Serum Markers of the Microbiome

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2 Serum Markers of the Microbiome Mark S. Riddle, MD, DrPH Professor and Chair Department of Preventive Medicine and Biostatistics Uniformed Services University of the Health Sciences Bethesda, MD Conflict of Interest Disclosure: Presenter is Principal investigator in US Government collaborations with Prometheus Laboratories, Janssen Pharmaceuticals, Mayo Clinic, Mt. Sinai and Cedars Sinai with the goals of using serum biomarkers in understanding immunemediated disease pathogenesis and diagnostic development.

3 Easy, peasy

4 Learning Objectives Understand the advantages and disadvantages of serum microbiome biomarkers Describe the current clinical laboratory assays available for use in GI disease diagnosis IBD IBS Appreciate additional research efforts and promise

5 Why Not Just Test the Fecal Microbiome? Describing the structural composition of host microbiome can be done and is relatively inexpensive (Tong, 2014) High-throughput sequencing and automated bioinformatics is bringing functional genomic characterization in reach (Kumar, 2016) Innovations in metabolomics are advancing ( Characterization of intestinal IgA s role in disease pathology is bridging a gap (Shapiro, 2015)

6 Because It s Complicated Fecal microbiome compartment is probably not as good as mucosal microbiome (Zhang, 2017) Exceptional diversity Structure/composition has not yielded predicted value in diagnosis or treatment decisions Does not effectively incorporate the interaction between the human host and the micriobiome in the disease process

7 What s Actually in a Drop of Blood? ~45% erythrocytes, leukocytes, thrombocytes 92% water 6-8% protein (1/3 IgX; 1% regulatory) 1% metabolites

8 Serum Markers of the Microbiome and IBD Following the discovery of anti-bodies against a food antigen in Celiac disease, parallel search commenced Led to the discovery of immune responses directed against a specific oligomannose epitope present on the cell wall of the yeast Saccharomyces cerevisiae strongly in CD patients (Main, 1988) Combined with panca associations seen with UC (Saxon, 1990), hope for a serum diagnostic for IBD launched. Growing list of research/commercially available markers: OmpC, Cbir, I-2, A4- Fla2,Fla-X, ACCA, ALCA, AMCA

9 Clinical Potential of Serum Biomarkers in IBD IBD v. healthy IBD v. other GI CD v. UC v. IBD-unclassif. Diagnosis Prognosis Aggressive v. non-aggressive Anti-TNF responsive v. step down modality Correlation with disease activity Signal support for recurrence Response to treatment

10 Diagnosis Most studies conclude that the specificity of serological markers for IBD is high but sensitivity is low, making them less useful as diagnostic tests Specificity and sensitivity plots for pooled ASCA IgG or IgA+, panca tests for the diagnosis of CD Sensitivity (95% Cl) 0.66 ( ) 0.56 ( ) 0.64 ( ) 0.48 ( ) 0.48 ( ) 0.54 ( ) 0.43 ( ) 0.54 ( ) Saibeni, 2003 Peeters, 2001 Llinskens, 2002 Lawrence, 2004 Kim BG, 2002 Montanelli, 2005 Lecis, 2002 Elitsur, 2005 Sensitivity (95% Cl) 0.98 ( ) 0.92 ( ) 0.94 ( ) 0.94 ( ) 0.87 ( ) 0.92 ( ) 0.91 ( ) 0.97 ( ) 55% Pooled Sensitivity = 0.55 ( ) X 2 = 10.95; df = 7 (P = ) Sensitivity Dubinksy & Bruan. 2016; Reese GE et al. Am J Gastroenterol Specificity Pooled Sensitivity = 0.93 ( ) X 2 = 7.81; df = 7 (P = ) 93%

11 Special Circumstances Diagnosis IBD-unclassified occurs in about 10-15% Accurate diagnosis has important treatment implications Antibodies may play a role in clarifying the diagnosis IBD-U / UC distinction in patients who who have failed medical therapy and may potentially require ileal pouch procedure True IBD-U do not do as well with procedure Most true IBD-U are negative for both ASCA and panca UC vs. Crohn s disease with colonic involvement (panca+) Distinction does not necessarily inform treatment impact / disease outcome, adding CBir1 may help to differentiate (CBir1+ in true CD) (Prideaux L. 2012; Dubinsky & Braun

