TOLLIP, MUC5B and the Response to N-acetylcysteine
among Individuals with Idiopathic Pulmonary Fibrosis
Justin Oldham, MD MS
Pulmonary and Critical Care Medicine
University of Chicago
Pulmonary Fibrosis Foundation Summit 2015
Disclosures
• No relevant commercial interests
Background
rs5743890 (TOLLIP)
rs35705950 (MUC5B)
GG
AA
AG
GA
Noth et al. Lancet RM 2013
Fingerlin et al. Nature Genetics 2013
Peljto et al. JAMA 2013
TOLLIP
• 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 antiinflammatory 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 Nacetylcysteine (NAC)
Shah et al.. J Immunol 2012
Saito et al. Cell and tissue research 2005
Janardhan et al. Histology and histopathology 2006
MUC5B
• 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.
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?
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
Raghu et al. NEJM 2012
Martinez et al. NEJM 2014
Martinez et al. NEJM 2014
Our Goal
• Determine whether single nucleotide polymorphisms
(SNPs) within TOLLIP and MUC5B modify the effect
of PANTHER interventions
Methods
• 5 SNPs genotyped in patients consenting to genetic analysis
–
–
–
–
–
rs5743890 (TOLLIP) (intronic)
rs5743894 (TOLLIP) (intronic)
rs3750920 (TOLLIP) (exonic)
rs5743854 (TOLLIP) (promoter)
rs35705950 (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)
Result
341 patients Enrolled in PANTHER
26 patients identifying as Hispanic
ethnicity or non-white race excluded
315 patients eligible for analysis
161 patients did not enroll
in genetics sub-study
154 patients enrolled
in genetics sub-study
54 assigned to
Placebo arm
60 assigned to
NAC arm
40 assigned to
PAN arm
Results – Baseline Characteristics and Outcomes
Genotyped (n=154)
Characteristic
Age, mean (±SD)
Male, n (%)
Ever Smoker, n (%)
FVC, % predicted (±SD)
DLCO, % predicted (±SD)
Death, n (%)
FVC Decline ≥ 10%, n (%)
Hospitalization, n (%)
Transplant, n (%)
Composite Endpoint**, n (%)
Placebo Arm
(n=54)
NAC Arm
(n=60)
PAN Arm
(n=40)
p-value
66.1 (7.9)
39 (72.2)
41 (75.9)
73.2 (14.7)
46.4 (11.7)
2 (3.7)
12 (22.2)
8 (14.8)
1 (1.9)
17 (31.5)
67.9 (8.7)
47 (78.3)
44 (73.3)
73 (15.7)
43.9 (10.9)
1 (1.7)
11 (18.3)
8 (13.3)
3 (5)
19 (31.7)
69.7 (6.8)
31 (77.5)
28 (70)
71.2 (15.2)
43 (10.6)
4 (10)
6 (15)
13 (32.5)
1 (2.5)
19 (47.5)
0.11
0.72
0.81
0.81
0.3
0.14
0.67
0.04
0.74
0.18
Results - Drug-Gene Interaction
Multivariable Cox Interaction Model Estimates*
Variable
Coefficient (95% CI)
0.84 (-0.32 - 1.99)
rs5743890 (TOLLIP)
NAC Therapy
0.17 (-0.58 - 0.92)
PAN Therapy
1.36 (0.56 - 2.17)
-1.17 (-2.91 - 0.56)
rs5743890*NAC interaction
-1.08 (-2.70 - 0.55)
rs5743890*PAN interaction
0.05 (-0.91 - 1.00)
rs5743894 (TOLLIP)
NAC Therapy
0.45 (-0.40 - 1.30)
PAN Therapy
0.74 (-0.30 - 1.78)
-1.42 (-2.89 - 0.06)
rs5743894*NAC interaction
0.58 (-0.82 - 1.97)
rs5743894*PAN interaction
rs3750920 (TOLLIP)
0.39 (-0.32 - 1.10)
NAC Therapy
1.34 (0.17 - 2.52)
PAN Therapy
0.89 (-0.49 - 2.27)
-1.47 (-2.45 - -0.48)
rs3750920*NAC interaction
0.19 (-0.81 - 1.19)
rs3750920*PAN interaction
rs35705950 (MUC5B)
0.61 (-0.53 - 1.75)
NAC Therapy
0.91 (-0.34 - 2.51)
PAN Therapy
0.98 (-0.42 - 2.37)
-1.40 (-2.89 - 0.09)
rs35705950*NAC interaction
0.13 (-1.48 - 1.74)
rs35705950*PAN interaction
p-value
0.16
0.66
0.001
0.19
0.19
0.93
0.30
0.17
0.06
0.42
0.28
0.03
0.20
0.001
0.71
0.29
0.15
0.17
0.07
0.87
• Significant interaction
detected between
NAC and rs3750920
(TOLLIP)
• Suggested interaction
observed between
NAC and rs5743894
(TOLLIP) and
rs35705950 (MUC5B)
