MMP1 and MMP7 as Potential Peripheral Blood Biomarkers in Idiopathic
Pulmonary Fibrosis
Ivan O. Rosas MD, Thomas J. Richards PhD, Kazuhisa Konishi MD, Yingze
Zhang PhD, Kevin Gibson MD, Anna E. Lokshin PhD, Kathleen O. Lindell MSN,
Jose Cisneros PhD, Sandra D. MacDonald RN, Annie Pardo PhD, Frank Sciurba
MD, James Dauber MD, Moises Selman MD, Bernadette R. Gochuico MD,
Naftali Kaminski MD.
Online Supplement
Methods
Study Population
Initial IPF Derivation cohort - This study included 74 patients with IPF
evaluated at the Dorothy P. and Richard P. Simmons Center for Interstitial Lung
Disease at the University of Pittsburgh Medical Center between 2002 and 2004,
and was approved by the Institutional Review Board at the University of
Pittsburgh School of Medicine. The diagnosis of IPF was determined similarly in
all cohorts and was based on established criteria[1]. Briefly, all patients
presented with progressive dyspnea of > 3 months, nonproductive cough,
restrictive pulmonary functional impairment with reduced diffusing capacity for
carbon monoxide (DLCO), and arterial hypoxemia exacerbated by exercise, and
Velcro-type inspiratory crackles on physical examination. Other known causes of
interstitial lung disease, such as drug toxicities, environmental exposures, and
connective tissue diseases were excluded. When HRCT findings typical of UIP
were found, the diagnosis of IPF was established and surgical lung biopsy was
avoided. HRCT findings were considered characteristic of IPF when exhibiting
bilateral basilar subpleural reticulation and honeycomb cysts, accompanied by
traction bronchiectasis and architectural distortion. When HRCT was considered
atypical, (for example showing typical features but more extensive ground glass
attenuation) the diagnosis of usual interstitial pneumonia was confirmed on
surgical lung biopsy as indicated[2]. All patients were seen by Simmons Center
Physicians (KFG, JD). Clinical data (demographics, smoking status, clinical
findings, drug treatment, pulmonary functions, and imaging studies) were
available through the Simmons Center Database. Smoking status was
characterized as “never”, “former” (patients with smoking history who quit
smoking at least 12 months before presentation), or “current” (patients with
smoking history who are either still smoking or quit less than a year before
presentation)[3]. Fifty-three controls were obtained from the Pulmonary Division
Sample Collection core. Baseline demographic information is detailed in Table 1
in manuscript. The mean age for IPF patients was 65.9 + 9.4 years, average
FVC% predicted was 61.9 + 20.8 and average DLCO% predicted was 42.1 + 17.4.
Eight IPF patients had been treated with prednisone, 3 with IFN, 16 with IFN +
prednisone, and 47 were untreated as of the sampling date.
COPD cohort - Samples from 73 patients with COPD who were evaluated at the
University of Pittsburgh were available for this study. Individuals were clinically
stable at the time of examination, had tobacco exposure of at least 10 pack-years
and had no clinical diagnosis of rheumatologic, infectious or other systemic
inflammatory disease. Exclusion criteria included a dominant restrictive
spirometric impairment, a significant allergic history, completely reversible airflow
obstruction or a history of clinical asthma. The study was approved by the
University of Pittsburgh Institutional Review Board.
GOLD stages were defined as previously described [4]:
Stage 0: Chronic cough and sputum production but normal lung function.
Stage I: Mild airflow limitation (FEV1/FVC < 70% and FEV1 80% predicted or
more).
Stage II: FEV1 50%-80% predicted.
Stage III: FEV1 30%-50% predicted.
Stage IV: FEV1<30% predicted or the presence of respiratory failure or clinical
signs of right heart failure.
The COPD cohort included 13 patients with GOLD 0-I, 21 patients with GOLD II
and 39 patients with GOLD III-IV.
Sarcoidosis cohort - 47 patients with sarcoidosis evaluated at the University of
Pittsburgh Medical Center between 2002 and 2004 were available for this study.
70% of patients were female and 21.3% African-American. The average age was
45.7 ± 9.7 years. The diagnosis and staging of disease was performed according
to American Thoracic Society/European respiratory Society criteria. The staging
was defined as: stage 1, adenopathy alone; stage 2, adenopathy plus infiltrates;
stage 3, infiltrates alone; stage 4, fibrosis. Patients with stages 2-4 (n=29)
showed FVC % 76.7 ± 22.1, and DLCO % 72.9 ± 25.5.
