p53 Immunohistochemistry Is a Reliable Method for Assessing TP53 Allelic and Functional Status in TP53‐Mutated Cases

To the Editor,

1.

TP53 alterations play an important role in the development, classification, and prognostication of myeloid neoplasms. Earlier studies of myelodysplastic neoplasms (MDS) have shown that biallelic TP53 alterations, rather than monoallelic mutations, are associated with complex karyotypes and poor outcomes [1]. These observations are consistent with the primary role of TP53 as a tumor suppressor gene, which often requires loss of heterozygosity (LOH) for complete functional inactivation. Currently, establishment of biallelic TP53 alteration status is based on: (1) a TP53 mutation + TP53 copy loss or copy‐neutral LOH; or (2) more than one TP53 mutation; or (3) one TP53 mutation with a variant allele frequency (VAF) of ≥ 50% [2, 3].

Mutation analysis by DNA sequencing, including next generation sequencing (NGS), is widely used to evaluate TP53 mutation status. p53 immunohistochemistry is readily available and has been proposed as a surrogate marker to indicate TP53 mutation status [4, 5, 6]. A wild‐type (normal) immunostaining pattern is characterized by heterogeneous p53 expression in a subset of cells with weak to moderate staining intensity. In contrast, a mutant (abnormal) pattern manifests in two distinct forms: (1) strong and uniform expression (overexpression), typically associated with missense mutations; or (2) a null (complete loss) pattern, commonly linked to frameshift, nonsense, deletion, or splice‐site alterations [4, 7].

Despite extensive investigation of TP53 mutations and p53 expression patterns, several important questions remain unresolved. Do TP53 mutations invariably result in abnormal p53 protein expression? If not, do TP53 mutations with a normal p53 expression pattern contribute to neoplastic transformation? What is the relationship between allelic status (monoallelic versus biallelic) and p53 expression patterns? Given that determination of biallelic TP53 status can be difficult or even impossible in some cases, can abnormal p53 expression by immunohistochemistry serve as a reliable surrogate for biallelic TP53 inactivation? In this study, we sought to address these questions by elucidating the relationship between p53 protein expression patterns and TP53 allelic status, and their association with disease status. The detailed experimental methods, including p53 immunohistochemistry, TP53 mutation analysis and cytogenetic analysis, are provided in the Methods S1.

In a recent study [8], we characterized 18 patients with Li‐Fraumeni syndrome (LFS) who developed hematologic neoplasms (group 1) and 5 LFS patients without hematologic neoplasms (group 2). All 23 patients were confirmed to have germline TP53 mutations. In the current study, we evaluated p53 expression patterns by immunohistochemistry on bone marrow biopsies from both cohorts and correlated the findings with TP53 mutation type, allelic status, and karyotype.

The detailed information for each case, including diagnosis, p53 expression pattern, TP53 mutation, allelic status, karyotype and FISH results, is provided in Tables S1 and S2. Briefly, p53 immunohistochemistry was performed in 14 cases in group 1 (4 excluded due to inadequate specimens) and all 5 cases in group 2. In group 1, all (100%) showed a mutant p53 protein expression pattern, including 11 with strong overexpression (Figure 1, upper) and 3 with a null pattern (Figure 1, middle). All cases with strong overexpression had missense TP53 mutations. In the 3 cases with a null expression pattern, 2 (cases 8 and 14) had frameshift mutations and 1 (case 10) had two mutations (one frameshift and one nonsense). Complex karyotypes were seen in 15/18 (83%) cases. Biallelic TP53 alterations were documented in 14/15 (93%) assessable cases. By contrast, all 5 cases in group 2 showed a normal p53 expression pattern (Figure 1, lower). All had a normal karyotype and none showed biallelic TP53 alterations. In summary, although patients in both groups harbor TP53 mutations, their p53 expression patterns differed markedly and were associated with distinct clinicopathologic features; only cases with hematologic neoplasms (group 1) showed mutant p53 expression patterns, which were associated with biallelic TP53 alterations and abnormal karyotypes.

FIGURE 1.

