1. TOLLIP, MUC5B and the Response to N-acetylcysteine among Individuals with Idiopathic Pulmonary Fibrosis
20 minutes
Justin Oldham, MD MS
Pulmonary and Critical Care Medicine
University of Chicago
Pulmonary Fibrosis Foundation Summit 2015

2. Disclosures
No relevant commercial interests

3. Background rs5743890 (TOLLIP) rs35705950 (MUC5B)AG GG GA AA
Contemporaneously several studies were published identifying gene polymorphisms associated with IPF susceptibilty as well as survival. These genes of course are TOLLIP and MUC5B
Noth et al. Lancet RM 2013
Fingerlin et al. Nature Genetics 2013
Peljto et al. JAMA 2013

4. TOLLIP encodes toll-like interacting protein (tollip)
Inhibitory adaptor protein acting downstream of toll-like receptors (TLR)
TLR-2 and TLR-4 are active in the lung
After interaction with TLR-2 and TLR-4 ligands, tollip protein increases anti-inflammatory IL-10 production and suppresses pro-inflammatory TNF-α and IL-6 production
TLR-4 is an oxidant dependent pathway
Can be modulated by the presence of anti-oxidants, including N-acetylcysteine (NAC)
Shah et al.. J Immunol 2012
Saito et al. Cell and tissue research 2005
Janardhan et al. Histology and histopathology 2006

5. MUC5B encodes a mucin-producing protein (Mucin-5b) Mucin-5b
Contributes to airway mucus production
Influences lower airway microbiome
Is necessary for airway defense
Molyneaux et al. AJRCCM 2014.
Roy et al. Nature 2014.

6. Research Question
Because TOLLIP and MUC5B play critical roles in lung host defense, do variants within these genes influence the effect of immunsuppressive and anti-oxidant therapy?

7. Randomized controlled trial comparing 3 treatment arms
Background
Randomized controlled trial comparing 3 treatment arms
Prednisone/Azathioprine/N-acetylcysteine combination (PAN)
N-acetylcysteine (NAC) monotherapy
Placebo
Trial stopped and PAN arm terminated after interim analysis showed an increased risk of death and hospitalization
Trial then resumed with NAC and placebo arms
So we are all of course familiar with the PANTHER clinical trial which compared 3 treatment arms – placebo, NAC and combination prednisone, aza and NAC. The combination arm was stopped early due to increased risk of death and hospitalization in this group and the placebo and NAC arms were continued until completion. Analysis showed that NAC was no different than placebo when comparing FVC decline over time. This figure from the paper showed something interesting however. Prior to the clinical alert that was issued for the combination arm, NAC was performing quite well compared to placebo, whereas after the clinical alert the opposite was true. This went for several other metrics as well.
Raghu et al. NEJM 2012
Martinez et al. NEJM 2014

8. Martinez et al. NEJM 2014

9. Our Goal
Determine whether single nucleotide polymorphisms (SNPs) within TOLLIP and MUC5B modify the effect of PANTHER interventions

10. Methods 5 SNPs genotyped in patients consenting to genetic analysis
rs (TOLLIP) (intronic)
rs (TOLLIP) (intronic)
rs (TOLLIP) (exonic)
rs (TOLLIP) (promoter)
rs (MUC5B) (promoter)
Drug-gene interaction tested with multivariable Cox regression model with interaction term
pinteraction<0.01 considered significant based on Bonferroni correction
Composite endpoint used
death, hospitalization, transplant or ≥10% decline in forced vital capacity (FVC)

11. Result 341 patients Enrolled in PANTHER
26 patients identifying as Hispanic ethnicity or non-white race excluded
315 patients eligible for analysis
154 patients enrolled
in genetics sub-study
161 patients did not enroll
in genetics sub-study
Figure S1. PANTHER Genetic Analysis
54 assigned to Placebo arm
60 assigned to NAC arm
40 assigned to PAN arm

