Activating KRAS mutations mark premalignant cystic structures in congenital pulmonary airway malformations

Background: Congenital Pulmonary Airway malformations (CPAM) may lead to malignant degeneration, and therefore many surgeons opt to resect CPAM even in asymptomatic patients. Previously, we identified Kirsten rat sarcoma virus (KRAS) mutations in a subset of CPAM patients, possibly indicating a pre-malignant state. In order to unify treatment strategy in (asymptomatic) patients we focused on KRAS mutations as a potential risk factor for developing malignancy in CPAM. Methods: Resected lung tissue of CPAM patients was separated in affected region (“cyst”) and non-affected region (“control”) to subsequently initiate airway organoids. Cyst and control organoids from the same patients with and without KRAS mutations (KRAS^POS vs. KRAS^NEG) (n = 3) where processed for single cell RNA sequencing (scRNA-Seq), and the cellular composition of the organoids was validated by immunofluorescent staining. The role of KRAS was identified by manipulating the expression in the organoids. Results: ScRNA-Seq data revealed differences in cell proportions between KRAS^POS and KRAS^NEG cyst, and control organoids. The significant differentially expressed genes in the KRAS^POS cyst are comparable to those identified in lung cancer patients with KRAS mutations. Manipulation of KRAS expression showed that KRAS^POS cyst organoids grew larger due to more proliferative cells and that KRAS directly affected the cell cycle. Conclusions: KRAS^POS cyst organoids show transcriptomic similarities with KRAS mutated lung cancers, show changes in cellular composition and have increased growth and proliferation. These findings support the hypothesis that KRAS mutated CPAM cysts belong to a group of CPAM patients at higher risk of developing a malignancy.