Netherlands

Rationale
IL-33 and its receptor IL-1RL1 are important asthma genes. T helper-2 (TH2) cells present in asthmatic airway wall contribute to chronic inflammation and epithelial remodeling. TH2 cells express IL-1RL1 and respond to IL-33 by increased cytokine production upon stimulation. We aimed to test the extent of the IL-33-driven modification of Th2 cell activation, and whether the asthma-susceptibility alleles of IL1RL affect this response. 
Methods
CD4+ T cells isolated from PBMCs of 16 healthy donors and 16 asthma patients, stratified for IL-1RL1 high and low risk-genotypes, were differentiated into TH2 cells in vitro, and re-stimulated by CD3/CD28-crosslinking in presence or absence of 100 ng/mL rhIL-33. Gene expression was assessed by RNA-sequencing, and subsequently analyzed using limma in R comparing high and low risk genotype and asthma versus controls. Membrane bound IL1-RL1-b protein levels were determined using FACS.
Results
Presence of IL-33 during CD3/CD28 stimulation of TH2 effector cells in vitro results in extensive changes in gene expression, affecting up to 40% of the transcriptomic changes induced by TH2 cell activation. The fold change in gene expression induced by IL-33 was significantly higher in TH2 cells carrying the IL-1RL1 risk haplotype compared to the IL-1RL protective haplotype. However, the identity of the IL-33 regulated genes was not different between the IL-1RL1 haplotype groups. In addition, significant differences in IL-33 regulated genes were observed between Th2 cells derived from healthy controls and asthma patients. At the protein level, the change in TH2 cells expressing the transmembrane IL-1RL1 receptor was higher in asthma patients than in controls . 
Conclusions
The asthma-associated IL-1RL1 haplotype strongly increases the sensitivity of TH2 effector cell for IL-33-mediated regulation of gene expression, while disease status also has an independent effect. IL-33 in the airway wall will contribute to chronic inflammation maintained by TH2 effector cells mainly in patients with asthma carrying the IL-1RL1 asthma risk alleles. 


Dutch Lung Congress

Enhanced IL-33 response in differentiated human TH2 cells carrying the IL-1RL1 asthma risk genotype

th2 cells

il-33/il-1rl1 pathway

asthma

[![](https://s3.eu-west-1.amazonaws.com/underline.prod/uploads/markdown_image/1/image/261902fdf187a88bfa74da226ff77186.png)](https://drive.google.com/file/d/1gUViQJJHCSgM38OhwpDoTdN89evCsZ-7/view?usp=sharing)
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### **Welcome Message**

Welcome to the inaugural **Dutch Lung Congress.**  We have all missed our opportunities to network and interact with our colleagues in the last twelve months or more.  While our desire was to meet face to face once again, when it became evident that this would not be possible until much later in 2021 (or even into 2022) the hard working committee organizing the Dutch Lung Congress found, what we hope will be, an excellent alternative to facilitate our annual meeting.

The Dutch Lung Congress aims to bring together everyone interested in research related to pulmonary diseases in the Netherlands.  We hope this event will provide opportunities for multidisciplinary interactions to enhance our approaches for addressing these challenging diseases.  Each session will include input from patients, clinical and basic and/or translational scientists discussing the latest information around the central themes chosen by the organizing committee.  One advantage of holding our congress as a virtual event this year is that we will have the opportunity to hear from world leading experts from all over the globe.  We hope you enjoy the programme the organizing committee have put together and will engage in dynamic discussions with all the speakers.

One highlight of our congress will be the Young Investigator Session.  Remember to join us for this exciting session when we will hear from the presenters who submitted the top scoring abstracts.  This promises to be a session full of exciting science that will display the strengths that constitute the future talent in our field.

When we are not listening to these great presentations we can all meet in Gathertown.  Within this interactive platform you will be able to meet colleagues and friends for networking. Enjoy a private chat over a virtual coffee, visit the booths of our sponsors and other parties, chat with the Young Investigator Board members or take some timeout in the relaxation spaces.  While in Gathertown take the opportunity to visit all the diverse posters on display. Chat live with the presenters or leave them a question on the discussion board for them to engage with you.  Make sure you check for any messages each time you enter Gathertown to make the most of your networking opportunities.

