A simple method to measure CFTR-dependent iodide transport in peripheral blood leukocytes: mutant HS-YFP assay

La fibrosi cistica \ue8 una malattia ereditaria multiorgano causata da una mutazione nel gene codificante per la proteina di regolatore di conduttanza transmembrana della fibrosi cistica (CFTR). Le diverse mutazioni del gene CFTR sono state divise in 6 classi sulla base di difetti molecolari e delle conseguenti alterazioni funzionali. A seconda del tipo di mutazione, la proteina pu\uf2 essere pi\uf9 o meno espressa e funzionante. Questo comporta diversi gradi di gravit\ue0 della malattia e non solo tra diverse mutazioni ma anche tra pazienti che hanno la stessa mutazione. La ricerca effettuata fino ad oggi per cercare di curare il difetto di base ha portato a risultati buoni ma non sufficienti. Al fine di testare farmaci promettenti in grado di aumentare l'espressione e la funzione di CFTR sono necessari metodi facili e veloci e l'uso di cellule primarie derivanti dai singoli pazienti. I leucociti sono riconosciuti nella letteratura scientifica come componente chiave degli eventi patogenetici associati alla fibrosi cistica. I leucociti, in particolare i monociti, rappresentano una fonte facilmente accessibile di cellule primarie che potrebbero essere sfruttate per monitorare l'espressione e l'attivit\ue0 di CFTR. Sulla base di lavori precedenti sono stata coinvolta nello sviluppo di un nuovo test per misurare l'attivit\ue0 di CFTR. Il risultato di questo metodo si basa sulla quantit\ue0 residua di ioduro presente nel surnatante, dopo opportuna stimolazione, quantificata dalla estinzione della fluorescenza della proteina a fluorescenza gialla ad alta sensibilit\ue0 (GST-HS-YFP). La specificit\ue0 del dosaggio per l'attivit\ue0 CFTR \ue8 stata testata utilizzando due diversi inibitori CFTR, CFTR-172 e PPQ-102, in cellule MM6 e dopo il silenziamento di MM6 con siRNA CFTR. Questi dati sono stati confermati anche nei leucociti primari, dove \ue8 stata registrata una differenza significativa tra WT e CF PBMC e Monociti (p <0,005). Il dosaggio GST-HS-YFP pu\uf2 rappresentare un metodo semplice e veloce per misurare la funzione CFTR nei leucociti da pazienti CF durante gli studi clinici e un'altra tecnica applicabile in diversi tipi di cellule per eseguire lo screening di nuovi farmaci.Cystic Fibrosis is a multi organ hereditary disease caused by a mutation in the gene coding for Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR).The different mutations of the CFTR gene were divided into 6 classes on the basis of molecular defects and the consequent functional alterations. Depending on the type of mutation, the protein may be more or less expressed and functioning. This involves varying degrees of disease severity and not only between different mutations but also between patients having the same mutation. Research carried out to date to try to cure the basic defect has led to good but not sufficient results. In order to test promising drugs able to rescue the expression and function of CFTR are necessary easy and fast methods and the use of primary cells from the individual patients. Leukocytes are recognized in the scientific literature as key component of the pathogenetic events associated to cystic fibrosis. Leukocytes, especially monocytes, represent an easily accessible source of primary cells that might be exploited to monitor CFTR expression and activity. Based on previous work I was involved in the development of a new assay to measure CFTR activity suitable to be used in primary cells. The readout of this method is based on residue quantity of iodide present in the supernatant, after proper stimulation, quantified by the quenching of the fluorescence of Halide Sensitivity Yellow Fluorescence Protein (GST-HS-YFP). The specificity of the assay for CFTR activity was tested using two different CFTR inhibitors, CFTR-172 and PPQ-102, in MM6 cells and after silencing of MM6 with CFTR siRNA. These data were confirmed also in primary Leukocytes, where we recorded a significant difference between WT and CF PBMCs and Monocytes (p<0,005). GST-HS-YFP assay can represent easy and fast methods to measure the CFTR function in Leukocytes from CF patients during clinical trials and another useful technique applicable in different cell types to perform new drugs screenin

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P &lt; .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

Intestinal epithelial organoids contribute to combination of CFTR functional tests supporting drug development and diagnosis

Introduction: In vivo and ex vivo measurements of CFTR function in human cells and tissues can be used for screening and monitoring new ther- apies and phenotyping of controversial CFTR genotypes. Among the tools currently available, a technique enabling intestinal stem cells to expand into closed organoids containing crypt-like structures and an internal lumen lined by differentiated cells, recapitulating the in vivo tissue architecture (Sato T, et al. Gastroenterology. 2011;141:1762-72) was set up in our lab and used for measuring CFTR function by a simple and quantitative assay. Material and Methods: We developed intestinal organoids from 14 non-CF and 13 CF subjects and measured forskolin-induced swelling (FIS) (Dekkers JF, et al. Nat Med 2013;19:939-45). Results: In non-CF organoids swelling was completely blocked by the CFTR (inh)-172 and significantly enhanced following treatment with the potentiator ivacaftor (VX-770). Swelling rates in CF organoids were variable, dependent on the CFTR mutation. Remarkably, in organ- oids from a CF patient carrying the W1282X/R117H CFTR genotype we observed swelling following 24h exposure to the premature termination corrector ataluren (PTC124), but not in its absence. We also performed the membrane depolarization assay in monocytes (Sorio C, et al. PLoS One. 2011;6:e22212) carrying different CFTR mutations and detected resto- ration of CFTR function following 24h exposure to the corrector VRT-325 or the combination of lumacaftor (VX-809)+VX-770 (F508del mutation) and to PTC124 (nonsense mutations). Nasal potential difference (NPD) was abnormal but intestinal current measurement (ICM) was normal in the patient with the W1282X/R117H CFTR mutations and in a CF patient with F508del/T5TG13 CFTR genotype while sweat Cl - was 70 and 79mEq/L, respectively. ICM was abnormal in all other CF patients tested. Conclusions: This study explored and compared several robust assays for individualized medicine approaches aiming at supporting diagnosis, CFTR functional studies and new drug development and monitoring. We have been able to grow intestinal organoids from CF and non-CF subjects and to evaluate CFTR function by FIS assays showing partial restoration of CFTR activity in response to a CFTR potentiator, a CFTR corrector and a premature termination corrector. We can use a combination of CFTR func- tional tests covering multiple tissues and cell types including leukocytes (membrane potential), native intestinal epithelium (ICM), intestinal organ- oids (FIS), nasal epithelium (NPD), sweat gland duct (Gibson-Cooke sweat test) and sweat gland coil (ratiometric sweat rate measurements: Wine JJ, et al. PLoS One. 2013;8:e77114) for individualized approaches for CF diag- nosis, drug development and monitoring of new therapies targeting the basic defect

WS18.3 A combination of CFTR functional tests supporting drug development and diagnosis: the contribution of intestinal epithelial organoids

CFTR functional test

ePS01.7 Supporting diagnosis with a combination of standardized and new CFTR functional tests

Cystic Fibrosi

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