Pulmonary infection associated with immune dysfunction is associated with poor prognosis in patients with myelodysplastic syndrome accompanied by TP53 abnormalities

The aim of this study was to examine the characteristics and prognosis of patients with myelodysplastic syndrome (MDS) accompanied by TP53 abnormalities and explore potential prognostic factors and treatment responses. This retrospective analysis included 95 patients with MDS and TP53 abnormalities and 173 patients with MDS without TP53 abnormalities at the Fujian Medical University Union Hospital between January 2016 and June 2023. Among patients with TP53 abnormalities, 26 (27.4%) developed AML during the disease course, with a median transformation time of 5.7 months. Complex karyotypes were observed in 73.1% of patients, and the proportions of -5 or del(5q), -7 or del(7q), +8, and -20 or del(20q) were 81.8%, 54.5%, 30.7%, and 25.0%, respectively. These patients exhibited poor survival, with a median overall survival (OS) of 7.3 months, and had 1- and 2-year OS rates of 42.2% and 21.5%, respectively. The complete response rates for azacitidine monotherapy, venetoclax combined with azacitidine, decitabine monotherapy, and decitabine combined with low-dose chemotherapy were 9.1%, 41.7%, 37.5%, and 33.3%, respectively. Long-term survival was similar among the four treatment groups. Patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) had a median OS of 21.3 months, which trended to be longer than that of patients who did not undergo allo-HSCT (5.6 months; P = 0.1449). Patients with pulmonary infection at diagnosis experienced worse OS than those without pulmonary infection (2.3 months vs. 15.4 months; P < 0.0001). Moreover, 61.9% of patients with pulmonary infection had immune dysfunction, with a ratio of CD4+ to CD8+ T lymphocytes below two. Pulmonary infections and complex karyotypes were independent adverse prognostic factors for OS. In conclusion, TP53 abnormalities in patients with MDS were frequently accompanied by complex karyotypes, and treatments based on hypomethylating agents or venetoclax have limited efficacy. Pulmonary infections associated with immune dysfunction is associated with poor prognosis.


Introduction
Since its identification in 1979 and the revelation of its role as a tumor suppressor gene in 1989, tumor protein 53 (TP53) has been a hot topic in the field of cancer research (1).TP53 is the most frequently mutated gene in cancer (2,3).The frequency of TP53 mutations is highly variable among the different types and stages of cancers (4).Myelodysplastic syndrome (MDS) is a group of acquired clonal stem cell disorders that is very heterogeneous in its morphology, clinical features, and survival (5).TP53 mutations are detected in approximately 10%-20% of patients with de novo MDS and 30%-40% of patients with therapy-related MDS (6,7), and TP53 abnormalities occur in 70-80% of patients with complex karyotypes or a loss of chromosome 17/17p, 5/5q, or 7/7q (8,9).TP53 abnormalities in MDS are associated with high-risk disease, rapid transformation to acute myeloid leukemia (AML), resistance to conventional therapies, and poor outcomes (10)(11)(12)(13).However, the clinical value of TP53 abnormalities in patients with MDS has not been fully investigated.
Despite being one of the most studied genes since its discovery, TP53 is considered "undruggable."Therefore, TP53-mutated MDS remains a long-standing therapeutic challenge, with a median survival of only 5-10 months, irrespective of the therapies administered (14).Hypomethylating agents (HMAs) are the current standard treatment for newly diagnosed high-risk MDS and offer an overall response rate (ORR) of 17%-77% in patients with TP53-mutated MDS, with International Working Group complete response (CR) in 10-25% of patients and a median overall survival (OS) of 8.2-12.4months (15,16).Venetoclax, a selective small-molecule B-cell lymphoma 2 inhibitor, is a promising agent for the treatment of myeloid malignancies.A retrospective study showed an ORR of 57.2% and a median OS of 14 months in patients with refractory/relapsed (R/R) MDS treated with venetoclax combined with HMAs (17).Recent clinical trials have shown that venetoclax with azacitidine is effective in patients with high-risk R/R MDS, provides clinically meaningful benefits, and improves OS (18,19).Presently, treatments based on HMAs or venetoclax are the main choices for high-risk MDS; however, the efficacy of these regimens in the treatment of MDS with TP53 abnormalities needs to be further clarified.
Additionally, immune dysfunction associated with TP53 mutation has recently been observed in patients with MDS.Sallman et al. concluded that the microenvironment of TP53-mutated MDS has an immune-privileged, evasive phenotype that may be a primary driver of poor outcomes and suggested that immunomodulatory therapeutic strategies may improve survival in this molecularly defined subpopulation (20).Recently, novel immunotherapeutic approaches have been developed for TP53mutated MDS and AML and have demonstrated promising results (21,22).Currently, few studies have focused on the relationship between immune dysfunction and infection in MDS with TP53 abnormalities.
The aim of this study was to examine the characteristics and prognosis of MDS patients with TP53 abnormalities and explore potential prognostic factors for OS in patients receiving HMA-or venetoclax-based treatments.We also evaluated the prognostic value of pulmonary infections associated with immune dysfunction in this cohort.gDNA was extracted from the patient's bone marrow (BM) sample, then amplified by multiplex polymerase chain reaction (PCR).The detection of a whole exon of the TP53 gene was performed by an illumina-based NEXTSeq550 sequencer.

