Effect of CFTR modulator therapy with elexacaftor/tezacaftor/ivacaftor on pulmonary ventilation derived by 3D phase-resolved functional lung MRI in cystic fibrosis patients

Objectives To investigate whether 3D phase-resolved functional lung (PREFUL)-MRI parameters are suitable to measure response to elexacaftor/tezacaftor/ivacaftor (ETI) therapy and their association with clinical outcomes in cystic fibrosis (CF) patients. Methods Twenty-three patients with CF (mean age: 21; age range: 14–46) underwent MRI examination at baseline and 8–16 weeks after initiation of ETI. Morphological and 3D PREFUL scans assessed pulmonary ventilation. Morphological images were evaluated using a semi-quantitative scoring system, and 3D PREFUL scans were evaluated by ventilation defect percentage (VDP) values derived from regional ventilation (RVent) and cross-correlation maps. Improved ventilation volume (IVV) normalized to body surface area (BSA) between baseline and post-treatment visit was computed. Forced expiratory volume in 1 second (FEV1) and mid-expiratory flow at 25% of forced vital capacity (MEF25), as well as lung clearance index (LCI), were assessed. Treatment effects were analyzed using paired Wilcoxon signed-rank tests. Treatment changes and post-treatment agreement between 3D PREFUL and clinical parameters were evaluated by Spearman’s correlation. Results After ETI therapy, all 3D PREFUL ventilation markers (all p < 0.0056) improved significantly, except for the mean RVent parameter. The BSA normalized IVVRVent was significantly correlated to relative treatment changes of MEF25 and mucus plugging score (all |r| > 0.48, all p < 0.0219). In post-treatment analyses, 3D PREFUL VDP values significantly correlated with spirometry, LCI, MRI global, morphology, and perfusion scores (all |r| > 0.44, all p < 0.0348). Conclusions 3D PREFUL MRI is a very promising tool to monitor CFTR modulator–induced regional dynamic ventilation changes in CF patients. Clinical relevance statement 3D PREFUL MRI is sensitive to monitor CFTR modulator–induced regional ventilation changes in CF patients. Improved ventilation volume correlates with the relative change of mucus plugging, suggesting that reduced endobronchial mucus is predominantly responsible for regional ventilation improvement. Key Points • 3D PREFUL MRI–derived ventilation maps show significantly reduced ventilation defects in CF patients after ETI therapy. • Significant post-treatment correlations of 3D PREFUL ventilation measures especially with LCI, FEV 1 %pred, and global MRI score suggest that 3D PREFUL MRI is sensitive to measure improved regional ventilation of the lung parenchyma due to reduced inflammation induced by ETI therapy in CF patients. • 3D PREFUL MRI–derived improved ventilation volume (IVV) correlated with MRI mucus plugging score changes suggesting that reduced endobronchial mucus is predominantly responsible for regional ventilation improvement 8–16 weeks after ETI therapy.


Introduction
Cystic fibrosis (CF) is an autosomal recessive genetic disorder of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which reduces chloride and sodium ion transport across cell membrane of epithelial barriers.Consequently, the airways are filled with thick mucus that restricts breathing.CF occurs in approximately 1 in 3000-4000 newborns among Caucasians [1].Thus, early diagnosis and monitoring of therapy is of significant interest.Recently, a new CFTR modulator combination medication, composed of a chloride channel potentiator (ivacaftor) and two CFTR correctors (elexacaftor and tezacaftor), has been introduced to treat CF.The first studies with this triplecombination therapy, henceforth abbreviated ETI, demonstrated significant improvements in clinical outcomes in CF patients [2][3][4].
The ventilation outcomes in CF are routinely measured using spirometry and nitrogen multiple-breath washout (N 2 -MBW).Both techniques estimate global pulmonary ventilation parameters.However, they cannot assess regional information.Chest CT allows for regional monitoring of early lung disease [5].In contrast to CT, MRI offers radiation-free imaging and therefore the possibility for follow-up measurements without accumulating radiation exposure.
In the last 15 years, several MRI techniques have been developed to assess regional ventilation during free breathing and without the usage of contrast-agent [6][7][8][9][10].Among them, 3D phase-resolved functional lung (PREFUL) MRI enables quantitative assessment of pulmonary ventilation during free breathing on a regional level of the total lung volume [11].In contrast to 2D alternatives, 3D techniques [11][12][13] offer whole lunge coverage (better spatial resolution and likely higher sensitivity for hypoventilated regions) and account for through-plane motion, which potentially improve the quality of functional ventilation maps.3D PREFUL ventilation parameters have been shown to correlate well with spirometry measurements and showed a good interscan repeatability in a study cohort consisting of healthy volunteers and chronic obstructive pulmonary disease patients [14].Recently, ETI therapy has been shown to improve global clinical ventilation parameters and semi-quantitative morphologic MRI scoring in CF patients [15].However, it is unknown whether 3D PREFUL ventilation parameters are sensitive to measure treatment changes in patients with CF.
The objective of this study was to investigate if the ventilation parameters derived by 3D PREFUL are suitable to measure response to ETI therapy and their association with improvements in clinical outcome measures in CF patients.

