Abstract 3778: Comprehensive analysis of single disseminated cancer cells identifies a stem-like phenotype with strong prognostic impact
Authors: Mederer Tobias , Spitzl Daniel , Elsner Felix , Polzer Bernhard , Ried Michael , Neu Reiner , Robold Tobias , Hofmann Hans-Stefan , Klein Christoph A. 
Published: March 2024

Introduction: Recent studies on disseminated cancer cells (DCCs) in Non-Small-Cell Lung Cancer (NSCLC) patients demonstrated their clinical relevance even in early tumor stages. However, their molecular and phylogenetic link to the primary tumor is currently unclear. Certain genetic alterations, such as TP53 or KRAS mutations, have been proposed to be clonal events and drivers of dissemination and metastasis. In this study, we analyzed the genome and transcriptome of single DCCs to identify the characteristics of early DCCs and their translational clinical value.

Materials and methods: From 2011 to 2019 we prospectively collected and analyzed bone marrow aspirates and lymph node samples from 296 newly diagnosed NSCLC patients undergoing tumor resection with curative intention. Presence of DCC was examined by i) immunocytology (IC) for Cytokeratin (clone A45-B/B3, bone marrow, BM-DCCs) or EpCAM (clone BerEp4, lymph node, LN-DCCs) and ii) immunofluorescent (IF) staining for EpCAM in a live cell suspension (clone HEA-125) from BM or LN. Single cells were isolated using micromanipulation and subjected to Whole Genome Amplification or combined Whole Genome/Transcriptome Amplification for IC and IF, respectively. Sanger sequencing for known hotspot mutations in TP53 and KRAS was performed on single DCCs. Gene expression profiles of DCCs were analyzed by single cell RNA sequencing and the results were compared with clinical data with a mean follow-up of 2.93 years (range 0.03-7.22).

Results: LN-DCCs were found in 44.8% and 59.6% by IC and IF, respectively. CKIC pos BM-DCCs were found in 45.2%, while EpCAMIF pos BM-DCCs were present in 52.8%. LN-DCCs correlated with

reduced tumor-specific survival (TSS, p=0.009) and progression-free survival (PFS, p=0.025). EpCAMIF pos, but not CKIC pos BM-DCCs correlated with reduced TSS (p=0.003) and PFS (p=0.083). Genetic analysis of single DCCs revealed that TP53 or KRAS mutations could be found in 3.6% and 2.8% of analyzed BM-DCCs and 5.0% and 29.3% of LN-DCCs for IF and IC, respectively. Single cell RNA-seq showed distinct transcriptomic profiles for LN- and BM-DCCs, and identified a DCC phenotype with high stemness score in the bone marrow. Further analysis of potential transcriptome-based biomarkers identified a novel candidate marker for activated EpCAMpos BM-DCCs that strongly correlates with PFS and TSS (p&lt;0.001).

Conclusions: DCCs in early-stage NSCLC have high prognostic value and predict reduced PFS and TSS. While genetic alterations in the TP53 and KRAS gene were not detected in the majority of analyzed DCCs, transcriptomic phenotypes provided relevant information and identified a subset of activated DCCs that correlate strongly with relapse and poor outcome.

Citation Format: Tobias Mederer, Daniel Spitzl, Felix Elsner, Bernhard Polzer, Michael Ried, Reiner Neu, Tobias Robold, Hans-Stefan Hofmann, Christoph A. Klein. Comprehensive analysis of single disseminated cancer cells identifies a stem-like phenotype with strong prognostic impact [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3778.