Acute Myeloid Leukemia and Myelodysplastic Neoplasms: Clinical Implications of Myelodysplasia-Related Genes Mutations and TP53 Aberrations

doi:10.21203/rs.3.rs-4974493/v1

Download PDF

Research Article

Acute Myeloid Leukemia and Myelodysplastic Neoplasms: Clinical Implications of Myelodysplasia-Related Genes Mutations and TP53 Aberrations

https://doi.org/10.21203/rs.3.rs-4974493/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Purpose

The fifth World Health Organization (WHO) classification (2022 WHO) and International Consensus Classification (ICC) of myeloid neoplasms have recently been published. We reclassified patients according to the revised classification and analyzed their prognosis to confirm the clinical utility of the new classifications.

Methods

We included 101 adult patients, including 77 with acute myeloid leukemia (AML) and 24 with myelodysplastic neoplasms (MDS), who underwent bone marrow aspiration and next-generation sequencing (NGS) between August 2019 and July 2023. We reclassified patients according to the revised criteria, then examined the differences and analyzed the prognosis using survival analysis.

Results

According to the 2022 WHO and ICC, 23 (29.9%) patients and 32 (41.6%) patients were reclassified into different groups, respectively, due to the addition of myelodysplasia-related (MR) gene mutations to the diagnostic criteria or the addition of new entities associated with TP53 mutations. The median overall survival (OS) of patients with AML and MR gene mutations was shorter than those of other AML group; however, the difference was not significant. Patients with AML and TP53 mutation had a significantly shorter OS than the other AML group (p = 0.0014, median OS 2.3 vs 10.3 months). They also had significantly shorter OS than the AML and MR mutation group (p = 0.002, median OS 2.3 vs 9.6 months).

Conclusion

The revised classifications allow for more detailed categorization based on genetic abnormalities, which may be helpful in predicting prognosis. AML with TP53 mutations is a new ICC category that has shown high prognostic significance in a small number of cases.

acute myeloid leukemia

gene mutations

International Concensus Classification

World Health Organization

The World Health Organization (WHO) classification of myeloid neoplasms has been revised several times to improve our understanding of the molecular features of this disease [1]. Development of molecular genetic technology has advanced our understanding of myeloid neoplasms by adding distinct groups to their classification, such as acute myeloid leukemia (AML) with genetic abnormalities [2–4]. The commercialization of next-generation sequencing (NGS) has introduced genetic information into the diagnostic criteria for AML and myelodysplastic neoplasm (MDS). Recent studies have provided a detailed census of genes mutated in myeloid neoplasms; thus, the number of gene mutations incorporated into AML diagnosis and risk stratification has increased [5]. The fifth edition of the WHO classification (2022 WHO) and the International Consensus Classification (ICC) of myeloid neoplasms have been published [6, 7]. Changes included lowering the blast threshold that defines AML and renaming “myelodysplastic syndrome” as “myelodysplastic neoplasm”.

One of the largest differences between the revised fourth WHO classification (2016 WHO) and the 2022 WHO/ICC is the change in diagnostic criteria for AML associated with myelodysplasia. In the 2016 WHO, the main diagnostic criteria of AML with myelodysplasia-related changes (AML-MRC) were morphological changes in the bone marrow (BM), a history of MDS, and chromosomal abnormalities [8]. In the 2022 WHO, morphological dysplasia alone was excluded from the criteria and mutations in eight myelodysplasia-related (MR) genes (ASXL1, BCOR, SF3B1, EZH2, SRSF2, STAG2, U2AF1, and ZSZR2) were included [7]. RUNX1 mutations were added to the ICC criteria in addition to the eight MR genes [6].

Another significant difference is the addition of MDS and AML groups associated with TP53 mutations [6, 7]. In the 2022 WHO, the subtype MDS with biallelic TP53 inactivation (MDS-biTP53) was identified when there are two or more TP53 mutations or when there is one mutation with evidence of TP53 copy number loss [7]. A subtype of MDS with mutated TP53 (MDS-TP53) was added to the ICC. AML with mutated TP53 (AML-TP53) was added only to the ICC [6]. Subsequently, the 2022 European Leukemia Net (ELN) risk stratification was revised to include new diagnostic classifications [4]. In addition to ASXL1 and RUNX1, which had already been classified as adverse in the 2017 ELN risk stratification, six other MR gene mutations were newly classified as adverse.

These changes have advanced our understanding of the molecular genetic characteristics of AML and MDS and helped apply this knowledge to clinical diagnosis and therapeutic strategies. Several studies have primarily focused on reclassification, examining differences in diagnostic criteria between 2016 WHO and the two new classifications [8–10]. However, recent studies have targeted the prognostic effects of specific AML subtypes, such as AML, myelodysplasia-related (AML-MR), and AML-TP53 [11–13]. In Korea, a study found that patients with AML-MR according to the 2022 WHO had a shorter overall survival (OS) similar to that of patients with AML-MRC according to the 2016 WHO [14], and a study of a small group of patients with MDS reported that those with TP53 mutations had a worse prognosis than those without TP53 mutations [15].

In this study, we reclassified patients with myeloid neoplasms who were initially classified according to the 2016 WHO at our institution according to the revised criteria. We identified gene mutation rates in AML and MDS and performed survival analyses for the subgroups, particularly focusing on AML with MR gene and TP53 mutations. We compared the clinical outcomes of these groups with those of other AML subgroups to evaluate the clinical usefulness of the new classifications.

