Cytogenetic and molecular data are recognized as the most valuable prognostic factors in AML both in National Comprehensive Cancer Network (NCCN) and European Leukemia Net (ELN) risk stratification models [2,3]. Most of the studies on cytogenetic profiling of AML are from Western countries [4] and similar data from the Indian subcontinents is lacking. We conducted this retrospective study to analyze the cytogenetic abnorma-lities in AML patients at a single cancer centre in India.

The diagnosis of AML was made according to the World Health Organization (WHO) classification of hematopoietic neoplasm, which requires identification of 20% or more leukemic blasts in the bone marrow or blood [6]. ELN classification was used to categorize divide the patients into three prognostic risk groups; favorable risk, intermediate risk and adverse risk [2]. Complex karyotype was defined as any karyotype with at least three chromosome aberrations, regardless of their type and the individual chromosomes involved, excluding recurrent cytogenetic abnormalities [7,8]. Conventional cytogenetic analyses were conducted on baseline bone marrow samples of patients at National Accreditation Board for testing and calibration laboratories (NABL). Bone marrow (BM) cells were cultured for 24 hours, then karyotype was analyzed using the standard G-banding technique. The karyogram were constructed, and chromosomal abnormalities were reported in accordance with the International system for human cytogenetic nomenclature (ISCN 2013) [5]. Fms-related tyrosine kinase 3 internal tandem duplication (FLT3-ITD) and nucleophosmin-1 (NPM1) mutation were performed using reverse transcriptase polymerase chain reaction (RT-PCR) from RNA extracted from BM/PB sample obtained at diagnosis from patients [9,10].

CR was defined as bone marrow blast <5%, absolute neutrophil count >1000/uL, platelet count >100000/uL, no residual evidence of extramedullary disease and the patient child independent of transfusion [11]. EFS were measured from the date of diagnosis until relapse or death. Relapse following CR is defined as reappearance of leukemic blast in peripheral blood or the finding of >5% blasts in the bone marrow, not attributable to another cause [11].

Statistical analysis: Differences between groups were assessed using Student t test for continuous variables and Pearson chi-square test for categorical variables. Kaplan-Meier curves were obtained for survival analysis for event free survival (EFS) and overall survival (OS) and the log rank test was used for comparison. OS was measured as the time from the date of diagnosis until death or last follow-up. The censoring date of the study was January 31, 2017. P<0.05 was considered to be statistically significant. Data were analyzed using the statistical software STATA 11.1 version (Texas; USA).

A total of 617 patients were registered during the study period; 145 patients were excluded from the study (16 had incomplete data, 31 were acute promyelocytic leukemia (APML), cytogenetic assessment was not done for 61 patients and cytogenetic assessment had failed in 37 patients). 472 (non M3, de novo AML) patients (320 boys) were eligible for the detailed analysis. The median (range) age was 10 (0.3, 18) years. Of these, 265 (56.1%) patients were in the intermediate risk group and 162 (34.3%) patients in the favorable risk group. There was no significant difference in baseline hemoglobin, platelet- and leucocyte count between the three risk groups. Gum hypertrophy was observed in 124 (26.2%) patients; most of these patients (66.9%) belonged to the intermediate risk group. Chloroma was present in 100 (21.1%) patients, and 54% of these belonged to the favorable risk category. Gum hypertrophy and chloroma were significantly different among the cytogenetic risk groups (both P<0.01). Rate of complete remission (P<0.01), EFS (P<0.01) and OS (P<0.01) were significantly different among three cytogenetic risk group (Table I).

