Clonal hematopoiesis in unexplained cytopenias.

Mutational landscape of 76 CCUS patients. Unexplained cytopenia is an abnormal hematopoietic state characterized by undetermined peripheral blood cell reduction. Clonal hematopoiesis (CH) is a phenomenon in which hematopoietic stem cells (HSCs) with molecular mutations form terminally differentiated blood cells carrying reproducible biological markers. Unexplained cytopenia without CH is referred to as idiopathic cytopenia of undetermined significance (ICUS). A special subtype of unexplained cytopenia with clonal gene mutations related to hematological malignancies, named clonal cytopenia of undetermined significance (CCUS), was shown to be different from ICUS in prognosis.1 Although the diagnosis and treatment of patients with unexplained cytopenia remain controversial, it is agreed that unexplained cytopenia patients have a propensity to develop myelodysplastic syndrome (MDS) or even acute myeloid leukemia (AML).2 Therefore, the identification of unexplained cytopenia patients that have more tendency to malignant transformation is of great clinical significance. In the present study, we conducted targeted next-generation sequencing (NGS) using a relatively large panel of MDS-AML-related genes in unexplained cytopenia patients from several medical centers, aiming to investigate the profile of CH in unexplained cytopenia and explore its significance in disease progression. We enrolled 273 patients with unexplained cytopenia from 7 tertiary hospitals in China between October 2016 and April 2022. Peripheral blood or bone marrow (BM) samples were collected for targeted sequencing. Unexplained cytopenia patients were diagnosed according to the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid and Histiocytic/Dendritic Neoplasms.1 Follow-up was initiated at the date of blood or BM sampling and ended in May 2022 or at the date of death. Informed consent was obtained from all patients before sampling. The study was approved by the Medical Ethics Committee of each institution. The protocols about NGS, the methods for statistical analysis, and risk prediction model construction are described in detail in Data S1. Among all included patients, 27.8% (76/273) cases were found to harbor clonal mutations (Figure 1A), which was lower than the previously reported frequencies of 38%–64%.2, 3 The median age of our patients was about 10 years lower than those in previous studies, which might account for the decreased percentage of CH in our cohort. In addition, our NGS panels included fewer genes than those used in previous studies, which could also cause the lower frequency of CH in our study. We found that patients with CH were significantly older than those without CH (p < .001; Figure 1B), and the percentage of CH in male patients was much higher than that in female patients (38.10%, 56/147 vs. 15.87%, 20/126, p < .001; Figure 1C). Moreover, the cumulative incidence of CH increased progressively with age in unexplained cytopenia patients (Figure 1D). Mutations were detected in 25 genes, among which, DNMT3A, ASXL1, U2AF1, and TET2, were the most frequently mutated genes (Figure 1E). More details of mutation types in each patient are shown in Figure S1. It has been reported that epigenetic regulators, DNMT3A, TET2, and ASXL1, were the most frequently mutated genes in age-related clonal hematopoiesis and several malignancies.4 Among the 76 patients with CH, 67.11% (51/76) patients had 1 mutated gene, 19.74% (15/76) cases harbored 2 different mutated genes, 11.84% (9/76) cases with 3, and 1.32% (1/76) patient with 4 (Figure 1F). The relationship between blood indices and CH status in treatment-naive unexplained cytopenia patients was also analyzed. It was observed that the lymphocyte counts in the CH group were significantly lower than those in the non-CH group (1.09 ± 0.45 × 109/L vs. 1.26 ± 0.68 × 109/L, p = .031; Figure 1G). By contrast, the platelet counts in the CH group were significantly higher than those in the non-CH group (67.3 ± 78.3 × 109/L vs. 44.7 ± 44.6 × 109/L, p = .003; Figure 1H). These statistical differences remained after correction for age and sex using linear regression models. No statistical difference was observed in leukocyte, neutrophil, hemoglobin, mean corpuscular volume, red blood cell distribution width, or reticulocyte between CH and non-CH groups (all p > .05; Table S1). Smoking, prior chemotherapy, or radiation exposure has been reported to have an influence on CH occurrence.5 Accordingly, our results showed that the proportion of smokers in CH patients was significantly higher compared with that in non-CH patients (41.5% vs. 24.8%, p = .034), and no patient in our research has been received for chemotherapy or radiation. As most genes function synergistically or counteractively with other genes, we further analyzed the co-mutations and exclusive mutations between different genes. The results indicated that co-mutations occurred frequently between ASXL1 and TET2, ASXL1 and U2AF1, BCOR and DNMT3A, SF3B1 and