Study participants
A total of 23 CF patients (13 female, mean age ± standard deviation (SD), 21.0 ± 9.3 years, mean FEV 1 % pred.at baseline 87.8%) were recruited in this prospective observational single-center study.The local ethics committee of Hannover Medical School (8922_B0_S_2020) approved the study and informed written consent was obtained prior to the examination from each study participant and/or their legal guardians.Participants were included when they (1) were older than 12 years, (2) were compound-heterozygous for F508del and a minimal function mutation or homozygous for F508del, and (3) had no prior exposure to ETI.The following exclusion criteria were applied: (1) pulmonary exacerbation at baseline or follow-up, (2) history of solid organ transplantation, (3) pregnancy, (4) claustrophobia, (5) metallic implantations incompatible with MR imaging.Morphological and functional 3D PRE-FUL MRI, PFT, and N 2 -MBW were evaluated at baseline and 8-16 weeks after initiation of ETI therapy.The morphological MRI images, spirometry data, and LCI values of the included CF patients have been reported in a previous study [15].

MR protocol
MR examinations were performed on a 1.5-T scanner (MAGNETOM Avanto; Siemens Healthcare).The MR protocol consisted of the following sequences [15]: (1) T2-weighted half-Fourier acquisition single-shot turbo spin echo (HASTE) sequences in axial and coronal orientation, acquired by breath-hold maneuvers; (2) T2-weighted turbo spin echo sequences in PROPELLER technique (BLADE), acquired in axial and coronal orientation by using respiratory navigator triggering; (3) T1-weighted volumetric interpolated breath-hold examination (VIBE) sequences, acquired in axial and coronal orientation.(4) 2D non-contrast-enhanced PREFUL technique to assess lung perfusion and 3D PREFUL technique to asses lung ventilation.

MR image analysis
Morphological MR images were evaluated based on a wellestablished semi-quantitative scoring system [16].Briefly, the global MRI score consists of morphology score and perfusion score.The morphology score is divided into the following subscores: bronchial wall thickening/bronchiectasis, mucus plugging, abscesses/sacculations, consolidations, and special findings, i.e., pleural affection.The perfusion scores were derived by the subtraction of the baseline images (contrastfree) from the images with maximal contrast in the lung tissue [16].All subscores including the perfusion score are evaluated for each lobe with values of (a) 0 (no abnormality), (b) 1 (< 50% of the lobe affected), and (c) 2 (≥ 50% of the lobe affected).Considering lingula as a sixth lobe, the global MRI score ranges from 0 (normal lung) to 72 (maximally affected lung).
The dynamically acquired 3D PREFUL data were reconstructed to ~40 breathing phases and each phase was registered towards end-inspiration image [17].Using 3D PREFUL registered images, ventilation defect maps derived from static regional ventilation (RVent) [18] and dynamic cross-correlation (CC) [9] maps based on previously published thresholds were calculated [14].CC maps assess the dynamics of ventilation using regional flow-volume loops during tidal breathing.Furthermore, ventilation defect percentage (VDP) percent values were computed from both ventilation defect maps as the number of voxels with ventilation below the threshold divided by the total number of voxels of the segmented lung volume.To analyze the treatment effect on a regional level, morphological images of the second visit were co-registered to the morphological end-inspiratory image of the first visit.The treatment response maps (TRMs) were then calculated by subtracting the ventilation defect map after therapy from the baseline ventilation defect maps.Post-treatment improved ventilation volume (IVV RVent and IVV CC ) was calculated as the number of positively changed voxels in TRMs (absolute amount of ventilation defect voxels at baseline measurement which resolved after ETI therapy in the follow-up measurement) multiplied by lung volume in milliliters.Both volumes were normalized to body surface area (BSA) at baseline.For image analysis, the lung parenchyma of the 3D PREFUL baseline end-inspiratory reconstructed image was automatically segmented using a 3D convolutional neuronal network (CNN) and manually corrected if needed.The details of the 3D CNN network were described previously [17].

