CANCER STEM CELLS AND CELL CYCLE GENES AS INDEPENDENT PREDICTORS OF RELAPSE IN NON-SMALL CELL LUNG CANCER: SECONDARY ANALYSIS OF A PROSPECTIVE COHORT STUDY.

Objective. Cancer stem cells (CSCs) are described as resistant to chemo-radio therapy. It has been shown that CSCs influence the disease-free survival in patients undergoing surgery for lung cancer (NCT04634630). Moreover, CSCs have been assessed for the expression of a genes panel, previously validated by Bueno et al. in epithelial lung cancer cells, to predict lung cancer mortality. We recently described an overexpression of these recurrence-related genes (RG), which are cell cycle genes, in CSCs for early and locally advanced stages (IIIA) in ACL and squamous cell carcinoma (SCCL) of the lung. This study aim to investigate CSC frequency and RG expression as independent predictors of relapse in lung cancer. METHODS. This secondary analysis of a prospective cohort study involved 22 surgical tumor specimens from 22 patients in early (I-II) and locally advanced (IIIA) stages of ACL and SCCL. Cell population frequency analysis of ALDHhigh (CSC) and ALDHlow (cancer cells) was performed on each tumor specimen. In addition, RG expression was assessed for 31 target genes separately in ALDHhigh and ALDHlow populations. CSC frequency and RG expression were assessed as predictors of disease- free survival by Cox analysis. RESULTS. Cox analysis showed that CSC frequency and RG expression were two independent predictors of disease-free survival. CSC frequency was not related to disease-free survival in early-stage patients (HR=0.84, 95%CI=0.53-1.33, p=0.454), whereas it was a risk factor in locally advanced stage patients (HR=1.22, 95%CI=1.09-1.35, p=0.000). RG expression – if measured in CSC - was related to a higher risk of recurrence (HR=1.19, 95%CI=1.03-1.39, p=0.021). If considering RG expression measured in cancer cells, its effect on disease-free survival was lower and was not statistically significant (HR=1.12, 95%CI=0.94-1.33, p=0.196). CONCLUSIONS. This first observational study showing a connection between CSCs and lung cancer recurrence relies on data gathered from the analysis of the frequency of CSCs, directly isolated through cell sorting of primary cell suspensions derived from surgical tumor specimens. We focused on the quantification of CSCs within the tumor of each patient, measuring their frequency by cytofluorimetric analysis as a percentage of ALDHhigh cells and connecting it with the time to tumor relapse in both ADL and SCCL. CSC frequency and RG expression are independent predictors of relapse in lung cancer. Considering these results, CSCs and RG may need to be considered for both target therapy and prognosis. If these findings are confirmed in larger cohorts of patients, a better CSC characterization might be considered in further studies.