TP53 Amplification, a Rare but Recurrent Alteration in Myeloid Neoplasms

ABSTRACT

The tumor suppressor gene TP53 is a key regulator of genomic stability, frequently altered across human cancers. In hematologic malignancies, TP53 lesions—typically mutations, deletions, and copy‐neutral loss of heterozygosity—are associated with complex karyotypes, therapy resistance, and dismal prognosis. Amplifications of TP53, however, have not been systematically described in this context. We retrospectively analyzed cases submitted to our laboratory (09/2005–10/2025) by interphase FISH in which we suspected a TP53 amplification. Among these, eight cases exhibited TP53 amplification, all classified as myeloid neoplasms (n = 6 acute myeloid leukemia, AML; n = 2 myelodysplastic neoplasms, MDS). Median patient age was 72 years (range 48–83). Chromosome banding revealed highly complex karyotypes in all cases (4–12 aberrations), including 5q deletions (in 7/8 patients) and −7/7q− (4/8). Targeted sequencing demonstrated concurrent TP53 mutations in all patients (median variant allele frequency 48%, range 18%–91%) with few additional co‐mutations. CNV (copy number variation) analysis confirmed focal TP53 copy number gains, but whole‐transcriptome sequencing showed only modest TP53 expression increases, indicating that amplification did not translate into overexpression. Clinical data available for four AML cases revealed a median overall survival of 1 month. These findings indicate that TP53 amplification, although rare, constitutes a recurrent event in myeloid neoplasms, uniformly accompanied by TP53 mutation and complex cytogenetics. The combination of mutation and amplification likely confers biallelic TP53 inactivation, aligning these cases with multi‐hit TP53 AML/MDS characterized by extreme genomic instability and highly adverse outcomes. Taken together, these data indicate that TP53 copy number assessment may be of potential diagnostic interest, although its role in risk stratification requires further studies.

1. Introduction

The tumor suppressor gene TP53 plays a central role in safeguarding genomic stability through regulation of DNA repair, cell cycle arrest, senescence, and apoptosis. Alterations in TP53—including mutations, deletions, copy‐neutral loss of heterozygosity (CN‐LOH) and complex structural aberrations—are among the most frequent genetic events across human cancers and are strongly associated with adverse clinical outcomes [1]. In hematologic malignancies, TP53 abnormalities are less common than in solid tumors, yet they carry profound biological and prognostic implications such as a strong association with poor prognosis and limited response to conventional treatment [2, 3, 4, 5, 6]. They may arise as primary drivers in disease initiation or, more frequently, as secondary events during clonal evolution, particularly under therapeutic pressure [7, 8]. The heterogeneity of TP53 lesions, ranging from missense hotspot mutations that exert dominant‐negative or gain‐of‐function effects to monoallelic and biallelic inactivation, contributes to the variability in pathogenesis and treatment resistance. Biallelic TP53 alterations lead to the inactivation of both TP53 alleles which can occur through several mechanisms: the presence of a TP53 mutation on one allele combined with deletion (usually 17p deletion or monosomy 17) or CN‐LOH affecting the other allele, or by ≥ 2 distinct TP53 mutations affecting both alleles. In hematologic malignancies (like acute myeloid leukemia, AML, or myelodysplastic neoplasms, MDS), biallelic TP53 alterations are associated with a complex karyotype, therapy resistance, and very poor prognosis [4, 5, 9, 10, 11]. The distinction between biallelic (“multi‐hit”) and monoallelic TP53 lesions is clinically important, as biallelic hits signify complete loss of normal p53 function, while monoallelic mutations or deletions may exert dominant negative or gain‐of‐function effects but still retain some wild‐type activity. While TP53 alterations in hematologic malignancies are primarily represented by deletions and mutations, amplifications of TP53 could potentially lead to increased wild‐type or mutant protein expression. Therefore, this study includes a descriptive case series to identify and characterize TP53‐amplified myeloid neoplasms and to explore their clinical/genetic context.