12 Frequency of Disease Behavior, % Prognosis Both the quantitative and qualitative expression of anti-microbial antibodies serve as a risk marker for IBD behavior and phenotype Nonpenetrating, nonstricturing Internal penetrating Stricturing Surgery Dubinsky n=199 n=262 n=194 n=57 Antibody sum (AS): number of positive antibodies (0,1,2 or 3) per individual

13 Hints of the future Journal of Gastroenterology and Hepatology. 32 (2017) Prognosis Those positive (n = 28) for all four Abs (anti-cbir1, anti-ompc, anti-a4-fla2, and anti-fla-x) at baseline were more likely to experience recurrence at 18 months than those negative (n = 32) for all four (82% vs 18%, P = 0.034) Among 931 newly diagnosed children with CD followed for 3 years, 9% had complications in which age, race, ASCA and CBir1 had a 94% negative predicted value

14 Pushing Diagnostics to the Limits! Prevention/ Interception Diagnosis Prognosis Response to treatment

15 Accumulating Injury Prevention/ Interception Intervention Time Torres, Riddle, Colombel. Evidence for Life Before Inflammatory Bowel Disease. Clin Gastroenterol Hepatol Jun;14(6):

16 Summary of studies so far conducted in preclinical phase of IBD Prevention/ Interception

17 PRoteomic Evaluation & Discovery in an IBD Cohort of Tri-Service Subjects (PREDICTS) Prevention/ Interception Study design: case-control 1000 UC cases 1000 CD cases 500 healthy controls (matched) Outcomes using ICD-9/CPT codes 2 encounters w/ same IBD subtype Colonoscopy and/or sigmoidoscopy 2 total; 1 prior to a IBD-related medical encounter Clinical, military and demographic data Four (4) serum samples from the repository ~ 4 years

18 PREDICTS Study 1: Association between pre-disease serology and complicated & uncomplicated CD at diagnosis Prevention/ Interception Disease behavior at diagnosis was defined by the Montreal classification based on diagnostic codes (ICD-9-CM & CPT codes) Uncomplicated CD: no evidence of stricturing or penetrating disease behaviors Complicated CD: intestinal penetrating (IP), stricturing (S), or surgical history of intestinal resection. CD associated Serologic antibodies (Prometheus Labs) ASCA IgA: ASCA IgG: Anti-A4-Fla2: Anti-FlaX: Anti-CBir1: Anti-OmpC: Anti-Saccharomyces Cerevisiae Antibody IgA Anti-Saccharomyces Cerevisiae Antibody IgG Anti-Clostridium cluster XIVa flagellin 2 antibody Anti-Clostridium cluster XIVa flagellin X antibody Anti-Bacterial flagellin CBir1 antibody Anti-Escherichia coli Outer Membrane Protein C Analytic Strategy Antibody sum (AS): number of positive antibodies over time and between complicated and uncomplicated CD groups Choung RS et al. Aliment Pharmacol Ther

19 PREDICTS Study 1: Association between pre-disease serology and complicated & uncomplicated CD at diagnosis Prevention/ Interception Results 100 patients 24 complicated phenotype 76 uncomplicated phenotype 15 (63%) had isolated stricturing disease 4 (17%) had internal penetrating (IP) 4 had both S and IP complications 5 patients (21%) underwent surgery. Choung RS et al. Aliment Pharmacol Ther

20 Proportion (%) Serologic Antibodies Are Present in Serum at Diagnosis Prevention/ Interception 59% 51% 46% 48% 37% 14% Choung RS et al. Aliment Pharmacol Ther

21 Proportion (%) Serologic Antibodies Are Present in Serum Before Diagnosis Prevention/ Interception 46% * 59% * 51% * * 46% 48% * 34% 26% 30% 21% 37% 11% 14% +, Earliest Available serum = around the clinical diagnosis, - 6 (-8.1, -5.6) years *, p< 0.05

22 Patients with Positive Test (%) Accumulations of biomarkers preceding diagnosis and median survival time to first complication is less in those with higher Antibody Sum scores (AS)* Prevention/ Interception ASCA-IgA titers increase as the time of CD diagnosis approached Anti-FlaX titers increase as the time of CD diagnosis approached Proportion of positivity of each antibody prior to diagnosis is higher in complicated CD compared to noncomplicated CD at diagnosis Year Choung RS et al. Aliment Pharmacol Ther

