Netherlands

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.


Dutch Lung Congress

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

immune response

allergy

single-cell analysis

allergic rhinitis

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Join the AMA Research Challenge poster symposium and semifinals to explore top research posters and score posters in the semifinals (an AMA member exclusive opportunity).

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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
 **Janette Burgess
 Chair, DLC Organizing Committee**
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### Special Session: Publieksmiddag
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**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)
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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 


Fibroblast-seeded lung extracellular matrix- derived hydrogels as an in vitro model for stromal bed in  Idiopathic Pulmonary Fibrosis (IPF)

Emphysema in patients with obstructive pulmonary disease (COPD) is characterized by progressive inflammation and causes irreversible alveolar tissue destruction. The mechanism leading to alveolar damage is not yet clear but to date consensus remains that neutrophils, monocytes and macrophages are key inflammatory cells mediating tissue destruction, and regulation involves various types of lymphocytes. Therefore, we performed a first mass cytometry (cytometry by time-of-flight, CyTOF) single cell analysis to compare immune cells derived from non-emphysematous (n=5) and severe emphysematous (n=15) lung tissue and paired blood samples. We applied a lymphoid and a myeloid antibody panel with 76 markers to obtain an overview of the immune cell profile in the lung and blood. Data were analyzed with Hierarchical Stochastic Neighbor Embedding (HSNE) and dual t-SNE algorithm to identify tissue-specific cell clusters. CyTOF analysis revealed distinct immune cell populations in human lung tissue (Fig. 1), which clustered differently from blood-derived cells. Moreover, CyTOF analysis revealed distinct features of several immune cell populations in emphysematous tissue, and we are now exploring emphysematous lung-specific cell clusters. To determine disease associated cellular interactions in emphysema, we are now applying imaging CyTOF, allowing simultaneous detection of up to 40 markers in a single lung tissue section.

High-dimensional mass cytometry analysis of immune cells in emphysematous lung tissue

Background: 

The Asthma Optimiser is a web-based application to support primary care health care professionals during their asthma consultations by structured guidance through step-by-step questioning and capture of patients’ answers. Using a built-in asthma management decision tree, the tool suggests how they may optimize patients’ asthma management or facilitate referral to asthma specialists. This is in accordance with GINA and NHG guidelines. Data can be stored in the EMR. The CAPTURE study aims to evaluate the feasibility and utility of the Asthma Optimiser in primary care. 

Examination of the feasibility:

Qualitative semi-structured interviews with 20 primary care nurses will be held to examine barriers and facilitators of using the Asthma Optimiser (also known as Refer ID). Purposive sampling will be adopted to attain a sample of primary care nurses who differ in work experience, age, and who work in different GP practices. Interviews will be held using a ‘topic guide’. Data will be collected in an iterative manner (i.e., by updating the topic guide after each interview). Experiences will be examined with thematic analysis and axial coding. This will result in an exhaustive taxonomy of ‘tips and tricks’. 

Examination of the utility: 

A prospective observational study of 354 adult primary care patients with asthma will be conducted. All patients receive standard medical care with additional blood eosinophilic measurement using point-of-care testing and complete the Work Productivity and Activity Impairment (WPAI) questionnaire. Primary outcome is the distribution of asthma patients over the management suggestions of the decision-tree. We will study the association of this distribution with blood eosinophil counts, self-reported WPAI, as well as other pre-existing patient characteristics. Patients referred to secondary care will be followed-up for an additional 26 weeks, analyzing these difficult-to-treat asthma cases.


Implementation of the Asthma Optimiser e-health tool in primary care: a mixed methods approach

