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24 Stratification for cystic fibrosis (CF) disease severity in adults with CF with homozygous F508del mutations by intestinal organoids
Posters / Journal of Cystic Fibrosis 15 (2016) S51–S120
in CFBE41o-, a bronchial epithelial cell line homozygous for F508del mutation, and 16HBE14o-, from healthy donor, that are used to test molecules targeting CFTR defect. As additional parameter we evaluated CFTR activity by single-cell fluorescence assay. Methods: CFBE41o- were treated with VRT325 (10mM), VX809 (5mM) or the drugs vehicle DMSO for 24 h. FC analysis was performed with the anti-CFTR monoclonal antibody CF3 (Abcam 2784, specific for the extracellular domain of CFTR: aa 103–117). CFTR expression was evaluated by Mean Fluorescence Intensity (MFI) ratio value and % CFTR positive cells. CFTR activity was assayed with the potentialsensitive probe (DiSBAC2(3), Invitrogen, USA). We defined “CF index” as a parameter that is positive in non-CF cells and negative in CF cells (Sorio et al, 2011). Results: A higher % of CFTR positive cells was recorded in CFBE41ocells after 24 h treatment with VRT325 respect to non-treated cells while VX809 apparently is not effective as VRT325 in these experimental conditions. These were related to the measure of CFTR function recorded with depolarization assay. The CF index value confirming a more effective correction by VRT325. Conclusion: Optimization of the protocol is in progress as variability of fluorescence values is present. Nevertheless the method described is potentially capable to detect correction of CF phenotype, is simple and rapid. It may be applied to primary cells to monitor the responses to drugs whose efficacy can depend on increased CFTR protein expression to the cell surface. 23 Stratifying young children with cystic fibrosis for disease severity using intestinal organoid swelling, intestinal current measurements or sweat chloride concentration as CFTR-dependent biomarker K.M. de Winter-de Groot1 , H.M. Janssens2 , R.T. van Uum1 , J.F. Dekkers1 , H.A.W.M. Tiddens2 , G. Berkers1 , A. Vonk1 , E. Kruisselbrink1 , R.G.J. Vries3 , H. Clevers3 , R.H.J. Houwen4 , J.C. Escher5 , J.M. Beekman1 , C.K. van der Ent1 . 1 Wilhelmina Children’s Hospital/University Medical Centre, Pediatric Pulmonology, Utrecht, Netherlands; 2 Sophia Children’s Hospital/Erasmus University Medical Centre, Pediatric Pulmonology, Rotterdam, Netherlands; 3 Hubrecht Institute for Developmental Biology and Stem Cell Research/ University Medical Centre, Utrecht, Netherlands; 4 Wilhelmina Children’s Hospital/University Medical Centre, Pediatric Gastroenterology, Utrecht, Netherlands; 5 Sophia Children’s Hospital/Erasmus University Medical Centre, Pediatric Gastroenterology, Rotterdam, Netherlands Objectives: Forskolin-induced swelling (FIS) can be used to measure individual CFTR residual function. Aim of this study is: 1. to compare CFTR residual function in intestinal organoids with current diagnostic markers sweat chloride concentration (SCC) and intestinal current measurement (ICM) and 2. to validate the predictive capacity of this model for clinical outcome parameters in infants with CF. Methods: Infants with CF, identified by newborn screening, are enrolled in a monitoring protocol for CF care, according to the AREST-CF protocol. At the age of 1 year patients undergo a diagnostic BAL, chest CT, infant lung function and rectal biopsy for ICM. After performing ICM, organoids are cultured from the residual materials and FIS is measured. Results: CFTR measurements and clinical parameters of 17 infants with 12 different genotypes are analysed. FIS correlates with ICM and SCC, r = 0.78 and −0.64, respectively, both p < 0.01. Children with high FIS values have higher values of IRT, are more often pancreatic sufficient and have lower CT PRAGMA scores compared to children with lower FIS values: 165 mg/ml (134–213) versus 123 mg/ml (79–141), p 0.05; 100% versus 22%, p 0.02 and 0.89% (0.62–2.85) versus 3.81% (1.76–7.62) disease, p 0.03, respectively. High versus low SCC is only significantly related to fecal elastase values (p < 0.01), while ICM has no relation with any clinical parameter. Conclusion: FIS of intestinal organoids correlates with currently established CFTR-dependent biomarkers SCC and ICM. Stratification for FIS appears to better identify subgroups that differ in pulmonary and gastrointestinal clinical outcome parameters when compared to SCC or ICM.
