5P - Circulating tumor cell-derived organoids from lung adenocarcinoma patients for assessment of EGFR and KRAS mutations

Background

Liquid biopsy serve as a potential alternative to repeat invasive biopsy for tumor genomic profiling in patients with metastatic cancer. Circulating tumor cells (CTCs), as a component of liquid biopsies, could be a source of cancer–specific DNA. With the advent of patient-derived organoid culture approaches, there is the possibility of in vitro expansion of CTCs and these could be employed to examine a range of somatic variants. The current study aimed to establish CTC-derived organoids (CTCDO) from patients with lung adenocarcinoma (LUDC) and explore them for the assessment of EGFR and KRAS mutation status.

Methods

Blood samples were collected from 20 LUDC patients, including 12 (60%) men and 8 (40%) women, 9 smokers (45%), 2 ex-smokers (10%) and 9 non-smokers (45%). CTCs were enriched from 4 mL blood by antibody-based negative depletion. Then, enriched CTC fractions were cultured in vitro (3D) under optimized conditions to expand organoids. Further, we examined the presence of EGFR (exons 19-21) and KRAS (exon 2) mutations in expanded CTCDOs using Sanger sequencing.

Results

Short-term CTC 3D cultures were successfully generated from isolated CTCs in 15 (75%) LUDC patients (passages 2-7), of which 2 (13.3%) were non-metastatic cases. Almost all CTCDOs showed positive staining of TFF1 and negative staining of CD45. EGFR mutation (Exon21-L858R) was detected in seven (46.6%) cases. In one patient harboring L858R mutation, with available paired primary and expanded CTC, this mutation was confirmed in CTCDO. Moreover, KRAS mutation (Exon2-G12D) was identified in 2 (25%) wild-type EGFR cases.

Conclusions

We have successfully isolated and expanded CTCs from patients with LUDC. CTCDOs culture allowed for expansion of cells to a critical mass and exploration of them to assess mutations using less sensitive techniques. This non-invasive way could be alternative to tissue biopsies in patients with small biopsy and/or requiring a rebiopsy for molecular testing. Further optimization of the culture methodology is required, concomitantly with the functional and molecular characterization, with the aim of establishing CTCDO models for treatment response prediction and studying tumor heterogeneity and metastatic cascade.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

M. Lahmadi.

Funding

The national Directorate-General for Scientific Research and Technological Development (DGRSDT).

Disclosure

The author has declared no conflicts of interest.