TP53 immunohistochemistry correlates with TP53 mutation status and clearance in decitabine-treated patients with myeloid malignancies

TP53 mutations are associated with adverse prognosis in both myelodysplastic syndromes (MDS) and in acute myeloid leukemia (AML). Clinical trials focused on therapeutic options for this group of high-risk patients require a rapid, inexpensive technique to identify patients with TP53 mutations and to closely monitor clinical responses. While sequencing-based assessment of TP53 mutation status is now widely available, the costs and turnaround times may not be optimal for prompt therapeutic selection, clinical trial enrollment, and monitoring of response. Using routine immunohistochemistry (IHC), a rapid, inexpensive and readily available test, we found that expression levels of TP53 were a reliable proxy for both the presence of TP53mutations and mutation clearance during clinical responses. Across samples with a wide range of TP53 variant allele frequencies (VAF), we show that TP53 IHC has both high sensitivity (86%) and high specificity (90%) to detect TP53 mutations in bone marrow (BM) cores and has a high concordance (r=0.7114, R=0.5061) with mutation levels across a range of post-therapy responses. TP53mutations in AML frequently produce dominantnegative proteins, which may stabilize mutant TP53 due to lack of negative feedback mechanisms, such as TP53-regulation of the ubiquitin ligase MDM2 that targets TP53 for degradation. Unlike the wild-typeTP53 protein, which is quickly degraded, mutant TP53 protein accumulates in the cell nucleus and is detectable by TP53 IHC. Detection of increased TP53 protein in acute leukemia has also been reported using flow cytometry, but this requires intracellular permeabilization and adequate aspirate, and is not a widely used clinical assay. In contrast, TP53 IHC is a commonly used clinical test that can be run on routine BM core or clot sections. We evaluated TP53 staining by IHC in serial samples from MDS and AML patients (Online Supplementary Table S1) treated on a previously published, prospective, openlabel study of 10-day decitabine therapy (clinicaltrials.gov identifier: 01687400). Formalin-fixed paraffin-embedded bone marrow (BM) trephine biopsies were available from TP53-mutated (n=21) and TP53-wild-type (n=51) patients. Three additional cases with truncating TP53 mutations were subsequently evaluated from patients collected on unrelated protocols. All patients consented to genome sequencing analysis and were treated in accordance with the Declaration of Helsinki. TP53 mutation status was established by paired tumor/normal high coverage next-generation sequencing. Serial BM samples obtained during therapy were available for 16 of the TP53-mutated cases (n=44 samples, median number of samples per patient = 4). TP53 and TP53/lineage marker IHC was performed using standard automated protocols [TP53 antibody clones D07 (all cases) and BP53-11 (selected cases), CD3 (2GV6), and CD34 (QBEnd/10), Ventana Medical Systems, Tucson, AZ, USA; CD61 (2f2), E-cadherin (EP700Y), and myeloperoxidase (polyclonal); Cell Marque, Rocklin, CA, USA]. Quantitative scoring of TP53 IHC slides was performed independently by three board-certified hematopathologists (MBR, EJD and YSL) by manually counting any nuclear staining in 500 BM hematopoietic cells. Statistical analysis was performed using GraphPad Prism (San Diego, CA, USA) and SPSS (Chicago, IL, USA). Similar to previous reports, we found significantly increased nuclear TP53 staining in TP53-mutated cases (48±5%) as compared to TP53-wild-type (10±1.5%) cases (P<0.0001) (Figure 1A-C). We also found significant TP53 overexpression in cases with complex cytogenetics and chromosome 5 and 17 abnormalities (Online Supplementary Appendix and Online Supplementary Figure S1A-C). A high degree of concordance in assessing TP53 counts was noted between observers (R =0.92 and 0.94) (Online Supplementary Figure S1D). A receiver operating characteristic (ROC) curve was constructed using different TP53 expression levels in order to establish the best TP53 cut-off value (Online Supplementary Figure S1E). The area under the ROC curve when using TP53 expression to detect TP53mutations was 0.872 and a cutoff value of >20% TP53-positive cells produced sensitivity of 86% and specificity of 90% compared to sequencing, in close agreement with a previously published study (area under ROC curve of 0.873, cut off value >10% of TP53-positive cells, with sensitivity of 75% and specificity of 91%). Co-staining with lineage markers was performed to determine whether mutated TP53 protein accumulated preferentially in specific BM lineages. No consistent pattern of TP53 overexpression was identified in erythroid (Figure 1D-G), myeloid (Figure 1E-H), or megakaryocytic (Figure 1F-I) elements (Table 1). Increased expression of TP53 was seen more consistently in CD34-positive (+) blasts (Table 1 and Online Supplementary Figure S1G), but cases with CD34-negative (-) blasts or no increase in blasts limit the usefulness of CD34/TP53 IHC. No significant TP53 overexpression was identified in T cells (Table 1 and Online Supplementary Figure S1H). Previous studies reported occasional BM samples with TP53 mutations that showed no corresponding elevation of TP53 expression. In our cohort, we identified three cases with TP53 mutations that showed no signifi-