12. Results – Baseline Characteristics and Outcomes
Genotyped (n=154)
p-value
Placebo Arm (n=54)
NAC Arm (n=60)
PAN Arm (n=40)
Age, mean (±SD)
66.1 (7.9)
67.9 (8.7)
69.7 (6.8)
0.11
Male, n (%)
39 (72.2)
47 (78.3)
31 (77.5)
0.72
Ever Smoker, n (%)
41 (75.9)
44 (73.3)
28 (70)
0.81
FVC, % predicted (±SD)
73.2 (14.7)
73 (15.7)
71.2 (15.2)
DLCO, % predicted (±SD)
46.4 (11.7)
43.9 (10.9)
43 (10.6)
0.3
Death, n (%)
2 (3.7)
1 (1.7)
4 (10)
0.14
FVC Decline ≥ 10%, n (%)
12 (22.2)
11 (18.3)
6 (15)
0.67
Hospitalization, n (%)
8 (14.8)
8 (13.3)
13 (32.5)
0.04
Transplant, n (%)
1 (1.9)
3 (5)
1 (2.5)
0.74
Composite Endpoint**, n (%)
17 (31.5)
19 (31.7)
19 (47.5)
0.18

13. Results - Drug-Gene Interaction
Multivariable Cox Interaction Model Estimates*
Variable
Coefficient (95% CI)
p-value
rs (TOLLIP)
0.84 ( )
0.16
NAC Therapy
0.17 ( )
0.66
PAN Therapy
1.36 ( )
0.001
rs *NAC interaction
-1.17 ( )
0.19
rs *PAN interaction
-1.08 ( )
rs (TOLLIP)
0.05 ( )
0.93
0.45 ( )
0.30
0.74 ( )
0.17
rs *NAC interaction
-1.42 ( )
0.06
rs *PAN interaction
0.58 ( )
0.42
rs (TOLLIP)
0.39 ( )
0.28
1.34 ( )
0.03
0.89 ( )
0.20
rs *NAC interaction
-1.47 ( )
rs *PAN interaction
0.19 ( )
0.71
rs (MUC5B)
0.61 ( )
0.29
0.91 ( )
0.15
0.98 ( )
rs *NAC interaction
-1.40 ( )
0.07
rs *PAN interaction
0.13 ( )
0.87
Significant interaction detected between NAC and rs (TOLLIP)
Suggested interaction observed between NAC and rs (TOLLIP) and rs (MUC5B)
No interaction observed between these SNPs and PAN combination therapy
One of the TOLLIP SNPs did not meet the proportional hazard assumption needed to conduct Cox modeling so only 4 SNPs were tested here. And what we found was significant interaction between NAC therapy and the TOLLIP exonic SNP. We also see that there was a suggestion of interaction between the 894 intronic TOLLIP SNP as well as the MUC5B promoter SNP. We did not observe any interaction between these SNPs and PAN therapy.

14. Results – rs (TOLLIP) genotype-stratified composite endpoint-free survival between NAC and placebo arms
NAC
Placebo
logrank p=0.01
HR 3.23; 95% CI ; p=0.10
logrank p=0.78
HR 0.76; 95% CI ; p=0.62
logrank p=0.06
HR 0.14 ; 95% CI ; p=0.03
Placebo
So based on this interaction analysis we constructed genotype stratified Cox models and KMs for the exonic TOLLIP SNP and found that those with a CC genotype had a trend towards worse outcomes compared to those receiving placebo with this genotype. We observed no difference between groups with a CT genotype and found that those in the NAC arm with a TT genotype did significantly better than their counterparts in the Placebo arm with a HR of 0.14 and p-value of 0.03.
CC: Unadjusted logrank p=0.01
CT: p=0.82
TT: p=0.06

