(390) Proof-of-concept of ionocytes’ CFTR content as a novel biomarker for cystic fibrosis

Background: Despite the huge progress made in cystic fibrosis (CF) knowledge and care, the exact relationships between primary defects and different manifestations of the disease are still poorly understood. More than 2.200 CFTR mutations are known, most of which are of varying clinical consequences or of unknown functional significance [1]. Also, high inter-individual variability of disease onset and progression, and cases of inconclusive diagnosis (CRMS/CFSPID) are also quite frequently observed.

For these reasons, CF is still a diagnostic and therapeutic challenge, and it is of paramount importance to identify more precise, specific, and disease-relevant biomarkers allowing a better understanding of CFTR mutations effects in vivo. Moreover, with the advent of effective CFTR modulators, such biomarkers are also required as outcome parameters to quantify the rescue of CFTR.

In this context, the recent identification of CFTR-rich ionocytes could highlight novel possibilities to monitor CFTR protein abundance, subcellular localization, and function in the airways [2].

The aim of this study was to evaluate if the ionocytes’ CFTR content, initially in terms of expression and localization of CFTR at the protein level, can be used as a novel biomarker for CF.

Methods: We collected nasal epithelial cells by nasal brushings in a cohort of non-CF subjects and CF patients with different mutations. Moreover, we enrolled CF patients starting treatment with ELX/TEZ/IVA. The ionocytes-specific expression of CFTR was quantified by immunofluorescence detection of CFTR and FOXI1 (as ionocytes marker) followed by confocal imaging. We set up the analysis to quantify the fraction of CFTR localized in the apical membrane and defined this parameter as ICC (ionocytes’ CFTR content).

Results: We found a wide difference in the ICC values between the control group and the CF cohort. ICC appeared relatively low in many patients, particularly those carrying two copies of F508del or one F508del and a minimal function variant. Interestingly, also patients under Orkambi therapy displayed very low CFTR expression, result that is in accordance with the low clinical efficacy of this drug. Instead, the ICC resulted significantly increased after ELX/TEZ/IVA treatment.

Conclusions: We found that ICC evaluation allows the fine quantification of CFTR protein in native airway samples, highlighting its potential as a novel biomarker for the follow-up of CF and CRMS/CFSPID patients. Importantly, ICC showed a robust and significant increase in patients treated with ELX/TEZ/IVA, thus indicating a potential use as a primary outcome parameter for novel therapies aimed at increasing CFTR.

For future developments, it would be desirable to devise assays for the functional interrogation of ionocytes’ CFTR, as well as to investigate the role of CFTR-rich ionocytes in airways physiology and CF disease.

Acknowledgements: This work was developed within the framework of the DINOGMI Department of Excellence of MIUR 2018-2022 (legge 232 del 2016).

References: [1] Sosnay PR, Salinas DB, White TB, Ren CL, Farrell PM, Raraigh KS, Girodon E, Castellani C. Applying Cystic Fibrosis Transmembrane Conductance Regulator Genetics and CFTR2 Data to Facilitate Diagnoses. J Pediatr 2017, 181S: S27-S32.e1. doi: 10.1016/j.jpeds.2016.09.063

[2] Plasschaert LW, Žilionis R, Choo-Wing R, Savova V, Knehr J, Roma G, Klein AM, Jaffe AB. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocytes. Nature 2018, 560, 377–381. doi: 10.1038/s41586-018-0394-6