Cytogenetics and Molecular Genetics

Cytogenetic profile of de novo acute myeloid leukemia: a study based on 1432 patients in a single institution of China

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Abstract

Geographic heterogeneity of cytogenetic patterns in hematological malignancies has been reported earlier, but few systematic studies of cytogenetic abnormalities in acute myeloid leukemia (AML) patients are available. We examined the karyotypic patterns in 1432 de novo AML patients from a single center in China and compared our data with reports from other regions of the world. Chromosomal abnormalities were detected in 746 (58%) cases. The most frequent cytogenetic abnormality was t(15;17), detected in 14% of successful cases, followed by t(8;21) in 8%, and t(9;22), +21 and +8 each in 2%. The mean age of patients with a translocation karyotype was significantly younger than that of patients with normal, deletion or trisomy karyotypes. A higher incidence of AML M3 and a lower frequency of M4 were observed in the Asian population and the frequencies of certain cytogenetic aberrations were different from those in the earlier reports. Population-based age-specific incidences of AML were calculated and compared with those in western reports.

Introduction

Acute myeloid leukemia (AML) is a heterogenous disease in terms of morphology, cytogenetics, immunophenotypes, molecular genetics and clinical features. The French-American-British (FAB) classification system categorizes AML into 11 subtypes, based on morphological aspects, the percentages and maturation of blasts, and cytochemistry. Owing to the development of immunotyping, cytogenetics and molecular genetics, several other subtypes marked with recurrent chromosomal abnormalities are identified. As a result, the World Health Organization classification defines AML with t(8;21)(q22;q22), t(15;17)(q22;q11–12), inv(16)(p13q22) and 11q23/MLL rearrangements as distinct subtypes.1

Diagnostic cytogenetics is regarded as one of the most valuable prognostic indicators in acute leukemia.2, 3, 4, 5 Some cytogenetic and molecular aberrations are found to be specifically associated with distinct morphological subtypes and thus are important for revealing the underlying disease mechanisms.6 However, such close relationships are not found in most of the cases because of relatively small sample sizes.

Chromosomal abnormalities occur in approximately 56% of de novo AML in adults5, 6, 7 and a little higher in children.8, 9 To date, most of the data reported were from western countries. Racial differences in various hematological malignancies were previously reported between Asian and western countries. However, no systematic study has been undertaken in large series of Chinese patients. In this study, cytogenetic patterns were analyzed in 1432 patients with AML, most of whom were from Zhejiang, a southeastern province of China. Our aim was to define the frequencies and subtypes of chromosomal abnormalities among AML patients in the Chinese population and to compare the cytogenetic profile as well as possible underlying causes with those reported from other populations.

Materials and methods

Patients

The current patient information was derived from the Department of Hematology, the First Affiliated Hospital, Zhejiang University College of Medicine. As the central hospital in Zhejiang Province, with a population of more than 45.9 million, a minimum of 95% of our patients were residents of this province. Since 1994, nearly 10 000 patients with suspected hematological disorders had cytogenetic analyses performed in our institution. All patients diagnosed with AML between 1994 and 2007 were included in this study, except those with a history of prior chemo- or radiotherapy or an antecedent hematological disorder. Some hematological patients may have been treated in other hospitals in Zhejiang Province, but our institution has the only central laboratory capable of cytogenetic analysis. Therefore, all bone marrow samples from other hospitals in this province were sent to our laboratory for karyotyping, allowing our study to cover almost the whole province.

Cytogenetic studies

Conventional cytogenetics was performed in all cases at the time of diagnosis. Chromosomes were R-banded on bone marrow cells from direct and/or 24-h unstimulated cultures. Karyotypes were described according to the International System for Human Cytogenetic Nomenclature criteria (2005).10 Cytogenetic analysis was considered successful if a clonal chromosomal abnormality was detected or a minimum of 20 metaphases were analyzed. If a clonal aberration was absent and 20 or more metaphases had a normal karyotype, the patient was considered to be cytogenetically normal. Owing to the lack of facilities and the high cost of the procedure, fluorescence in situ hybridization was not performed as a routine analysis.

