Chronic rhinosinusitis (CRS) has been divided into two subtypes: CRS with (CRSwNP) and
without nasal polyps (CRSsNP), which not only differ in terms of presence of polyps, but also
appear to have distinct pathogenesis and clinical presentations. It is known that CRSwNP
patients have a greater disease burden compared with those suffering from CRSsNP with respect
to disease severity and poor treatment. More specifically, approximately 85% of CRSwNP
patients are characterized by severe symptoms, recurrent disease and a dominant Th2 endotype
associated with a marked infiltration of eosinophils and mast cells, goblet hyperplasia and
increased levels of Th2 inflammatory cytokines including Interleukin IL-4, IL-5, and IL-13.
An additional hallmark of CRSwNP is the loss of healthy barrier function in sinonasal
epithelial cells, increased permeability, decreased epithelial resistance, and a high degree
of tissue remodeling compared with cells from CRSsNP patients and control individuals. This
loss of barrier function is reflective of a general inflammatory process, though it is
unclear if the epithelial cells are inherently abnormal or if the state is induced. Treatment
for CRS is most frequently glucocorticoid-based, but response is quite variable in patients
with nasal polyps and side-effects from oral steroids limit their long-term efficacy. An
inverse relationship between glucocorticoid receptor β expression in nasal polyp tissue and
steroid efficacy has been observed. Furthermore, neutrophil accumulation in nasal polyp
tissue has been related to corticosteroid insensitivity. Some individuals exhibit a very high
level of resistance to steroid therapy thus underscoring the need for therapeutics targeting
non-steroid-responsive pathophysiologic mechanisms involved in sinus polyp formation.
Asthma is frequently a comorbid condition sharing similar pathophysiology in CRSwNP patients,
which affects 20-60% of diseased individuals. Yet, specific subsets of patients such as those
with IL-5-enriched nasal polyps are characterized by a greater percentage of asthma and
revision surgery. Clinically, CRSwNP with comorbid asthma (CRSwNP + AS) is associated with
even more severe sinonasal symptoms and worse quality of life, and it is more difficult to
treat both medically and surgically. Correspondingly, asthma in the presence of nasal
polyposis is harder to control, being more exacerbation prone, with increased airway
obstruction and more extensive eosinophilic inflammation.
Although a clear correlation apparently exists between sinonasal and lower airway
inflammation in patients with CRSwNP+AS, the definitive underlying mechanism(s) remains
poorly elucidated. The airways microbiota, i.e. the niche-specific communities of microbes
including bacteria, fungi, archaea and viruses that inhabit the respiratory tract, has been
proved to play a critical role in airway health and immune cells homeostasis -including
eosinophils regulation- through its constant interaction with the mucosal immune system.
Alterations in the composition and diversity of microbiome across the respiratory tract may
contribute to the observed inflammatory crosstalk in CRSwNP + AS, and perhaps influence
patients' response to treatment. Nasal and lower airway microbiota dysbiosis have been proved
to be implicated in the persistence of characteristic inflammatory endotypes in both CRSwNP
and asthma. It has been shown that bacterial dysbiosis is correlated with CRS status and that
specific microbiota taxonomic classifications are correlated with patient phenotypes,
including the presence of nasal polyps. A high proportion of patients with CRSwNP are
colonized with Staphylococcus (S.) aureus and IgE antibodies to S. aureus enterotoxins are
frequently found in diseased tissue specimens. Both S. aureus and Pseudomonas aeruginosa
bacteria can disrupt the epithelial barrier contributing to presumed physiologic mechanisms
for CRSwNP development. It has been previously demonstrated that S. aureus is able to drive
Th2 type inflammation in CRSwNPand that the expression of IL-5 and of IgE against S. aureus
superantigens (SE-IgE) within polyp tissue is associated with comorbid asthma and CRSwNP
recurrence. Furthermore, antimicrobial compounds including lysozyme, S100 proteins, and
β-defensins all are decreased in CRSwNP patients compared to matched controls. This reduction
in natural defenses could play a key role in shifting the balance towards dysbiosis.
Furthermore, in addition to bacterial microbiome which has been the focus of most recent
studies, the contribution of fungal microbiota to allergic airway diseases has been recently
emerged. An alternative proposed pathogenic mechanism for Th2-biased CRS is that T-cells are
allergically sensitized to fungi in the ambient environment, leading to allergic inflammation
characterized by a Th2-high state. Overall, a distinct microbiome role in CRSwNP and asthma
pathogenesis is actually recognized. However, the significance of interactions between the
lower/upper airways flora and the host local and systemic inflammatory response has not yet
been well defined neither such a knowledge is exploited for a more accurate patients
classification and selection of best possible therapeutic manipulation to change disease
progression.
