Forskolin-induced swelling of intestinal organoids predicts long-term cystic fibrosis disease progression

ABSTRACTPatient-derived organoids hold great potential as predictive biomarker for disease expression or therapeutic response. Here, we used intestinal organoids to estimate individual cystic fibrosis transmembrane conductance regulator (CFTR) function of people with cystic fibrosis, a monogenic life-shortening disease associated with more than 2000 CFTR mutations and highly variable disease progression. In vitro CFTR function in CF intestinal organoids of 176 individuals with diverse CFTR mutations was quantified by forskolin induced swelling and was strongly associated with longitudinal changes of lung function and development of pancreatic insufficiency, CF-related liver disease and diabetes. This association was not observed when the commonly used biomarker of CFTR function sweat chloride concentration was used. The data strongly exemplifies the value of an organoid-based biomarker in a clinical disease setting and supports the prognostic value of forskolin induced swelling of intestinal organoids, especially for people with CF who have rare CFTR genotypes with unclear clinical consequences.

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Stratifying infants with cystic fibrosis for disease severity using intestinal organoid swelling as a biomarker of CFTR function

European Respiratory Journal ◽

10.1183/13993003.02529-2017 ◽

2018 ◽

Vol 52 (3) ◽

pp. 1702529 ◽

Cited By ~ 18

Author(s):

Karin M. de Winter-de Groot ◽

Hettie M. Janssens ◽

Rick T. van Uum ◽

Johanna F. Dekkers ◽

Gitte Berkers ◽

...

Keyword(s):

Cystic Fibrosis ◽

Disease Severity ◽

Gastrointestinal Disease ◽

Pancreatic Insufficiency ◽

Chloride Concentration ◽

Individual Level ◽

Outcome Parameters ◽

Sweat Chloride ◽

Intestinal Organoids ◽

Residual Capacity

Forskolin-induced swelling (FIS) of intestinal organoids from individuals with cystic fibrosis (CF) measures function of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein mutated in CF.We investigated whether FIS corresponds with clinical outcome parameters and biomarkers of CFTR function in 34 infants diagnosed with CF. Relationships with FIS were studied for indicators of pulmonary and gastrointestinal disease.Children with low FIS had higher levels of immunoreactive trypsinogen (p=0.030) and pancreatitis-associated protein (p=0.039), more often had pancreatic insufficiency (p<0.001), had more abnormalities on chest computed tomography (p=0.049), and had lower z-scores for maximal expiratory flow at functional residual capacity (p=0.033) when compared to children with high FIS values. FIS significantly correlated with sweat chloride concentration (SCC) and intestinal current measurement (ICM) (r= −0.82 and r=0.70, respectively; both p<0.001). Individual assessment of SCC, ICM and FIS suggested that FIS can help to classify individual disease severity.Thus, stratification by FIS identified subgroups that differed in pulmonary and gastrointestinal outcome parameters. FIS of intestinal organoids correlated well with established CFTR-dependent biomarkers such as SCC and ICM, and performed adequately at group and individual level in this proof-of-concept study.

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Correction of CFTR function in intestinal organoids to guide treatment of cystic fibrosis

European Respiratory Journal ◽

10.1183/13993003.02426-2019 ◽

2020 ◽

Vol 57 (1) ◽

pp. 1902426 ◽

Cited By ~ 2

Author(s):

Anabela S. Ramalho ◽

Eva Fürstová ◽

Annelotte M. Vonk ◽

Marc Ferrante ◽

Catherine Verfaillie ◽

...

Keyword(s):

Cystic Fibrosis ◽

Clinical Data ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Vast Number ◽

Sweat Chloride ◽

Intestinal Organoids ◽

Rectal Biopsies ◽

Cftr Modulators

RationaleGiven the vast number of cystic fibrosis transmembrane conductance regulator (CFTR) mutations, biomarkers predicting benefit from CFTR modulator therapies are needed for subjects with cystic fibrosis (CF).ObjectivesTo study CFTR function in organoids of subjects with common and rare CFTR mutations and evaluate correlations between CFTR function and clinical data.MethodsIntestinal organoids were grown from rectal biopsies in a cohort of 97 subjects with CF. Residual CFTR function was measured by quantifying organoid swelling induced by forskolin and response to modulators by quantifying organoid swelling induced by CFTR correctors, potentiator and their combination. Organoid data were correlated with clinical data from the literature.ResultsAcross 28 genotypes, residual CFTR function correlated (r2=0.87) with sweat chloride values. When studying the same genotypes, CFTR function rescue by CFTR modulators in organoids correlated tightly with mean improvement in lung function (r2=0.90) and sweat chloride (r2=0.95) reported in clinical trials. We identified candidate genotypes for modulator therapy, such as E92K, Q237E, R334W and L159S. Based on organoid results, two subjects started modulator treatment: one homozygous for complex allele Q359K_T360K, and the second with mutation E60K. Both subjects had major clinical benefit.ConclusionsMeasurements of residual CFTR function and rescue of function by CFTR modulators in intestinal organoids correlate closely with clinical data. Our results for reference genotypes concur with previous results. CFTR function measured in organoids can be used to guide precision medicine in patients with CF, positioning organoids as a potential in vitro model to bring treatment to patients carrying rare CFTR mutations.