S57
24 Stratification for cystic fibrosis (CF) disease severity in adults with CF with homozygous F508del mutations by intestinal organoids K.M. de Winter-de Groot1 , R. van der Meer2 , R.E.M. van der Wilt1 , J.F. Dekkers1 , M. Geerdink1 , S. Michel1 , E. Kruisselbrink1 , A. Vonk1 , G. Berkers1 , R.G.J. Vries3 , H. Clevers3 , E.A. van de Graaf4 , F.P. Vleggaar5 , H.G.M. Heijerman2 , C.K. van der Ent1 , J.M. Beekman1 . 1 Wilhelmina Children’s Hospital/University Medical Centre, Pediatric Pulmonology, Utrecht, Netherlands; 2 Haga Teaching Hospital, Department of Pulmonology & Cystic Fibrosis, The Hague, Netherlands; 3 Hubrecht Institute for Developmental Biology and Stem Cell Research/University Medical Centre, Utrecht, Netherlands; 4 University Medical Centre, Pulmonology, Utrecht, Netherlands; 5 University Medical Centre, Gastroenterology & Hepatology, Utrecht, Netherlands Introduction: Forskolin-induced swelling (FIS) of intestinal organoids can be used to measure individual CFTR residual function. The aim of this study is to analyse relations between FIS and clinical outcome parameters in adult homozygous F508del subjects. Methods: Multicentre observational study. During a study visit subjects underwent a rectal biopsy as well as chest CT and pulmonary function tests. Relevant diagnostic and clinical parameters were collected from all outpatient visits during the five years before the study visit and at the age of 12. Forskolin-induced swelling (FIS) is measured in cultured intestinal organoids as parameter for CFTR activity. Results: Data of 38 adult patients with CF were collected and analysed. Subjects whose organoids demonstrate higher FIS values (n = 19) versus lower FIS values have higher BMI (19.6 (17.9–22.5) versus 18.2 (16.4– 19.2) kg/m2 , p0.008), and tend to have lower Crispin-Norman scores at chest X-ray (18.0 (11.5–20.5) versus 21.5 (17.3–23.8), p = 0.059) and higher mean FEV1 during the last year (2.6 (2.1–3.2) versus 1.9 (1.3–3.0) ltr, p = 0.08). Conclusion: Preliminary data suggest that FIS of intestinal organoids can define clinically distinct subgroups in adult homozygous F508del subjects, implicating CFTR residual function as modifier of disease in subjects with identical CF-causing mutations. 25 Real-world outcomes in young (6- to 12-year-old) patients (pts) with cystic fibrosis (CF) treated with ivacaftor (IVA): analysis of 2014 US and UK CF registries data Y. Bai1 , M. Higgins2 , N. Volkova1 , L. Bengtsson1 , S. Tian1 , A. Sewall3 , S. Nyangoma4 , A. Elbert3 , D. Bilton4,5 . 1 Vertex Pharmaceuticals Incorporated, Boston, United States; 2 Vertex Pharmaceuticals (Europe) Limited, London, United Kingdom; 3 US CF Foundation, Bethesda, United States; 4 Imperial College London, London, United Kingdom; 5 UK CF Registry, London, United Kingdom Objective: Literature suggests that early therapeutic intervention benefits pts with CF. This ongoing, long-term, post-approval observational safety study uses data from UK and US CF registries to evaluate IVA treatment outcomes in a real-world setting; here we present results of a 2014 analysis focusing on pts aged 6–12 years. Methods: Annual risk of death, organ transplant, hospitalization, and pulmonary exacerbations (PEx) and CF complication prevalence were compared between all IVA-treated pts in the 2014 registries and comparators (COMP) never treated with IVA matched on age, sex, and genotype severity. Outcome
