CFTR function is impaired in a subset of patients with pancreatitis carrying rare CFTR variants.

BACKGROUND: Many affected by pancreatitis harbor rare variants of the cystic fibrosis (CF) gene, CFTR, which encodes an epithelial chloride/bicarbonate channel. We investigated CFTR function and the effect of CFTR modulator drugs in pancreatitis patients carrying CFTR variants. METHODS: Next-generation sequencing was performed to identify CFTR variants. Sweat tests and nasal potential difference (NPD) assays were performed to assess CFTR function in vivo. Intestinal current measurement (ICM) was performed on rectal biopsies. Patient-derived intestinal epithelial monolayers were used to evaluate chloride and bicarbonate transport and the effects of a CFTR modulator combination: elexacaftor, tezacaftor and ivacaftor (ETI). RESULTS: Of 32 pancreatitis patients carrying CFTR variants, three had CF-causing mutations on both alleles and yielded CF-typical sweat test, NPD and ICM results. Fourteen subjects showed a more modest elevation in sweat chloride levels, including three that were provisionally diagnosed with CF. ICM indicated impaired CFTR function in nine out of 17 non-CF subjects tested. This group of nine included five carrying a wild type CFTR allele. In epithelial monolayers, a reduction in CFTR-dependent chloride transport was found in six out of 14 subjects tested, whereas bicarbonate secretion was reduced in only one individual. In epithelial monolayers of four of these six subjects, ETI improved CFTR function. CONCLUSIONS: CFTR function is impaired in a subset of pancreatitis patients carrying CFTR variants. Mutations outside the CFTR locus may contribute to the anion transport defect. Bioassays on patient-derived intestinal tissue and organoids can be used to detect such defects and to assess the effect of CFTR modulators.

CFTR Modulators Rescue the Activity of CFTR in Colonoids Expressing the Complex Allele p.[R74W;V201M;D1270N]/dele22_24.

An Italian, 46-year-old female patient carrying the complex allele p.[R74W;V201M;D1270N] in trans with CFTR dele22_24 was diagnosed at the Cystic Fibrosis (CF) Center of Verona as being affected by CF-pancreatic sufficient (CF-PS) in 2021. The variant V201M has unknown significance, while both of the other variants of this complex allele have variable clinical consequences, according to the CFTR2 database, with reported clinical benefits for treatment with ivacaftor + tezacaftor and ivacaftor + tezacaftor + elexacaftor in patients carrying the R74W-D1270N complex allele, which are currently approved (in USA, not yet in Italy). She was previously followed up by pneumologists in northern Italy because of frequent bronchitis, hemoptysis, recurrent rhinitis, Pseudomonas aeruginosa lung colonization, bronchiectasis/atelectasis, bronchial arterial embolization and moderately compromised lung function (FEV1: 62%). Following a sweat test with borderline results, she was referred to the Verona CF Center where she presented abnormal values in both optical beta-adrenergic sweat tests and intestinal current measurement (ICM). These results were consistent with a diagnosis of CF. CFTR function analyses were also performed in vitro by forskolin-induced swelling (FIS) assay and short-circuit currents (Isc) in the monolayers of the rectal organoids. Both of these assays showed significantly increased CFTR activity following treatment with the CFTR modulators. Western-blot analysis revealed increased fully glycosylated CFTR protein after treatment with correctors, in line with the functional analysis. Interestingly, tezacaftor, together with elexacaftor, rescued the total organoid area under steady-state conditions, even in the absence of the CFTR agonist forskolin. In conclusion, in ex vivo and in vitro assays, we measured a residual function that was significantly enhanced by in vitro incubation with CFTR modulators, especially by ivacaftor + tezacaftor + elexacaftor, suggesting this combination as a potentially optimal treatment for this case.

In silico analysis and theratyping of an ultra-rare CFTR genotype (W57G/A234D) in primary human rectal and nasal epithelial cells.

Mutation targeted therapy in cystic fibrosis (CF) is still not eligible for all CF subjects, especially for cases carrying rare variants such as the CFTR genotype W57G/A234D (c.169T>G/c.701C>A). We performed in silico analysis of the effects of these variants on protein stability, which we functionally characterized using colonoids and reprogrammed nasal epithelial cells. The effect of mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein was analyzed by western blotting, forskolin-induced swelling (FIS), and Ussing chamber analysis. We detected a residual CFTR function that increases following treatment with the CFTR modulators VX661±VX445±VX770, correlates among models, and is associated with increased CFTR protein levels following treatment with CFTR correctors. In vivo treatment with VX770 reduced sweat chloride concentration to non-CF levels, increased the number of CFTR-dependent sweat droplets, and induced a 6% absolute increase in predicted FEV1% after 27 weeks of treatment indicating the relevance of theratyping with patient-derived cells in CF.

Effectiveness of endoscopic vacuum therapy as rescue treatment in refractory leaks after gastro-esophageal surgery.

The treatment of leak after esophageal and gastric surgery is a major challenge. Over the last few years, endoscopic vacuum therapy (E-VAC) has gained popularity in the management of this life-threatening complication. We reported our initial experience on E-VAC therapy as rescue treatment in refractory anastomotic leak and perforation after gastro-esophageal surgery. From September 2017 to December 2019, a total of 8 E-VAC therapies were placed as secondary treatment in 7 patients. Six for anastomotic leak (3 cervical, 1 thoracic, 2 abdominal) and 1 for perforation of the gastric conduit. In 6 cases, E-VAC was placed intracavitary; while in the remaining 2, the sponge was positioned intraluminal (one patient was treated with both approaches). A total of 60 sponges were used in the whole cohort. The median number of sponge insertions was 10 (range: 5-14) with a median treatment duration of 41 days (range: 19-49). A complete healing was achieved in 4 intracavitary (67%) and in 1 intraluminal (50%) E-VAC. We observed only one E-VAC-related complication: a bleeding successfully managed endoscopically. E-VAC therapy seems to be a safe and effective tool in the management of leaks and perforations after upper GI surgery, although with longer healing time when it is used as secondary treatment.

Cystic fibrosis transmembrane conductance regulator functional evaluations in a G542X+/- IVS8Tn:T7/9 patient with acute recurrent pancreatitis.

BACKGROUND: Acute recurrent pancreatitis (ARP) is characterized by episodes of acute pancreatitis in an otherwise normal gland. When no cause of ARP is identifiable, the diagnosis of "idiopathic" ARP is given. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene increase the risk of ARP by 3- to 4-times compared to the general population, while cystic fibrosis (CF) patients present with a 40- to 80-times higher risk of developing pancreatitis. CASE SUMMARY: In non-classical CF or CFTR-related disorders, CFTR functional tests can help to ensure a proper diagnosis. We applied an individualized combination of standardized and new CFTR functional bioassays for a patient referred to the Verona CF Center for evaluation after several episodes of acute pancreatitis. The CFTR genotype was G542X+/- with IVS8Tn:T7/9 polymorphism. The sweat (Cl-) values were borderline. Intestinal current measurements were performed according to the European Cystic Fibrosis Society Standardized Operating Procedure. Recent nasal surgery for deviated septum did not allow for nasal potential difference measurements. Lung function and sputum cultures were normal; azoospermia was excluded. Pancreas divisum was excluded by imaging but hypoplasia of the left hepatic lobe was detected. Innovative tests applied in this case include sweat rate measurement by image analysis, CFTR function in monocytes evaluated using a membrane potential-sensitive fluorescent probe, and the intestinal organoids forskolin-induced swelling assay. CONCLUSION: Combination of innovative CFTR functional assays might support a controversial diagnosis when CFTR-related disorders and/or non-classical CF are suspected.