Definition of pulmonary infection and ratio of CD4+ to CD8+ lymphocyte
The diagnosis of pulmonary infection was based on the patient's clinical symptoms and signs, in combination with a computed tomography (CT) scan of the lung and pathogenic indicators, such as sputum culture and nucleic acid testing of pathogens.Pleural effusion was diagnosed according to a CT scan of the lung.

Treatment protocols
Among the patients with MDS and TP53 abnormalities, fiftyeight patients received HMA-based therapies, including 18 who received azacitidine and 40 who received decitabine.Twelve patients were administered a combination of venetoclax and azacitidine.Ten patients underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT).Details of the initial treatment protocols for these patients are summarized in Table 1.

Definition of response and outcomes
The therapeutic effects were evaluated according to the revised International Working Group 2023 response criteria for high-risk MDS (24).Peripheral blood counts were obtained for each cycle before treatment.BM aspiration and biopsy were performed every one or two cycles for response assessment.The ORR included CR and partial remission (PR) rates.Duration of response (DOR) was calculated as the time from CR or PR to progression or relapse, and OS was calculated from diagnosis to death or the last follow-up visit.

Statistical analysis
Differences between patient subgroups were analyzed using the chi-square test, t-test, or nonparametric test, as appropriate.Survival analysis was performed using the Kaplan-Meier method, and survival curves were compared using the log-rank test.The impact of prognostic factors such as sex, age, cytogenetic abnormalities, and other parameters on OS was analyzed using the Cox regression model.Parameters that had significant impact on OS in the univariate analysis were further included in the multivariate analysis, which was performed with the Cox regression model.All data were analyzed using GraphPad Prism