UK CF Registry
US CF Registry RR (95% CI)
n (%)
Death Transplant Hospitalization PEx Complication CFRD Hepatobiliary Bone/Joint Pulmonary GI
IVA (N = 215)
COMP (N = 1109)
0 (0) 0 (0) 25 (11.6) 20 (9.3) 126 (58.9) 16 (7.5) 3 (1.4) 3 (1.4) 61 (28.4) 49 (22.8)
2 (0.2) 1 (0.1) 338 (30.5) 307 (27.7) 780 (70.3) 131 (11.9) 62 (5.6) 23 (2.1) 392 (35.4) 425 (38.3)
− − 0.38 0.34 0.84 0.63 0.25 0.67 0.80 0.59
(0.26–0.56) (0.22–0.52) (0.74–0.94) (0.38–1.03) (0.08–0.79) (P=0.79)* (0.64–1.01) (0.46–0.77)
RR (95% CI)
n (%) IVA (N = 72)
COMP (N = 325)
0 (0) 0 (0) 15 (20.8) 16 (22.2) 42 (58.3) 1 (1.4) 6 (8.3) 1 (1.4) 23 (32.4) 9 (12.5)
1 (0.3) 1 (0.3) 115 (35.4) 129 (39.7) 213 (66.2) 11 (3.4) 58 (17.9) 5 (1.5) 124 (38.8) 41 (12.6)
− − 0.59 (0.37–0.95) 0.56 (0.36–0.88) 0.88 (0.71–1.09) 0.41 (P=0.70)* 0.47 (0.21–1.05) 0.90 (P=1.00)* 0.84 (0.58–1.21) 0.99 (0.50–1.94)
*Fisher’s exact test P values given when the expected value is <5 in ≥1 cell of the contingency table.
Results: No deaths or transplants were reported in IVA-treated pts and risk of hospitalization and PEx was significantly lower in IVA than COMP
in CFBE41o-, a bronchial epithelial cell line homozygous for F508del mutation, and 16HBE14o-, from healthy donor, that are used to test molecules targeting CFTR defect. As additional parameter we evaluated CFTR activity by single-cell fluorescence assay. Methods: CFBE41o- were treated with VRT325 (10mM), VX809 (5mM) or the drugs vehicle DMSO for 24 h. FC analysis was performed with the anti-CFTR monoclonal antibody CF3 (Abcam 2784, specific for the extracellular domain of CFTR: aa 103–117). CFTR expression was evaluated by Mean Fluorescence Intensity (MFI) ratio value and % CFTR positive cells. CFTR activity was assayed with the potentialsensitive probe (DiSBAC2(3), Invitrogen, USA). We defined “CF index” as a parameter that is positive in non-CF cells and negative in CF cells (Sorio et al, 2011). Results: A higher % of CFTR positive cells was recorded in CFBE41ocells after 24 h treatment with VRT325 respect to non-treated cells while VX809 apparently is not effective as VRT325 in these experimental conditions. These were related to the measure of CFTR function recorded with depolarization assay. The CF index value confirming a more effective correction by VRT325. Conclusion: Optimization of the protocol is in progress as variability of fluorescence values is present. Nevertheless the method described is potentially capable to detect correction of CF phenotype, is simple and rapid. It may be applied to primary cells to monitor the responses to drugs whose efficacy can depend on increased CFTR protein expression to the cell surface. 23 Stratifying young children with cystic fibrosis for disease severity using intestinal organoid swelling, intestinal current measurements or sweat chloride concentration as CFTR-dependent biomarker K.M. de Winter-de Groot1 , H.M. Janssens2 , R.T. van Uum1 , J.F. Dekkers1 , H.A.W.M. Tiddens2 , G. Berkers1 , A. Vonk1 , E. Kruisselbrink1 , R.G.J. Vries3 , H. Clevers3 , R.H.J. Houwen4 , J.C. Escher5 , J.M. Beekman1 , C.K. van der Ent1 . 