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Prediction of Real-World Long-Term Outcomes of People with CF Homozygous for the F508del Mutation Treated with CFTR Modulators

Journal of Personalized Medicine ◽

10.3390/jpm11121376 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1376

Author(s):

Danya Muilwijk ◽

Marlou Bierlaagh ◽

Peter van Mourik ◽

Jasmijn Kraaijkamp ◽

Renske van der Meer ◽

...

Keyword(s):

Cystic Fibrosis ◽

Real World ◽

Target Therapy ◽

Chloride Concentration ◽

Sweat Chloride ◽

Limited Effectiveness ◽

Pulmonary Exacerbations ◽

Cftr Modulators

The clinical response to cystic fibrosis transmembrane conductance regulator (CFTR) modulators is variable within people with cystic fibrosis (pwCF) homozygous for the F508del mutation. The prediction of clinical effect in individual patients would be useful to target therapy to those who would benefit from it. A multicenter observational cohort study was conducted including 97 pwCF (F508del/F508del), who started lumacaftor/ivacaftor (LUM/IVA) treatment before June 2018. In order to assess the associations of individual in vivo and in vitro biomarkers with clinical outcomes, we collected clinical data regarding sex, age, and sweat chloride concentration (SwCl) at baseline and after six months of LUM/IVA; the percent predicted forced expiratory volume in 1 s (ppFEV1) and the number of pulmonary exacerbations (PEx) during the three years before up to three years after modulator initiation; and the forskolin-induced swelling (FIS) responses to LUM/IVA, quantified in intestinal organoids. On a group level, the results showed an acute change in ppFEV1 after LUM/IVA initiation (2.34%, 95% CI 0.85–3.82, p = 0.003), but no significant change in annual ppFEV1 decline in the three years after LUM/IVA compared to the three years before (change: 0.11% per year, 95%CI: −1.94–2.19, p = 0.913). Neither of these two outcomes was associated with any of the candidate predictors on an individual level. The median number of pulmonary exacerbations (PEx) per patient year did not significantly change in the three years after LUM/IVA compared to the years before (median: 0.33/patient year, IQR: 0–0.67 before vs. median: 0/patient year, IQR: 0–0.67 after p = 0. 268). The PEx rate after modulator initiation was associated with the PEx rate before (IRR: 2.26, 95%CI: 1.67–3.08, p < 0.001), with sex (males vs. females IRR: 0.36, 95%CI: 0.21–0.63, p = 0.001) and with sweat chloride concentration (SwCl) at baseline (IRR: 0.96, 95%CI: 0.94–0.98, p = 0.001). The change in SwCl was also significant (−22.9 mmol/L (95%CI: −27.1–−18.8, p < 0.001) and was associated with SwCl at baseline (−0.64, 95%CI: −0.90–−0.37, p < 0.001) and with sex (males vs. females 8.32, 95%CI: 1.82–14.82, p = 0.013). In conclusion, ppFEV1 decline after CFTR modulator initiation remains difficult to predict in individual patients in a real-world setting, with limited effectiveness for double CFTR modulator therapies. The PEx rate prior to CFTR modulator treatment initiation, sex and SwCl at baseline could be potential predictors of long-term PEx rate and of changes in SwCl after modulator initiation.

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Letter to the Editor

PEDIATRICS ◽

10.1542/peds.43.5.905b ◽

1969 ◽

Vol 43 (5) ◽

pp. 905-906

Author(s):

Aubrey Milunsky

Keyword(s):

Cystic Fibrosis ◽

Bone Marrow ◽

Clinical Presentation ◽

Pancreatic Insufficiency ◽

Chloride Concentration ◽

Letter To The Editor ◽

Patient Reported ◽

Sweat Sample ◽

Chloride Concentrations

The patient reported in the foregoing letter is of particular interest in view of the recent observations on patients with tnisomy 21 and cystic fibrosis. The multiple possibilities that could explain the clinical presentation have no doubt been considered by the authors. However, the description of "hypoplastic thrombocytopenia" and later pancytopenia in this patient, associated with pancreatic insufficiency, leads to the serious consideration of Shwachman's syndrome (pancreatic insufficiency and bone marrow dysfunction). The wide discrepancy between the sodium and chloride concentrations in the sweat reported in their letter indicates that evaporation of water may have occurred from the sweat sample, leading to an apparently higher sodium and chloride concentration.

