Cytogenetic and Genetic Mutation Features of de novo Acute Myeloid Leukemia in Elderly Chinese Patients

Objectives: The present study aimed to examine the cytogenetic and genetic mutation features of acute myeloid leukemia (AML) in elderly Chinese patients. Methods: A retrospective analysis of cytogenetics and genetic mutations was performed in 113 cases (age range 50-82 years) with de novo AML. Results: The most frequent cytogenetic abnormality was t (15;17) (q22;q21), detected in 10.0% (n = 9) of successfully analyzed cases, followed by t (8;21) (q22;q22) in 8.89% (n = 8), and complex karyotypes in 5.56% (n = 5). Those with complex karyotypes included 4 cases (4.44%) of monosomal karyotypes. The frequencies of NPM1, FLT3-ITD, c-kit, and CEBPA mutations were 27.4% (31/113), 14.5% (16/110), 5.88% (6/102), and 23.3% (7/30), respectively. The complete remission rates of patients in low, intermediate, and high risk groups were 37.5%, 48.6%, and 33.3%, respectively ( χ 2 = 0.704, P = 0.703) based on risk stratification. Conclusion: Cytogenetics and genetic mutations alone may not be sufficient to evaluate the prognoses of elderly AML patients. The search for a novel model that would enable a more comprehensive evaluation of this population is therefore imperative.


Introduction
Acute myeloid leukemia (AML) is a highly heterogeneous disease. Cytogenetics and genetic mutations play important roles in the diagnoses, risk stratification, and treatment of AML. In general, the clonal chromosomal abnormalities and genetic alterations observed in the leukemic cells from AML patients can be used to categorize cases into groups with similar clinical features and prognoses (Moorman et al., 2001). However, the prognosis of elderly patients with AML is very poor, and a higher incidence of adverse karyotypes is one of the factors associated with such unfavorable outcomes (Estey, 2007;Quintás-Cardama et al., 2012). In addition, we previously reported that the occurrence rates of favorable karyotypes decreased with age, whereas that of unfavorable karyotypes increased . Furthermore, a number of genetic mutations have a particularly significant impact on AML prognosis, refining the disease's risk stratification, especially in patients with a normal karyotype. Mutations in the nucleophosmin (NPM1), CCAAT/enhancer binding protein alpha (CEBPA), and c-kit genes, as well as the internal tandem duplications of Fms-like tyrosine kinase 3 gene (FLT3-ITD) are listed as molecular predictors for AML in both, the European LeukemiaNet (ELN) classification and Long Su 1 , Xian Li 2 , Su-Jun Gao 1 *, Ping Yu 1 , Xiao-Liang Liu 1 , Ye-Hui Tan 1 , Ying-Min Liu 1 that the frequencies of NPM1 and FLT3-ITD mutations decreased in patients older than 60 years of age (Schneider et al., 2012;.
Although cytogenetics and molecular mutations of AML have been studied for many years, most investigations were conducted in young patients. Therefore, information available on elderly patients is scant. Furthermore, cytogenetic and molecular mutation data on elderly Chinese patients are even less readily available although China accounts for over 20% of the world's population. In the present study, we retrospectively analyzed the cytogenetics and genetic mutations in 113 consecutive Chinese patients with de novo AML.

Patients and induction therapy
From January 1, 2010 to August 31, 2013, 113 patients (62 men and 51 women), aged 50-82 years (median age, 59 years), with de novo AML were enrolled in this study. All patients were from the northeast region of China, including the Jilin, Heilongjiang, and Liaoning provinces. Those with a previous diagnosis of myelodysplastic syndrome (MDS) or chronic myeloproliferative disorder were excluded. Non-acute promyelocytic leukemia (APL) patients were treated with the standard "3 + 7" regimen for initial induction (daunarubicin/idarubicin + cytarabine). Some patients were also treated with CAG (cytarabine + aclarubicin + granulocyte colonystimulating factor) regimen or decitabine. APL patients were treated with arsenic trioxide and all-trans retinoic acid (ATRA). Response was assessed by bone marrow aspiration performed on days 14 and 28 after treatment initiation. All participating patients provided informed consent prior to enrolment. This study was approved by the ethics committee of Jilin University and conducted in accordance with the Declaration of Helsinki.

Cytogenetic analyses
Cytogenetic analysis was conducted as previously described (Mir Mazloumi et al., 2013). Briefly, patients' bone marrow samples were cultured in RPMI 1640 medium containing 15% fetal bovine serum (Invitrogen Co., CA, USA) for 24 hours. Cells were then treated with colcemid, followed by trypsin, and stained with Giemsa. Clonal abnormalities were defined and described according to the International System for Human Cytogenetic Nomenclature (Shaffer et al., 2009).