Patient selection and data collection

We included all patients aged 18 years or older who had a BM examination and targeted NGS between August 2019 and July 2023. During this period, 288 patients underwent BM examination with suspicion of AML or MDS, of which 105 were tested with NGS at the time of initial diagnosis. The electronic medical records were retrospectively reviewed with respect to each patient’s demographic data and laboratory findings, including BM examination results, treatment information, and survival outcomes. After excluding four patients who subsequently relapsed and received the same NGS results as before, 101 patients were enrolled. This study was approved by the Institutional Review Board of Inje University, Busan Paik Hospital (No. 2023-10-026), which waived the need for informed consent. After reviewing the data, we reclassified the patients according to the 2022 WHO and ICC [6, 7].

Molecular tests including targeted next-generation sequencing

For fusion gene detection, RNA was isolated from BM using a QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany). Multiplex reverse transcription polymerase chain reaction was performed using the HemaVision-28N Panel (DNA Diagnostic, Risskov, Denmark).

NGS was performed using a Miseq Dx sequencing platform (Illumina Inc., San Diego, CA, USA). We identified 48 genes associated with AML and MDS (Supplementary Table S1). The targeted specimen was genomic DNA isolated from BM aspirates, and the target enrichment method was hybridized with oligonucleotide probes. The panel version and bioinformatics pipeline were NGB-Wet-V2.0 and NGB-DNA-somatic-V1.3, respectively. The sequence was aligned with the human reference genome GRCh37. The average depth of coverage was 592.8X. Among the NGS test results, gene mutations with Tier 3, unknown clinical significance, were excluded [16].

Karyotyping

BM samples were processed after 24 and 48 hours of unstimulated culture using GTL-banding (Giemsa banding using trypsin and Leishman stain). The band resolution was 300 to 400 bands. Karyotypes were interpreted according to the International System for Human Cytogenetic Nomenclature [17].

Statistical analysis

Chi-square and Fisher’s exact tests were used to detect differences in the gene mutation distribution in each classification system. We divided with AML patients into prognostic groups based on ELN risk stratifications [4]. The OS was defined as the period from the time of diagnosis to death or the end of follow-up. The endpoint was October 31, 2023. We used the Kaplan-Meier curve and log-rank test to analyze the OS of the patients in each group. All statistical analyses were performed using MedCalc (Ver 12.4, MedCalc Software, Ostend, Belgium) and R (R version 4.4.1; Rstudio version 2024. 04.2–764); p < 0.05 was considered statistically significant.

Human Ethics and Consent to Participate declarations

This study was a retrospective medical record analysis using only general clinical characteristics, molecular genetic test results, and charts of patients. No unnecessary blood collection or tests were performed. In addition, personal identification information was anonymized and managed. The study was conducted following the principles adopted in the Declaration of Helsinki, and was approved by the Institutional Review Board of Inje University, Busan Paik Hospital, Busan, Korea (2023-10-026).

Reclassification of AML and MDS based on the revised criteria

Seventy-seven patients with AML and 24 with MDS were enrolled in this study. The median age for the AML group was 67 years (range, 19–88), and that of the MDS group was 73 years (range, 54–86). Fifty-eight patients were male (57.4%).

Among the 77 patients with AML, 23 (29.9%) and 32 (41.6%) patients were reclassified into other groups based on the 2022 WHO and ICC, respectively (Fig. 1A). Using the revised criteria, 33 patients (42.9%) with AML and recurrent genetic abnormalities were classified the same as the 2016 WHO, with only the group names changing (Table 1). Of the 17 patients (22.1%) with AML-MRC in the 2016 WHO, 15 were reclassified as AML-MR based on the 2022 WHO. Seven of these had TP53 mutations and were reclassified as AML-TP53 according to the ICC. Of the 18 patients (23.4%) diagnosed with AML, not otherwise specified (NOS) according to the 2016 WHO, 11 (61.1%) were reclassified as AML-MR based on the 2022 WHO. For the ICC, 13 (72.2%) were reclassified as AML-MR due to the difference in MR gene mutations between the two classifications. One patient with t(1;11)(p32;q23) was reclassified as AML with KMT2A rearrangement (AML-KMT2A) using the 2022 WHO and AML with other KMT2A rearrangements using the ICC.

