Introduction/Background Tumor spheroids in the ascites of high-grade serous cancer (HGSC) patients are poorly described since all research has focused on 3D in vitro models. Our objective was to describe HGSC spheroids in the ascites, with emphasis on their morphological features, cell composition, and PD-1 and PD-L1 expression on spheroid-associated tumor- and immune cells.

Methodology Patients with histologically confirmed HGSC and present malignant ascites were included in the study. Spheroid density and size in the ascites were assessed from Giemsa smears, the cell composition of spheroid-associated immune cells, tumor cells and endothelial cells, as well as PD-1 and PD-L1 expression were assessed from immunocytochemically stained cell block sections for CD3, CD4, CD8, CD20, CD68, CD34, ERG, PD-1, and PD-L1 (22C3, SP263 and SP142 clones). The analysis of all parameters was performed semi-quantitatively. Obtained data were used to calculate if there is any correlation with progression-free survival (PFS) and overall survival (OS). Furthermore, a cut-off value for low and high density of tumor spheroids in the ascites that could predict better/poorer survival outcome in the patients was determined. Survival analysis was based on a 3.5-year patient follow-up.

Results Forty-seven patients were included in the study. Three different spheroid-size categories were observed in the ascites, (small, medium, large) but the size did not correlate with survival outcome. However, a 10% cut-off for spheroid density was established, that significantly predicted better/worse PFS and OS. Furthermore, our results confirmed the presence of spheroid-associated lymphocytes (mostly T lymphocytes), macrophages, and endothelial cells. PD-1 expression was observed only on spheroid-associated lymphocytes. PD-L1 expression was seen on both spheroid-associated tumor cells, lymphocytes, and macrophages assessed by 22C3 and SP263 clones but not with the SP142 clone.

• Download figure
• Open in new tab
• Download powerpoint

Abstract #271 Figure 1

(A-C) Size and density of the spheroids, detected in ascites Giemsa smears from HGSC patients. Representative images of A) small (100 x magnification), B) medium (100 x magnification) and C) large spheroids (50 x magnification). The red ruler represents 100 µm length. (D-E) Two different density groups of spheroids in the Giemsa smear, D) low-density and E) high density. (F-G) Correlation of the density of tumor spheroids with patient survival. F) Progression-free survival. G) Overall survival.

Conclusion Our results highlight the potential of cytopathology to analyze ascites spheroids as potential predictive markers for HGSC patients.

Disclosures The authors made no disclosures. The study was funded by the research program P3–0289 of the Slovenian Research Agency.