br Time months br survival br Overall br Patients at
Patients at Risk
free survival 0.75
Patients at Risk
Patients at Risk
Abbreviations: ALDH ¼ aldehyde dehydrogenase; EGFR ¼ epidermal growth factor receptor; HES1 ¼ hairy and enhancer of split 1; NSCLC ¼ nonesmall-cell lung cancer; OS ¼ overall survival; PFS ¼ progression-free survival.
with Aldefluor assay results, the triple-drug combination was able to decrease the expression of all investigated CSC markers in H1975 cells, while in the PC9 298-93-1 only expression of cleaved intracellular NOTCH3 was lowered (Figure 3B). Our results indicate heterogeneous parallel and downstream signaling path-ways in these cell lines, reflecting the differences in the tumor resistance mechanism in patients. In a patient cohort of EGFR-mutationepositive NSCLC, we found that CSC-marker mRNA expression can predict survival in EGFR TKI-treated patients. The 4 markers investigated, HES1, Bmi-1, and 2 ALDH1A isoforms, when highly expressed, were shown to be indicators of worse outcome to EGFR TKIs. Importantly, these findings
confirm the relevance of CSC presence after EGFR TKI treat-ment and their correlation with development of therapy resis-tance. It can be concluded that different tumors initiate distinct resistance pathways, stressing the importance of mutation anal-ysis and personalized treatment plans. Furthermore, this study highlights the importance of CSCs in resistance to EGFR TKI treatments and presents an opportunity to stratify patients based on mRNA expression of prognostic CSC markers.
Further research should focus on developing new (combination) therapies to eliminate CSCs, although caution should be taken when treating different tumor types because of heterogeneous resistance pathways.
Cancer Stem Cell Biomarkers
Figure 6 Model. (A) CSCs are Thought to be Subset of EGFR-MutationePositive Cancer Cells That are Resistant to Single EGFR Blockade and Finally Lead to Relapse of Disease. (B) Concomitant EGFR, STAT3, and Src Blockade May Improve Clinical Outcomes by Reducing Portion of CSCs Most Likely to Persist Through Single EGFR Inhibition and Lead to Tumor Regression
EGFR TKI EGFR TKI
EGFR-mutant tumor Enrichment in ALDH+ cells Resistance and tumor relapse
EGFR-mutant tumor + Eradica on of ALDH+ cells
Tumor degenera on
Cancer stem cell
Apopto c cell
Abbreviations: CSC ¼ cancer stem cell; EGFR ¼ epidermal growth factor receptor; STAT3 ¼ signal transducer and activator of transcription 3.
Clinical Practice Points
Single therapy with EGFR TKIs for EGFR-mutationepositive NSCLC patients is insufficient and activates parallel signaling pathways that ultimately cause resistance.
In culture, we demonstrated the enrichment in ALDHþ cells upon application of gefitinib, afatinib, or osimertinib in EGFR-mutationepositive models. By Western blot analysis, we confirmed the increase in several CSC markers when the cells are treated with single EGFR TKIs.
The combination of first-, second-, or third-generation EGFR TKIs with a STAT3 and Src inhibitor prevents the increase of ALDHþ cells and CSC markers in one EGFR-mutatione positive model.
The baseline evaluation of CSC markers, like ALDH1A1, ALDH1A3, Bmi-1, and HES1, predicted shorter PFS and OS in EGFR-mutationepositive patients treated with EGFR TKIs.
Combination therapy may be of benefit for the poor prognostic subgroup of EGFR-mutationepositive NSCLC patients defined in our study.
We thank Isabel Crespo and the Cytomics core facility of the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDI-BAPS) for technical assistance. Work in R.R.’s laboratory is partially supported by a grant from La Caixa Foundation, an Instituto de Salud Carlos III grant (RESPONSE, PIE16/00011), a Marie Skłodowska-Curie Innovative Training Networks European grant (ELBA 765492), and a Spanish Association Against Cancer (AECC) grant (PROYE18012ROSE). The funders had no role in the design
and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the report for publication.
The authors have stated that they have no conflict of interest.
2. Jung MJ, Rho JK, Kim YM, et al. Upregulation of CXCR4 is functionally crucial for maintenance of stemness in drug-resistant nonesmall cell lung cancer cells. Oncogene 2013; 32:209-21.