Various p53 expression patterns in TP53‐mutated cases. The upper panel shows an AML case harboring a TP53 missense mutation with biallelic alterations. H&E stain shows increased immature cells, which are large with dispersed chromatin, consistent with blasts. p53 immunohistochemistry shows a mutant pattern with strong nuclear overexpression. The middle panel shows an AML case harboring a TP53 frameshift mutation with biallelic alterations. H&E stain shows sheets of immature cells, consistent with blasts. p53 immunohistochemistry shows a mutant pattern with null expression. A single cell with dim to moderate expression (red circle) serves as an internal positive control. The lower panel shows an unremarkable bone marrow specimen harboring a TP53 missense mutation without biallelic alterations. H&E stain shows orderly trilineage hematopoiesis. p53 immunohistochemistry shows a wild‐type pattern with heterogeneous dim‐to‐moderate nuclear staining in a subset of cells.

Next, we sought to determine whether this association is also observed in non‐LFS patients with TP53 mutations. We searched our pathology archives from the past 2 years and identified 10 cases with TP53 mutations (VAF > 15%) but with no evidence of hematologic neoplasms based on bone marrow aspiration and biopsy (group 3). For comparison, we randomly selected 51 cases of myeloid neoplasms with TP53 mutations (group 4) from the same study period. The detailed information for each case, including diagnosis, p53 expression pattern, TP53 mutation, TP53 allelic status, and cytogenetic results, is provided in Tables S3 and S4. As summarized in Table 1, p53 immunohistochemistry demonstrated a wild‐type pattern in all 10 cases in group 3. In contrast, all 51 cases in group 4 exhibited a mutant p53 expression pattern, including strong p53 overexpression in 49 cases and a null pattern in 2 cases (cases 16 and 48). Both null‐pattern cases harbored nonsense TP53 mutations. Of the 49 cases with p53 overexpression, one case (case 42) harbored a frameshift mutation in exon 10, and all of the remaining 48 cases harbored missense TP53 mutations, including 11 cases with a concurrent second TP53 mutation of a different type (Table S4). There was no significant difference in TP53 mutation VAF between the two groups (Table 1). In both groups, TP53 mutations predominantly occurred in the DNA binding domain (exons 4–8). However, striking differences were observed in cytogenetic and allelic features. Only 1 case in group 3 had an abnormal karyotype with loss of chromosome Y, which may be age‐related. In contrast, all cases in group 4 exhibited abnormal karyotypes, with 49 (96%) cases showing complex karyotypes. Biallelic TP53 alterations were not identified in group 3, compared with 94% in group 4.

TABLE 1.

Comparison of TP53‐mutated normal and neoplastic cases.

Diagnosis		Normal (Group 3, n = 10)	Neoplastic (Group 4, n = 51)	p
Age (median, range)		69 (36–84)	70 (32–89)	0.82
Gender (female%)		80% (8/10)	53% (27/51)	0.17
TP53 mutations	VAF (median, range)	34% (15%–47%)	33% (5%–92%)	0.92
	Region (exons 4–8)	100% (10/10)	91% (71/78)	> 0.99
Karyotype (%abnormal)		10% (1/10)	100% (51/51)	< 0.0001
FISH (%loss of TP53)		0% (0/10)	50% (18/36)	0.0036
%biallelic TP53		0% (0/10)	94% (48/51)	< 0.0001
p53 IHC (%mutant pattern)		0% (0/10)	100% (51/51)	< 0.0001

Note: Statistical analysis: variables were compared between groups 3 and 4 using chi‐square or Fisher's exact tests for categorical variables and Mann–Whitney U tests for numeric variables. Statistical analyses were performed using GraphPad Prism 10.6.1 (La Jolla, CA, USA).

In summary, we identified two distinct p53 protein expression patterns using immunohistochemistry in TP53‐mutated cases. The wild‐type/normal p53 expression pattern is primarily associated with monoallelic TP53 alteration (Table 1). This finding can be explained by the retention of a functional wild‐type TP53 allele, which is sufficient to maintain normal p53 protein regulation and expression. Conversely, the abnormal p53 expression pattern is consistently associated with biallelic TP53 alterations. These expression patterns are associated with distinct clinicopathologic features. Although all cases in this study harbored TP53 mutations, cases with a wild‐type p53 expression pattern (groups 2 and 3) had normal karyotypes and unremarkable bone marrow findings. In contrast, cases with a mutant p53 pattern (groups 1 and 4) exhibited abnormal karyotypes (commonly complex) and the presence of hematologic neoplasms. These findings demonstrate that p53 immunohistochemistry can discriminate preserved and impaired TP53 functional status in TP53‐mutated cases.