Lung function testing
Spirometry outcomes (forced expiratory volume in 1 second (FEV 1 ) and mid-expiratory flow at 25% of forced vital capacity (MEF25)) were obtained according to American Thoracic Society (ATS)/European Respiratory Society (ERS) guidelines [19] with global lung function initiative (GLI) reference values [20].Lung clearance index (LCI) was assessed using the N 2 -MBW technique according to ATS/ERS guidelines [21].

Statistical analysis
For the 3D PREFUL regional parameters of RVent and CC, mean values and SD values inside lung parenchyma were computed.Further ventilation impairment was assessed using 3D PREFUL by a total lung VDP and post-treatment IVV values in milliliters.
The treatment effect on all derived MRI and clinical outcome parameters was analyzed using paired Wilcoxon signed-rank tests.
Treatment changes and post-treatment agreement between 3D PREFUL and clinical parameters were evaluated using Spearman's correlation (r) analyses.
For all comparisons, p values < 0.05 were considered statistically significant.Statistical analyses were performed using JMP Pro 16 (SAS Institute) and MATLAB 2020b (MathWorks).

Study participants
MRI scans and PFT measurements were successfully performed in all 23 CF patients.Two out of 23 CF patients did not comply with N 2 -MBW measurement at the follow-up visit.Due to coronavirus pandemic-related restrictions, the follow-up measurement of seven out of 23 CF patients (30%) had to be postponed up to 19 weeks with one patient exception (28 weeks).

Comparison of treatment changes between 3D PREFUL and lung function data
There was no significant correlation between absolute and relative treatment changes of 3D PREFUL MRI ventilation parameters (mean RVent, SD RVent, mean CC, SD CC, VDP RVent , and VDP CC ) with FEV 1 , MEF25, LCI, and MRI scores (p > 0.05), except relative change of SD RVent parameter, which was significantly correlated with relative change of LCI (r = 0.48, p = 0.0275).Figure 3 shows regional TRMs derived by 3D PREFUL for two study participants.Looking only at positive changes in TRMs, the mean improved ventilation volume normalized to BSA was 147 (SD = 94) and 129 (SD = 114) mL/m 2 for RVent (IVV RVent ) and CC (IVV CC ) parameters, respectively.The IVV RVent normalized to BSA was significantly correlated with relative treatment changes of MEF25 and mucus plugging MRI score (all |r| > 0.48, all p < 0.219, Table 2).