1 Wilhelmina Children’s Hospital/University Medical Centre, Pediatric Pulmonology, Utrecht, Netherlands; 2 Sophia Children’s Hospital/Erasmus University Medical Centre, Pediatric Pulmonology, Rotterdam, Netherlands; 3 Hubrecht Institute for Developmental Biology and Stem Cell Research/ University Medical Centre, Utrecht, Netherlands; 4 Wilhelmina Children’s Hospital/University Medical Centre, Pediatric Gastroenterology, Utrecht, Netherlands; 5 Sophia Children’s Hospital/Erasmus University Medical Centre, Pediatric Gastroenterology, Rotterdam, Netherlands Objectives: Forskolin-induced swelling (FIS) can be used to measure individual CFTR residual function. Aim of this study is: 1. to compare CFTR residual function in intestinal organoids with current diagnostic markers sweat chloride concentration (SCC) and intestinal current measurement (ICM) and 2. to validate the predictive capacity of this model for clinical outcome parameters in infants with CF. Methods: Infants with CF, identified by newborn screening, are enrolled in a monitoring protocol for CF care, according to the AREST-CF protocol. At the age of 1 year patients undergo a diagnostic BAL, chest CT, infant lung function and rectal biopsy for ICM. After performing ICM, organoids are cultured from the residual materials and FIS is measured. Results: CFTR measurements and clinical parameters of 17 infants with 12 different genotypes are analysed. FIS correlates with ICM and SCC, r = 0.78 and −0.64, respectively, both p < 0.01. Children with high FIS values have higher values of IRT, are more often pancreatic sufficient and have lower CT PRAGMA scores compared to children with lower FIS values: 165 mg/ml (134–213) versus 123 mg/ml (79–141), p 0.05; 100% versus 22%, p 0.02 and 0.89% (0.62–2.85) versus 3.81% (1.76–7.62) disease, p 0.03, respectively. High versus low SCC is only significantly related to fecal elastase values (p < 0.01), while ICM has no relation with any clinical parameter. Conclusion: FIS of intestinal organoids correlates with currently established CFTR-dependent biomarkers SCC and ICM. Stratification for FIS appears to better identify subgroups that differ in pulmonary and gastrointestinal clinical outcome parameters when compared to SCC or ICM.
S57
24 Stratification for cystic fibrosis (CF) disease severity in adults with CF with homozygous F508del mutations by intestinal organoids K.M. de Winter-de Groot1 , R. van der Meer2 , R.E.M. van der Wilt1 , J.F. Dekkers1 , M. Geerdink1 , S. Michel1 , E. Kruisselbrink1 , A. Vonk1 , G. Berkers1 , R.G.J. Vries3 , H. Clevers3 , E.A. van de Graaf4 , F.P. Vleggaar5 , H.G.M. Heijerman2 , C.K. van der Ent1 , J.M. Beekman1 . 1 Wilhelmina Children’s Hospital/University Medical Centre, Pediatric Pulmonology, Utrecht, Netherlands; 2 Haga Teaching Hospital, Department of Pulmonology & Cystic Fibrosis, The Hague, Netherlands; 3 Hubrecht Institute for Developmental Biology and Stem Cell Research/University Medical Centre, Utrecht, Netherlands; 4 University Medical Centre, Pulmonology, Utrecht, Netherlands; 5 University Medical Centre, Gastroenterology & Hepatology, Utrecht, Netherlands Introduction: Forskolin-induced swelling (FIS) of intestinal organoids can be used to measure individual CFTR residual function. The aim of this study is to analyse relations between FIS and clinical outcome parameters in adult homozygous F508del subjects. Methods: Multicentre observational study. During a study visit