We look forward to seeing you during the DLC and the great scientific (and other) discussions we are anticipating during our 2021 meeting.

On behalf of the DLC organizing Committee
<br>**Janette Burgess
<br>Chair, DLC Organizing Committee**
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### Special Session: Publieksmiddag
![](https://s3.eu-west-1.amazonaws.com/underline.prod/uploads/markdown_image/1/image/77e768dd500e6205175e5cafba59abdc.png)






![](https://s3.eu-west-1.amazonaws.com/underline.prod/uploads/markdown_image/1/image/f461fb275d9667fb2a76a30813388e90.png)
**More about the committee team:**:
[Barbro N. Melgert, PhD] (https://drive.google.com/file/d/1q_IIH3JvTivumCUzzsZGX7BgD41HxQmk/view?usp=sharing)
// [Bas Holverda, PhD] (https://drive.google.com/file/d/1QKXHmTTQjgs4OZVuM8doXU055S-fu_uU/view?usp=sharing)
// [Frits Franssen, PhD] (https://drive.google.com/file/d/1d7HRUJaqGuRFzkPN3SVgkbBfqFkDkWq2/view?usp=sharing)
// [Janette Burgess, PhD] (https://drive.google.com/file/d/1VHFlrEN6uCQGeeW3Lj4I8aTw7HsB0GFr/view?usp=sharing)
// [Michel van den Heuvel, PhD] (https://drive.google.com/file/d/1JeOEP8MHxiK_4lONr8UM2HaxdxpkAmA2/view?usp=sharing)
// [Patrick Bhairosing] (https://drive.google.com/file/d/1xI8ORq-g1bbVcrlRDviZ9NhAwanc4dFl/view?usp=sharing)
// [Gimano Amatngalim, PhD] (https://drive.google.com/file/d/1fyVDDKARLNjRk_JZk8_GyhGMjmp6DWHp/view?usp=sharing)
// [Peter Kunst, PhD] (https://drive.google.com/file/d/1QhtB4nQezpAjbfUAcBnINc-Bm3Shy2Lf/view?usp=sharing)
// [Dr. Michel van den Heuvel, Prof., MD, PhD] (https://drive.google.com/file/d/1JBdNEBXZCEikEZhJ6MoD5DhQfXMz3bP6/view?usp=sharing)
![](https://s3.eu-west-1.amazonaws.com/underline.prod/uploads/markdown_image/1/image/f461fb275d9667fb2a76a30813388e90.png)

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poster

Introduction
Cystic Fibrosis (CF) is a severe inherited disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel, resulting in a severe muco-obstructive lung disease. It is proposed that potentiation of airway epithelial fluid secretion by the alternative chloride channel TMEM16A may act as therapy for all individuals with CF, independent of their mutation type. Therefore, we aimed to design a TMEM16A-dependent fluid secretion assay using nasal organoids from individuals with CF.

Methods
Nasal organoids from individuals with CF were derived from nasal brushing. Organoid culture conditions were optimized for TMEM16A-dependent fluid secretion with the cytokine IL-4. This was validated by qPCR, western blot, an organoid swelling assay using chemical inhibitors, and with CRISPR gene knock-out organoids. 

Results
IL-4 enhanced TMEM16A mRNA and protein expression in CF nasal organoids. Furthermore, IL-4 treatment increased nasal organoid swelling, induced by the TMEM16A activator ATP. This was inhibited by the chemical TMEM16A inhibitors ani9 and niclosamide. 

Conclusion
A TMEM16A-dependent functional fluid secretion assay was developed which can be used to identify TMEM16A potentiating compounds in future. These compounds may act as lead for novel therapies that promote epithelial fluid secretion in individuals with CF, independent of their mutation type.