Our study demonstrated a strong association between aberrant p53 expression patterns and biallelic TP53 alterations. Among TP53‐mutated cases with aberrant p53 expression, biallelic TP53 alterations were frequently identified, whereas TP53‐mutated cases with a normal p53 expression pattern consistently lacked evidence of biallelic TP53 alterations. Based on the data shown in Table 1, p53 immunohistochemistry showed a positive predictive value of 94% and a negative predictive value of 100% for detecting biallelic TP53 alterations. This indicates that aberrant p53 expression can potentially be used as a surrogate marker to indicate biallelic alterations. Demonstration of biallelic TP53 alterations can be challenging in some cases, such as those with copy‐neutral LOH or micro‐deletions, which can be missed by FISH and aCGH. Additionally, in some cases, there might not be material available for evaluation of copy‐number status. In these scenarios, p53 immunohistochemistry is valuable, and the demonstration of an abnormal p53 expression pattern is suggestive of biallelic TP53 alterations and loss of TP53 function. In this study, 4 cases (1 in group 1 and 3 in group 4, Supporting Information Tables) showed aberrant p53 expression in the absence of detectable biallelic alterations, likely representing undetected TP53 deletion due to the possibilities mentioned above.

In the current International Consensus Classification, AML with TP53 mutation is defined by AML harboring any somatic TP53 mutation with a VAF ≥ 10% [3]. This 10% cutoff is empiric and biallelic TP53 alterations are not required in this definition. This is not without controversy. First, there is no clear consensus regarding the optimal VAF threshold, and various cutoffs (ranging from 1% to 20%) have been used and shown clinical significance [9, 10, 11]. Further complicating the issue is that the VAF can be artifactually underestimated due to sampling issues such as hemodilution, bone marrow fibrosis, or patchy disease distribution. Second, previous studies have shown that AML patients with biallelic TP53 alterations are associated with significantly poorer survival compared to those with monoallelic TP53 alterations [4, 12]. Our current study demonstrates that p53 protein retains a “wild‐type” (normal) expression pattern when TP53 alteration is monoallelic. Its expression became aberrant when an additional hit (biallelic) occurs. These findings support incorporating biallelic TP53 alteration into the diagnostic criteria for AML with mutated TP53. Given VAF variability and the critical role of biallelic alterations, detection of a TP53 mutation at any VAF should prompt further evaluation to clarify whether the alteration is monoallelic or biallelic. In cases in which allelic status cannot be assessed by molecular or cytogenetic techniques, p53 immunohistochemistry should be considered. The abnormal protein expression pattern is highly suggestive of biallelic alterations.

In conclusion, our findings highlight the value of p53 immunohistochemistry in the evaluation of TP53 allelic (monoallelic versus biallelic) and functional status in TP53‐mutated cases. p53 immunohistochemistry should be considered when a TP53 mutation is detected, regardless of VAF, particularly in cases lacking sufficient data to establish TP53 allelic status (Figure 2). A wild‐type p53 pattern is indicative of monoallelic alteration with intact TP53 function, whereas an abnormal pattern is consistent with biallelic alterations, indicating TP53 function loss, which commonly triggers chromosome instability and neoplastic transformation.

FIGURE 2.

The proposed algorithm for the workup of TP53‐mutated cases. p53 immunohistochemistry is recommended in TP53‐mutated cases, especially when allelic status is indeterminate. A normal p53 expression pattern supports monoallelic TP53 alteration with retained function, whereas a mutant pattern (overexpression or null) supports biallelic TP53 inactivation and functional loss, which frequently induce chromosomal instability and promote neoplastic transformation.

Author Contributions

W.W. and H.F. designed the concept, developed the study, and wrote the original draft. All other coauthors performed the experimental studies and revised the manuscript. All authors contributed to and approved the final version of the manuscript.

Funding

The authors have nothing to report.

Ethics Statement

This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Institutional Review Board (IRB) of The University of Texas MD Anderson Cancer Center.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Clinicopathologic findings in patients with Li‐Fraumeni syndrome (LFS) who had hematologic diseases.

Table S2: Clinicopathologic findings in patients with LFS who had no hematologic diseases.

Table S3: Clinicopathologic findings in TP53‐mutated non‐LFS patients who had no hematologic diseases.

Table S4: Clinicopathologic findings in TP53‐mutated non‐LFS patients who had hematologic diseases.

Methods S1: The methodologies for p53 immunohistochemistry, TP53 mutation analysis, conventional karyotyping, and fluorescence in situ hybridization.

Data Availability Statement

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