23 What can we learn from our RA colleagues? OP0182 Prevention of Rheumatoid Arthritis by B Cell Directed Therapy in The Earliest Phase of The Disease: The Prairi Study Design. Multi-center, RDBPCT, among 82 subjects with arthralgia but no clinically manifest arthritis and no prior disease-modifying therapies Eligibility. positive for both rheumatoid factor & anti-citrullinated protein antibodies AND they had CRP levels 3 mg/l and/or subclinical synovitis US/MRI Treatment groups. Single iv infusion of 1000 mg rituximab (anti-cd20) or PBO Results. 37% developrf clinically manifest arthritis (27% efficacy at 27 months) 53% reduction of clinically manifest arthritis at 18 months Prevention/ Delay in onset, effect attenuated over time Interception Gerlag D et al. Annals of the Rheumatic Diseases 2016;75:

24 Univ. of Alabama Microbiota Microarray 8 Bacteroidetes 24 Firmicutes 8 Proteobacteria 7 metabolic functions 13 flagellin/motility 6 transcription/ translation 12 others are cell-surface Firmicutes flagellin are known to be immunodominant in patients with CD Christmann et al Phylum Name Class Name Protein ID Function Primary species Bacteroidetes Bacteroidia cbir23 Elongation factor 1A Transc/transl Alistepes shahii keto Transketolase Metabolism Bacteroides fragilis p3 HSP90 Other/unknown Bacteroides fragilis pora Prevotella oralis Firmicutes Clostridia 14-2 Flagellin from 14-2 isolate Motility Roseburia intestinalis flagellin 28-4 Flagellin Motility Lachnospiraceae Flagellin Motility Lachnospiraceae Cbir1 Flagellin Motility Butyrivib. fibrisolvens Cbir11 Flagellin Motility Rose. inulinivorans Cbir66 Flagellin Motility Roseburia intestinalis Fla2 Flagellin 2 from A4 isolate Motility Roseburia intestinalis Fla3 fla3 Motility Rose. inulinivorans Flax Flagellin Motility Rose. inulinivorans Mdr254 Flagellin Motility Flavonifractor plautii Firmicutes Clostridia Cbir63 Ig-like surface protein Other/unknown Roseburia intestinalis Rib12 Homoserine dihydrogen. Metabolism Flavonifractor plautii Rib4 Relaxase Transc/transl Clostr. asparagiforme Rib8 Glycosyltransferase Metabolism Lachnospirac Proteobacteria Delta/epsil Cbir5 Methyl-accepting chemotax Motility Helicobacter cinaedi Cbir56 Methyl-accepting chemotax Motility Helicob. Canadensis Gamma Ompc OmpC from UNC E coli Other/unknown Escherichia coli Firmicutes Clostridia Rib1 RecN Transc/transl Clostridium citroniae RIB10 Nucleotidyltransferase Transc/transl Roseburia intestinalis Proteobacteria Beta RIB20 ABC transporter Other/unknown Verminephr. eiseniae Firm. & Bacteroid. Rib2 SAM domain protein Transc/transl Blautia hansenii

25 Average Fluorescence Intensity Trends of Seroreactivity to Multiple Phyla Clusters in the Preclinical Stage of Crohn s Disease * Earliest available sample 2 years before diagnosis Just before diagnosis * * * 0 Firmicutes Flagellin Firmicute Bacteroidetes Proteobacteria Universal * P<0.0001; mean ± SEMs Choung et al. DDW 2016.

26 Average Fluorescence Intensity Changes of Adaptive Immune Response to Flagellin Fimicutes Antigens in CD Earliest available time 2 years prior to diagnosis Just before diagnosis *, a median of 6.0 (IQR, ) years before diagnosis

27 Autoimmunity Links Vinculin to the Pathophysiology of Chronic Functional Bowel Changes Following Campylobacter jejuni Infection in a Rat Model CdtB - cytolethal distending toxin, B subunit CdtB ICCs Pmentel et al. Digestive Disease Sciences Vinculin - human membrane cystoskeletal protein