TET2, SRSF2 and TET2, SRSF2 and WT1, and TET2 and WT1 (Table S2, all p < .05). On the contrary, mutations in DNMT3A and TET2, SF3B1 and U2AF1, as well as SRSF2 and U2AF1 exhibit mutual exclusivity within their respective gene function groups (Table S3, all p < .05). We further analyzed overall survival (OS) and progression-free survival (PFS) of the included patients using Kaplan–Meier analysis. The results showed that CH patients had a shorter OS compared with non-CH patients (p = .033; Figure 1I). We also compared the OS between CH and non-CH in the young (≤50 years old) and elderly ICUS groups separately. The results showed that CH patients have significantly poorer OS compared with non-CH patients in the young ICUS group (p = .034, Figure S2), while in the elderly unexplained cytopenia group, no significant difference was found (p = .264, Figure S3). Moreover, CH patients also presented a shorter PFS in comparison with non-CH patients (p = .008; Figure 1J). To further investigate the roles of mutations in disease transformation, we followed 6 patients who performed NGS after MDS transformation, including 3 patients from the CH group and 3 patients from the non-CH group. The follow-up periods of these 6 patients ranged from 6 months to 26 months. Three patients in the non-CH group showed new emergence of TET2, ASXL1, or PDGFRA mutations after MDS transformation (Figure 1K–M). The other three patients in the CH group also showed VAF changes in already existing mutations or emergence of new mutations after disease transformation. The first patient had a relatively stable level of ASXL1 mutation, elevated ETV6 mutation, and emergence of CBL and NRAS mutations during follow-up (Figure 1N). By contrast, U2AF1 and TP53 mutation VAFs of the second patient increased after MDS transformation (Figure 1O). MDS transformation of the third patient was accompanied by a VAF reduction in U2AF1 mutation and new occurrence of RUNX1 mutation (Figure 1P). Since there is no model available for the dynamic prediction of survival in CCUS patients, we developed a clinical prognostic signature for CCUS based on patient's blood indices. Z score was calculated to normalize the data, and leukocyte and reticulocyte were selected as indicators related to prognosis through univariate analysis after statistical correction for age and sex (Table S4). The risk scores of each patient were calculated on the basis of the following formula: Risk score = −1.814 × Expr (leukocyte) −1.898 × Expr (reticulocyte). Kaplan–Meier analysis showed that CCUS patients with a high score had a worse OS than those with a low score (p = .004, Figure 1Q). In addition, patients in the high-risk group had more gene mutations compared with those in the low-risk group (p = .043, Figure 1R). However, further validation of the risk prediction model is needed in more CCUS patients. In summary, our study revealed a decreased survival in patients with CH as compared to patients without CH in unexplained cytopenia. Unexplained cytopenia is a rare hematological disorder, while caution should be taken in unexplained cytopenia patients because of the possible transformation of the disease into MDS or even AML. Consistent with previous studies about Caucasian patients,4, 6 the most common mutated genes in our cases were also DNMT3A, ASXL1, and TET2. Moreover, U2AF1 mutations, especially in hotspots of S34 and Q157, were also common in Chinese ICUS (Figure S4). Our follow-up results showed that several gene mutations, like ASXL1, TET2, and TP53, had closer relations with disease transformation than other mutated genes. Our study highlights the importance of mutation analysis for the diagnosis and follow-up of patients with unexplained cytopenia. Our risk score model provides an estimation tool for CCUS. Therefore, close monitoring and risk evaluation of malignant transformation of CCUS patients is of great importance. Han-yang Wu was responsible for conducting the research, extracting and analyzing data, interpreting results, and writing and editing this article. Chao-yang Gu was responsible for conducting the research, extracting and analyzing data, interpreting results, and editing this article. Shuang Liu, Qiao-feng Dong, Lin Dong, Liang Wang, Xin-ru Wang, Si-yuan Cui, and Zhao Li were responsible for providing essential material and method support. Xin-guang Liu was responsible for designing the project, interpreting results, and editing this article. Dao-xin Ma was responsible for designing the project, interpreting results, editing this article, and supervision. Chun-yan Ji was responsible for supervision. This work was supported by the National Natural Science Foundation of China (Nos. 32241005, 81873439, 82170123, and 81570103), the Cultivation Project of Qilu Sanitation and Health Leading Talents (2019), and the Natural Science Foundation of Shandong Province (ZR2021MH132). The authors declare no competing financial interests. For original data, please contact [email protected]. Data S1. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.