Acute myeloid leukemia cases were categorized into four mutually exclusive karyotype groups: translocation, deletion, trisomy and normal, as described earlier by Moorman et al.11 Cases were also grouped according to a modified Chicago Classification,2 using the hierarchy: t(15;17), t(8;21), t(9;22), −5/del(5q), −7/del(7q), 11q23 rearrangements, +21 and +8. Cases with a normal karyotype and with a complex aberrant karyotype (defined as three or more clonal abnormalities12) represented their own categories. inv(16)/t(16;16) was not included, because R-banding is unsuitable for detecting this aberration. As M4EO was rare in our cohort (n=8), it would have minimal effect on the results.

Statistics

For each cytogenetic subgroup, the percentage of the total number of cases successfully karyotyped in the corresponding age category was calculated and prevalence was evaluated by χ2 analysis. The mean ages of the subgroups were compared using one-way analysis of variance, with Scheffe post hoc testing to determine where differences existed. P-values less than 0.05 were considered statistically significant. All statistical analyses were evaluated using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA).

Results

Patient characteristics

Between December 1994 and November 2007, a total of 1432 patients with de novo AML registered in our institutional cytogenetic laboratory, including 778 male and 654 female individuals. The median age was 42 years (range 4–84 years). These patients were further categorized into FAB subtypes based on morphological diagnoses, and the prevalence was listed in Table 1. The most common subtype in this cohort was M2 (30%, n=426), followed by M3 (25%, n=376) and M5 (23%, n=336).

Table 1 Number (%) of AML patients by FAB subtype, age, gender and cytogenetics

Cytogenetic results

Successful cytogenetic analyses were achieved in 1293 (90%) patients, among whom 746 (58%) had detectable clonal abnormalities, whereas 547 (42%) were considered cytogenetically normal. The most frequent cytogenetic abnormality was t(15;17), detected in 14% of successful cases, followed by t(8;21) in 8%, and t(9;22), +21 and +8 each in 2%. By Moorman classification,11 30% had translocational karyotypes, 9% had deletional karyotypes and 19% had trisomic karyotypes. Considering FAB subtype, M3 cases had the highest incidence of translocation (58%); M1 had the highest incidence of deletion (18%); and M5 had the highest incidence of trisomy (29%). A complex karyotype was more common in the M0 subgroup (17%), detailed in Table 2.

Table 2 Cytogenetic results of different FAB subtypes

Age and sex distribution

The mean ages of different subtypes were compared. Patients with M3 were younger than those with any other FAB subtypes, significantly when compared with M1, M2 and M5. By the Moorman classification, the translocation population was significantly younger than any other, with no significant differences between the other groups (that is, deletion, trisomy and normal). Such trends were not observed between Chicago groups, except that patients with t(15;17) and t(8;21) were significantly younger than the normal karyotype group. Table 1 showed the incidence of each cytogenetic abnormality by age decade.

In this series, AML occurred in all age ranges, with an age peak of incidence at 40–49 years. Age distributions of FAB subtypes were similar, except for M1 and M6, which both had a peak at 50–59 years (P<0.05). By Moorman classification, the age distributions of patients with normal, deletion and trisomy karyotype patterns were similar to the total distribution, whereas translocation was more common in a younger population (P<0.01). Overall, the age distributions of patients with specific chromosomal abnormalities were similar to those of the corresponding Moorman group. The only noted difference was +21, which had higher incidence at 10–19 years (P<0.05).

Three subgroups showed an uneven sex distribution (that is, male:female2 or 0.5): M6 and −7/del(7q) had a male predominance, whereas −5/del(5q) had a female predominance.

Population-based incidence

The population-based absolute incidences of AML with different cytogenetic abnormalities were defined. The age-specific incidences were calculated on the basis of the age distribution of the general population of Zhejiang Province in 2000.13 In this population, the incidence of AML increased with age and ranged from 0.3 to 6.2 cases per 100 000 between 0 and 60 years and began to decrease with age from 4.9 to 1.4 cases per 100 000 above 60 years. The incidences of cytogenetic abnormalities in the Moorman classification had similar age distribution (Figure 1), that is increased with age below 60 years and decreased with age above 60 years, with the exception that the deletion group had the highest incidence at 60–69 years and decreased after that.