Discussion
This study assessed the effects of ETI therapy on pulmonary ventilation parameters derived by 3D PREFUL MRI in patients with CF. 3D PREFUL-derived ventilation parameters showed significantly reduced ventilation defects after initiation of ETI therapy and the improved ventilation volume measure was significantly correlated to the relative change in the morphological parameter mucus plugging and MEF25.
To our best knowledge, this is the first study to determine the effects of ETI therapy on pulmonary ventilation function using patient-friendly non-contrast-enhanced 3D MR acquisition during tidal breathing.As reported previously [15,22], the improvements after initiation of ETI were also found for clinical outcome parameters, including spirometry and N 2 -MBW ventilation parameters, as well signal generation differences, breathing conditions (free tidal breathing for 3D PREFUL vs. inspiration breath-hold for 19 F), spatial resolution (4 x 4 x 4 mm 3 for 3D PREFUL compared to 6.25 × 6.25 × 15 mm 3 for 19 F), and scanner field strengths (3D PREFUL at 1.5T vs. 19 F at 3T).
As 3D PREFUL covers the whole lung volume, it is feasible to register pre-and post-treatment acquisitions and calculate treatment response ventilation maps, which enable a detailed longitudinal regional treatment change analysis [24].Regarding relative treatment change, we found significant correlations of 3D PREFUL MRI-derived improved ventilation volume (IVV RVent normalized to BSA) with relative change of MEF25 and the mucus plugging MRI score.These findings suggest that reduced endobronchial mucus is predominantly responsible for regional ventilation improvement 8-16 weeks after initiation of ETI therapy.The positive changes in MRI-derived mucus plugging and wall thickening/bronchiectasis score have been shown to be associated with the improvement of CFTR function, which might reflect the resolution of inflammation in CF patients [15].De Vuyst et al [25] demonstrated that the ETI therapy reduces airway inflammation in CF.Although 3D PREFUL MRI is not able to assess inflammation directly, the previous studies support the hypothesis that the improved regional ventilation derived by 3D PREFUL is related to the reduction in inflammation induced by ETI therapy.Also, the correlation of the regional ventilation changes with the relative change of MEF25 may indicate that 3D PREFUL MRI is sensitive to airflow changes in the small airways.This appears reasonable as 3D PREFUL MRI measures regional ventilation in the lung parenchyma.However, this finding warrants further investigation in future studies.
Absolute and relative changes of 3D PREFUL parameters were not directly correlated to changes in FEV 1 , LCI, and MRI scoring system.The missing correlations might be ), which uses the information of the whole respiratory cycle, with spirometric measures.This finding suggests increased treatment response sensitivity of CC when compared to static RVent parameters derived by 3D PREFUL.Nonetheless, the significant post-treatment correlations of 3D PREFUL ventilation measures especially with FEV 1 , LCI, and global MRI score demonstrate that 3D PREFUL MRI is sensitive to measure improved regional ventilation of the lung parenchyma due to reduced inflammation and endobronchial mucus in response to ETI therapy in CF patients.In addition, the improved perfusion score in the presented cohort may also be indicative of resolving inflammatory changes in the lung parenchyma post-ETI treatment.
In addition to the small sample size, we acknowledge further limitations.Firstly, the ventilation parameters derived by PREFUL (or other Fourier-decomposition-based methods) are considered indirect measures for ventilation.The key assumption of these methods is that the signal intensity variations of the lung parenchyma are induced by different lung volumes [6].This idea has been validated for 2D PREFUL with direct 19 F MRI and 129 Xe ventilation measurement [26,27] but not (yet) for 3D methods.Secondly, the repeatability of 3D PRE-FUL-derived dynamic parameters in CF patients has not been examined yet.Recent repeatability results in chronic obstructive pulmonary disease patients and stable CF patients showed no significant bias between repeated measurements of 3D and 2D PREFUL, respectively [14,28].This supports our results that the differences in 3D PREFUL parameters between baseline and post-treatment acquisition are a true response to ETI therapy.Additional useful information might be provided by assessment of improvement in ventilation on the lobar level, however was beyond the scope of this work.Future multicenter validation studies are necessary to investigate if 3D PREFUL MRI regional ventilation assessment may add value to current clinical standard techniques such as spirometry, N 2 -MBW, semi-quantitative thoracic MRI scores, or gas-based MRI techniques.

Conclusions
In summary, our study demonstrates that ETI therapy leads to improvement in lung ventilation determined by 3D PRE-FUL MRI in CF patients.Our data suggest that endobronchial mucus reduction is mainly responsible for regional ventilation improvement 8-16 weeks after initiation of ETI therapy.

Fig. 4
Fig. 4 Spearman's correlation analysis of improved ventilation volume IVV RVent normalized to BSA with relative change of MEF25 % pred.(top row (a)) and relative change of mucus plugging score (bottom row (b)).Black circles represent the patients (n = 23) and dashed black lines represent simple linear regression, r = Spearman's correlation coefficient

Table 1
Analysis of 3D PREFUL, spirometry, N 2 -MBW, and morphological MRI parameters in response to ETI therapy.Baseline and posttreatment ventilation values are expressed as average with standard deviation in brackets.Significant results are marked with *

Table 2
Spearman's correlation analysis of improved ventilation volumes (IVV RVent normalized to body surface area (BSA) and IVV CC normalized to BSA, respectively) derived by 3D PREFUL and % change of clinical parameters.Statistically significant correlations are marked with * (p value < 0.05)