Table 1

Reclassification of acute myeloid leukemia and myelodysplastic neoplasm based on the 2022 WHO and ICC
	2016 WHO	N	%	2022 WHO	N	%	ICC	N	%
AML	RUNX1-RUNX1T1	3	3.9%	RUNX1::RUNX1T1	3	3.9%	RUNX1::RUNX1T1	3	3.9%
	CBFB-MYH11	2	2.6%	CBFB::MYH11	2	2.6%	CBFB::MYH11	2	2.6%
	PML-RARA	3	3.9%	PML::RARA	4	5.2%	PML::RARA	4	5.2%
	DEK-NUP214	2	2.6%	DEK::NUP214	2	2.6%	DEK::NUP214	2	2.6%
	MLLT3-KMT2A	4	5.2%	KMT2A rearrangement	6	7.8%	MLLT3::KMT2A	4	5.2%
							Other KMT2A	2	2.6%
	NPM1	13	16.9%	NPM1 mutation	13	16.9%	mutated NPM1	13	16.9%
	biCEBPA	6	7.8%	CEBPA mutation	6	7.8%	bZIP CEBPA	6	7.8%
	Myelodysplasia-related changes	17	22.1%	Myelodysplasia-related	30	39.0%	MR gene	24	31.2%
							MR cytogenetic abnormalities	1	1.3%
	Panmyelosis with myelofibrosis	4	5.2%	Blast-phase MPN	3	3.9%	NA
	NA			NA			mutated TP53	10	13.0%
	Not otherwise specified	18	23.4%	Defined by differentiation	8	10.4%	Not otherwise specified	6	7.8%
	Therapy-related	5	6.5%	NA			NA
MDS	Single lineage dysplasia	1	4.2%	Low blasts	10	41.7%	NOS with single lineage dysplasia	1	4.2%
	Multilineage dysplasia	9	37.5%				NOS with multilineage dysplasia	9	37.5%
	Excess blasts-1	2	8.3%	Increased blasts-1	2	8.3%	Excess blasts	2	8.3%
	Excess blasts-2	5	20.8%	Increased blasts-2	6	25.0%	MDS/AML	2	8.3%
							MDS/AML with MR gene mutations	4	16.7%
							MDS/AML with TP53	3	12.5%
	Ring sideroblasts	1	4.2%	Low blasts and SF3B1	1	4.2%	SF3B1	1	4.2%
	NA			biTP53	5	20.8%	mutated with TP53	2	8.3%
	Unclassifiable	1	4.2%	NA			NA
	Therapy-related	5	20.8%	NA			NA
Abbreviations: WHO, World Health Organization; ICC, International Consensus Classification; AML, acute myeloid leukemia; bZIP, basic leucine zipper; MR, myelodysplasia-related; NA, not applicable; MDS, myelodysplastic neoplasm.

In the 2022 WHO and ICC, therapy-related AML (t-AML) was removed and added as an additional qualifier to other AML categories. Of the five patients who were diagnosed with t-AML in the 2016 WHO, three were reclassified as AML-MR, one as acute promyelocytic leukemia (APL), and one as AML-KMT2A according to the 2022 WHO. In the ICC, two patients diagnosed with t-AML according to 2016WHO were reclassified as the same with the 2022 WHO, whereas three patients with AML-MR according to the 2022 WHO were reclassified as AML-TP53, AML-MR and AML with MR cytogenetic abnormalities in the ICC, respectively. Patients with panmyelosis and fibrosis was also excluded, and three patients had a history of myeloproliferative neoplasm (MPN). Therefore, they were reclassified as blast-phase MPN according to the 2022 WHO. In the ICC, these patients were reclassified as AML-MR and AML-TP53, respectively, according on the presence of genetic mutations.

Among the 24 patients with MDS, 7 (29.2%) and 12 (50.0%) patients were reclassified into other groups based on the 2022 WHO and ICC, respectively (Fig. 1B). Despite adjustment of the blast threshold in the 2022 WHO and ICC schemes, no cases of MDS were reclassified as AML because of the absence of defined genetic abnormalities or rare gene fusion. Instead, in the ICC, one patient with MDS with excess blasts 2 (MDS-EB2) and one patient with t-MDS according to the 2016 WHO were reclassified into the MDS/AML group, and four patients with MDS-EB2 were reclassified into MDS/AML with MR gene mutations. One patient with MDS with multilineage dysplasia (MDS-MD) and one with MDS with excess blast 1 (MDS-EB1) in the 2016 WHO was reclassified as MDS-biTP53 based on the 2022 WHO. Based on the ICC, they were reclassified as MDS-TP53. Three patients with t-MDS were reclassified as having MDS/AML with mutated TP53 (MDS/AML-TP53) in the ICC and MDS-biTP53 in the 2022 WHO. One patient with MDS, unclassifiable (MDS-U) was reclassified as having MDS with low blasts (MDS-LB) in the 2022 WHO and MDS, NOS with multilineage dysplasia in the ICC. MDS with ring sideroblasts (MDS-RS) was revised, and the name was changed to MDS with low blasts and SF3B1 mutation (MDS, LB and SF3B1) in the 2022 WHO and MDS with mutated SF3B1 (MDS-SF3B1) in the ICC.

Genetic mutation landscape in AML and MDS

Approximately 84.2% (85/101) of all patients had mutations in 29 genes. In total, 89.6% (69/77) of the patients with AML and 66.7% (16/24) of those with MDS had 211 mutations (median 2, range 0–9 per patient) and 36 mutations (median 2, range 0–7 per patient), respectively (Fig. 2). Among 69 patients with AML and mutations, 16 (20.8%) had 2 mutations and 36 patients (52.2%) had ≥ 3 mutations. The most common AML mutations were ASXL1, IDH2 and NRAS (14 patients, 18.2%), followed by DNMT3A, FLT3-ITD and NPM1 (13 patients, 16.9%). ASXL1 and IDH2 mutations were predominantly identified in AML-MR patients, whereas NRAS mutation were frequently detected in patients with AML, defining genetic abnormalities in the 2022 WHO. DNMT3A and FLT3-ITD mutations were most commonly observed in patients with AML harboring NPM1 mutation. In a survival analysis of gene mutations present in more than 10% of patients with AML, we detected a statistically significant difference in median OS based on only FLT3-ITD and TP53 mutations (p < 0.05; median OS 7.2 and 2.3 months with mutation vs. 11.7 and 10.3 months without mutation, respectively).

In patients with MDS, the most frequently observed gene mutations were ASXL1 and TP53, each found in five patients (20.8%, supplementary Fig. 1). Gene mutations in ASXL1, RUNX1, and STAG2 were significantly more common in patients with MDS with increased blasts (MDS-IB) than in those with MDS-LB in the 2022 WHO (p < 0.05, Table 2).