Hypersensitivity
Pneumonitis
cohort:
Sera
from
41
HP
with
subacute/chronic disease were included in this study (49.8 + 12.8 years).
Diagnosis of HP was made as previously described[5,6]. Briefly, patients showed
the following features: a) antecedent of bird exposure and positive serum
antibodies against avian antigens; b) clinical and functional features of an
interstitial lung disease; c) HRCT showing diffuse centrilobular poorly defined
micronodules, ground glass attenuation, focal air trapping and mild/moderate
fibrotic changes and d) >35% lymphocytes in bronchoalveolar lavage (BAL) fluid.
Forty-four percent of the patients were biopsied and in all of them lung histology
was compatible with the diagnosis of HP.
Validation cohort: 20 controls, 8 patients with sub-clinical ILD, 16 patients with
familial IPF and 9 patients with sporadic IPF were evaluated at the Clinical
Center, National Institutes of Health (NIH), in Bethesda, MD. Subjects, at least 18
years old, were recruited by advertisements, and were enrolled in protocols 99H-0068 and/or 04-H-0211, approved by the National Heart, Lung, and Blood
Institute Institutional Review Board. Subjects were eligible if they had an open
lung biopsy demonstrating usual interstitial pneumonia or HRCT scan findings
consistent with IPF as outlined by the American Thoracic Society/European
Respiratory Society guidelines. Written informed consent was obtained from
subjects. The cohorts have been recently described by us [7,8].
Average ages for subjects with sporadic and familial IPF were 66 + 8
years and 64 +11 years, respectively. Eight patients with familial IPF were
diagnosed with early asymptomatic interstitial lung disease using HRCT[8], the
mean age in this group was 49 +11 years. Twenty normal volunteers with a mean
age of 39 +17 years were used as a control group. Gender, ethnic origin and
smoking status for the four groups are presented in Table 2.
Samples for oligonucleotide microarrays were obtained from the University
of Pittsburgh Health Sciences Tissue Bank as previously described by us[9]. The
use of archived tissue has been approved by the local Institutional Review Board.
Diagnosis of IPF was supported by history, physical examination, pulmonary
function studies, chest high-resolution computed tomography (HRCT), and
corroborated by open lung biopsy. The morphologic diagnosis of IPF was based
on typical microscopic findings consistent with usual interstitial pneumonia[2].
The patients fulfilled the criteria of the American Thoracic Society and European
Respiratory Society [10]. Twenty-three samples obtained from surgical remnants
of biopsies or lungs explanted from patients with IPF who underwent pulmonary
transplant and 15 normal histology lung samples resected from patients with lung
cancer were used for microarray analysis.
Collection and Storage of Blood.
45 ml of peripheral blood were drawn from subjects using standardized
phlebotomy procedures, after informed consent. Handling and processing was
similar for patients and controls. Cells, plasma or serum were separated by
centrifugation, and all specimens were immediately aliquoted, frozen and stored
in a dedicated –80°C freezer. No more than two freeze-thaw cycles were allowed
per sample.
Bronchoalveolar lavage (BAL)
BAL was performed through flexible fiber-optic bronchoscopy under local
anesthesia as previously described by us [9]. Briefly, 300 ml of normal saline
were instilled in 50-ml aliquots, with an average recovery of 60-70%. The
recovered BAL fluid was centrifuged at 250 X g for 10 min at 4°C. The cell pellet
was resuspended in 1 ml phosphate buffer saline and an aliquot was used to
evaluate the total number of cells. Other aliquots were fixed in carbowax, stained
with hematoxylin & eosin and used for differential cell count. Supernatants were
kept at -70oC until use.
BAL fluids from 22 IPF patients (age 62.2 + 7.2 years) and 10 normal
controls (age 41.5 + 5 years) were available for this study. These patients belong
to a cohort of IPF individuals studied and followed up at the National Institute of
Respiratory Diseases, Mexico and their inclusion was approved by the local
Ethics Committee. Diagnosis of IPF was made based on established criteria [1]
and confirmed by lung biopsy in 40% of the patients. In IPF patients BAL was
performed as part of the diagnostic process as previously described by us
[6,9,11]. Cells were stained with hematoxylin & eosin for differential cell counts,
and supernatants were frozen at -70°C until use.
All studies were approved by the Institutional Review Board at the
University of Pittsburgh, the National Heart, Lung, and Blood Institute or the
National Institute of Respiratory Diseases, Mexico. Informed consent was
obtained from all patients.