• No interaction
observed between
these SNPs and PAN
combination therapy
Results – rs3750920 (TOLLIP) genotype-stratified composite endpointfree survival between NAC and placebo arms
NAC
Placebo
Placebo
NAC
Placebo
Placebo
NAC
logrank p=0.01
HR 3.23; 95% CI 0.79-13.16; p=0.10
logrank p=0.78
HR 0.76; 95% CI 0.27-2.19; p=0.62
logrank p=0.06
HR 0.14 ; 95% CI 0.02-0.83; p=0.03
Results – Sensitivity Analysis of genotype-stratified endpoint risk
Table 2. Sensitivity Analysis of NAC-associated endpoint risk after rs3750920 genotype stratification*
Primary Composite Endpoint Hospitalization-Free Survival Progression-Free Survival
SNP Genotype (Gene)
HR (95% CI)
p-value
HR (95% CI)
p-value
HR (95% CI)
p-value
rs3750920 (TOLLIP) CC
3.22 (0.79-13.16)
0.1
3.74 (0.35-39.80)
0.27
1.27 (0.21-7.55)
0.79
rs3750920 (TOLLIP) CT
0.76 (0.27-2.19)
0.62
0.45 (0.10-2.15)
0.32
0.63 (0.17-2.30)
0.48
rs3750920 (TOLLIP) TT
0.14 (0.02-0.83)
0.03
**
**
0.09 (0.01-0.76)
0.03
* 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 rs3750920 (TOLLIP) CC genotype
driven primarily by hospitalizations
• Benefit associated with NAC therapy in those with rs3750920 (TOLLIP) TT
genotype consistent across all endpoints
Conclusion
• NAC may be an efficacious therapy for those with
rs3750920 (TOLLIP) TT genotype (~25% of IPF population)
• NAC may be harmful for those with an rs3750920 CC
genotype (~25% of IPF population)
• Replication needed
Replication Cohort
Replication Cohort (n=405) Baseline Characteristics and Genotypes*
NAC
Non-NAC
Characteristic
p-value
(n=47)
(n=358)
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 (%)
33 (70.2)
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)
132 (36.9)
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
Replication Cohort Interaction Model Estimates
Variable
Coefficient (95% CI)
p-value
rs5743894 (TOLLIP)
-0.06 (-0.34 - 0.23)
0.70
NAC Therapy
0.46 (0.06 - 0.98)
0.08
rs3750920*NAC interaction
-0.33 (-1.26 - 0.60)
0.48
rs3750920 (TOLLIP)
0.01 (-0.19 - 0.22)
0.90
NAC Therapy
1.12 (0.47 - 1.79)
0.001
rs3750920*NAC interaction
-0.73 (-1.3 - -0.16)
0.012
rs35705950 (MUC5B)
-0.23 (-0.54 - 0.09)
0.31
NAC Therapy
1.0 (0.38 - 1.62)
0.001
rs35705950*NAC interaction
-1.07 (-1.95 - -0.19)
0.02
* Adjusted for azathioprine use, prednisone use, FVC (% predicted)
and DLCO (% predicted)
Interaction present when pinteraction < 0.017
- Adjusts for multiple testing
Replication Cohort Genotype-stratified Composite Endpoint
Risk* Associated with NAC Therapy
Combined Cohort
Genotype
Log rank
HR
95% CI
p-value
p-value
rs3750920 CC
0.004
3.11
1.25-7.72
0.01
rs3750920 CT
0.002
2.18
1.14-4.13
0.02
rs3750920 TT
0.51
0.23
0.06-0.94
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
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
Acknowledgments
The University of Chicago
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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
References
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Noth I, Zhang Y, Ma S-F, Flores C, Barber M, Huang Y, et al. Genetic variants associated with
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Fingerlin TE, Murphy E, Zhang W, Peljto AL, Brown KK, Steele MP, et al. Genome-wide association
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Peljto AL, Zhang Y, Fingerlin TE, Ma SF, Garcia JG, Richards TJ, et al. Association between the MUC5B
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Thank You!
Pulmonary Fibrosis Foundation Summit 2015