Table 2

Patient characteristics and gene mutations in myelodysplastic neoplasm
	All MDS	2022 WHO
	All MDS	Low blasts	Increased blasts	biTP53	P
Patients	24	11	8	5
Sex, male: female	15:9	6:5	6:2	3:2	0.656
Age, years (range)	73(54–86)	73 (56–83)	70 (54–85)	70 (58–80)	0.930
Laboratory findings, median value
White blood cell, ×10⁹/L, median (range)	2.48 (0.31–34.12)	2.44 (0.31–3.78)	2.27 (1.05–34.12)	3.46 (1.70–7.87)	0.406
Hemoglobin, g/dL, median (range)	8.3 (4.1–12.7)	9.1 (5.5–11.0)	6.9 (5.9–12.7)	7.5 (4.1–9.7)	0.537
Platelet, ×10⁹/L, median (range)	67.5 (6.0-264.0)	77.0 (6.0-259.0)	57.5 (10.0-264.0)	62.0 (21.0-102.0)	0.931
Blasts in peripheral blood, %, median (range)	0 (0–9)	0 (0–1)	1 (0–9)	3 (0–7)	0.004
Blasts in bone marrow, %, median (range)	4.8 (0-19.2)	2.2 (0-4.5)	14.5 (5.1–19.2)	13.0 (2.6–13.4)	< 0.001
Cytogenetics
Abnormal karyotype, N (%)	11 (45.8%)	4 (36.4%)	3 (37.5%)	4 (80.0%)	0.226
Complex karyotype, N (%)	6 (25.0%)	2 (18.2%)	0 (0%)	4 (80.0%)	0.003
Gene mutations
N, median (range)	2 (0–7)	0 (0–3)	4 (0–7)	0 (0–2)	0.040
Tumor suppressor
TP53	5 (20.8%)	0	0	5 (100%)	< 0.001
Transcription factors (except MR gene)
RUNX1	4 (16.7%)	0	4 (42.9%)	0	0.008
CEBPA	2 (8.3)	0	2 (28.6%)	0	0.113
Myelodysplasia related genes
ASXL1	5 (20.8%)	0	5 (62.5%)	0	0.002
BCOR	3 (12.5%)	1 (9.1%)	2 (25.0%)	0	0.373
SF3B1	2 (8.3%)	1 (9.1%)	0	0	0.725
SRSF2	2 (8.3%)	0	2 (25.0%)	0	0.113
STAG2	4 (16.7%)	0	4 (50.0%)	0	0.008
U2AF1	1 (4.2%)	0	1 (12.5%)	0	0.352
ZRSR2	1 (4.2%)	1 (9.1%)	0	0	0.540
DNA methylation
DNMT3A	3 (12.5%)	2 (18.2%)	1 (12.5%)	0	0.595
IDH2	1 (4.2%)	0	1 (12.5%)	0	0.352
TET2	1 (4.2%)	0	1 (12.5%)	0	0.352
RNA helicase
DDX41	2 (8.3%)	2 (18.2%)	0	0	0.276
Abbreviations: MDS, myelodysplastic neoplasm; WHO, World Health Organization; MR, myelodysplasia-related.

Patient characteristics and clinical outcomes in AML with MR gene mutations based on 2022 WHO classification

Among 25 patients with AML and MR gene mutations, three had gene fusions, two had a previous history of myeloproliferative neoplasm, and one had an NPM1 mutation. Six patients were not classified as AML-MR according to the 2022 WHO. Thirty patients were classified as AML-MR according to the 2022 WHO, and MR gene mutations were present in 66.7% (20/30). The remaining nine patients had complex karyotypes, and one patient had a history of MDS.

In our study, ASXL1 mutation (N = 9, 30.0%) was the most frequent among MR genes, followed by SRSF2 mutation (N = 6, 20.0%). Although RUNX1 mutation is included in MR genes only in the ICC, most of the RUNX1 mutations (75%, 6/8) were found in patients with AML-MR in the 2022 WHO. Patients with AML-MR were significantly older, had lower white blood cell (WBC) counts, and were more likely to have complex karyotypes than patients in the other groups (Table 3). When analyzing the frequency of gene mutations between the AML-MR and other groups, TP53 and SRSF2 mutations showed significantly higher rate in patients with AML-MR (p < 0.05).

