74 patients had complete cytogenetic analysis through conventional chromosome analysis and fluorescence in-situ hybridization. Results Patients were divided into two cohorts, the TP53-mutated type (TP53Mut) group (n = 19) and TP53 wild type (TP53WT) group (n = 55). Compared with the TP53WT group, patients in the TP53Mut group had higher ratios of cytogenetic abnormalities (82.4% vs. 30.8%, P < 0.001), with 5q- karyotype (64.70% vs. 38.5%, P < 0.001), complex karyotype(CK) (64.70% vs. 38.5%, P < 0.001), HR-MDS (94.7% vs. 61.8%, P = 0.008), and acute myelogenous leukemia (AML) transformation (26.3% vs. 12.7%, P < 0.001). Interestingly, patients in the TP53Mut group had lower median MCV than the TP53WT group (94.40 fl vs. 101.90 fl, P = 0.008). Furthermore, MCV = 100 fl as cutoff, and found that MCV ≤ 100 fl was more common in the TP53Mut group (73.7% vs. 38.2%, P < 0.001). After 1–4 courses of HMA ± chemotherapy, the overall response rate of the TP53Mut group was higher than the TP53WT group (83.3% vs. 71.4%, P = 0.012). With the median follow-up 12.0 months (1–46 months), the results show that the median OS and leukemia-free survival (LFS) of TP53Mut group was significantly shorter than the TP53WT group (P = 0.0018; P = 0.0310). Results of multivariate Cox proportional hazard analyses show TP53 mutation was an independent prognostic factor for the OS (HR 2.724, 95%CI 1.099–6.750, P = 0.030). Conclusion TP53-mutated primary MDS patients were associated with higher frequency of cytogenetic abnormalities, with 5q- karyotype, CK, AML transformation, higher risk IPSS-R, lower MCV and sensitive to HMA treatment, but worse survival.

TP53, a tumor suppressor gene that spans 19,144 bp on chromosome 17p13.1 and contains 11 exons, which is associated with various cancers including hematological malignancies, and it was reported to be an independent prognostic factor for MDS patients [5,6].TP53 mutations were observed in 20% of MDS patients, but the mutations reached up to 30-40% in therapyrelated MDS cases [4,7,8].Moreover, TP53 mutations occurred frequently in MDS patients with complex karyotype (CK), strongly correlated with aberrations of chromosome 5, also predicting poor prognosis [9,10].This retrospective study analyzed 74 de novo primary MDS patients in our single center, especially the gene mutations through NGS, and further enriched the characteristics of MDS patients with TP53 mutation.

Patients
This study included 74 newly diagnosed primary MDS between January 2018 and September 2021.Including the complete blood counts (CBC), LDH, mean corpuscular volume(MCV), morphology (at least two puncture sites), biopsy and cytogenetics (conventional chromosome analysis or fluorescence in-situ hybridization), the target NGS panel using bone marrow (BM) samples was also performed.The CBC and MCV should exclude the influence of blood transfusion factors.The diagnostic criteria of MDS were according to the 2016 revision of the WHO classification [1].Prognostic model of MDS was based on the revised International Prognostic Scoring System (IPSS-R).Patients in very low-, low-or intermediate-risk groups were regarded as lower-risk MDS patients, and high-and very high-risk groups as higher-risk MDS.The presence of three or more distinct numerical or structural cytogenetic abnormalities was considered as CK.This study was approved by the Ethics Committee of the Affiliated Huaian No.1 People's Hospital of Nanjing Medical University.

Statistical analysis
Statistical analysis was performed with SPSS 26.0 (IBM Corporation, Armonk, NY, USA) and Graphpad prism 8 (Graph Pad Software Inc., La Jolla, CA, USA).The twosided χ 2 test was used for categorical variables and the Mann-Whitney U test was used for continuous variables.The Kaplan-Meier analysis was adopted to assess the associations of TP53 mutation with leukemia-free survival (LFS) and overall survival (OS), and log-rank test was introduced to compare both curves.Univariate and multivariate Cox proportional hazards model were performed to identify significant prognostic predictors.Hazard ratio (HR) and 95% confidence interval (CI) were calculated.The significant variables with P < 0.1 defined in univariate survival analyses were included for the multivariate analyses to validate the prognostic value of TP53 mutation.Two-tailed P < 0.05 was considered significant.
In addition, the parameter of MCV in blood routine was also surveyed, and results showing that the median MCV of patients in the TP53 Mut group was much lower than the TP53 WT group (94.40 fl vs. 101.90fl, P = 0.008) (Table 1).According to the normal range of MCV in blood routine, using MCV = 100 fl as cutoff, we found that 15 of 19 patients in the TP53 Mut group with MCV ≤ 100 fl, while there were 21 of 55 patients in the TP53 WT group, indicating that MCV ≤ 100 fl was more common in the TP53 Mut group (73.7% vs. 38.2%,P < 0.001) (Table 1).Based on the IPSS-R, there were 18 cases with higher risk IPSS-R except 1 case in the TP53 Mut group, while 34 cases with higher and 21 cases with lower risk in the TP53 WT group.Thus, much more patients had higher risk IPSS-R in the TP53 Mut group (P = 0.008, Table 1).Furthermore, compared with the TP53 WT group, patients in the TP53 Mut group had higher ratios of cytogenetic abnormalities (82.4% vs. 30.8%,P < 0.001), with 5q-karyotype (64.70% vs. 38.5%,P < 0.001), and CK (64.70% vs. 38.5%,P < 0.001) (Table 1).