TABLE I Baseline Patient Characteristics and Outcomes Among Different AML Cytogenetic Risk Groups 

The most common cytogenetic abnormality was the loss of Y chromosome observed in 61 (12.9%) patients. In the cohort of 472 patients, trisomy 8 was most frequent gain; while among the losses, the loss of Y chromosome was most commonly observed (n=61) (Web Fig. 1). Among the chromosomal gains, trisomy 8 (P<0.01) and trisomy 21 (P<0.01) were found to be significantly different between all these groups. On analyzing the chromosomal losses, monosomy 5 (P<0.01), monosomy 7 (P<0.01), monosomy 9 (P=0.03), loss of X chromosome (P<0.01) and loss of Y chromosome (P<0.01) were significantly different in the three cytogenetic risk groups (Table II).

TABLE II Various Cytogenetic Abnormalities Across ELN Groups

The information on t (8;21) by cytogenetics was available in all 472 patients. Out of these, 141 (29.9%) patients were positive for t (8; 21). WBC count (P=0.01), gum hypertrophy (P<0.01) and chloroma (P<0.01) were significantly different between patients with and without t (8;21). Choloromas were more frequently noted in t (8; 21) positive patients (P<0.01) (Table III). Significant difference was observed for trisomy 8 (P<0.01), loss of X chromosome (P<0.01) and loss of Y chromosome (P<0.01) status between the two groups with or without t (8;21). There was no significant difference in the EFS however, significant difference was observed in OS (P=0.04) of the patients with and without t (8;21) (Table III).

TABLE III Baseline Parameters, Outcomes and Other Cytogenetic Abnormalities With and Without  t (8; 21)

Survival and relapse information for all the 472 patients included in this study was available (Table I). EFS and OS were statistically significantly different for the three risk groups identified using the ELN criteria (Fig.1).

Fig. 1 Kaplan-Meier survival curves showing Event-free survival and Overall survival (OS) in three cytogenetic risk group patients.

In the current study, cytogenetic abnormalities were detected in about two-thirds of AML cases. Gum hypertrophy, chloroma and rate of complete remission were found to be significantly different between ELN cytogenetic risk groups. Translocation t (8; 21), loss of Y chromosome and trisomy 8 were the most common cytogenetic abnormalities. Event-free survival (EFS) and overall survival (OS) were found to be significantly different among the three risk groups identified using the ELN criteria.

Our institute is a major referral center for pediatric AML and caters to a major portion of patients from northern part of India. As this is not a population-based study, the data presented here may not be representative of the Indian population. Our study shows significant difference in overall survival but does not show any significant difference in event free survival of the patients differing by t (8;21) status, as we lacked molecular data for all patients. The data on molecular abnormalities is somewhat fragmented because of the retrospective nature of the study.

There are only a few population-based studies on AML patients and most have selection bias (regarding age, treatment protocol etc). In general, karyotypic pattern and frequency of specific chromosomal abnormalities were similar to those reported in previous large series except for few remarkable differences [1,12-18]. The median age in our analysis was less than other studies that have included both pediatric and adult patients. Another important finding of this study is an increased frequency (29.9%) of t (8; 21) in our population. This compares well with the data published by Amare, et al. [18] who had reported a similar frequency among their 567 pediatric patients from a tertiary care cancer center from Western India. Nakase, et al. [4] have also reported a higher frequency of t (8;21) in the Japanese patients. However, this is in stark contrast to studies from other parts of the world [14]. The 21.2% occurrence of chloromas in our study was significantly higher than the incidence of myeloid sarcoma reported in literature (2-8%) [3]. Out of these, 33.9% had favourable risk cytogenetic. The reason for the association of t (8;21) with chloroma is unknown.

Our study has shown an increased frequency of t (8; 21) and its association with chloroma. Further studies using advanced molecular tools like Next generation sequencing (NGS) would pave the way to better understanding of the biology of this disease.

Contributors: AT,RP,SB: contribution to design, acquisition of data, analysis, interpretation of data, drafting the manuscript and critical review the intellectual content of the manuscript; SC,AC: contribution to acquisition of data, interpretation of data and drafting the manuscript; VS: was the statistician. AT and RP contributed equally to this work. The final draft was approved by all.

Funding: None; Competing interest: None stated.

