Clinical and molecular characteristics of acute myeloid leukemia and the dismal prognosis of TP53 mutations in a real-world setting

ABSTRACT Objectives: This study aimed to evaluate the incidence and prognostic significance of common cytogenetic and molecular abnormalities in patients with TP53-mutated and non-TP53-mutated acute myeloid leukemia (AML). Methods: We retrospectively analyzed the clinical data of 326 patients with newly diagnosed AML hospitalized in our institution between October 2015 and June 2021. Classification variables were reported as percentages and compared by χ2 tests. Survival rates were evaluated by the Kaplan-Meier method. Results: The incidence of TP53 mutations in AML patients in this clinic was 9.8%, of whom 87.5% patients were over 50 years old. The common concurrent mutations of TP53 were DNMT3A, IDH2, NRAS and TET2. Patients with a TP53 variant allele frequency (VAF) ≤ 40% had better overall survival (OS) than patients with a VAF >40%. Compared with non-TP53-mutated patients, significantly more TP53-mutated patients were gene-fusion negative, +mar, – 7/del (7q), – 5/del (5q), – 17/17p-, – 12/12p-, incomplete (inc) karyotype, or complex karyotype (CK), and had FLT3-ITD or IDH2 mutations, as well as a lower complete remission (CR) rate (31.3%) and higher recurrence rate (80.0%). The 2-year OS rates of TP53-mutated and non-TP53-mutated patients were 18.8% and 47.3%, respectively (P < 0.001). Univariate analysis showed that non-TP53-mutated patients with MLL family gene fusion, +mar or – 17/17p – karyotype, and FLT3-ITD mutations had a poor prognosis, while t(8; 21) karyotype was associated with a better prognosis. TP53-mutated patients with – 7/del (7q) or – 5/del (5q) karyotype had a poor prognosis. Conclusions: The cytogenetic and molecular landscapes differed between TP53-mutated and non-TP53-mutated patients, and some abnormalities had different values between them.

Cytogenetic and molecular abnormalities may affect treatment efficacy and prognosis, and their occurrence and prognostic significance show diverse characteristics in different populations.Research into the prognosis of AML patients remains a hot topic.Improved understanding of the molecular characteristics of AML and the recent development of next-generation gene sequencing (NGS) mean that molecular detection, based on gene expression, has improved the prognostic evaluation and treatment strategies of AML.Most molecular abnormalities, such as TP53 mutations, affect the prognosis of AML but lack specific treatments, and their occurrence are mostly related to age [6][7][8].TP53 mutations are one of the most common alterations in cancer.TP53 plays important regulatory roles in apoptosis and cell cycle integrity, and its inactivation may lead to uncontrolled cell proliferation and promote cancer development [9].Approximately 5% -10% of newly diagnosed cases of AML have mutations in the TP53 gene, which tends to be associated with a poor prognosis [10][11][12][13][14][15][16].
In this study, we investigated the clinical characteristics, cytogenetic and molecular landscapes, probability and diversity of co-occurring mutations, and prognostic significance of these abnormalities in AML patients with and without TP53 mutations in a realworld setting.

Patients and diagnosis
We retrospectively analyzed 326 patients with newly diagnosed AML and available cytogenetic and molecular data, who received treatment at The Affiliated Huaian No.1 People's Hospital of Nanjing Medical University between October 2015 and June 2021.AML was diagnosed according to the 2016 revision of the WHO classification of myeloid neoplasms and acute leukemia [17].Patients with acute promyelocytic leukemia and patients lacking baseline mutation data were excluded.The clinical characteristics of the patients were collected, including age, sex, white blood cell count (WBC), hemoglobin (Hb), platelet count (PLT), cytogenetic and molecular abnormalities, initial and subsequent treatment strategies, and overall survival (OS).Bone marrow examination was performed before each consolidation chemotherapy or when the disease condition changed.All the patients provided written informed consent.The study was approved by the relevant ethics committee and was performed in accordance with the Declaration of Helsinki.
Complete remission (CR) and morphologic leukemia-free state were defined according to the European Leukemia Network (ELN 2017) criteria, and grouping was defined by the ELN 2017 criteria for combined gene mutations and karyotype abnormalities [18].CR referred to bone marrow blasts <5%, neutrophil count ≥1 × 10^9/L, PLT ≥100 × 10^9/L, and no evidence of extramedullary leukemia.OS was defined as the time from diagnosis to death from any cause or censored at last follow-up.

Statistical analysis
Data were analyzed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA).Continuous variables were expressed as median and range and compared using t-tests, and categorical variables were reported as percentages and compared using χ 2 tests.OS rates, determined from diagnosis to death, were estimated by the Kaplan -Meier method and curves were compared using log-rank analysis.Risk factors was analyzed by Cox regression.A p value <0.05 was considered to indicate a significant difference.