2. Materials and Methods

We retrospectively analyzed cases that were sent to our laboratory between 09/2005 and 10/2025 for potential TP53 amplifications, that is, inspected all cases with available information regarding the copy number state of TP53 using interphase FISH (fluorescence in situ hybridization, totally 223 698 cases). Diagnoses (from peripheral blood and/or bone marrow) were established based on cytomorphology, immunophenotype, cytogenetics, and molecular genetics as previously published according to respective WHO classifications [12, 13, 14]. All patients had given written informed consent to the use of genetic and clinical data according to the Declaration of Helsinki. The study was approved by our internal institutional review board. Cases were selected by the copy number state of TP53 using interphase FISH. Amplification by FISH analysis was defined by an increased number of the respective specific signals that cannot accurately be counted due to its large number (see Supporting Information S1 for details). Additionally, chromosome banding analysis (CBA) was performed according to standard methods [15]. Targeted sequencing (> 1500× coverage) was used to assess TP53 mutation status and load [16]. All samples were subjected to targeted panel sequencing analyzing mutations in 40 myeloid genes as described before [17]. For analyzing copy number variations (CNV), the xGen Human Copy Number Variant Backbone Hybridization Panel was added (see Supporting Information S1). For four samples, whole transcriptome sequencing (WTS) was performed to analyze expression of TP53. For this, 2 × 100‐bp paired‐end reads were sequenced on the NovaSeq 6000 (Illumina, San Diego, CA, USA) with a median of 50 million reads per sample. For gene expression analysis estimated read counts for each gene were normalized applying Trimmed mean of M‐values (TMM) normalization method and the resulting log2 counts per million (CPMs) were used. SPSS (version 19.0.0) software (IBM Corporation, Armonk, NY, USA) was used for overall survival analysis (Kaplan–Meier). Continuous variables were compared using a two‐sided Student's t‐test; when variances were unequal, Welch's correction was applied. A p < 0.05 was considered statistically significant.

3. Results

We found eight cases with a TP53 amplification detected by FISH within our patient database (Figure 1). Interestingly, all cases with TP53 amplification were diagnosed as myeloid neoplasm: n = 6 were diagnosed as AML (all AML‐MR, myelodysplasia‐related, according to WHO‐HAEM5), the remaining n = 2 as MDS (all de novo). The median age of patients was 72 years with a range of 48–83 years. The cohort comprised five female and three male patients. At diagnosis, patients showed a median WBC of 7.3 × 10⁹/L (range 3.9–22.8 × 10⁹/L), a median hemoglobin of 9.0 g/dL (range 6.6–9.1 g/dL) and median platelets were 38 × 10⁹/L (range 17–94 × 10⁹/L). Median blast count of AML patients was 61.5% (range 25%–63%). CBA was available for all eight cases and revealed a complex aberrant karyotype in all cases, with a 5q deletion in 7/8 and a chromosome 7 aberration (−7 or 7q−) in 4/8. The number of detected chromosomal aberrations ranged from 4 to 12, thus the majority of cases was highly complex. Structural abnormalities involving 17p were visible in all cases (Table S1). Molecular genetic data for TP53 was available for all patients revealing an accompanying TP53 mutation in all 8/8 cases. Median VAF (variant allele frequency) of TP53 was 48% with a range of 18%–91%. No recurrent additional molecular mutations were detected applying a panel of ≥ 40 genes (co‐mutations: Case 1: KRAS, Case 4: NRAS, Case 5: DNMT3A, Case 6: MPL, U2AF1; Table 1). The complex chromosomal aberrations including the TP53 amplification were also visible on CNV plots derived from targeted panel sequencing (Figure 1 and Table S2). Of note, CN‐LOH covering 17p/TP53 was not detected in any sample. CNV analysis confirmed TP53 copy‐number gains in all cases and showed that the altered regions were either focal or embedded in broader 17p/chromosome 17 abnormalities, consistent with the complex structural context suggested by chromosome banding and FISH (Table S1). WTS was available for four TP53‐amplified cases (TP53 in all AML cases [5]: 4.46; in cases with TP53 amplification: 5.35, 4.03, 3.40, and 3.80, respectively; for this, estimated read counts were normalized applying TMM normalization method and the resulting log2 CPMs were used; Table 1; RNA expression was compared with an AML reference cohort analyzed on the same platform [772 AML cases]). Thus, TP53 expression in the amplified cases was within the range of the control group and did not show a marked increase using an unpaired, two‐sided t‐test on normalized log2 CPM values; however, in the absence of protein‐level validation, these data are restricted to RNA expression. Clinical data was available for four AML cases and is reported descriptively. Although the median overall survival in this limited subset was 1 month, incomplete clinical annotation precludes any firm conclusions regarding prognosis or treatment resistance. Of note, this OS is comparable to the previously described dismal outcome of AML cases with biallelic TP53 alterations.

FIGURE 1.

Two examples of TP53 amplification as detected by FISH analysis and CNV plots derived from targeted panel sequencing. In example 1, a probe for TP53/DLEU was used (TP53: green; DLEU: red), in example 2 a probe for TP53/NF1 was applied (TP53: red; NF1: green). In both examples two interphase nuclei are exemplarily shown. Further, CNV plots of all chromosomes (left) and in more detail of chromosome 17 (right) are depicted. Location of TP53 is highlighted in red.

TABLE 1.

Summary of cases detected with TP53 amplifications including patient characteristics, cytogenetics, WTS results, co‐mutations, and TP53 VAF (variant allele frequency).