15. Results – Sensitivity Analysis of genotype-stratified endpoint risk
Table 2. Sensitivity Analysis of NAC-associated endpoint risk after rs genotype stratification*
SNP Genotype (Gene)
Primary Composite Endpoint
Hospitalization-Free Survival
Progression-Free Survival
HR (95% CI)
p-value
rs (TOLLIP) CC
3.22 ( )
0.1
3.74 ( )
0.27
1.27 ( )
0.79
rs (TOLLIP) CT
0.76 ( )
0.62
0.45 ( )
0.32
0.63 ( )
0.48
rs (TOLLIP) TT
0.14 ( )
0.03 **
0.09 ( )
* Models adjusted for age, gender, FVC (% predicted) and DLCO (% predicted)
** No events observed in this group so HR could not be estimated
Primary Composite Endpoint = death, transplant, hospitalization or 10% FVC decline
Hospitalization-Free Survival = death, transplant or hospitalization
Progression-Free Survival = death, transplant or 10% FVC decline
Harm associated with NAC therapy in those with rs (TOLLIP) CC genotype driven primarily by hospitalizations
Benefit associated with NAC therapy in those with rs (TOLLIP) TT genotype consistent across all endpoints
You’ll notice that the heterozygotes for the 94 TOLLIP SNP and MUC5B promoter SNP are lumped together. This was because of the small number of individuals who were homozgyous minor for these SNPs so a dominant model was used for them whereas an additive model was used for the 920 TOLLIP SNP.

16. Conclusion
NAC may be an efficacious therapy for those with rs (TOLLIP) TT genotype (~25% of IPF population)
NAC may be harmful for those with an rs CC genotype (~25% of IPF population)
Replication needed

17. Replication Cohort

18. NAC (n=47) Non-NAC (n=358) p-value
Replication Cohort (n=405) Baseline Characteristics and Genotypes*
Characteristic
NAC (n=47)
Non-NAC (n=358)
p-value
Age, mean (±SD)
68.3 (8.3)
66.4 (7.8)
0.12
Male, n (%)
33 (70.2)
267 (74.6)
0.52
Ever Smoker, n (%)
235 (70.4)
0.98
Azathioprine therapy, n (%)
5 (10.9)
10 (2.8)
0.02
Prednisone therapy, n (%)
23 (50.0)
132 (36.9)
0.09
FVC, % predicted (±SD)
62.6 (18.6)
71.1 (14.2)
<0.001
DLCO, % predicted (±SD)
39.1 (10.5)
48.0 (11.9)
0.003
Death, n (%)
19 (40.4)
78 (21.8)
0.01
FVC Decline ≥ 10%, n (%)
17 (36.2)
0.93
Transplant, n (%)
2 (4.3)
11 (3.1)
0.46
Composite Endpoint**, n (%)
27 (57.5)
167 (46.7)
0.6
Abbreviations: NAC = N-acetylcysteine; FVC = forced vital capacity; DLCO = diffusion capacity of the lung for carbon monoxide; SNP = single nucleotide polymorphism
*Non-Hispanic white individuals by self-report
** Death, transplant or 10% FVC decline
So we identified patients in each cohort who received NAC therapy then combined cohorts to perform the same analysis as we performed with the PANTHER trial. As opposed to the PANTHER trial, which compared equivalent groups, with these cohorts we see that patients who received NAC were substantially sicker than those who did not. So it’s perhaps not surprising that we saw a much higher mortality rate among those receiving NAC. Some of this may have been due to the fact that the patients who received NAC also received prednisone and azathioprine, which we now know is harmful in IPF. So not an ideal dataset with which to validate our findings but it was the best we had. As for the SNPs, we limited the analysis to those SNPs which had significant or near significant interaction in our previous modeling, specifically one of the intronic TOLLIP SNPs, the exonic TOLLIP SNP and the MUC5B promoter SNP.

19. Replication Cohort Interaction Model Estimates Variable
Coefficient (95% CI)
p-value
rs (TOLLIP)
-0.06 ( )
0.70
NAC Therapy
0.46 ( )
0.08
rs *NAC interaction
-0.33 ( )
0.48
rs (TOLLIP)
0.01 ( )
0.90
1.12 ( )
0.001
-0.73 ( )
0.012
rs (MUC5B)
-0.23 ( )
0.31
1.0 ( )
rs *NAC interaction
-1.07 ( )
0.02
* Adjusted for azathioprine use, prednisone use, FVC (% predicted) and DLCO (% predicted)
What we found was that significant interaction was again present with the TOLLIP exonic SNP.
Interaction present when pinteraction < 0.017
- Adjusts for multiple testing