subjects underwent a rectal biopsy as well as chest CT and pulmonary function tests. Relevant diagnostic and clinical parameters were collected from all outpatient visits during the five years before the study visit and at the age of 12. Forskolin-induced swelling (FIS) is measured in cultured intestinal organoids as parameter for CFTR activity. Results: Data of 38 adult patients with CF were collected and analysed. Subjects whose organoids demonstrate higher FIS values (n = 19) versus lower FIS values have higher BMI (19.6 (17.9–22.5) versus 18.2 (16.4– 19.2) kg/m2 , p0.008), and tend to have lower Crispin-Norman scores at chest X-ray (18.0 (11.5–20.5) versus 21.5 (17.3–23.8), p = 0.059) and higher mean FEV1 during the last year (2.6 (2.1–3.2) versus 1.9 (1.3–3.0) ltr, p = 0.08). Conclusion: Preliminary data suggest that FIS of intestinal organoids can define clinically distinct subgroups in adult homozygous F508del subjects, implicating CFTR residual function as modifier of disease in subjects with identical CF-causing mutations. 25 Real-world outcomes in young (6- to 12-year-old) patients (pts) with cystic fibrosis (CF) treated with ivacaftor (IVA): analysis of 2014 US and UK CF registries data Y. Bai1 , M. Higgins2 , N. Volkova1 , L. Bengtsson1 , S. Tian1 , A. Sewall3 , S. Nyangoma4 , A. Elbert3 , D. Bilton4,5 . 1 Vertex Pharmaceuticals Incorporated, Boston, United States; 2 Vertex Pharmaceuticals (Europe) Limited, London, United Kingdom; 3 US CF Foundation, Bethesda, United States; 4 Imperial College London, London, United Kingdom; 5 UK CF Registry, London, United Kingdom Objective: Literature suggests that early therapeutic intervention benefits pts with CF. This ongoing, long-term, post-approval observational safety study uses data from UK and US CF registries to evaluate IVA treatment outcomes in a real-world setting; here we present results of a 2014 analysis focusing on pts aged 6–12 years. Methods: Annual risk of death, organ transplant, hospitalization, and pulmonary exacerbations (PEx) and CF complication prevalence were compared between all IVA-treated pts in the 2014 registries and comparators (COMP) never treated with IVA matched on age, sex, and genotype severity. Outcome
UK CF Registry
US CF Registry RR (95% CI)
n (%)
Death Transplant Hospitalization PEx Complication CFRD Hepatobiliary Bone/Joint Pulmonary GI
IVA (N = 215)
COMP (N = 1109)
0 (0) 0 (0) 25 (11.6) 20 (9.3) 126 (58.9) 16 (7.5) 3 (1.4) 3 (1.4) 61 (28.4) 49 (22.8)
2 (0.2) 1 (0.1) 338 (30.5) 307 (27.7) 780 (70.3) 131 (11.9) 62 (5.6) 23 (2.1) 392 (35.4) 425 (38.3)
− − 0.38 0.34 0.84 0.63 0.25 0.67 0.80 0.59
(0.26–0.56) (0.22–0.52) (0.74–0.94) (0.38–1.03) (0.08–0.79) (P=0.79)* (0.64–1.01) (0.46–0.77)
RR (95% CI)
n (%) IVA (N = 72)
COMP (N = 325)
0 (0) 0 (0) 15 (20.8) 16 (22.2) 42 (58.3) 1 (1.4) 6 (8.3) 1 (1.4) 23 (32.4) 9 (12.5)
1 (0.3) 1 (0.3) 115 (35.4) 129 (39.7) 213 (66.2) 11 (3.4) 58 (17.9) 5 (1.5) 124 (38.8) 41 (12.6)
− − 0.59 (0.37–0.95) 0.56 (0.36–0.88) 0.88 (0.71–1.09) 0.41 (P=0.70)* 0.47 (0.21–1.05) 0.90 (P=1.00)* 0.84 (0.58–1.21) 0.99 (0.50–1.94)
*Fisher’s exact test P values given when the expected value is <5 in ≥1 cell of the contingency table.
Results: No deaths or transplants were reported in IVA-treated pts and risk of hospitalization and PEx was significantly lower in IVA than COMP