Treatment response and survival
Treatment regimens of the 74 patients were diversified, including supportive care (SC), hypomethylation agents (HMA) ± chemotherapy (HMA ± chemo) and allogeneic hematopoietic stem cell transplantation (allo-HSCT).A total of 37 patients received SC, 1 allo-HSCT, and other 36 patients underwent HMA ± chemo regimens, of which 12 patients in the TP53 Mut group, and 21 patients in the TP53 WT group respectively.However, 2 patients have terminated HMA ± chemo regimens due to serious infection complications, 1 patient died of intracranial hemorrhage during chemotherapy.After 1-4 courses of HMA ± chemo regimens, much more patients got complete response (CR) or partial response (PR) in the TP53 Mut group than the TP53 WT group (83.3% vs. 71.4%,P = 0.012) (Table 1).Post HMA ± chemo regimens, 3 patients received allo-HSCT in the TP53 WT group.

Univariate analyses for LFS and OS
Univariate analyses were performed to investigate the prognostic factors affecting disease transformation and death (Table 3).We found that TP53 mutation (HR 3.480, 95%CI 1.506-8.041,P = 0.004) and LDH level (HR 1.001, 95%CI 1.000-1.002,P = 0.001) were potential risk factors for poor OS.TP53 mutation was also prognostic for LFS (HR 3.374, 95%CI 1.04-10.901,P = 0.042).Five patients without complete cytogenetic data, which could affect the IPSS-R stratification, thus IPSS-R was not a potential risk factor for OS in this study.

Multivariate analyses for OS
All variables with P < 0.1 in univariate analyses were included in multivariate analyses.TP53 mutation, LDH level BM dysplasis (%) were the prognosticrelated risk factors for OS.We performed multivariate Cox regression analyses with the clinical variables based on the risk factors above.The results showed that TP53 mutation was an independent unfavorable factor for OS in primary MDS patients (HR 2.724, 95% CI 1.099-6.750,P = 0.030, Table 4).

Discussion
With the widespread availability of high throughput technology like NGS, multiple mutations have been revealed as significant factors in MDS.TP53 mutational analysis by NGS has proven to be a fundamental step in the diagnosis and prognosis of MDS.In this study, the frequency of mutated TP53 was up to 25.7%.Gu et al [11] conducted a cohort of 63 MDS patients, found that TP53-mutated patients accounted for 18.5%.In our single-center study, we also found that patients in the TP53 Mut group presented higher ratio of cytogenetic abnormalities, with 5q-karyotype, CK and higher risk IPSS-R than the TP53 WT group (P < 0.05) (Table 1).Kulasekararaj AG et al [12] analyzed 318 MDS patients, found that TP53 mutations were present in 5/ 26(19%) patients with isolated 5q-, and 21/29 (72%) with CK with 5q abnormalities.Yan et al [13] summarized 425 MDS patients, with a result that mutational frequency of TP53 in IPSS-R cytogenetic prognostic subsets was 0 in very good, 2 in good (3.0%), 3 in intermediate (6.0%), 7 in poor (11.5%), and 16 in very poor (41.0%), indicating that TP53 mutational frequency ascended as karyotype risk increased (P < 0.001).In addition, we found that DNA binding domains (exons 5-8) were the most common mutation sites, and missense variant was the most common variant, consistent with literature reports [14].Based on the WHO 2022 guidelines for MDS, we found that 10 patients had biTP53 mutations, and we will study their clinical characteristics in the subsequent multi-center study.
In the past decades, the approved HMAs, azacytidine (AZA) and decitabine (DAC), have become the standard front-line treatment option in patients with higher-risk MDS [15].Some preclinical studies have demonstrated that TP53 mutations could increase the novo primary MDS.TP53 mutation correlates with high frequency of cytogenetic abnormalities, with 5q-karyotype, CK, AML transformation, higher risk IPSS-R, lower MCV and sensitive to HMA treatment, but survival.Allo-HSCT is the only curative approach for TP53-mutated MDS patients.Hunter et al [21] showed a trend of improved OS in those who achieved TP53 clonal clearance before the allo-HSCT (median 25.2 vs. 11.7 months; P = 0.10), and a serial VAF < 5% was recognized as TP53 clearance, proven to be an informative biomarker for patients proceeding to allo-HSCT.In addition, TP53-mutated MDS patients have normal or lower MCV levels inversely, increasing difficulty for clinician in the differential diagnosis of cytopenias through the blood routine.Thus, the application of an MCV cutoff (100 fl) might help us to make a more accurate risk assessment of MDS patients with TP53 mutation.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.The subtypes of MDS based on WHO 2016 classification.(A) The TP53 Mut group; (B) The TP53 WT group.

Table 1 .
Clinical and laboratory characteristics of patients in TP53 Mut group and TP53 WT group.

Table 2 .
Cytogenetic and mutation characteristics of TP53-mutated patients.