A personalized nasal organoid fluid-secretion assay to identify TMEM16A potentiators as treatment for cystic fibrosis

Rationale: COPD is characterized by a progressive and irreversible loss of lung function, to which emphysema is a contributor. Emphysema is characterized by destruction of alveolar walls, that are composed of the alveolar epithelium, the endothelium and mesenchymal cells. It is increasingly realized that microvascular endothelial cell (dys)function contributes toemphysema development. Therefore, the aim is to study the role of that pulmonary microvascular endothelial cells in alveolar repair and regeneration. 
Methods: EpCAM+ (i.e. epithelial) cells isolated from mouse lungs (BALB/c) were seeded in matrigel and deposited on a monolayer of the pulmonary microvascular endothelial cell line (HPMEC.ST1.R), resulting in both airway and alveolar epithelial organoids. Human resected lung tissue was used to isolate HTII-280+ alveolar cells and CD31+ endothelial cells. Endothelial cells (P2) were allowed to form a monolayer in the bottom of a culture plate and freshly isolated HTII-280+ alveolar cells were seeded in basement membrane matrix (BME2) inside the insert. 
Results and Conclusion: The presence of HPMEC.ST1.R cells increased alveolar organoid number but not airway organoids isolated from murine lungs. Similarly, human primary lung endothelial cells cultured and expanded in vitro (P2), induced an increase (1.7X) in human alveolar organoid number, at day 10 after isolation from human lung tissue. Collectively these results show that pulmonary endothelial cells support alveolar progenitor cell function as shown by an increased number of alveolar organoids. We hypothesize that this function is disturbed in emphysema, and therefore we are currently analyzing the impact of COPD-relevant triggers such as cigarette smoke and inflammatory mediators on endothelial cell-driven alveolar repair using this co-culture model. Furthermore, we are expanding our model to include human induced pluripotent stem cell (hiPSC)-derived lung cells.


The role of pulmonary endothelial cells in alveolar repair and regeneration

Rationale: Chronic obstructive pulmonary disease (COPD) is characterized by several phenotypes, including emphysema resulting from the destruction of alveolar tissue in the lung parenchyma. Emphysema is likely partly due to a reduced repair capacity of the alveolar epithelial type II cell (AECII). Previously we showed in mouse lung organoids that amphiregulin (AREG), which is ligands of the epidermal growth factor receptor (EGFR) that is present on AECII, regulates epithelial cell differentiation and proliferation(1). In this study, we evaluated the hypothesis that impaired alveolar regeneration in patients with COPD could be explained by aberrant AREG-EGFR signaling in COPD-derived AECIIs in an organoid model.

Method: We examined lung tissue from Stage IV COPD patients (n=12) and non-COPD subjects (n=9) for expression of AREG, EGF, EGFR and SPC by immunohistochemical staining. Next, we investigated whether the function of COPD AECII was impaired using a human organoid model. AECII isolated from lungs with COPD (n=2) and non-COPD subjects (n=2) were cultured as organoids and cultured with or without AREG.

Result: COPD patients had a lower number of SPC positive cells per surface of lung parenchyma and EGFR-expressing AECII per total cell number of lung parenchyma when compared to non COPD controls. No differences were found in the number of AREG-expressing cells per lung parenchyma. AECII-derived organoids from COPD patients were significantly smaller in size than organoids from controls. In non COPD controls, AREG treatment appeared to result in smaller organoids compared to untreated organoids. SPC immunofluorescent staining revealed that the AECII proportion within the total cell number was not different between COPD or AREG-treated groups. 

Conclusions: The lower number of EGFR-expressing AECII in COPD could explain a possible impaired ability to form organoids, which might imply an impaired repair capacity. More insight regarding the growth and formation of AECII-derived organoids in COPD is necessary to establish the link between EGFR signaling and AECII proliferation.


Alveolar epithelial type II cell behavior in COPD.

Introduction Pharmacological treatment is the cornerstone of asthma management. Asthma patients may also exhibit treatable traits (TTs) like persistent smoking, hyperventilation (PaCO2<4.7 kPa), frequent exacerbations, overweight (BMI ≥25 kg/m2), exercise intolerance (6MWD ≤70%pred), physical inactivity (≤7000 steps/day), fatigue (CIS ≥36 points), depressive symptoms (measured using the Beck Depression Inventory (BDI ≥4 points), dyspnea (Modified Medical Research Council ≥2), qualifying for non-pharmacological interventions. The aim of this study was to investigate the prevalence of these nine non-pharmacological TTs in asthma patients at the time of first referral to a pulmonologist for an outpatient consultation.