	Sex	Age (years)	Diagnosis	WTS available	Co‐mutations	TP53 VAF	OS data available	Cytogenetics
Case 1	Female	83	AML	Yes (TP53: 3.80)	KRAS	30	No	Complex
Case 2	Male	73	MDS	No	—	32	Yes	Complex
Case 3	Female	75	AML	No	—	91	Yes	Complex
Case 4	Female	80	AML	Yes (TP53: 3.40)	NRAS	25	No	Complex
Case 5	Female	60	AML	Yes (TP53: 5.35)	DNMT3A	62	Yes	Complex
Case 6	Male	69	AML	No	MPL, U2AF1	48	Yes	Complex
Case 7	Female	70	MDS	No	—	18	No	Complex
Case 8	Male	48	AML	Yes (TP53: 4.03)	——	89	No	Complex

Note: An accompanying TP53 mutation was found in all cases; additionally, all cases showed a complex aberrant karyotype. For analysis of TP53 gene expression by WTS, estimated read counts were normalized applying Trimmed mean of M‐values (TMM) normalization method and the resulting log2 counts per million (CPMs) were used and are depicted.

4. Discussion

TP53 amplifications are a very rare but recurrent aberration in myeloid neoplasms that, in this cohort, consistently co‐occurred with TP53 mutations, complex karyotype, and frequent 5q and chromosome 7 abnormalities, indicating a highly adverse genetic background. This type of aberration was to our knowledge not described before in hematological malignancies. Of note, gene amplification typically leads to activation of oncogenes such as MYC or KMT2A. The median TP53 VAF of ~50% together with copy‐number gain suggests functionally bi‐allelic TP53 alterations in many cases, aligning these patients biologically with established multi‐hit TP53 AML/MDS [4, 5, 6, 10, 11]. Thus, TP53 amplification may represent an additional mechanism of allelic TP53 deregulation in myeloid neoplasms, but it does not appear to drive marked overexpression, suggesting that copy gain alone is unlikely to be biologically neutral or beneficial. Rather, its tight association with TP53 mutation and complex karyotype supports a model of advanced clonal evolution in which amplification contributes to functional TP53 disruption within an already genomically unstable background. These findings broaden the spectrum of clinically relevant TP53 abnormalities and underscore the need to assess both mutation and copy‐number status when defining multi‐hit TP53 disease. The coexistence of TP53 mutation and amplification suggests allelic imbalance and may represent multi‐hit TP53 disease in some cases, but definitive proof of biallelic TP53 inactivation requires allele‐specific analyses that were not available here. Clinically, the extremely short median OS of 1 month in evaluable AML cases resembles outcomes reported for biallelic TP53 alterations and further supports classifying TP53‐amplified myeloid neoplasms as a very high‐risk subgroup with pronounced treatment resistance [7, 8, 9]. Given that current therapeutic strategies for TP53‐mutated disease—such as hypomethylating agents with or without targeted combinations or allogeneic transplantation—remain largely unsatisfactory in multihit settings, TP53 amplification may identify patients in particular need of novel, p53‐directed or alternative approaches. In summary, TP53 mutation with concurrent amplification indicates substantial TP53 genomic complexity and may contribute to multi‐hit biology, but the absence of allele‐specific data prevents definitive assignment of biallelic TP53 inactivation in this cohort. Although this study is limited by the small number of cases and incomplete clinical follow‐up data, which restrict firm conclusions regarding mechanism, prevalence, and treatment implications, the findings nonetheless argue for systematic assessment of TP53 copy number and allelic state in routine diagnostics and for incorporating TP53‐amplified myeloid neoplasms into a multi‐hit TP53 AML/MDS ultra‐high‐risk entity.

Author Contributions

A.S. and W.W. designed the study, A.S., M.M., and W.W. interpreted the data, A.S. and W.W. wrote the manuscript. A.S. was responsible for chromosome banding and FISH analyses, M.M. and W.W. for molecular and bioinformatic analyses, T.H. for cytomorphologic analyses. All authors read and contributed to the final version of the manuscript.

Funding

The authors have nothing to report.

Ethics Statement

The study adhered to the tenets of the Declaration of Helsinki and was approved by the laboratory's institutional review board and the Ethics Committee of the Bavarian Medical Association for the use of archived DNA/RNA samples and associated clinical information. The clinical data were retrieved and the samples were collected and analyzed with the endorsement of the Ethics Committee of the Bavarian Medical Association.

Consent

All patients gave their informed consent for genetic analyses and the use of laboratory results for research purposes.

Conflicts of Interest

T.H. declares part ownership of Munich Leukemia Laboratory (MLL). A.S., M.M., and W.W. are employed by the MLL.

Supporting information

Data S1: Material and methods.

Table S1: Genomic coordinates of the amplified segment, and approximate segment size based on the CNV profile.

Table S2: Karyotype description of all cases.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.