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Test for the Concentration of Electrolytes in Cystic Fibrosis of the Pancreas Utilizing Pilocarpine by Iontophoresis, by Lewis E. Gibson and Robert E. Cooke,Pediatrics; 1959;24:545–549

PEDIATRICS ◽

10.1542/peds.102.s1.230 ◽

1998 ◽

Vol 102 (Supplement_1) ◽

pp. 230-231

Author(s):

Victor Chernick

Keyword(s):

Cystic Fibrosis ◽

Filter Paper ◽

Direct Injection ◽

Heat Stroke ◽

Chloride Concentration ◽

Hot Water ◽

Sweat Test ◽

High Concentration ◽

Sweat Chloride ◽

Plastic Bag

Aim. To develop a method for stimulating sweating that is rapid, painless, and avoids the risk of heat stress. Background. Since the discovery that there is a high concentration of sodium and chloride in the sweat of patients with cystic fibrosis of the pancreas in 1953, the sweat test has been performed by placing the patient's body in a plastic bag with or without hot water bottles to stimulate sweating. This method is unsatisfactory because of complications such as hyperpyrexia and heat stroke. Direct injection of a cholinergic agent intradermally is painful and therefore not practical. Methods. A rheostat with a milliampere meter was constructed at a cost of ∼$7 that allowed the iontophoresis of pilocarpine into the skin using negative and positive (2-cm diameter) electrocardiography electrodes. The positive electrode was placed on the flexor surface of the arm over a filter paper soaked in 0.2 mL of 0.2% pilocarpine nitrate. Current (0.2 mA) was applied for 5 minutes and then sweat was collected onto a preweighed filter paper for 30 minutes. Sweat chloride was determined by a polarographic method. Sweat tests were performed on 25 patients with cystic fibrosis (CF), 17 asymptomatic relatives and 27 control patients. Patients with CF had sweat chloride concentration >80 mEq/L; relatives, 32.5 mEq/L (highest 57 mEq/L); and control subjects, 21.1 mEq/L (highest 60 mEq/L). Conclusions. The iontophoresis of pilocarpine into the skin is a rapid, painless, safe, and reliable method for stimulating sweating and facilitating the determination of sweat chloride concentration.

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Negative Effects of Oral Fatty Acid Supplementation on Sweat Chloride in Cystic Fibrosis

PEDIATRICS ◽

10.1542/peds.64.1.50 ◽

1979 ◽

Vol 64 (1) ◽

pp. 50-52

Author(s):

John D. Lloyd-Still ◽

Stuart H. Simon ◽

Hans U. Wessel ◽

Lewis E. Gibson

Keyword(s):

Cystic Fibrosis ◽

Fatty Acid ◽

Sweat Rate ◽

Chloride Concentration ◽

Control Group ◽

Safflower Oil ◽

Negative Effects ◽

Fatty Acid Supplementation ◽

Acid Supplementation ◽

Sweat Chloride

Essential fatty acid supplementation with oral safflower oil (1 gm/kg/day) to 11 cystic fibrosis patients (aged 6 months to 14 years) for one year produced no significant change in sweat chloride concentration (mEq/liter) or sweat rate (gm/min/m2). Addition of vitamin E (10 mg/kg/day) to the safflower oil had no effect on sweat chloride concentration or rate compared to placebo. No clinical improvement could be detected compared to a control group. These results do not support previous reports of the effects of fatty acid supplementation on sweat electrolyte concentrations in cystic fibrosis.

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Clinical Presentation of the c.3844T>C (p.Trp1282Arg, W1282R) Variant in Russian Cystic Fibrosis Patients

Genes ◽

10.3390/genes11101137 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1137

Author(s):

Nika V. Petrova ◽

Nataliya Y. Kashirskaya ◽

Stanislav A. Krasovskiy ◽

Elena L. Amelina ◽

Elena I. Kondratyeva ◽

...

Keyword(s):

Lung Function ◽

Severe Disease ◽

Pancreatic Insufficiency ◽

Clinical Manifestations ◽

Chloride Concentration ◽

Class I ◽

Sweat Test ◽

Microbial Infection ◽

Sweat Chloride ◽

In Trans

The goal was to study the phenotypic manifestations of c.3844T>C (p.Trp1282Arg, W1282R) variant, a CF-causing mutation, in patients from the Russian Federation. Clinical manifestations and complications (the age at CF diagnosis, sweat test, pancreatic status, lung function, microbial infection, body mass index (BMI), the presence of meconium ileus (MI), diabetes, and severe liver disease) were compared in four groups: group 1—patients carrying c.3844T>C and severe class I or II variant in trans; group 2—3849+10kbC>T/F508del patients; group 3—F508del/F508del patients; and group 4—patients with W1282R and “mild” variant in trans. Based on the analyses, W1282R with class I or II variant in trans appears to cause at least as severe CF symptoms as F508del homozygotes as reflected in the early age of diagnosis, high sweat chloride concentration, insufficient pancreatic function, and low lung function, in contrast to 3849+10kbC-T/F508del compound heterozygotes having milder clinical phenotypes. The W1282R pathogenic variant is seemed to lead to severe disease phenotype with pancreatic insufficiency similarly to the F508del homozygous genotype.

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