Baseline characteristics of patients with and without TP53 mutations
A total of 326 out of 396 AML patients were available for analysis, including 294 patients without TP53 mutations and 32 with TP53 mutations.The average age of the 326 patients was 57 years (range, 14-88 years) and the median follow-up time was 10 months (range, 1-67 months).The median survival times were 6 months (range, 1-47 months) for the 32 TP53mutated patients and 11.4 months (range, 1-67  1. M2 and M5 were the most common subtypes, and there were no significant differences in sex, WBC, Hb, and PLT between the TP53mutated and non-mutated patients.The proportion of peripheral blood primordial cells was higher in nonmutated patients.TP53 mutations were more common in patients aged ≥50 years(87.5%)(P < 0.001).Patients with TP53 mutations were all highrisk according to the 2017 ELN criteria.These patients were also more likely to be fusion-gene negative; to have + mar (marker chromosome), -7/del (7q), -5/ del (5q), -17/17p-, -12/12p-, and inc karyotypes (one or more incomplete karyotypes), CK (complex karyotype, three or more abnormalities), and FLT3-ITD mutations; and to have a lower CR rate (31.3%) and higher recurrence rate (80.0%).Patients without TP53 mutations were more likely to be lowor mediumrisk according to the 2017 ELN stratification, to have higher probabilities of t(8; 21) karyotype and IDH2 mutations, to receive standard chemotherapy, and to have a higher CR rate (58.8%) and lower recurrence rate (56.6%).
Given that age was an important factor affecting the therapy and prognosis of patients with AML, we focused on the survival of patients in different age groups (Figure 5A).Patients aged <30 years, 30-39 years, and 40-49 years had significantly better OS than patients aged 50-59 years, 60-69 years, 70-79 years, and ≥80 years.Patients aged ≥50 years were a high-risk population with similar OS rates.Taking the age 50 years as the cut-off, 104 patients were <50 years and 222 were ≥50 years old.The 1-year OS rates were 75.0% and 46.8%, respectively (P < 0.001), and the 2-year OS rates were 68.3% and 33.3%, respectively (P < 0.001).OS was significantly better in patients aged <50 years compared with patients ≥50 years(Figure 5B).Among patients aged ≥50 years, there was a significant difference in OS between TP53-mutated (n = 28) and non-TP53-mutated (n = 194) patients.However, among patients aged <50 years(n = 104), there was no significant difference in survival between patients with (n = 4) and without(n = 100) TP53 mutations(P = 0.924), possibly because of the small number of cases.

Discussion
Cytogenetic and molecular abnormalities have received increasing attention in terms of their roles  in AML prognosis.TP53 mutations are somewhat rare in AML, but TP53-mutated patients have been identified as an important molecula subgroup with dismal outcomes.The current study showed that the unique clinical biological characteristics of TP53-mutated AML patients were often accompanied by fusiongene negativity, +mar, -7/del (7q), -5/del (5q), -17/ 17p-, -12/12p-, and inc karyotypes, and CK, with a poor treatment response and a higher recurrence rate.However, significantly more non-TP53-mutated patients showed lowand medium-risk in the 2017 ELN stratification, t(8; 21) karyotype, and IDH2 mutations, and more patients received standard chemotherapy, with a higher CR rate and lower recurrence rate.Among the adverse prognostic factors in patients with TP53 mutations, CK occurred in 10% -15% of adult AML cases and was closely associated with TP53 mutations.TP53 mutations and deficiency were previously shown to allow the development of karyotype aberrations and heterogeneity in myeloid cells by perpetuating chromosome segregation errors [19][20][21].Alwash et al. found that TP53 mutations emerged during treatment in 15% of refractory or relapsed AML patients without initial TP53 mutations and in 45% of patients with CK.Patients with CK, chromosome 5 abnormality or IDH2 mutations at initial diagnosis, intensive treatment, and hematopoietic stem cell transplantation were more likely to have TP53 mutations (P < 0.05) [15].In the current study, patients with TP53 mutations had a high incidence of other karyotypes with a poor prognosis, in addition to CK, which was one of the adverse prognostic factors in these patients.Among TP53-mutated patients in our center, 37.5% had CK, which was a significantly higher percentage than in non-TP53mutated patients.The present study also showed that TP53 mutations were more common in patients aged ≥50 years, with a lower response rate to chemotherapy, and the efficacy did not depend on the type or intensity of chemotherapy drugs.Even highdose chemotherapy failed to improve the prognosis of patients with TP53 mutations, which was consistent with other studies [19,[22][23][24][25][26].Notably, even allogeneic hematopoietic stem cell transplantation had limited significance in patients with TP53-mutant AML [13,22].Patients who achieved complete or partial remission after frontline treatment had better survival than patients without remission, irrespective of transplantation.
In 2020, Short et al. explored the prognostic impact of the TP53 VAF on AML [14].In multivariate analysis, a TP53-mutated VAF >40% was independently associated with a significantly higher cumulative recurrence rate (P = 0.003) and poorer recurrence-free survival (P  ).The median survival times of the two groups were 6.1 and 3.5 months, respectively (P < 0.05), and the prognosis was poor [15].The concurrent-mutation status and mutual influence of TP53 mutations and other AML mutations remained unclear.TP53 mutations rarely co-existed with FLT3-ITD, CEBPA, or IDH1 mutations, and the most common concurrent mutations were DNMT3A, IDH2, NRAS and TET2, which were consistent with other retrospective studies [15,16,22,27].However, concurrent mutations were enriched in patients with AML marked by a non-CK.The majority of concurrent mutations are known to contribute to age-related clonal hematopoiesis, which has shown a lack of prognostic significance [20,22,27].
TP53-mutated AML still lacks targeted drugs, and shows inferior responses to high-intensity chemotherapy, hypomethylating agents, venetoclax, CPX-351 (a liposomal form of cytarabine and daunorubicin), and immunotherapy.The response rates of patients with TP53 mutation range from 20% to 42%, with a median OS of 4-9 months [28,29].Outcomes of allogeneic hematopoietic stem cell transplantation in the TP53-mutated population also tend to be among the poorest, with a 2-year OS of 24.9% and lower relapse-free survival of 23.7% [28,29].New treatment strategies are needed to overcome the adverse prognosis of TP53 mutations.Klimovich et al. have modeled partial TP53 reactivation by using knock-in mice with inducible expression of the TP53 variant E177R.Owing to the high sensitivity of cancer cells to even small changes in TP53 activity, partial p53 reactivation was sufficient to induce cancer regression [30].