28 Development and Validation of a Biomarker for Diarrhea- Predominant Irritable Bowel Syndrome in Human Subjects Objective: Compare plasma Ab to vinculin and CdtB in cross-section of IBS-D, Celiac Dz & IBD relative to HC Case selection (clincally comfired except IBS-D by Rome III): IBS-D (TARGET 3 Study) IBD/CeD same health system; no matching Control selection: No prior Hx of GI disease or active GI symptoms; no matching Pimentel M et al Case/control Characteristics: Healthy controls 43 D-IBS 2375 CD b 73 UC c 69 IBD (UC+CD) 142 Celiac disease 121 Number of Subjects IBS subjects on average 3.9 years older than non-ibs groups (P<0.001) More females in HC, IBS and CeD compared to IBD group (IBS / non- IBS similar)

29 S1 Table. Sensitivity and specificity for the diagnosis of D- IBS vs. healthy controls at different OD cutoffs OD Specificity % Sensitivity % CdtB Vinculin Interpretation CdtB/Vinculin Abs able to differentiate IBS-D from IBD and HCs, with reasonable degree of accuracy Many people, still with IBS, will test negative The utility of results in terms of provider / patient decision support needs consideration

30 Immunological Biomarkers of Postinfectious Irritable Bowel Syndrome DoD Serum Repository: pilot study Groups: 1 ❶ 1 & ❸ 3 PI-IBS ❷ 2 healthy (GI infx) ❹ 4 idiopathic-ibs Serum at time of initial ICD-9-CM diagnosis for IBS onset (or matched time of censure for healthy controls) Biomarkers: cytokines, microbiome antigens (C. Elson) & vinculin Pike BL et al. Immunological Biomarkers in Postinfectious Irritable Bowel Syndrome. J Travel Med. 2015

31 Antibodies Directed against Antigens of Gut Commensals Differed between PI-IBS Cases and Those IBS Cases Lacking a Prior IGE Episode (Univ. of Alabama microarray) idiopathic IBS [Mean (SD)] n = 30 PI-IBS (all cause) [Mean (SD)] n = 60 FC p-value MDR254 (IgG) (18.93) (7.35) CBir8 (IgA) 3.07 (10.80) 9.33 (28.52) EF20 (IgG) (8.19) (6.05) rib16 (IgG) (8.89) (3.62) P3 (IgG) (5.43) (2.32) rib19 (IgG) (2.79) (1.73) Geometric Mean and Std. Deviation by Group; Fold-Changes (FC) <1 are presented as x = -1/FC. False Discovery Rate p-value

32 Anti-vinculin antibody higher in Campylobacter specific PI-IBS vs other PI-IBS Post- Other IBS [Mean (SD)] n = 20 Post-Campy IBS [Mean (SD)] n = 10 FC p-value Vinculin (IgG) (5.51) << (3.27)

33 Take Home Points No antibody, individually or in combination, has sufficient sensitivity to replace conventional tools (endoscopy, histology, radiology) for the diagnosis of IBD May be useful as an adjunct lab, especially in cases of diagnostic uncertainty Antibodies towards CdtB and vinculin can distinguish between IBS-D (and IBS-M) and IBD and healthy controls. First diagnostic available to be able to rule-in IBS Validation in non-us populations and primary care settings as well as costeffectiveness analyses are needed (and underway) Supports underlying concept of PI etiology and offers hope for targeted Rx Novel platforms are continuing to be developed and applied in microbiomemediated GI diseases with promise

34 Acknowledgements PRoteomic Evaluation and Discovery in an IBD Cohort of Tri-Service Subjects (PREDICTS) Study team NAVAL MEDICAL RESEARCH CENTER Chad K. Porter Ramiro Gutierrez Renee Laird Vicky Chapman Mark S. Riddle PROMETHEUS Fred Princen Tom Stockfish MAYO CLINIC Rok Seon Choung Joseph A. Murray UNIVERSITY OF ALABAMA Chuck Elson Peter Mannon A Joint Government, Academic, & Industry Cooperative Research & Development Agreement JANSSEN PHARMACEUTICALS Francisco Leon Shannon Telesco Carrie Brodmerkel Paul Dunford Scott Plevy Richard Strauss ICHAN SCHOOL OF MEDICINE, MT. SINAI Joana Torres Kristen Swithers Anabella Castillo Carmen Argmann Brian Kidd Jean Frédéric Colombel We thank the US military service personnel and the Armed Forces Health Surveillance Center (Silver Spring, MD) for their significant contributions in providing data / serum specimens