Table 3

Patient characteristics and gene mutations in acute myeloid leukemia
	All AML	2022 WHO			ICC
	All AML	AML-MR	AML-others	P	AML-TP53	AML-MR	AML-others	P
Patients	77	30	47		10	25	42
Sex, male: female	43:34	20:10	23:24	0.196	8:2	14:11	21:21	0.229
Age, years (range)	67 (19–88)	71 (32–87)	62 (19–88)	0.002	69.5 (57–87)	71 (32–83)	62 (19–88)	0.034
Laboratory findings, median value
WBC, ×10⁹/L, median (range)	5.94 (0.81–231.1)	3.25 (1.03-224.18)	13.71(0.81-231.05)	0.005	3.29 (1.57–26.92)	3.33 (1.02-224.18)	15.27 (0.81-231.05)	0.009
Hemoglobin, g/dL, median (range)	8.0 (2.6–15.0)	7.9 (2.7–11.0)	8.3 (2.6–15.0)	0.154	8.3 (5.3–9.8)	7.8 (2.7–11.0)	8.3 (2.6–15.0)	0.647
Platelet, ×10⁹/L, median (range)	40.0 (3.0-344.0)	39.5 (3-143)	42.0 (6-344)	0.711	36.0 (3.0–48.0)	51.0 (4.0-143.0)	35.0 (6.0-344.0)	0.458
Blasts in PB, %, median (range)	27 (0–96)	18.5 (0–90)	30 (0–96)	0.339	4.5 (0–90)	27.0 (0–88)	30.0 (0–96)	0.218
Blasts in BM, %, median (range)	57.4 (13.7–92.7)	52.0 (21.2–92.7)	60.2 (13.7–91.8)	0.137	43.7 (23.2–90.3)	52.0 (21.2–92.7)	60.2 (13.7–91.8)	0.511
Cytogenetics
Abnormal karyotype, N (%)	45 (58.4%)	20 (66.7%)	25 (53.2%)	0.458	8 (80.0%)	13 (52.0%)	24 (57.1%)	0.060
Complex karyotype, N (%)	14 (18.2%)	13 (43.3%)	1 (2.1%)	< 0.001	8 (80.0%)	5 (20.0%)	1 (2.4%)	< 0.001
Gene mutations
N, median (range)	2 (0–9)	2.5 (0–6)	2 (0–9)	0.557	1 (1–6)	3 (0–6)	2 (0–9)	0.082
RAS pathway-related
NRAS	14 (18.2%)	4 (13.3)	10 (21.3)	0.563	0	4 (16.0%)	10 (23.8%)	0.202
KRAS	5 (6.5%)	2 (6.7)	3 (6.4)	0.671	0	2 (8.0%)	3 (7.1%)	0.667
KIT	2 (2.6%)	0	2 (4.3)	0.682	0	0	2 (4.8%)	0.425
FLT3-ITD	13 (16.9%)	4 (13.3)	9 (19.1)	0.725	0	4 (16.0%)	9 (21.4%)	0.264
FLT3-TKD	7 (9.1%)	1 (3.3)	6 (12.8)	0.319	0	2 (8.0%)	5 (11.9%)	0.487
PTPN11	5 (6.5%)	0	5 (10.6)	0.170	1 (10.0)	0	4 (9.5%)	0.276
Tumor suppressor
TP53	11 (14.3%)	8 (26.7)	3 (6.4)	0.032	10 (100.0)	0	1 (2.3)	< 0.001
PHF6	1 (1.3%)	1 (3.3)	0	0.820	0	1 (4.0%)	0	0.349
WT1	6 (7.8%)	1 (3.3)	5 (10.6)	0.465	0	1 (4.0%)	5 (11.9%)	0.311
Transcription factors (except MR gene)
RUNX1	8 (10.4%)	6 (20.0)	2 (4.3)	0.068	0	8 (32.0%)	0	< 0.001
CEBPA	6 (7.8%)	0	6 (12.8)	0.109	0	0	6 (14.3%)	0.067
SETBP1	1 (1.3%)	1 (3.3)	0	0.820	0	1 (4.0%)	0	0.349
GATA2	4 (5.2%)	1 (3.3)	3 (6.4)	0.951	0	1 (4.0%)	3 (7.1%)	0.624
Myelodysplasia related
ASXL1	14 (18.2)	9 (30.0)	5 (10.6)	0.065	0	10 (43.5)	4 (9.5%)	0.002
BCOR	3 (3.9)	3 (10.0)	0	0.108	0	3 (12.0%)	0	0.039
SF3B1	2 (2.6)	2 (6.7)	0	0.290	0	2 (8.0%)	0	0.118
SRSF2	7 (9.1)	6 (20.0)	1 (2.1)	0.024	1 (10.0)	5 (20.0%)	1 (2.4%)	0.051
STAG2	2 (2.6)	1 (3.3)	1 (2.1)	0.682	0	1 (4.0%)	1 (2.4%)	0.791
U2AF1	3 (3.9)	2 (6.7)	1 (2.1)	0.689	1 (10.0)	2 (8.0%)	0	0.148
ZRSR2	3 (3.9)	3 (10.0)	0	0.108	0	3 (12.0%)	0	0.039
DNA methylation
DNMT3A	13 (16.9)	5 (16.7)	8 (17.0)	0.786	1 (10.0)	4 (16.0%)	8 (19.0%)	0.782
IDH1	4 (5.2)	2 (6.7)	2 (4.3)	0.951	0	3 (12.0%)	1 (2.4%)	0.168
IDH2	14 (18.2)	6 (20.0)	8 (17.0)	0.978	1 (10.0)	8 (32.0%)	5 (11.9%)	0.092
TET2	9 (11.7)	4 (13.3)	5 (10.6)	0.996	1 (10.0)	4 (16.0%)	4 (9.5%)	0.716
Risk group by ELN 2022 guideline
Favorable, N (%)	20 (26.0)	0	20 (42.6%)	< 0.001	0	0	20 (47.6%)	< 0.001
Intermediate, N (%)	18 (23.4)	0	18 (38.3%)		0	0	18 (42.9%)
Adverse, N (%)	39 (50.6)	30 (100%)	9 (19.1%)		10 (100%)	25 (100%)	4 (9.5%)
Abbreviations: AML, acute myeloid leukemia; WHO, World Health Organization; ICC, International Consensus Classification; MR, myelodysplasia-related; PB, peripheral blood; BM, bone marrow; ELN, European Leukemia Net.

