P08.02.B BEYOND THE EXPECTED: CIRCULATING TUMOR CELLS IN GLIOBLASTOMA REVEAL HIDDEN HETEROGENEITY AND BECOME POTENTIAL DIAGNOSTIC BIOMARKERS
Authors: Lessi F , Morelli M , Di Gangi A , Di Lorenzo F , Franceschi S , Aretini P , Pastore A , Ferri G , Menicagli M , Pasqualetti F , Gambacciani C , Pieri F , Grimod G , Di Stefano A , Santonocito O S , Mazzanti C M 
Published: October 2024

Abstract

BACKGROUND
For many years, glioblastoma (GBM) was thought to be restricted to the brain. Nevertheless, a growing body of evidence indicates that, like many other cancers, hematogenic dissemination is a reality. Detection of circulating tumor cells (CTCs) in the blood of GBM patients offers a non-invasive means to assess tumor-related information. Our aim is to use GBM CTCs to obtain knowledge about the tumor and improve the current GBM diagnosis and prognosis.

MATERIAL AND METHODS
Blood samples were collected from 18 primary tumor cases, 16 recurrences, 5 healthy controls, and 4 special cases. CTCs were enriched with Parsortix Cell Separation System and analysed on DEPArray system. The CTCs have been counted for all the samples. Copy Number Alterations (CNAs) analysis was performed on some of the isolated CTCs, examining a minimum of 1 to a maximum of 10 cells per sample. We performed also single-cell RNA sequencing (scRNAseq) analysis on a primary cancer cell line derived from a GBM primary tumor and we anlayzed CNAs in all the cell populations with Scevan tool.

RESULTS
Cell counting demonstrated in both primary and recurrent GBM patients’ blood a statistically significantly higher number of CTCs compared to healthy controls (average of CTCs number 15.4 vs 1). Notably, primary GBM cases exhibited a greater number of CTCs compared to recurrent GBM cases, with averages of 18.2 and 12.4 CTCs, respectively. Based on these results provided by the cell count, we established a cut-off of 3 CTCs to identify a tumor sample. Moreover, among the four special cases examined, which included one lymphoma, one case of radionecrosis, and two specific tumors (an oligodendroglioma and a xanthoastrocytoma), our established cut-off was validated, affirming the accuracy of our CTCs detection method. Furthermore, CNAs analysis has revealed a completely novel finding observed thus far in GBM, specifically that approximately 51% of the CTCs isolated and analyzed harbored alterations, while the remainder 49% exhibited a wild-type profile at level of copy number alterations. This novel finding was corroborated by scRNAseq analysis of the GBM primary cancer cell line analyzed, wherein both wild-type cells and CNAs were observed with a percentage of CNAs of 51% and 49% respectively, mirroring the distibution observed in CTCs

CONCLUSION
Our study demonstrates the utility of CTCs as a non-invasive diagnostic tool for GBM. Elevated CTCs counts, particularly in primary GBM cases, underscore their potential as biomarkers for disease progression. Additionally, our identification of copy number wild-type CTCs highlights the heterogeneity within CTC populations, which reflects the heterogeneous structure typical of GBM itself offering valuable insights into tumor biology and in understanding CTCs functions in the bloodstream.