Multiple studies have indicated that growing up on a traditional farm is associated with protection against virus-induced wheezing, allergies and asthma, suggesting protective effects on the airway epithelial barrier. Environmental triggers such as allergens, pollutants and pathogens can damage the airway epithelial barrier and contribute to both the development and the progression of asthma. This is mediated in part by airway epithelial alarmins such as IL-25, IL-33 and TSLP, that activate an array of innate immune cells. The aim of the present study is to investigate if (pre-)treatment using farm dust extract (FD) inhibits expression of IL-25, IL-33 and TSLP in primary bronchial epithelial cells (PBEC). Both submerged (S) (n=5 donors) and differentiated (ALI-)PBEC (n=3 donor mixes; 5 donors/mix) were pre-treated with FD for 24 h. Next, cells were stimulated with TNF-α/IFN-γ and poly(I:C)/IL-4 (in S-PBEC only) for 6 h to induce alarmin expression in presence or absence of FD. FD reduced TNF-α/IFN-γ-mediated gene expression of IL33 and IL25 and Poly(I:C)/IL-4-mediated gene expression of TSLP after 6 h in S-PBEC, whereas in ALI-PBEC there was a trend towards a decrease in TNF-α/IFN-γ-mediated gene expression of IL33 and no effect on IL25. The results indicate that FD inhibits inflammation-mediated induction of expression of alarmins in PBEC. However, mechanisms underlying the inhibitory effects of FD on alarmin expression require further investigation. 
This study was supported a grant from the Lung Foundation Netherlands: AWWA grant # 12.0.17.001


Gene expression of epithelial alarmins is decreased by farm dust exposure in primary bronchial epithelial cells

Background:
With the continued advancement of CFTR modulator therapies there is likely to be a burgeoning population of adult cystic fibrosis (CF) patients unable to expectorate sputum. Consequently, the detection and surveillance of pulmonary colonisation, previously reliant on sputum culture, needs re-examining. We hypothesised that cough swabs analysed with culture-independent analysis of the 16S gene could serve as a surrogate for colonisation of the lower airways. 
Methods:
Cough swabs and sputum samples were prospectively collected from consecutive adults and children with CF across two sites at regular outpatient appointments. Conventional culture analysis and next generation sequencing were used to compare paired same day samples. 
Results:
Twenty-two adults and 8 paediatric patients provided 75 paired cough swabs and sputum samples. Alpha diversity measures showed increased bacterial richness in sputum, while evenness and Simpson’s index were higher in cough swabs. Within each sampling technique, microbial composition showed greater similarity when considering intra-patient variation. Poor concordance was observed between culture independent cough swabs and culture dependent/independent sputum analysis for specific pathogens, with cough swabs unable to accurately identify commonly associated CF pathogens (AUROCC range: 0.51 to 0.64). 

Conclusion:
Culture independent analysis of cough swabs provides an inaccurate diagnosis of lower respiratory tract colonization and should not be used as a diagnostic test in patients with CF.


Comparison of microbial composition of cough swabs and sputum for pathogen detection in patients with cystic fibrosis

Prednisone is first-line treatment for pulmonary sarcoidosis and leads to improvement of pulmonary function and symptoms on short-term. However, prednisone can also lead to side-effects, which may negatively affect quality of life. Hence, patients and doctors report an urgent need for better evidence based first line treatment options. Methotrexate is considered as second-line treatment and seems to have fewer side-effects as reported in a patient survey. In the PREDMETH study we investigate the effectiveness of methotrexate compared with prednisone as first line treatment for pulmonary sarcoidosis. Furthermore, we examine blood biomarkers to assess whether response to therapy can be predicted in individual patients.

Methods
The PREDMETH study is initiated by the Erasmus Medical Center and St. Antonius Hospital Nieuwegein and developed in collaboration with the sarcoidosis patient association. This non-inferiority study takes place in 18 hospitals across the Netherlands. Patients will be randomized to treatment with prednisone or methotrexate in a predefined schedule for 24 weeks. Data will be collected at baseline, 4 weeks, 16 weeks, 24 weeks, 1 year and 2 years. Additionally, patients perform weekly home spirometry with a handheld spirometer and report symptoms and side-effects through an online home monitoring program (developed in collaboration with patients with sarcoidosis) during the first six months of the study. The primary endpoint is change in forced vital capacity at six months. Secondary endpoints are adverse events, quality of life and blood biomarkers. The study is currently recruiting patients. 

Discussion
This unique national study is developed by doctors, researchers and patients to provide better evidence based treatment for pulmonary sarcoidosis. If our hypothesis is confirmed and methotrexate is as effective as prednisone with less side-effect this will initiate a change in practice. Furthermore, the translational research will provide more insight to tailored treatment and the pathophysiological mechanisms of sarcoidosis.

Design of a randomized controlled trial to evaluate effectiveness of methotrexate versus prednisone as first-line treatment for pulmonary sarcoidosis: the PREDMETH study

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

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