An Open-label Study to Identify Molecular Markers of Steroid Resistance
An Open-label Study to Identify Molecular Markers of Steroid Resistance
Aim 1: To assess steroid sensitivity to mometasone furoate (MF) in cultured nasal polyp
 explant tissue in vitro.

 Aim 2: To assess steroid sensitivity in vivo in each subject by comparing symptom scores,
 nasal endoscopic findings before and following 4 weeks of treatment with mometasone furoate
 nasal spray (MFNS) and by comparing tissue immunohistology in NP biopsies pre- and
 post-treatment withA MFNS.

 Aim 3: To characterize the molecular signature of gene mRNA expression in "steroid-sensitive"
 and "steroid-resistant" NP using microarray on NP tissue pre- and post-MFNS treatment.

 Hypothesis 1: Genes that regulate apoptosis are dysregulated in nasal polyp (NP) inflammatory
 cells, epithelial cells and smooth muscle actin myofibroblasts leading to persistence of
 inflammatory cell infiltration and abnormal epithelial and myofibroblast cellular
 proliferation. These can be corrected by mometasone. Apoptosis-regulating genes that cannot
 be corrected by mometasone are upregulated in steroid-resistant NP.

 Elucidation of this dysregulation may prove insightful in understanding the mechanism of
 action of mometasone in NP and identifying potential molecular targets that will increase
 steroid sensitivity or, conversely, overcome steroid resistance.

 Hypothesis 2: There is a molecular signature of gene expression in NP that signifies steroid
 sensitive NP (SS-NP). This signature is altered in steroid resistant NP (SR-NP).

 Elucidation of differences in the molecular signature of SS-NP versus SR-NP before and after
 treatment with mometasone furoate (MFNS) will provide novel insight into treatment of NP with
 steroids.
Visit 1: Baseline evaluation and nasal endoscopy

 At this visit, each subject will sign an informed consent, the subject's eligibility for the
 study will be confirmed, a medical history will be obtained, the subject will undergo a
 physical examination and nasal endoscopy. A urine pregnancy test will be performed on all
 females of childbearing potential. NP size will be recorded using the NP scoring system (see
 below).

 Allergy skin testing will only be performed if it has not been done in the past 3 years.

 Visit 2: Pre-treatment nasal polyp (NP) biopsies

 At this visit, each subject will undergo the pre-treatment NP biopsies. The biopsies will be
 obtained by Dr. Eric Holbrook at Massachusetts Eye & Ear Infirmary, located adjacent to MGH.

 Visit 3: Initiation of treatment with MFNS

 At this visit, each subject will have a nasal speculum examination to confirm that there is
 no residual bleeding from the NP biopsies. Then, the subject will be given a 4 week supply of
 MFNS (Nasonex ®) and be instructed to take 2 sprays/nostril BID. Subjects will be given a NP
 symptom diary and instructed to record NP symptoms daily for 4 weeks.

 Each subject will be assigned a unique number (screening or baseline number) for
 identification purposes. Under no circumstances will a patient be assigned more than one
 allocation number. The Nasonex bottles will be dispensed from the MGH Research Pharmacy.

 Visit 4: On-treatment NP biopsy

 At this visit, each subject will undergo the on-treatment NP biopsies. The biopsies will be
 obtained by Dr. Eric Holbrook at Massachusetts Eye & Ear Infirmary, located adjacent to MGH.
 The subject will be asked to withhold Nasonex treatment for 48 hours following this biopsy.

 Visit 5: Post-biopsy Exam At this visit, each subject will have a nasal speculum examination
 to confirm that there is no residual bleeding from the NP biopsies. If bleeding has ceased,
 the subject will resume Nasonex treatment.

 Visit 5 6: Final visit and nasal endoscopy

 At this visit, each subject will return their used and unused bottles of Nasonex and submit
 their NP symptom diary and undergo a nasal endoscopy. NP size will be recorded using the NP
 scoring system (see below). Subjects will also complete a global assessment of efficacy of
 MFNS for their NP symptoms.

 EXPERIMENTAL PROCEDURES:

 Pre-biopsies explant cultures: we will obtain NP biopsies for in vitro explant culture before
 steroid treatment from each patient. We will culture the explants in MF at 0, 10-9, 10-8,
 10-7, 10-6, 10-5M and analyze inflammatory cell infiltrate and steroid sensitivity based on
 induction of apoptosis (activated caspase-3 expression) in inflammatory cell subsets,
 alpha-smooth muscle cell actin+ myofibroblasts and epithelial cells. Each condition will be
 performed in duplicate so that one sample can be processed for IHC and one for microarray.

 Explant cultures: NP explants will be cultured in minimal essential medium for 24 hours at
 37oC using flat-bottom tissue culture plates. Following 24 hours in culture, 1 explant per
 condition will be processed for IHC and 1 for microarray. We will also save culture
 supernatant from each explant condition for confirmation of important differences in protein
 production, based on data from the IHC and microarrays.

