p53 Immunohistochemistry As a High-Specificity Biomarker to Predict Allelic State and Overall Survival in Myelodysplastic Syndrome or Acute Myeloid Leukemia with Mutated TP53
Authors: Patel Shyam Ajay , Khedr Salwa , Gordon Caroline D , Littman Noah , Woda Bruce , Hutchinson Lloyd , Cerny Jan , Li Shaoguang , Selove William , Gerber Jonathan M. 
Published: December 2024

Optimal front-line management of myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML) with mutated TP53 remains a matter of debate, and early recognition of TP53 mutation is critical given the poor response to standard chemotherapy, particularly for multi-hit TP53. Results of TP53 mutational status by next-generation sequencing (NGS) may take weeks, precluding timely selection of front-line therapeutics. Early detection of TP53 mutations in myeloid neoplasms may thus significantly impact clinical decision-making.

In this study, we queried theUMass Leukemia Registry and UMass Hematopathology archives from 2014 to 2024 and identified 127 clinically annotated MDS or AML bone marrow specimens with mutated TP53, for which there was concurrent information for both p53 IHC (index test) and TP53 NGS (referent test). IHC was performed using VENTANA CONFIRM anti-p53 (DO-7), a murine monoclonal antibody targeting recombinant wild-type human p53. Exome sequencing was performed for the entire coding region for p53 isoform A (codons 1-393) with coverage depth ranging from 500X to 6500X, as previously described (Patel SA, ..., Gerber JM. Leuk Lymphoma 2021) (Patel SA, ..., Gerber JM. EJHaem 2023).

For patients with MDS, mean turnaround time for a finalized hematopathology report for p53 IHC was significantly shorter compared to that of TP53 NGS: 4.9 ± 0.36 days vs. 10.4 ± 0.39 days (p = 3.4E-19), respectively. For patients with AML, mean turnaround time for p53 IHC vs. TP53 NGS was 3.3 ± 0.30 days vs. 12.9 ± 1.17 days (p = 4.4E-11), respectively. Costs incurred by IHC testing totaled $9.00 per bone marrow specimen, in contrast to the billable gross charge of NGS testing (CPT code 81450) of $8,051.00. The mean percentage of cells with positive p53 stain was higher for AML specimens compared to MDS specimens (29.9% ± 4.00% vs. 9.2% ± 1.83%, p = 6.3E-7). We assessed the value of IHC as a crude predictor of multi-hit TP53 status. In TP53-mutant MDS, the mean percentage of cells with positive p53 stain was higher in the multi-hit cases than those without multi-hit status (11.2% ± 2.24% vs. 1.56% ± 0.54%, p = 0.016). In TP53-mutant AML, the mean percentage of cells with positive p53 stain was also higher for multi-hit cases (31.8% ± 3.98% vs. 0.0% ± 0.0%, p = 0.029).

Specificity of the index test was 95.3% and 100% for MDS and AML, respectively. Sensitivity of IHC was only 75.7%, and we thus sought to understand whether the relatively high rate of false negatives was attributable to the specific mutant codons, functional domains, or types of aberrations within TP53.None of the specimens with sole nonsense or frameshift mutations (n = 8) stained positive for p53 by IHC, despite the presence of TP53 aberrations by NGS testing (i.e., false negatives by IHC). IHC positivity was found exclusively in cases with at least 1 missense mutation, typically in the DNA-binding domain.

Finally, we explored whether p53 IHC could serve as an early biomarker for clinical outcomes. We assessed differential median overall survival (OS) across dichotomized IHC thresholds. OS was inversely correlated with percentage of positive cells by p53 IHC. For example, median OS was 29.1 ± 3.24 months in patients with p53 IHC ≤20% vs. only 13.8 ± 3.57 months for those with p53 IHC &gt;20% (p &lt; 0.001). Given the importance of allelic state considerations, we dichotomized the IHC results into &lt;50% and &gt;50% categories, as TP53 VAF &gt;50% in patients with MDS or AML represents an obligatory biallelic hit in at least a fraction of cells. For patients with p53 IHC &lt;50%, median OS was 27.0 ± 1.79 months vs. 10.4 ± 3.30 months in those with p53 IHC &gt;50% (p &lt; 0.001). These findings suggest that p53 IHC serves as a high-specificity, cost-effective, and rapid biomarker for the detection of TP53 missense mutations in MDS or AML, permitting timely decisions on choice of therapy. Additionally, IHC provides some early insight into TP53 allelic state and type of mutation. IHC for p53 might particularly benefit patients in resource-constrained settings that lack ready access to NGS.