Figure 1
figure1

Population-based incidences of the cytogenetic subtypes in acute myeloid leukemia.

Discussion

Acute myeloid leukemia is a heterogenous group of diseases, each of which has characteristic clinical, morphological, phenotypic and cytogenetic features. Chromosomal abnormalities and the resultant unique molecular rearrangements may provide insights into the pathogenesis of each disease. The presence of geographic heterogeneity of cytogenetic abnormalities in hematological malignancies has been described,14, 15, 16 and a better understanding of the genetic and environmental factors could reveal etiological factors involved in the development of leukemia. However, cytogenetic data of AML patients are lacking from many parts of the world. The reported data were mostly based on small case numbers. To our knowledge, this study is the largest consecutive series of AML cases reported in China.

In our study, chromosomal abnormalities were detected in 58% of the AML cases, comparable with earlier reports from other geographic regions (ranging from 41.8 to 67%).6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 The results of this study compared with several population-based and regional studies are detailed in Table 3. These represent most large-scale published studies that deal exclusively with de novo AML. In this cohort, 29.9% of our AML patients were diagnosed with M2, comparable with that reported from Germany (39.6%), America (37%) and Singapore (32%).6, 18, 19 The incidence of M3 was higher in our study (25.3%) and Singapore (14%) than that in Germany (5%) and America (2%). In contrast, the frequency of M4 was lower in this cohort and Singapore than that in Germany and America (5.4 and 2.7% vs 20.4 and 23%). Notably, 81.3% of the patients in the Singaporean study were Chinese, whereas most of the patients analyzed in German and American reports were Caucasian, suggesting that the differences mentioned above may be because of ethnic factors.

Table 3 Comparison of the karyotypic patterns in AML patients

Balanced translocations were generally found in proportions comparable with other large series and correlated well with FAB subtypes. The t(15;17) was seen in 14.5% of our AML patients, comparable with that observed in Taiwan (15%), Singapore (11%) and Japan (11%).14, 19, 24 The frequency of t(15;17) in M3 in our series was only 56.0%, much lower than that reported in Japan (75.4%), Singapore (82.5%) and some western countries (around 90%). A possible explanation would be the methodology employed as most of the other countries, except France, use G-banding for cytogenetic analysis, whereas we use R-banding because of historical reasons. However, because the telomeres were heavily painted using R-banding, theoretically, it should be a more sensitive method than G-banding in detecting abnormalities that involve the ends of chromosomes, such as t(15;17). In support of this, a French report showed a high incidence of t(15;17) in their M3 cases (97%),27 indicating that this abnormality would seldom be missed by the technique. Thus, the discrepancies between the frequencies of t(15;17) might be attributed to other factors: (1) Molecular analysis was not initially applied for the detection of cytogenetic abnormalities. It was reported that leukemic cells with t(15;17) do not proliferate in culture and hence do not enter into metaphase,28 suggesting that it could be underestimated by conventional cytogenetic analysis. Owing to this consideration, reverse transcription PCR was performed in 264 of our M3 cases. A total of 232 (87.9%) samples were positive for a PML-RARα rearrangement, whereas a t(15;17) was detected in only 143 (54.2%) cases of this group using conventional cytogenetic analysis. This suggests that reverse transcription PCR is helpful in detecting cryptic t(15;17). In relation to this issue, Grimwade et al.29 studied 60 cases of M3 lacking t(15;17). By using fluorescence in situ hybridization/reverse transcription PCR, they found an underlying PML/RARα rearrangement in the majority (42/60) of the patients, either due to insertions (28/40) or due to complex aberrations (14/42). In their study, the cytogenetically undetectable PML/RARα rearrangement accounted for more than 5% of the whole M3 group. Taking into account the population-based nature of our study, it is likely that a considerable proportion of PML/RARα in our cohort resulted from cryptic insertion. In this case, as suggested by Grimwade's study, PML/RARα is formed on 15q, 17q or a third chromosome, whereas chromosomes 15 and 17 are apparently normal by both conventional cytogenetic analysis and fluorescence in situ hybridization using whole chromosome probes. Alternatively, some rare or even unreported complex aberrations could be very difficult to reveal by karyotyping. Unfortunately, insufficient material of most cases was preserved to perform further metaphase fluorescence in situ hybridization to define the precise location of the PML-RARα fusion gene. (2) Owing to the high incidence of M3 in our population, we studied 362 cases with M3, whereas most other reports were based on less than 70 cases. Such a large difference might cause a statistical variation. (3) Ethnic differences should also be considered. Nakase et al.14 observed that the incidence of t(15;17) in M3 was lower in Japanese compared with Australians, though the difference was not significant, and the result was limited because of a small number of cases. At present, there were insufficient data for a definite conclusion. More cases should be collected to clarify this.