20. Genotype Log rank p-value HR 95% CI p-value
Replication Cohort Genotype-stratified Composite Endpoint Risk* Associated with NAC Therapy
Genotype
Combined Cohort
Log rank p-value HR
95% CI
p-value
rs CC
0.004
3.11
0.01
rs CT
0.002
2.18
0.02
rs TT
0.51
0.23
0.04
* Adjusted for age, gender,prednisone use, azathioprine use, FVC (% predicted), DLCO (% predicted) and trial/center
Conclusion
NAC may be beneficial for genetically susceptible individuals, but…….
Recommendation regarding the use of NAC to treat IPF cannot be made until a prospective, genotype-stratified clinical trial can be conducted
And so we again calculated composite endpoint risk stratified by the TOLLIP exonic SNP genotype and we again found that those with a TT genotype had a significant reduction in endpoint risk when receiving NAC therapy.

21. Secondary post-hoc analysis
Limitations
Secondary post-hoc analysis
treatment arms were not randomized on genotype
Relied upon self-reported ancestry
~ 50% did not consent to genetic analysis
Small sample size
Unclear whether those analyzed are representative of those who were not

22. The University of Chicago Weill Cornell Medical College
Acknowledgments
The University of Chicago
Shwu Fan Ma, PhD
Yong Huang, MD
Rekha Vij, MD
Eleanor Valenzi, MD
Cathryn Lee, MD
Leah Witt, MD
Mary Strek, MD
Imre Noth, MD
Weill Cornell Medical College
Fernando J. Martinez, MD
Duke University
Kevin Anstrom, PhD
The University of Washington
Ganesh Raghu, MD
The University of Colorado
David Schwartz, MD

23. References
Noth I, Zhang Y, Ma S-F, Flores C, Barber M, Huang Y, et al. Genetic variants associated with idiopathic pulmonary fibrosis susceptibility and mortality: a genome-wide association study. The Lancet. 2013;1(4):
Fingerlin TE, Murphy E, Zhang W, Peljto AL, Brown KK, Steele MP, et al. Genome-wide association study identifies multiple susceptibility loci for pulmonary fibrosis. Nat Genet. 2013;45(6):
Peljto AL, Zhang Y, Fingerlin TE, Ma SF, Garcia JG, Richards TJ, et al. Association between the MUC5B promoter polymorphism and survival in patients with idiopathic pulmonary fibrosis. JAMA : the journal of the American Medical Association. 2013;309(21):
Idiopathic Pulmonary Fibrosis Clinical Research N, Martinez FJ, de Andrade JA, Anstrom KJ, King TE, Jr., Raghu G. Randomized trial of acetylcysteine in idiopathic pulmonary fibrosis. N Engl J Med. 2014;370(22):
Raghu G, Anstrom KJ, King TE, Jr., Lasky JA, Martinez FJ. Prednisone, azathioprine, and N-acetylcysteine for pulmonary fibrosis. N Engl J Med. 2012;366(21):
Roy MG, Livraghi-Butrico A, Fletcher AA, McElwee MM, Evans SE, Boerner RM et al. Muc5b is required for airway defence. Nature 2014;505:
Molyneaux PL, Cox MJ, Willis-Owen SA, Mallia P, Russell KE, Russell AM et al. The role of bacteria in the pathogenesis and progression of idiopathic pulmonary fibrosis. American journal of respiratory and critical care medicine 2014;190:
Shah JA, Vary JC, Chau TT, Bang ND, Yen NT, Farrar JJ et al. Human tollip regulates tlr2 and tlr4 signaling and its polymorphisms are associated with susceptibility to tuberculosis. J Immunol 2012;189:
Saito T, Yamamoto T, Kazawa T, Gejyo H, Naito M. Expression of toll-like receptor 2 and 4 in lipopolysaccharide-induced lung injury in mouse. Cell and tissue research 2005;321:75-88.
Janardhan KS, McIsaac M, Fowlie J, Shrivastav A, Caldwell S, Sharma RK, Singh B. Toll like receptor-4 expression in lipopolysaccharide induced lung inflammation. Histology and histopathology 2006;21:

24. Thank You!
20 minutes
Pulmonary Fibrosis Foundation Summit 2015