According to the 2022 ELN risk stratifications, all patients with AML-MR were classified into the adverse group. The median OS of the AML-MR group was shorter than that of other AML groups (p = 0.464, median OS 7.5 vs. 10.3 months, Fig. 3A). The median OS of patients with AML-MRC in the 2016 WHO and the AML-MR group in the 2022 WHO was shorter than that of patients with AML-NOS in the 2016 WHO classified as AML-MR in the 2022 WHO (P = 0.174, median OS 4.6 vs 9.6 months, Fig. 3B). In particular, the AML-MRC group in the 2016 WHO and the AML-MR group in the 2022 WHO demonstrated shorter OS than the group classified as AML-MRC in the 2016 WHO but not as AML-MR in the 2022 WHO (Fig. 3C). When examining the OS of patients with AML those with cytogenetic abnormalities showed a relatively shorter median OS than those with MR gene mutations (p = 0.473, median OS 2.4 vs. 9.6 months, Fig. 3D). The median OS of patients with SRSF2 mutation (3.85 months) was the shortest among the patients with MR gene mutations; however, statistical significance was not reached because of the small sample size (Table 4).

Table 4

Comparison of survival outcomes in patients with acute myeloid leukemia based on the presence of myelodysplasia-related gene mutations.
Gene	N	Median OS (months)	Range
ASXL1	14	7.8	0.5–28.3
BCOR	3	NA	8.2–26.6
SF3B1	2	10.4	2.6–18.2
SRSF2	7	3.85	0.4–8.2
STAG2	2	6.7	3.3–10.1
U2AF1	3	NA	0.6–6.7
ZRSR2	3	7.8	7.2–14.1
Abbreviations: OS, overall survival; NA, not applicable

Patient characteristics and clinical outcomes in AML and MDS patients with TP53 mutations based on the ICC classification

TP53 mutations were found in 11 patients with AML (14.3%) and 5 with MDS (20.8%). The median variant allelic frequencies of TP53 mutation in AML and MDS were 53.97% and 38.16%, respectively. Biallelic TP53 mutations were observed in only four patients with MDS. Ten patients with AML and TP53 mutations were classified as AML-TP53 according to the ICC. One patient with MLLT3-KMT2A fusion was classified as AML with MLLT3::KMT2A. In the ICC, AML was divided into three groups; AML-TP53, AML-MR and AML. The AML-TP53 and AML-MR groups were significantly older, had lower WBC counts, and exhibited higher rates of complex karyotypes than the other groups. We identified a significant negative correlation between TP53 mutation and the expression of other genes. According to the ICC, 25 patients with AML-MR showed a significantly higher frequency of mutations in ASXL1, BCOR, and ZRSR (p < 0.05). Ten patients with AML-TP53 were classified into the adverse group according to the 2022 ELN risk stratification. The median OS of the AML-TP53 group was significantly shorter than that of the non-AML-TP53 group (p = 0.0014, median OS 2.3 vs. 10.3 months, Fig. 4A). They also had a significantly shorter OS than the AML-MR group (p = 0.002, median OS 2.3 vs. 9.6 months, Fig. 4B). Patients with MDS and TP53 mutations had shorter median OS than patients without TP53 mutations (p = 0.1792; median OS 9.5 vs. 21.9 months).

We conducted a retrospective study on the newly revised classification of myeloid neoplasms, focusing on two key genetic alterations, MR genes and TP53 mutations, at a single institution to identify differences and assess their clinical utility.

The major changes in the 2022 WHO were the exclusion of morphologic dysplasia from the diagnostic criteria for AML-MR and the addition of eight MR gene mutations [7]. In this study, 11 patients were reclassified from AML, NOS to AML-MR in the 2022 WHO, resulting in a larger AML-MR group than the AML group. Based on the ICC, the RUNX1 mutation was added to the AML- MR criteria [6], and 13 patients in the AML, NOS group were reclassified as AML-MR. Differences in chromosomal abnormalities were observed in the diagnostic criteria for myelodysplasia-related AML included in the 2022 WHO and ICC, such as 11q deletion, monosomy 13, or 13q deletion in the 2022 WHO and trisomy 8 or 20q deletion in the ICC. In this study, one patient was reclassified as having AML with MR cytogenetic abnormalities based on an abnormal karyotype. In a similar single-center study, Zhou et al. [13] reported that the most common gene mutations in AML-MR were in TP53, RUNX1 and ASXL1. Our results are consistent with these findings, and ASXL1 and TP53 mutations were the most frequently found in patients with AML-MR. Among the MR genes, mutations in SRSF2 were the most common after ASXL1, similar to the frequencies reported in the Korean study by Park et al. [14]. In survival analysis, patients with AML-MR showed worse outcome. When comparing the survival rates between patients classified as AML-MRC in the 2016 WHO but not AML-MR in the 2022 WHO and patients classified as AML-MRC and AML-MR in the 2022 WHO, we found that MR-associated cytogenetic abnormalities and the presence of MR genes were more significant prognostic factors than morphological abnormalities [18, 19]. Additionally, patients with AML-MR with only cytogenetic abnormalities showed shorter survival outcome than those with only MR gene mutations. This finding aligns with those of previous reports, although statistical significance was not observed owing to the small sample size. Previous studies have reported a significantly shorter OS in patients with AML and ASXL1, SRSF2, and ZRSR2 mutations among MR genes than those without these mutations [14, 19, 20]. In our study, ZRSR2 mutation was detected in only three patients with AML-MR, and was found together with ASXL1 mutation in all cases. Consequently, despite its low detection frequency among MR genes, it significantly influences survival outcomes when co-occurring with ASXL1. In our study, although none of the MR gene mutations showed a significant difference in median OS, there were differences in the median OS based on the MR genes. Even though RUNX1 mutations are only included as MR genes in ICC, we observed a shorter survival time in patients with AML and RUNX1 mutations than patients with other MR gene mutations in this study, and it was the second most frequent mutations after TP53 in AML-MR in the 2022 WHO. Large-scale studies are needed to assess the difference in frequency and prognosis of MR gene mutations, including RUNX1.