Methods Data from asthma patients were collected in two Dutch hospitals (Amphia Breda, RadboudUMC Nijmegen). Data from a third hospital (Franciscus & Vlietland Rotterdam) were used for validation. All adult asthma patients who were referred for an integral diagnostic pathway without exacerbation <6 weeks were deemed eligible. 

Results 346 adults were studied (59% women, age 48 ±16 yrs, FEV1: 88 ±17%pred). Most patients (n=190, 58%) displayed uncontrolled asthma (ACQ >1.5 points). Median number of TTs was 2 (range 0-8). A majority of 174 (53%) patients had ≥3 TTs. Patients with uncontrolled asthma had significantly more TTs compared to patients with controlled and partially controlled asthma (3.4±1.8 vs 1.7±1.3 and respectively 2.2±1.4; P<0.001). 

Conclusion Treatable traits for non-pharmacological interventions frequently occur in asthma patients referred for the first time to a pulmonologist. Asthma control is associated with the number of non-pharmacological treatable traits.

Prevalence of non-pharmacological treatable traits in patients with asthma

Immune cells play a key role in preserving mucosal homeostasis. Here, we investigated their role at nasal mucosa following challenge with house dust mite. We combined single cell proteome and transcriptome profiling on immune cells from nasal biopsy cells of allergic rhinitis and non-allergic subjects, before and after repeated nasal allergen challenge.
At baseline, clear differences were observed between study groups: a GATA3+/IL13+ Th2 population was observed in patients which was absent in controls, while IL17A+/INFG+ T-cells were present at much higher frequencies in controls. 
Three days after repeated allergen challenge, patients displayed a clinical nasal response and cardinal features of allergic inflammation, including increased eosinophil and innate lymphoid cells (ILC)2 numbers. The 
Furthermore, patients showed infiltrating inflammatory HLA-DRhi CD14+ monocytes and CD16+ monocytes, and transcriptional changes in resident CD1C+ CD1A+ conventional dendritic cells (cDC)2 following challenge. Importantly, although clinically silent, non-allergic individuals displayed a distinct innate myeloid response to allergen challenge: predominant infiltration of myeloid-derived suppressor cells (HLA-DRlow CD14+ monocytes), as well as cDC2 clusters expressing increased inhibitory/tolerogenic transcripts. 
Taken together, we identified not only clusters involved in airway inflammation but also a non-inflammatory, homeostatic blueprint of innate MPS responses to allergens in non-allergic individuals. Future therapies should target innate MPS for treatment of inflammatory airway diseases.


Nasal systems immunology identifies inflammatory and tolerogenic cells that determine allergic outcomefollowing challenge

We studied the effect of severity of smoking on survival in patients with progressive, fibrosing interstitial lung disease (PF-ILD). A total of 377 patients fulfilled the criteria of PF-ILD as stated in the INBUILD trial. Patients were stratified into never, mild-moderate (pack years = 0.1-19.9), and severe smokers (20 pack years or more). Median transplant-free survival was 3.03 years in severe, 3.84 years in mild-moderate, and 4.77 years in never smokers. Survival differences between severe and mild-moderate and severe and never smokers were significant. Furthermore, it was demonstrated that pack years was an independent prognostic factor after adjusting for age, sex, and lung function. 
In summary, our data strongly suggests that early smoking cessation is beneficial for patients with PF-ILD.

The detrimental effect of severe smoking in progressive, fibrosing interstitial lung disease

Biologicals are a promising treatment for severe eosinophilic asthma. Self-administration by patients of biologicals using an injection pen at home is likely to facilitate their accessibility. However, little is known about patients’ and clinicians’ perceptions and experiences with regard to home administration of biologicals. The Perceptions on Self-Administration Injections as a Biological Therapy (POSSIBILITY) study aims to elucidate these. 