Statistical analyses
The Statistical Package for the Social Sciences (SPSS) version 16.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analysis. The chi-square test or Fisher exact test was employed to assess the statistical significance of the differences between groups, whereas independent sample t-test was used to compare between two groups. P-values < 0.05 were considered significant.

Therapeutic responses
APL patients treated with arsenic trioxide and ATRA were excluded from this analysis owing to their favorable outcomes. Of the 99 non-APL patients with available cytogenetic information, 35 did not elect chemotherapy, and the remaining 64 were induced with one course. Thirty cases achieved complete remission (CR), yielding a CR rate of 46.9%, whereas 16 patients achieved partial remission. The overall response rate was thus 71.9%.

Risk stratification based on cytogenetics and mutations
According to cytogenetic results, 13 patients (14.4%) were stratified as low risk, and the remaining 77 (85.6%) as intermediate (80.0%, n = 72) and high (5.56%, n = 5) risk (Table 1). Since genetic mutations could refine the risk stratification based on cytogenetics, we performed such risk analysis using both, cytogenetics and molecular mutations. Twenty-nine patients (32.2%) were classified as low risk, whereas 54 (60.0%) were of intermediate risk, and the remaining 7 (7.78%) were of high risk.
The CR rates of patients in the favorable, intermediate, and high risk groups were 37.5%, 48.6%, and 33.3%, respectively based on risk stratification. However, no statistically significant difference was observed (χ 2 = 0.704, P = 0.703).

Discussion
The prognosis of elderly patients with AML is poor, with a median survival of 4-7 months despite intensive chemotherapy (Quintás-Cardama et al., 2012). A high frequency of adverse karyotypes was considered one of the factors associated with unfavorable outcomes. Considering the differences in genetic and ethnic backgrounds, the present study was conducted to investigate the prevalence of different karyotypes and genetic mutations in elderly Chinese patients with AML.
In the present study, the incidence rates of NPM1 and FLT3-ITD mutations in patients with a normal karyotype were 36.2% and 17.5%, respectively, which were significantly lower than those reported in the United States (US) (56.1% and 31.1%) (Becker et al., 2010) and Germany (42.4% and 23.3%) (Schneider et al., 2012). However, CEBPA mutations were detected in 31.8% of those enrolled in this study, which was significantly higher than that reported in the US (11.5%) (Becker et al., 2010) and Germany (10.0%) (Schneider et al., 2012).
The above-mentioned observations may indicate that compared to those in western countries: 1) the proportion of Chinese patients with favorable cytogenetics was higher, whereas that with unfavorable cytogenetics was lower; 2) the frequencies of NPM1 and FLT3-ITD mutations were lower, and that of CEBPA mutations was higher in Chinese patients with AML. Such phenomena were also observed in a younger cohort . The differences may arise from study cohort heterogeneity or diverse ethnic and environmental backgrounds.
Based on cytogenetics, 14.4% of our patients were of low risk, whereas the remainder were intermediate and high risk, consistent with a previous report from China (low: 16.0%; intermediate and high: 84.0%) (Liu et al., 2011). Furthermore, both Liu's and our studies demonstrated that, compared to younger patients (< 60 years), the proportion of elderly patients in the low risk group decreased (14.4-16.0% versus 30.2%). Such observation was consistent with previous reports (Estey, 2007;Quintás-Cardama et al., 2012) and indicated poor prognoses for elderly AML patients.
However, our data suggested that the CR rates were not significantly different among the 3 risk groups. Additionally, different molecular mutations had no influence on the CR rates after induction therapy. Since achieving CR is the prerequisite for long-term survival in AML patients, our results indicated that the poor prognoses of elderly AML patients may be attributed to many other factors, such as comorbid conditions, a higher incidence of secondary AML or evolution from MDS, and poor performance status. Unfortunately, the long term outcome was not assessed in the present study due to relatively short follow-up periods. Nevertheless, our findings suggested that cytogenetics and genetic mutations alone were not sufficient to evaluate the prognoses of elderly patients with AML.
In conclusion, although cytogenetic and genetic mutation features have been reported to be powerful prognostic markers for young patients with AML, they may not be sufficient to evaluate the prognoses in elderly patients. The search for a novel model that would enable a more comprehensive evaluation of elderly AML patients is therefore imperative.