Treatment outcomes
Seventy patients (73.9%) who received HMA-or venetoclaxbased treatments were included in the efficacy analysis.Among the patients treated with HMA-based therapies, 25.9% achieved CR, with an ORR of 31.5%.The treatment-related mortality rate was 15.5%, and no significant difference was observed between the azacitidine and decitabine groups (P > 0.9999).The CR rate of azacitidine monotherapy was only 9.1%, which was lower than those observed for decitabine monotherapy (37.5%;P = 0.1197) and decitabine combined with low-dose chemotherapy (33.3%;P = 0.5147).Patients receiving venetoclax combined with azacitidine achieved a CR rate of 41.7%, which was superior to that of azacitidine monotherapy (P = 0.1550) and similar to that of decitabine-based treatment (P > 0.9999).Importantly, the median DOR was only 4.4 months in the venetoclax and azacitidine treatment group, which was significantly inferior to that in the decitabine monotherapy treatment group (11.2 months; P = 0.0093).The response rate of patients with TP53 abnormalities was lower than those without TP53 abnormalities when treating with HMA-or venetoclax-based therapies (Table 3).
Patients with MDS accompanied by TP53 abnormalities had poor long-term survival when treated with HMA-based therapies, with a median OS of 7.3 months, 1-year OS rate of 42.2%, and 2year OS rate of 21.5% (Figure 1).Patients who received azacitidineor decitabine-based treatment had similar outcomes, with a median OS of 7.3 months and 9.0 months, respectively (P = 0.9167; Figure 1).The median OS of patients treated with venetoclax and azacitidine was 5.6 months, which was not superior to that of patients treated with HMA-based regimens (Figure 1).Patients who underwent allo-HSCT trended to have favorable survival, with a median OS of 21.3 months, whereas patients who did not undergo allo-HSCT had a median OS of 5.6 months (P = 0.1449; Figure 1).Patients who developed AML had an extremely poor prognosis, with a median OS of only 1.8 months, 1-year OS rate of 14.7%, and 2-year OS rate of 7.4%.The long-term survival of patients with TP53 abnormalities was significantly inferior to those without TP53 abnormalities when treating with HMA-or venetoclax-based therapies (Table 4).
Forty-one patients (43.2%) had pulmonary infection at the time of diagnosis, and these patients had a worse prognosis than those without pulmonary infection (median OS, 2.3 months vs. 15.4 months; P < 0.0001).The median neutrophil counts were 1.24 (0.13-6.67) × 10 (9)/L and 0.99 (0.07-5.23) × 10 (9)/L in patients with and without pulmonary infection, respectively (P = 0.6377).Pleural effusion was observed in 36 of 41 (87.8%) patients with pulmonary infection.Additionally, myeloid blasts were detected by flow cytometry in some patients with massive pleural effusion, indicating that MDS with TP53 abnormalities may present with extramedullary invasion.A T cell subpopulation analysis was

B C D A
Overall survival in MDS patients with TP53 abnormalities.Survival analysis was performed using the Kaplan-Meier method, and survival curves were compared using the log-rank test.performed in 21 patients with pulmonary infection, and 13 (61.9%)had a ratio of CD4+ to CD8+ T lymphocytes below two, which revealed immune dysfunction in these patients.