Evidently, an optimal diagnostic approach for CRSwNP + AS would include use of very specific
biomarkers ensuring a detailed endotyping, whereas, there is a growing consensus that both
diseases should be treated to improve therapeutic outcome. However, the management of CRSwNP
- AS patients who remain uncontrolled despite medical and often surgical intervention poses a
great challenge to clinicians. Fortunately, there has been significant innovation and
expansion in the treatment armamentarium since the advent of biological therapies. Targeted
biologics (monoclonal antibodies against IL-4, IL-5, IL-13 and IgE) for treating asthma are
now being used for CRSwNP with encouraging results.
Recently, mepolizumab (Nucala; GlaxoSmithKline), an anti-IL5 humanized mab known to
efficiently down-regulate the eosinophilic inflammatory pathway and to exhibit clinical
benefit in patients with severe, eosinophilic asthma, has completed a phase 3 trial (SYNAPSE;
NCT03085797) including 413 subjects with CRSwNP. Early results showed treatment with
mepolizumab had a significant difference in median nasal polyp score compared to baseline
(-0.73; 95% CI: -1.11 to -0.34) and nasal obstruction visual analog score compared to
baseline (-3.14; 95% CI: -4.09 to -2.18; unpublished data). However, there are only limited
longitudinal studies evaluating this biologic's efficacy in CRSwNP+AS patients, while the
assessment of action mode and biomarkers predicting responsiveness, remain to be elucidated.
Despite the persuasive rationale for systemic targeting of shared pathways in CRSwNP+AS with
novel biologics, in clinical practice the nose and lungs are often treated as separate
entities and these therapeutics are considered rather challenging for the clinicians due to
their high cost and necessity for careful selection of patients and right treatment.
Furthermore therapeutic decision-making is still based on a rather trial-and-error approach,
resulting in treatment failures or relapses. There is clearly a need for a personalized
medicine approach that would allow for a more accurate prediction of the appropriate choice
of the drug at the initial assessment, and ideally would communicate chances of long-term
success to an individual patient.
The principal goal of this three arms RWE study (CRSwNP+AS, CRSwNP-AS, healthy controls)
study is to develop signatures of host-microbiome biomarkers of both diagnostic and
predicting value in order to provide a rational guideline for mepolizumab selection in
precision treatment of patients with CRSwNP+AS.
In this frame, mepolizumab therapeutic potential will be assessed in relation to the rather
heterogeneous presentations of Th2 inflammation and airways microbiome structure. The
protocol will take on the question of microbiome dysbiosis involvement in immune activation
and dysfunction contributing to CRSwNP + AS diversity and delving into the complex patterns
of host-microbiota molecular interactions in the upper and lower airways that may shape
patients' clinical predisposition to mepolizumab therapy.
To address the study objectives a longitudinal design is proposed combining clinical
assessments with high-throughput multi-omics analyses of patients' samples and advanced
bioinformatics for data integration. The starting point of the project will be the consent
recruitment of healthy controls, CRSwNP + AS and CRSwNP - AS participants. Collection of
biological samples from patients and clinical assessment/measurements of cell counts will
take place at baseline, i.e. the day of treatment initiation before mepolizumab
administration (T0) and at 3 (T3), 6 (T6) and 12 (T12) months after mepolizumab initiation.
Samples from healthy participants will be collected only at baseline (T0) and analyzed in
parallel with the corresponding samples from CRSwNP patients. DNA and RNA will be isolated
from induced sputum and nasal samples collected at T0 and T3 for subsequent 16S rRNA gene
amplicon sequencing, DNA shot-gun sequencing (applied only in selected number of patients and
healthy controls) and bulk RNA sequencing (RNAseq), to identify airways microbiota
modifications and differential gene expression, respectively. Peripheral blood mononuclear
cells (PBMC) from a representative number of patients and healthy controls will be also
analyzed at the same time points by single-cell RNA sequencing (scRNAseq). In parallel, the
dynamic pattern of cytokines/chemokines in serum/airways samples will be determined by xMAP
immunoassays at T0 and T3. Subsequently, multiple comparisons at the level of microbiome and
host parameters will be undertaken mainly through two paths. First, between patients and
healthy controls at T0 to assess differences of disease versus "normal" condition; Second,
pairwise in each patient at T0 and at time intervals, mostly at T3, after treatment
intervention, to capture potential alterations in response to mepolizumab. Next, a thorough
integrative analysis engaging clinical data and multi-omics data (from microbiome, bulk
RNAseq, scRNAseq as well as cytokinome/chemokinome analyses) will be undertaken to
investigate possible microbiome-host interactions. Finally, this integrative analysis will be
exploited to gain a global view of hub genes, inflammatory mediators and microbial taxa
involved in key interactions and to build biomarker signatures that might serve as indicators
of specific disease subtypes and/or predictors of mepolizumab treatment outcome.