Reports in Japan and Taiwan showed a higher incidence of t(8;21) in their AML M2 patients (33.1 and 34%).14, 24 This was observed in only 22.1% of our subjects, closer to that observed in Singapore (14.5%)22 and an American report (19.6%).18 However, only 8.8% were reported in a German study,6 suggesting that some geographic differences exist.

The most frequent numerical abnormality was gain of chromosome 8, occurring in 3.8% of our subjects (with or without additional abnormalities). This frequency was lower than that seen in most western countries (6–13.9%).6, 18, 20, 21 The partial and/or complete deletion of chromosomes 5 and 7 accounted for 0.8 and 1.4% of our cases. This has been seen in 6–15% of de novo AML in other reports.18, 19, 20, 21 Abnormalities of 11q23 were observed in 1.2% of cases, similar to earlier studies in which it occurred in no more than 4% of AML patients.18, 19, 20, 21

The age distribution is different from population to population. The age-specific incidence of AML in our cohort was calculated based on the population of Zhejiang Province in China. The incidence increased with age and ranged from 0.3 to 6.2 cases per 100 000 between 0 and 60 years, comparable with that reported by Schoch et al.30 (0.7–3.9 cases per 100 000). However, in contrast to the increased incidence above 60 years in that report (6.7–19.2 cases per 100 000), the incidence of AML in our study decreased with age from 4.9 to 1.4 cases per 100 000 above 60 years.

To date, few population-based studies on AML patients are available in the literature. Majority of the studies included patients enrolled in particular treatment protocols or patients of a specified age. Hence there are limitations in comparison between these studies. In this study, we reported the pattern of cytogenetics in a large series of AML patients in China. This is the largest cohort reported in the Chinese population. More epidemiological studies involving different ethnic populations and geographic regions of the world should help unfold the true nature of environmental and genetic interplay in the development of AML.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Dr Janet D Rowley from the University of Chicago Medical Center, IL, USA, for critical review of the manuscript. We also thank Dr Gang Ye for grammatical and cosmetic polishing in English writing. This work was supported in part by the Chinese National High Tech Program (863) (2006AA02Z413) (Principal Investigator: Saijuan Chen; E-mail: sjchen@stn.sh.cn), the National Natural Science Foundation of China, Zhejiang Province Commission for Science and Technology research Foundation (Principal Investigator: Jie Jin; E-mail: jiej@hzcnc.com). Yizhi Cheng designed and performed research, analyzed data and wrote the manuscript; Yungui Wang was responsible for the RT-PCR section; Huanping Wang analyzed data and performed research; Zhimei Chen performed research; Jiyu Lou performed research; Huan Xu performed research; Huafeng Wang collected and reviewed cytogenetic data; Wenbin Qian collected and reviewed the medical records; Haitao Meng collected and reviewed the medical records; Maofang Lin reviewed the medical records; Jie Jin designed research and reviewed the manuscript.

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Correspondence to J Jin.

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Keywords

  • acute myeloid leukemia
  • cytogenetics
  • population based

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