1. Byrd JC, Mrózek K, Dodge RK, Carroll AJ, Edwards CG, Arthur DC, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: Results from Cancer and Leukemia Group B (CALGB 8461). Blood. 2002;100:4325-36.

2. Döhner H, Estey E, Grimwade D, Amadori S, Appelbaum FR, Büchner T, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129:424-47.

3. Avni B, Koren-Michowitz M. Myeloid sarcoma: current approach and therapeutic options. Ther Adv Hematol. 2011;2:309-16.

4. Nakase K, Bradstock K, Sartor M, Gottlieb D, Byth K, Kita K, et al. Geographic heterogeneity of cellular characteristics of acute myeloid leukemia: A comparative study of Australian and Japanese adult cases. Leukemia. 2000;14:163-8.

5. Shaffer LG, McGowan-Jordan J, Schmid M, editors. An international system for human cytogenetic nomenclature (ISCN). Basel: Karger; 2013. p. 88-95.

6. Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM, et al. The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia. Blood. 2016;127:2391-405.

7. Mrózek K, Marcucci G, Nicolet D, Maharry KS, Becker H, Whitman SP, et al. Prognostic significance of the European leukemia net standardized system for reporting cytogenetic and molecular alterations in adults with acute myeloid leukemia. J Clin Oncol. 2012;30:4515-23.

8. Mrózek K. Cytogenetic, molecular genetic and clinical characteristics of acute myeloid leukemia with a complex karyotype. Semin Oncol. 2008;35:365-77.

9. Sharawat S.K, Raina V, Kumar L, Sharma A, Bakhshi R, Vishnubhatla S, et al. High fms-like tyrosine kinase-3 (FLT3) receptor surface expression predicts poor outcome in FLT3 internal tandem duplication (ITD) negative patients in adult acute myeloid leukaemia: A prospective pilot study from India. Indian J Med Res. 2016;143:11-6.

10. Chopra A, Soni S, Pati H, Kumar D, Diwedi R, Verma D, et al. Nucleophosmin mutation analysis in acute myeloid leukaemia: Immunohistochemistry as a surrogate for molecular techniques. Indian J Med Res. 2016;143:763-68.

11. O’Donnell MR, Tallman MS, Abboud CN, Altman JK, Appelbaum FR, Arber DA, et al. Acute Myeloid Leukemia, Version 3.2017, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2017;15:926-57.

12. Grimwade D, Walker H, Harrison G, Oliver F, Chatters S, Harrison CJ, et al. The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): Analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial. Blood. 2001;98:1312-20.

13. Enjeti AK, Tien SL, Sivaswaren CR. Cytogenetic abnormalities in de novo acute myeloid leukemia in adults: Relation to morphology, age, sex and ethnicity-a single center study from Singapore. Hematol J. 2004;5:419-25.

14. Bacher U, Kern W, Schnittger S, Hiddemann W, Schoch C, Haferlach T. Further correlations of morphology according to FAB and WHO classification to cytogenetics in de novo acute myeloid leukemia: A study on 2,235 patients. Ann Hematol. 2005;84:785-91.

15. Cheng Y, Wang Y, Wang H, Chen Z, Lou J, Xu H, et al. Cytogenetic profile of de novo acute myeloid leukemia: A study based on 1432 patients in a single institution of China. Leukemia. 2009;23:1801-6.

16. So CC, Wan TS, Chow JL, Hui KC, Choi WW, Lam CC, et al. A single-center cytogenetic study of 629 Chinese patients with de novo acute myeloid leukemia-evidence of major ethnic differences and a high prevalence of acute promyelocytic leukemia in Chinese patients. Cancer Genet. 2011;204:430-8.

17. Li X, Li X, Xie W, Hu Y, Li J, Du W, et al. Comprehensive profile of cytogenetics in 2308 Chinese children and adults with de novo acute myeloid leukemia. Blood Cells Mol Dis. 2012;49:107-13.