(P 430) UNIQUE HISTOLOGIC MORPHOLOGY OF EWING SARCOMA CELLS THAT HARBOR TP53 MUTATIONS

Objective: Ewing sarcoma (EWS) often harbors TP53 mutations in 4% to 20% of cases. Patients with TP53 mutations are reported to have a poor prognosis, with more invasive and less chemoresponsive tumors. Immunohistochemistry (IHC) staining for TP53 is not routinely performed at diagnosis and while molecular testing reliably detects TP53 mutations, results are not immediately available. There have been no reports on the morphology of EWS cells that harbor TP53 mutation. The objective of this retrospective study is to describe and compare the histologic appearance of TP53 mutated (mut) EWS cells from patients’ tumor biopsy samples to tumors that do not harbor TP53 mutations.

Methods: A pilot single-institution retrospective medical record and histopathologic review of a small cohort of patients with EWS who underwent tumor biopsy from 2017-2023. Hematoxylin and eosin (H&E) stained slides were reviewed by an expert pathologist to select non-necrotic, tumor dense region of interest (ROI). Slides were scanned and reviewed independently by a researcher who was blinded to EWS TP53 mutation status. QuPath software is then used to measure nuclear area (NA) (µm^2) and nuclear optical density range (NODR) for each cell. The median and interquartile range (IQR) were calculated to summarize the measurements for NA and NODR. A Mann-Whitney test was used to evaluate the differences by TP53 mutation status. The study was approved by the Institutional Review Board prior to data collection (CCI-06-00198).

Results: Twelve patients with EWS were selected, 6 (50%) were metastatic at diagnosis; Ten (83%) had EWS::FLI fusion and 2 (17%) had EWS::ERG. A total of 14 tumor slides were assessed, with 9 (64%) obtained at diagnosis; 4 (29%) at 1st recurrence; 1 (7%) at 2nd recurrence. Eight (57%) were primary tumor biopsies; 1 metastatic and 5 relapse/recurrent biopsies. Half of the tumors (n=7) harbored TP53 mutations (mut-TP53). All TP53-wild-type (wt-TP53) EWS cells showed monotonous oval nuclei with fine chromatin pattern and inconspicuous nucleoli (Figure 1). In contrast, all mut-TP53 EWS cells demonstrated enlarged oval-to-round nuclei with a coarse chromatin pattern with 1-2 prominent nucleoli (Figure 2). Summary statistics of NA were comparable between wt-TP53 and mut-TP53 cells with a median NA median of 26.28 µm^2 (21.99-30.58 µm^2) and 24.77 µm^2 (18.45-29.06 µm^2), respectively; p=0.52, and a median NA IQR of 19.46 µm^2 (12.64-27.04 µm^2) vs. 17.06 µm^2 (14.15-24.01 µm^2), respectively; p=0.7 (Figure 3A/B). Summary statistics of NODR were comparable between wt-TP53 and mut-TP53 cells with a median of median NODR of 1.03 (0.83-1.29) vs. 0.94 (0.67-1.36), respectively; p=0.34, and a median NODR IQR of 0.44 (0.31-0.52) vs. 0.48 (0.33-1.00), respectively; p=0.57 (Figure 3C/D).

Conclusion: In this limited sample study, there was no significant difference in NA and NODR distributions of mut-TP53 compared to wt-TP53 EWS (Figure 4). However, EWS cells with mut-TP53 have a unique appearance on H&E morphology to those with wt-TP53, losing their classic monotonous small round blue cell appearance. As outcomes for patients with localized EWS continue to improve, the ability to predict TP53 mutational status at diagnosis will be an important tool to risk stratify patients, pre-treatment. We are expanding this pilot study to increase statistical power in assessing the prognosis of nuclear morphologic characteristics, NA and NODR.