Discussion
In our study, 25.9% of patients treated with HMA-based regimens achieved CR, with an ORR of 31.5%.Treatment-related mortality rates were similar between the azacitidine and decitabine groups.The CR rate of azacitidine monotherapy was lower than those of venetoclax combined with azacitidine, decitabine monotherapy, and decitabine combined with low-dose chemotherapy.Our data showed that patients with TP53 abnormalities had poor long-term survival when treated with HMA-based therapies, with median OS, 1-year survival, and 2year survival that were significantly inferior to those of patients without TP53 abnormalities in our studies (25).A previous study revealed that venetoclax plus azacitidine improved remission rates, but not DOR or OS, compared with azacitidine alone in patients with TP53-mutated MDS with high-risk cytogenetics (26).Our results also confirmed that despite a higher remission rate in the venetoclax with azacitidine and decitabine-based treatment groups compared to the azacitidine monotherapy group, no significant difference in long-term survival was observed.Hence, a higher CR rate did not directly lead to better long-term survival in patients with MDS and TP53 abnormalities, and subsequent therapies are needed after patients respond to these initial treatment protocols.
MDS is characterized by a high risk for transformation to AML, and approximately 30% of patients with MDS eventually progress to AML as reported in previous studies.In our study, 27.4% of patients developed AML during the disease course with a median transformation time of 5.7 months, which was significantly shorter than that in patients without TP53 abnormalities in previous studies (27).These patients had an extremely poor prognosis, with a median survival period of only 1.8 months.Therefore, improving the outcomes in this population remains challenging.
Although several drugs have been found to improve disease control in MDS, allo-HSCT remains the only curative treatment (28).However, multiple analyses have shown that patients with TP53-mutated MDS and AML harbor an 80-90% higher risk of relapse and death after allo-HSCT than patients with TP53 wildtype MDS (13, 29-32).The majority of these relapses and deaths following allo-HSCT occur in patients with concomitant chromosome 17 abnormalities or complex karyotypes, leading to multi-hit disease (33).Our results demonstrated that patients with MDS accompanied by TP53 abnormalities who underwent allo-HSCT trended to show better survival than those who only received HMAs or venetoclax-based treatments, but this advantage was limited and not as good as that in patients without TP53 abnormalities, as reported previously (34).
Most MDS prognostic scoring systems are based on the BM blast percentage, depth of cytopenia, and cytogenetics, and these three major features have been shown to have a significant impact on the prognosis and risk of AML transformation (35).Recently, TP53 and other genes mutations were incorporated into the Prognostic factors of patients with MDS accompanied by TP53 abnormalities.The impact of prognostic factors was analyzed using the Cox regression model, and the results are presented as hazard ratios.HR, hazard ratio; IB-2, increase blast-2; MF, myelofibrosis; DEC, decitabine; AML, acute myeloid leukemia; allo-HSCT, allogeneic hematopoietic stem cell transplantation; MDS, myelodysplastic syndrome; TP53, tumor protein 53.
Molecular International Prognostic Scoring System (36).It is well established that a complex chromosomal karyotype is an adverse prognostic factor for MDS, and the absence of TP53 mutations in patients with complex karyotypes is associated with much better survival than that in patients with TP53 mutations (37).Our data showed that 73.1% of patients with MDS accompanied by TP53 abnormalities had complex chromosomal karyotypes, and complex karyotypes were identified as an independent adverse prognostic factor of OS in patients treated with HMA-or venetoclax-based therapies.Thus, TP53 abnormalities were frequently associated with complex chromosomal karyotypes and dismal prognosis in patients with MDS.
TP53-mutated MDS is associated with a risk of severe infection.Neutropenia was previously believed to be the main predisposing factor for this increased risk, but recent studies have discovered immune abnormalities in these patients (38,39).Previous studies have demonstrated that patients with TP53-mutated MDS with infection have worse survival than those without infection (40).In our study, patients with pulmonary infection had extremely poor survival, and pulmonary infection was identified as an independent adverse prognostic factor.Importantly, patients with pulmonary infection had neutrophil counts similar to those in patients without pulmonary infection, and the T cell subpopulation analysis revealed that the majority of these patients had immune dysfunction.Therefore, immune dysfunction might be the main reason for uncontrollable infection and contribute to poor prognosis.However, the specific mechanisms responsible for this phenomenon need to be studied further.Pleural effusion was observed in 87.8% of patients with pulmonary infection, and myeloid blasts were detected by flow cytometry in some patients with massive pleural effusion.Whether patients with pleural infiltration and a percentage of BM blasts less than 20% should be diagnosed with MDS/AML, as proposed by the International Consensus Classification 2022 classification, requires further examination (41).
This study has some limitations that must be considered.Due to the single-center retrospective design and long observation period, some information bias was unavoidable.Additionally, our patients were heterogeneous with several therapeutic regimens used; therefore, the stratification of this population into small groups undermines the statistical power of the study.Because of the limited number of cases and the difficulty in detecting some microorganisms, detecting all pathogens and classifying them according to bacteria, fungi, and virus was impossible, which may have resulted in an additional source of bias.Therefore, prospective studies are still needed to confirm these findings.
In conclusion, TP53 abnormalities in MDS patients are frequently accompanied by complex karyotypes.Decitabine-based therapies are associated with a higher CR rate but similar long-term survival rates compared to azacitidine-based therapies.Venetoclax plus azacitidine improved the CR rate but did not improve survival due to the short DOR in this population.Pulmonary infection and complex karyotypes are independent adverse prognostic factors for OS.Further studies are required to overcome the poor survival of these patients.

TABLE 1
Initial treatment protocols..0software (GraphPad Software, San Diego, CA, USA), the Statistical Package for the Social Sciences software (SPSS version 21.0;IBM Corp., Armonk, NY, USA), and R software (Version 4.03, R Project for Statistical Computing, Vienna, Austria).Statistical significance was set at P < 0.05.

TABLE 2
Patients characteristics of patients with TP53 abnormalities.

TABLE 3
Treatment response of patients with MDS with or without TP53 abnormalities.
HMAs, hypomethylating agents; DEC, decitabine; AZA, azacitidine; CR, complete remission; ORR, overall response rate.P1-value: comparison of mortality rate between the two groups.P2value: comparison of CR rate between the two groups.P3-value: comparison of ORR rate between the two groups.

TABLE 4
Long-term survival of patients with MDS with or without TP53 abnormalities.
HMAs, hypomethylating agents; DEC, decitabine; AZA, azacitidine; OS, overall survival; NA: not available; Undefined: the median OS has not yet been reached by the end of follow-up.