Another major change in the 2022 WHO and ICC classification is the new category for TP53 mutations. TP53 mutations are commonly associated with complex karyotype, negative correlation with other gene mutations, and poor prognosis in AML and MDS [6, 21, 22]. In our study, patients with AML and TP53 mutations showed similar characteristics and poor survival outcome. In the 2022 WHO, a distinct category of myeloid neoplasm with mutated TP53 is defined for MDS, but not for AML. Therefore, the AML-MR group included some patients with TP53 mutations, as they frequently occur in patients with complex chromosomal abnormalities. MR gene mutations are also associated with poor survival in AML; however, the survival analysis between the AML-MR and other AML groups did not show a statistically significant difference. In contrast, the ICC separated AML-MR and AML-TP53, revealing a significant difference in the survival outcome between the two groups. This suggests that the AML-TP53 classification may provide a more precise stratification of patients with a poor prognosis based on the ICC. The MR genes has been reported to have better prognostic significance than the TP53 mutations and our study was consistent with that [12].

In our data, patients with AML-TP53 presented with extremely poor survival and a median OS of only 2.3 months. This is significantly lower than the survival rates reported in previous studies of AML and TP53 mutations [12]. For patients with high-risk AML, intensive chemotherapy, followed by allogenic hematopoietic stem-cell transplantation (allo-HSCT), is recommended as a potentially curative approach [23]. However, patients with AML harboring TP53 mutations exhibit a lower probability of achieving remission, and allo-HSCT has not been shown to improve OS [24]. In our study, all 10 patients with AML-TP53 received chemotherapy, with only one patient undergoing allo-HSCT. Although the median OS of the patients who underwent HSCT was longer than that of patients who received chemotherapy alone (10.8 vs 2.3 months), this difference was not statistically significant. Therefore, an optimal treatment strategy for patients with AML-TP53 remains to be established.

This study had several limitations. First, although treatment guidelines may affect prognosis, we did not classify patients based on their treatment regimens or account for treatment modification according to age. Consequently, it was not possible to perform survival analysis stratified by treatment modality. Additionally, because the data were derived from a single institution with a short observation period, some subgroups analyses were limited by the small number of cases that showed statistical significance. Finally, we only used NGS data without conducting fluorescence in situ hybridization to identify TP53 mutations and related chromosomal abnormalities.

In conclusion, the two newly revised classification criteria allowed us to further categorize patients diagnosed with AML, NOS as AML-MR or AML-TP53. The AML-TP53 group, a new classification for ICC, had highly significant prognostic value, even with a small number of patients. As more data are accumulated and analyzed, the revised criteria will be more useful in predicting prognosis and facilitate a more detailed categorization of myeloid neoplasms at the gene mutation level.

Acknowledgements: The authors declare that no funds, grants, or other support were received during the preparation of this article.

Funding Declaration: No funding

Competing interests: The authors have no conflict of interest to declare.

Human Ethics and Consent to Participate declarations: Fully approved protocol and informed consent exemption. This study was a retrospective medical record analysis using only general clinical characteristics, molecular genetic test results, and charts of patients. No unnecessary blood collection or tests were performed. In addition, personal identification information was anonymized and managed. The study was conducted following the principles adopted in the Declaration of Helsinki, and was approved by the Institutional Review Board of Inje University, Busan Paik Hospital, Busan, Korea (2023-10-026).

Author contributions: Yu S performed next-generation sequencing analysis; Lee S and Lee WS collected clinical data; Kim H analyzed data and wrote the original manuscript; Lee JY conceptualized, designed, supervised the study and reviewed and edited the manuscript; You E and Kim HR reviewed the manuscript. All authors have read and approved the final manuscript.

Availability of data and materials: all data generated or analyzed during this study are included in this article and supplementary information files.

Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016;127:2391-405.
Bernard E, Tuechler H, Greenberg PL, Hasserjian RP, Arango Ossa JE, Nannya Y, et al. Molecular International Prognostic Scoring System for Myelodysplastic Syndromes. NEJM Evid 2022;1:EVIDoa2200008.
Cancer Genome Atlas Research N, Ley TJ, Miller C, Ding L, Raphael BJ, Mungall AJ, et al. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med 2013;368:2059-74.
Dohner H, Wei AH, Appelbaum FR, Craddock C, DiNardo CD, Dombret H, et al. Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN. Blood 2022;140:1345-77.
Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND, et al. Genomic Classification and Prognosis in Acute Myeloid Leukemia. N Engl J Med 2016;374:2209-21.
Arber DA, Orazi A, Hasserjian RP, Borowitz MJ, Calvo KR, Kvasnicka HM, et al. International Consensus Classification of Myeloid Neoplasms and Acute Leukemias: integrating morphologic, clinical, and genomic data. Blood 2022;140:1200-28.
Khoury JD, Solary E, Abla O, Akkari Y, Alaggio R, Apperley JF, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid and Histiocytic/Dendritic Neoplasms. Leukemia 2022;36:1703-19.
Wang X, Wang J, Wei S, Zhao J, Xin B, Li G, et al. The latest edition of WHO and ELN guidance and a new risk model for Chinese acute myeloid leukemia patients. Front Med (Lausanne) 2023;10:1165445.
Chopra S and Bailey NG. Application of the International Consensus Classification and World Health Organization 5th edition classification to a series of myeloid neoplasms. Am J Clin Pathol 2023;160:566-70.
Falini B and Martelli MP. Comparison of the International Consensus and 5th WHO edition classifications of adult myelodysplastic syndromes and acute myeloid leukemia. Am J Hematol 2023;98:481-92.
Attardi E, Savi A, Borsellino B, Piciocchi A, Cipriani M, Ottone T, et al. Applicability of 2022 classifications of acute myeloid leukemia in the real-world setting. Blood Adv 2023;7:5122-31.
Chen Y, Zheng J, Weng Y, Wu Z, Luo X, Qiu Y, et al. Myelodysplasia-related gene mutations are associated with favorable prognosis in patients with TP53-mutant acute myeloid leukemia. Ann Hematol 2024;103:1211-20.
Zhou Q, Zhao D, Zarif M, Davidson MB, Minden MD, Tierens A, et al. A real-world analysis of clinical outcomes in AML with myelodysplasia-related changes: a comparison of ICC and WHO-HAEM5 criteria. Blood Advances 2024;8:1760-71.
Park HS, Kim HK, Kim HS, Yang Y, Han HS, Lee KH, et al. The new diagnostic criteria for myelodysplasia-related acute myeloid leukemia is useful for predicting clinical outcome: comparison of the 4th and 5th World Health Organization classifications. Ann Hematol 2022;101:2645-54.
Lee C, Kim HN, Kwon JA, Yoon SY, Jeon MJ, Yu ES, et al. Implications of the 5(th) Edition of the World Health Organization Classification and International Consensus Classification of Myeloid Neoplasm in Myelodysplastic Syndrome With Excess Blasts and Acute Myeloid Leukemia. Ann Lab Med 2023;43:503-7.
Kim J, Park WY, Kim NKD, Jang SJ, Chun SM, Sung CO, et al. Good Laboratory Standards for Clinical Next-Generation Sequencing Cancer Panel Tests. J Pathol Transl Med 2017;51:191-204.
Jean M SA, Schmid M. ISCN, 2016: An International System for Human Cytogenomic Nomenclature. In: Basel, Switzerland: Karger, 2016.
Fuhrmann I, Lenk M, Haferlach T, Stengel A, Hutter S, Baer C, et al. AML, NOS and AML-MRC as defined by multilineage dysplasia share a common mutation pattern which is distinct from AML-MRC as defined by MDS-related cytogenetics. Leukemia 2022;36:1939-42.
Gao Y, Jia M, Mao Y, Cai H, Jiang X, Cao X, et al. Distinct Mutation Landscapes Between Acute Myeloid Leukemia With Myelodysplasia-Related Changes and De Novo Acute Myeloid Leukemia. Am J Clin Pathol 2022;157:691-700.
Pratcorona M, Abbas S, Sanders MA, Koenders JE, Kavelaars FG, Erpelinck-Verschueren CA, et al. Acquired mutations in ASXL1 in acute myeloid leukemia: prevalence and prognostic value. Haematologica 2012;97:388-92.
Weinberg OK, Siddon A, Madanat YF, Gagan J, Arber DA, Dal Cin P, et al. TP53 mutation defines a unique subgroup within complex karyotype de novo and therapy-related MDS/AML. Blood Adv 2022;6:2847-53.
Zhao D, Eladl E, Zarif M, Capo-Chichi JM, Schuh A, Atenafu E, et al. Molecular characterization of AML-MRC reveals TP53 mutation as an adverse prognostic factor irrespective of MRC-defining criteria, TP53 allelic state, or TP53 variant allele frequency. Cancer Med 2023;12:6511-22.
Cornelissen JJ and Blaise D. Hematopoietic stem cell transplantation for patients with AML in first complete remission. Blood 2016;127:62-70.
Daver NG, Iqbal S, Renard C, Chan RJ, Hasegawa K, Hu H, et al. Treatment outcomes for newly diagnosed, treatment-naïve TP53-mutated acute myeloid leukemia: a systematic review and meta-analysis. J Hematol Oncol 2023;16:19.

No competing interests reported.

SupplementaryFig.1.jpg
Supplementary Fig.1. Molecular landscape of patients with myelodysplastic neoplasm.
SupplementaryTableS1.docx

Download PDF

Editorial decision: Revision requested
02 Oct, 2024
Reviews received at journal
02 Oct, 2024
Reviews received at journal
12 Sep, 2024
Reviewers agreed at journal
11 Sep, 2024
Reviewers agreed at journal
06 Sep, 2024
Reviewers invited by journal
06 Sep, 2024
Editor assigned by journal
06 Sep, 2024
Submission checks completed at journal
06 Sep, 2024
First submitted to journal
25 Aug, 2024

You are reading this latest preprint version

Acute Myeloid Leukemia and Myelodysplastic Neoplasms: Clinical Implications of Myelodysplasia-Related Genes Mutations and TP53 Aberrations

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Patient selection and data collection

Molecular tests including targeted next-generation sequencing

Karyotyping

Statistical analysis

Human Ethics and Consent to Participate declarations

Results

Reclassification of AML and MDS based on the revised criteria

Genetic mutation landscape in AML and MDS

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1