Study Design/Methodology:
Several gastroenterologists at our center have agreed to assist with biopsy collection at our
hospitals: UCSD: Singh, Boland, Patel, VA: Rivera-Nieves). Dr. Brian Behm who cares for most
IBD patients at the University of Virginia has also agreed to assist with biopsy/PBMC
collection.
During two visits (T0, T24-32) we will collect biopsies, endoscopies, histology, fecal and
blood samples (Table 1), prospectively from a total of 10 UC patients scheduled for
colonoscopy (before therapy with ozanimod and at 24-32 weeks post-treatment). A sample size
of 10 patients per condition should be sufficient to achieve significant differences using
CITRUS, based on required sample size determinations (see below).
The effects of the drug on cellular subsets will be assessed by comparing the two-time points
(24-32 weeks post therapy) with its pretreatment control (T0). We will additionally compare
the mass cytometry results to clinical parameters that are collected at our IBD center during
every visit (Table 1). The clinical samples and parameters from patients recruited at UVA
will be sent de-identified through a secure mailbox managed by the UVA.
Sample collection, storage, testing and analyses
After IV placement for conscious sedation, 10 mL of blood will be drawn on
EDTA-containing tubes and sent to the lab on ice for further processing. Specimen
transport is provided by staff from UCSD Center for Translational Research (CTRI). Upon
arrival to the wet lab blood is spun, plasma separated and leukocytes isolated by
gradient centrifugation. Cryopreservation media is added to the leukocyte fraction.
Plasma and leukocytes are stored at -80 C.
Standard biopsies will also be obtained from ileum (control effect of drug on uninvolved
segment), as well as in involved and uninvolved colonic segments and suspended in
complete media for cell isolation. Specimen transport is provided by staff from UCSD
Center for Translational Research. Upon arrival to the wet lab, cryopreservation media
is added to the biopsies and stored at -80 C.
We have found significant batch to batch variation during pilot studies , even controlled
using veri-cells. For that reason all cell/tissue processing and acquisition on the cytometer
will all be performed together, upon completion of collection.
No study specific visits will be conducted for IBD patients. All visits and sample
acquisitions will be at standard of care visits. No specific treatment related research
interventions will be performed on IBD patients for this study. We thus categorize the the
study as non-interventional.
Data Analysis:
We have been analyzing CyTOF data now for several years as illustrated in the two published
manuscripts by Tyler et al.. In addition we have worked with Amir El-Ad who runs the AstoLabe
platform for high dimensional cytometery analyses. To examine differences in population
abundance and specific cell marker expression, we have identified CITRUS as one of many
appropriate algorithms to perform our analysis of IBD biopsies. CITRUS identifies clusters of
phenotypically similar cells in an unsupervised manner, characterizes the behavior of
identified clusters using biologically interpretable metrics, and leverages regularized
supervised learning algorithms to identify subset clusters whose behavior is predictive of
the samples endpoint. CITRUS strongly encourages the use of a minimum of 8 samples per group.
Thus, for our current project, we will obtain a minimum of 10 samples for UC before and after
ozanimod therapy. As per estimates, based on our preliminary data, this sample size will be
sufficient to accurately identify even rare populations and to determine changes in abundance
between cell subsets. It is sensitive enough to assess differential expression of any marker
in a three-way analysis. Bioinformatics comparisons between the cell subsets present in
ileum, colon and PBMC at times 0 and 24-32 weeks for each patient will be performed for the
principal endpoint of assessing the effect of the drug on relevant cell subsets.
Most recently, all of our data is stored in the Omiq platform, which provides multiple
algorithms for data analyses.
For the univariable association of continuous independent variables with response, either an
unpaired t-test or a Wilcoxon two-sample test will be used, while for categorical variables
Fisher's exact test will used. All comparisons will be two-sided and performed at the 0.05
level of significance. As these analyses are exploratory, no adjustment will be made for
multiple comparisons.
Data Management Plan: All data will be stored on password-protected computers at UCSD. All
hard files and raw data will be stored in locked cabinets on UCSD campus within the lab of
Jesus Rivera-Nieves. Patients will be given study identifiers as part of the IBD Biobank and
no personal identifiers will be used. The Data Management plan will follow that of the UCSD
IBD Biobank which has been extensively reviewed by the IRB and Committee for Protection of
Human Subjects at UCSD.
At the UVA, data will be collected by the local study coordinator and de-identified. All data
will be sent electronically using a secure mailbox. This has been approved by the UVA IRB.