Conclusion
This study found that TP53 mutations were more common in AML patients aged ≥50 years.TP53mutated patients had specific clinicobiological features and cytogenetic changes associated with dismal outcomes.Newer molecular targeted therapies are urgently needed to enhance the remission rate and outcomes in patients with TP53-mutated AML.

Figure 1 .
Figure 1.The 2-year OS of patients with and without TP53 mutations.

Figure 2 .
Figure 2. The 2-year OS with different prognostic factors in patients without TP53 mutations.A: OS in patients with and without MLL family gene fusion.B: OS in patients with and without + mar karyotypes.C: OS in patients with and without -17/17p karyotype.D: OS in patients with and without FLT3-ITD mutations.E: OS in patients with and without DupMLL gene fusion.F: OS in patients with and without t (8; 21) karyotype.

Figure 3 .
Figure 3.The 2-year OS with different prognostic factors in patients with TP53 mutations.A: OS of patients with and without -7/ del (7q) karyotype.B: OS of patients with and without -5/del (5q) karyotype.C: OS of patients with and without CK.D: OS of patients with and without IDH1 mutations.

Figure 4 .
Figure 4.The 2-year OS with different prognostic factors for all patient.A: OS of patients with and without RUNX1-RUNX1T1 gene fusion.B: OS of patients with and without + mar karyotypes.C: OS of patients with and without -7/del (7q) karyotype.D: OS of patients with and without -5/del (5q) karyotype.E: OS of patients with and without -17/17p karyotype.F: OS of patients with and without -12/12p karyotype.G: OS of patients with and without inc karyotypes.H: OS of patients with and without CK.I: OS of patients with and without FLT3/ITD mutations.J: OS of patients with and without DupMLL gene fusion.

Figure 5 .
Figure 5. Age distribution and 2-year OS of AML patients in different age groups.A: Distribution of patients in different age groups.B: The OS of patients of different ages.C: OS of patients aged ≥50 years and <50 years.

Figure 6 .
Figure 6.The 2-year OS of patients with TP53 mutations.A: OS of patients with different treatment regimens.B: OS of patients weather CR or nor.

Figure 7 .
Figure 7.The 2-year OS of patients without TP53 mutations.A: OS of patients with different treatment regimens.B: OS of patients weather CR or nor.

Figure 8 .
Figure 8.The 2-year OS of patients with TP53 mutations in different situations.A: Patients with and without concurrent mutations in addition to TP53 mutations.B: OS of TP53-mutated patients VAF ≥40% and <40%.

Figure 9 .
Figure 9.A chart showing the characteristics, concurrent mutations, treatment, and outcomes of TP53-mutated patients.Each column represented one patient, and the presence of aberrations was represented by different colors.

Table 1 .
Baseline characteristics of AML patients with and without TP53 mutations.
months) for the 294 non-TP53-mutated patients.The basic clinical characteristics of the two groups of patients are listed in Table

Table 2 .
Univariate analysis of 2-year OS in patients with and without TP53 mutations.