 Immunohistochemistry (IHC) and confocal laser immunofluorescence microscopy (CLM): Frozen NP
 samples from SS-NP and SR-NP patients before and after MFNS treatment will be analyzed by
 immunofluorescence confocal laser microscopy (IF-CLM) for T lymphocyte (CD3 and CD4), MBP+
 eosinophils, alpha-smooth muscle actin (marker of activated myofibroblasts, monoclonal, R & D
 Systems, Inc.) and cleaved caspase-3 (polyclonal, CST, Beverly, MA). The number of
 inflammatory cells of each type and cleaved caspase-3 positive and negative cells of each
 type will be counted by two observers blinded to treatment and order of collection. A similar
 quantification of epithelial staining for PCNA (a marker of epithelial cell proliferation)
 and cleaved caspase-3 will be performed using semiquantitative grading of extent and
 intensity of staining as in our previous studies. The intensity and distribution of
 immunostaining will be quantified on a semi-quantitative grading scale of: 0 = absent
 staining, 1 = mild diffuse or moderate patchy staining; 2 = moderate diffuse staining; 3 =
 intense diffuse staining in the epithelium, stroma and glands by 2 independent observers.

 Histologic steroid-sensitive and steroid-resistant phenotype designation: Designation of NP
 subjects as histologically "steroid-sensitive" or "steroid-resistant" will be based on the
 following measures pre- and 1-week post-treatment with MFNS:

 1. extent of reduction in NP eosinophils improvement in NP inflammation,

 2. extent of induction of apoptosis in eosinophils, T lymphocytes and epithelial cells,

 3. extent of suppression of inflammatory gene expression based on microarray analysis.

 Subjects will be ranked in order from most sensitive to least sensitive to treatment. Linear
 regression will be used to examine correlations between these independent continuous
 variables.

 Microarray analysis: microarray analysis will be used to assess global gene mRNA expression
 and also capture information on pro- and anti-apoptotic gene expression in "steroid
 sensitive" (SS-NP) and "steroid resistant" SR-NP biopsies obtained before and 1 week
 following treatment with intranasal mometasone furoate (MFNS). RNA isolation for microarray
 is done using the Arcturus picopure RNA isolation system. Plastic LCM collecting caps are
 incubated at 42oC for 30 minutes in 25 microliter of the kit's GITC-containing extraction
 buffer, centrifuged briefly to collect the extracted solution, then frozen at -80oC. Samples
 are thawed and pooled prior to purification on picopure RNA columns, and genomic DNA is
 removed via RNAse-free DNAse (Qiagen, Hilden, GmbH) digestion on the columns. Total cellular
 RNAs from each column are eluted in 11 microliter of elution buffer and used for preparation
 of the RNA-Seq library, or converted into cDNA-for the QRTPCR assay, using a 20 microliter
 total reaction volume via the standard superscript III (Invitrogen, Carlsbad, CA) protocol.
 We also have microarray data from 8 healthy control middle turbinates obtained in a previous
 study that will be used for comparison of gene expression to nasal polyps.

 RNA will be purified from one NP biopsy per patient using the ArrayPure™ Nano-scale RNA
 Purification Kit, (EPICENTRE Biotechnologies, Madison, WI) which includes ScriptGuard™ RNase
 Inhibitor to maintain the integrity of purified RNA. Purified RNA quality will be checked
 using RNA Nano Analysis (Agilent Bioanalyzer, Quantum Analytics, Inc., Foster City, CA). The
 lower limit sensitivity is 200 pg/ml, allowing us to verify the quality of RNA extracted from
 about 1250 cells.

 QRTPCR: Genes that are differentially expressed by RNA expression profiling will be further
 investigated by quantitative real-time RT-PCR from a separate small sample of purified RNA
 from each tissue compartment. RT-PCR will be performed using the Multiplex MX3000P
 quantitative RT-PCR system (Stratagene, La Jolla, CA). Baseline gene expression will be
 quantified by QRTPCR relative to GAPDH using the delta-delta Ct (2-∆∆) method. Forward and
 reverse primers will be selected based on primerbank software
 (http://pga.mgh.harvard.edu/primerbank/). To confirm gene-expression within tissues we will
 use immunohistochemistry as in previous studies.
Inclusion Criteria:

 - The subject must fulfill all of the following conditions or characteristics to be
 considered for enrollment:

 1. Male or female between ages 21 - 70 years residing in the Boston area

 2. History of chronic rhinosinusitis (symptoms for at least 3 months). Subject must have
 two or more of the following:

 -  Facial pain/pressure or headache

 -  Nasal congestion

 -  Anterior or posterior nasal drainage

 -  Hyposmia/anosmia

 3. Abnormal CT scan in at least 2 sinuses areas within 3 months

 4. Evidence of bilateral polyps or polypoid mucosa (on nasal endoscopy) with minimum
 polyp/polypoid score of 4 (see scoring system below).

 Exclusion Criteria:

 -  4. History of suggestive of immunodeficiency (i.e. those who have had > one
 pneumonia in the past 12 months or those with known immune deficiency).

 5. History of cystic fibrosis, Kartagener's syndrome, immotile cilia syndrome,
 hypogammaglobulinemia or bleeding disorder

 6. URI within six weeks prior to enrollment

 7. Intranasal cocaine use

 8. Pregnancy (if applicable

 9. History of fainting

 MEDICATION EXCLUSIONS prior to NP biopsies:

 10. Use of prescription blood thinners

 11. Use of systemic glucocorticoids for two weeks prior to enrollment

 12. Use of intranasal corticosteroids and anticholinergics for three days prior to
 enrollment

 13. Use of an antibiotic for three days prior to enrollment

 14. Use of antihistamines for one week prior to enrollment