Methods/Approach

A qualitative cross-national study is ongoing in the Netherlands, Northern-Ireland, the United States, and Australia. Per country, two separate Online Focus Groups (OFGs) are held with 6-8 patients, i.e., starters and recent users vs. current users. Prior to the OFGs, patients will be prompted by presenting them with themes on an online forum. OFG discussions will be held using a topic guide to assess a thematic coding tree. Data will be collected iteratively (i.e., updating the topic guide and expanding the thematic coding tree after each OFG). For triangulation purposes, interviews will be held with chest physicians and nurses to discuss salient findings from the OFGs and prescribing decisions. Data will be analyzed with qualitative content analysis. A novel artificial intelligence approach using automated coding of transcripts will be adopted to support the analysis.

Results

In the Netherlands, the OFGs have been completed. A total of 26 patients responded to the themes on the online forum while 17 participated in the OFGs. Patients used three different brands of home administered biologicals. The topic guide elicited a wide range of experiences and perceptions and the thematic coding tree could be expanded. Salient statements such as “Biologicals are like an intensive rehabilitation treatment in a syringe” also came to the fore. 

Conclusion

These findings are a first step of the unravelling of patient perceptions and experiences with regard to self-administration of biologicals at home.


Perceptions on Self-Administration Injections as a Biological Therapy (POSSIBILITY): a multi-country qualitative study of patients with severe asthma

Introduction
Resistance to osimertinib inevitably occurs in the majority of patients. We aimed to identify the resistance mechanism (RM) using paired liquid and tissue biopsies in patients that progressed on 2nd line osimertinib and to determine progression free survival (PFS) of post-osimertinib therapy. 

Methods
From 09-2019 to 02-2021, 50 patients that progressed on 2nd line osimertinib treatment were enrolled. Liquid biopsies were analyzed using AVENIO ctDNA Expanded Panel, and tissue biopsies underwent DNA and RNA NGS. Results were discussed in a Molecular Tumor Board (MTB), leading to tailored treatment advise. Post-MTB treatment was monitored every 6-8 weeks with CT. 

Results
The EGFR mutation (m) was detected in 37/50 plasma samples (74%) and 48/50 tumor biopsies (96%). Matched results were available for 35/50 patients (70%). For 39/50 patients (78%) ≥1 RM could be identified in plasma and/or tissue, of which 15 showed ≥2 RMs. Most prevalent RMs were MET amplification (38%) and EGFR C797Ss (16%). 
23 patients received targeted therapy (TT) and 16 patients chemotherapy (CTX). Four received best supportive care. Six continued osimertinib, 3 of whom received additional local therapy. One had not yet started CTX. Median PFS was 5.2 months for CTX (95%CI 3.9 – 6.5 months), 3.3 months for TT (95%CI 1.4 – 5.2 months). Median FU was 9.1 months for (CTX) and 7.2 months for TT. For patients receiving TT after progression on osimertinib, the number of identified RMs impacted PFS. PFS for patients who had 1 RM was 5.8 months (95%CI 4.2 – 7.5 months), 3.2 months for those with ≥2 RMs (95%CI 0.2 – 6.2 months).
Ten patients were biopsied after progression on TT. The targeted RM was no longer detected in 7/10 (70%) biopsy samples, while a new RM was detected in 5/10 (50%).

Conclusion
When paired liquid and tissue biopsies were used to guide treatment after progression on 2nd line osimertinib, no large difference in PFS was observed between CTX and TT in an exploratory analysis on clinical data. A relatively short duration of response to TT after progression on osimertinib may be due to the prevalence of multiple RMs.

Paired liquid and tissue biopsies to guide treatment for patients that progress on 2nd line osimertinib treatment (OSIRIS)

Introduction
Despite high response rates to osimertinib in patients with EGFRm+ NSCLC, resistance inevitably develops. HER2 overexpression and amplification is detected in 13% of NSCLC-patients with acquired TKI-resistance. Here, we evaluated the efficacy and toxicity of T-DM1 and osimertinib in patients with HER2 overexpression and/or amplification after progression on osimertinib. We report a pre-planned interim analysis.

