Mutational Burden in Acute Myeloid Leukemia Is Largely Age Dependent

Risk stratification of acute myeloid leukemia (AML) is important for prognosis and for guiding treatment decisions. The National Comprehensive Cancer Network (NCCN) classifies AML into three risk categories (favorable, intermediate, or poor) based on recurrent cytogenetic or molecular abnormalities. Clinical next-generation sequencing (NGS) is beginning to clarify the relationship between these categories and the spectra of oncogenic mutations, and leading to the development of new targeted therapies. The role of NGS data in favorable risk disease and the interaction of the NCCN classification with other prognostic risk factors such as age are not fully understood.

Here, we assess 32 favorable risk [12 inv(16), 2 t(8;21), and 18 normal karyotype NPM1 mutated/FLT3 ITD negative] and 88 poor risk (33 transformed from documented MDS, 47 with complex or monosomal karyotype without previous documented MDS, and 8 therapy-related with normal karyotype) cases of AML. Results of NGS using a 37 gene, myeloid neoplasm-specific panel were incorporated with conventional cytogenetics and FISH results, as well as clinical history including patient age and survival data. Genes were arranged into four functional groups: splicing, epigenetic, signaling, and transcription (see Table 1).

Analysis of the mutational spectra categorized by NCCN risk category and by age reveals several patterns (see Table 1). TP53 mutations were associated with a low total mutation number, as previously reported. Core binding factor (CBF) AMLs were primarily seen in young patients (<60 years) and were associated with a predominance of signaling mutations, without splicing or epigenetic mutations. A similar pattern was seen in young patients with favorable risk, NPM1-positive cases as well as in young patients with non-TP53-mutated poor risk AML. Older patients (≥60 years) with non-TP53-mutation poor risk AML had a high number of splicing and epigenetic mutations, as expected, but so did older patients with favorable risk, NPM1-positive disease.

For favorable risk and non-TP53-mutated poor risk AML, stratification by age (younger/older than 60 years) provided significant differentiation between total mutations and mutation categories superior to stratification by risk group (see Table 2). Stratifying mutation spectrum by age demonstrates no significant difference between younger and older poor risk TP53-mutated cases.

To determine the degree to which the effect of age and risk category on survival is driven by the increased likelihood of splicing or epigenetic mutations, Cox regression analysis was performed on retrospective overall survival data for favorable risk and non-TP53-mutated poor risk cases. This analysis demonstrated the expected significant independent effects of risk category and age, but showed no significant independent effect of the presence of splicing or epigenetic mutations (see Table 3).

These preliminary findings suggest that the mutation spectrum of AML is more a function of age than of NCCN risk category. Additionally, in our analysis, the primary effect of age on survival is independent of mutation profile.

Table 1

Category	CBF	NPM1 pos, FLT3-ITD neg, normal karyotype		Known MDS		Complex cytogenetics with no known history of MDS		Treatment related, normal karyotype		TP53 mutated poor risk
Age	<60	<60	≥60	<60	≥60	<60	≥60	<60	≥60	<60	≥60
N	13	5	13	4	19	9	17	3	5	11	20
Total mutations	1.2±0.3	2.6±0.4	3.5±0.2	2.8±0.8	3.5±0.3	2.0±0.3	2.8±0.5	0.3±0.3	3.4±1.2	1.7±0.2	1.9±0.3
Mutations by gene group
Splicing1	0	0	5	0	13	0	9	0	2	1	4
Epigenetic2	0	2	16	2	23	3	17	0	4	1	7
Signaling3	10	4	5	3	8	8	4	1	7	4	2
Transcription4	2	0	2	2	9	0	10	0	1	0	1

¹: SRSF2, U2AF1, ZRSR2, SF3B1

²: DNMT3A, IDH1, IDH2, TET2, EZH2, ASXL1

³: FLT3, KIT, KRAS, NRAS, PTPN11, WT1, CBL, MYD88

⁴: GATA2, PHF6, RUNX1, BCOR, BCORL1, ETV6

Table 2

Data set	TP53-mutated Poor Risk	Favorable Risk and Non-TP53-mutated Poor Risk
Comparison	Younger vs. Older	Younger vs. Older	Favorable vs. Poor Risk
Total mutations, P value1	NS	< 0.0001	NS
Mutations by gene group, P value2
Splicing	NS	< 0.0001	0.04
Epigenetic	NS	< 0.0001	NS
Signaling	NS	0.05	NS
Transcription	NS	0.01	0.03

NS: Not significant.

¹: T-test

²: Fisher's exact test

Table 3

	P value	Hazard Ratio
NCCN Risk Category (Favorable vs. Poor)	<0.001	3.637
Age (< or ≥60)	0.038	2.006
Presence of Splicing or Epigenetic Mutations	0.788	0.922

Cox regression analysis of overall survival