Methods
In this multicenter single arm phase II study (NCT03784599), patients with EGFRm+ NSCLC, progressing on EGFR TKI treatment and with HER2 IHC ≥2+ in ≥10% of the cells were included. Patients were treated with T-DM1 3.6 mg/kg (IV) every 3 weeks and osimertinib 80 mg QD. Primary endpoints were to assess the ORR after 12 weeks and to evaluate safety. 
Responses were assessed every 6 weeks (RECIST 1.1). Treatment was continued until progression or unacceptable toxicity. Sample size was calculated using Simon’s two stage minimax design (H0=41%, H1>55%, 80% power, one-sided type I error rate of 10%, sample size: 58 patients, cohort A ORR 16/36 in order to proceed to cohort B).

Results
From 01-2019 until 04-2021, 27 patients were included and started with T-DM1. ORR and DCR after 12 weeks of treatment were 13% (3/23) and 43% (10/23). Four patients are not yet evaluable for the primary endpoint. Median DCR in HER2 IHC2+ and 3+ cohorts were was 36% (5/14) and 55% (5/9), respectively. PFS was 2.8 months (95% CI, 2.3-3.3 months), median duration of disease control was 20 weeks (range 4-47 weeks). Grade 2/3 adverse events (AEs) occurred in 29%/21% of patients, including pneumonitis, LVEF decrease, fatigue, erythema and CK elevation. There were no grade 4 or 5 therapy-related AEs.

Conclusion
TRAEMOS is the first trial combining T-DM1 and osimertinib in EGFRm+ NSCLC patients to target HER2 bypass track resistance. Safety profile was favorable compared to cytotoxic chemotherapy, however this treatment strategy showed limited efficacy. Further clinical evaluation of this regimen is not warranted.

Trastuzumab-emtansine (T-DM1) and osimertinib (TRAEMOS) combination treatment to target HER2 bypass track resistance in EGFR mutation positive (EGFRm+) NSCLC

INTRODUCTION: Idiopathic Pulmonary Fibrosis (IPF) is characterized by aberrant ECM deposition and remodeling of the lung – structural changes which further drive a pro-fibrotic cellular microenvironment. Decellularized lung ECM-derived hydrogels resemble the mechanical properties of native decellularized tissues, potentially providing a 3D model mimicking native cell-ECM interactions. We aimed to characterize this 3D human lung microenvironment model, with respect to stiffness and viscoelastic properties, in the presence and absence of primary human lung fibroblasts.
METHODS: Lyophilized powders of decellularized IPF and control lung matrices (pooled from 6 donors per group) were pepsin digested, and formed to hydrogels seeded with control primary lung fibroblasts (n=6 donors), and cultured for 14 days. Stiffness and viscoelastic relaxation were measured by Low-Load Compression Testing (20% strain). 
RESULTS: IPF hydrogels showed a trend towards higher stiffness compared to control hydrogels and cell-seeded IPF hydrogels were significantly stiffer than cell-seeded controls (1.85 ± 0.48 kPA vs 1.04 ± 0.22 kPA, p=0.0183, Kruskal-Wallis test) (Fig. 1). Time to reach 100% viscoelastic relaxation was similar in cell-seeded compared to native hydrogels for both IPF and control hydrogels. Total percentage relaxation of cell-seeded IPF hydrogels was significantly lower than of cell-seeded control hydrogels (108.4% ± 2.39% vs 117.6% ± 3.63%, p=0.0495, Kruskal-Wallis test) while empty hydrogels reached comparable values. The percentage relaxation was in general lower in IPF hydrogels than control hydrogels (cell-seeded or not). 
DISCUSSION & CONCLUSIONS: The mechanical properties of the ECM hydrogels were modified by fibroblasts and the source of the ECM influenced these modifications. These data suggest that higher stiffnesses and altered relaxation patterns of the ECM may be involved in fibrotic responses in IPF. Fibroblast-seeded ECM-derived hydrogels can provide more insight on cell-ECM interactions 


Enhanced IL-33 response in differentiated human TH2 cells carrying the IL-1RL1 asthma risk genotype

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A personalized nasal organoid fluid-secretion assay to identify TMEM16A potentiators as treatment for cystic fibrosis

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