Multiplex Analysis
The Luminex xMAP technology (Luminex Corp., Austin, TX) combines the
principle of a sandwich immunoassay with fluorescent-bead-based technology
allowing individual and multiplex analysis of up to 100 different analytes in a
single microtiter well. The Luminex xMAP plasma assays were done in 96-well
microplate format according to the protocol by Biosource International (Camarillo,
CA). A filter-bottom, 96-well microplate (Millipore, Billerica, MA) was blocked for
10 minutes with PBS/bovine serum albumin. To generate a standard curve, 5-fold
dilutions of appropriate standards were prepared in serum diluent. Standards and
patient sera were pipetted at 50 µL per well in duplicate and mixed with 50 µL of
the bead mixture. The microplate was incubated for 1 hour at room temperature
on a microtiter shaker. Wells were then washed thrice with washing buffer using a
vacuum manifold. Phycoerythrin (PE)-conjugated secondary antibody was added
to the appropriate wells. The wells were incubated for 45 minutes in the dark,
with constant shaking. Wells were washed twice, assay buffer was added to each
well, and samples were analyzed using the Bio-Plex suspension array system
(Bio-Rad Laboratories, Hercules, CA). Fluorescence measures were converted to
protein concentrations using a five-parameter logistic curve (5-PL).
Sources of beads based immunoassays.
34-plex assay for IL1A, IL1B, IL2, IL2R, IL4, IL5, IL6, IL7, IL8, L10,IL12B
(IL-12p40), IL13, IL15, IL17, TNFA, IFNA, IFNG, ,GMCSF, EGF, TNFR10B,
VEGF, GCSF, FGF2, HGF, CCL5 , CXCL10 , CCL11 , CCL3, CCL5, CCL2,
CXCL9, TNFRS1A, TNFRS1B, purchased from Biosource International
(Camarillo, CA). MMP assays for MMP1, MMP2, MMP3, MMP7, MMP8, MMP9,
MMP12, MMP13 were obtained from R&D Systems (Minneapolis, MN). Assay for
FAS, EGFR, FASL, CKRT19, IGFBP1, KLK10, was developed in our Pittsburgh
Clinical Proteomics Core Facility[13].
The assays were validated as described previously[13]. Inter-assay
variability within replicates presented as an average coefficient of variation in the
range of 5.4 to 15%. Intra-assay variability was between 7.0 and 15%[13]. Each
assay was further validated in comparison with appropriate ELISA and
demonstrated 97-99% correlation[13].
ELISA
Quantitative sandwich enzyme immunoassay for human AGER was
performed as recommended by the manufacturer (R& D Systems, Minneapolis,
MN). Patient samples were prepared by diluting 1:2 in Calibrator Diluent provided
with the kit. The AGER standard was reconstituted in 1 ml of distilled water to
50,000 pg/mL. Standards assembled on the plate ranged from 0 pg/mL to 5000
pg/mL, with the appropriate calibrator diluent. All samples and standards were
run in duplicate and incubated for two hours at room temperature with Assay
Diluent RD1-60. Following four washes, Conjugate was added to each well and
incubated. After 2 hours at room temperature, the reagents were aspirated and
the plate was washed an additional four times. Shielded from light, the samples
were incubated with substrate solution at room temperature for 30 minutes. After
color development to the bound AGER, the reaction was stopped and the optical
density of the samples measured at 450 nm on a microplate reader.
The quantitative sandwich enzyme immunoassay for Human MMP1 and MMP7
was performed similarly as recommended by the manufacturer (R&D Systems,
Minneapolis, MN).
Oligonucleotide microarray experiments
Lung samples were lysed in ice cold Trizol and total RNA was extracted
and used as a template for double stranded cDNA synthesis. RNA quantity was
determined by OD measurement at 260 nm and RNA integrity by Agilent
Bioanalyzer. Labeling was performed using the Agilent Low RNA Input Linear
Amplification Kit PLUS, (One-Color) (Agilent Technologies, Santa Clara, CA).
Briefly, double stranded cDNA synthesis was performed using an oligo(dT)24
primer containing a T7 RNA polymerase promoter site. The cDNA was used as a
template to generate Cy3 labeled cRNA that was used for hybridization. After
purification and fragmentation aliquots of each sample were hybridized to Agilent
Whole Mouse Genome 4 X 44K multi pack arrays (Agilent Technologies, Santa
Clara, CA). After hybridization, each array was sequentially washed and scanned
(Agilent DNA microarray scanner). Arrays were individually visually inspected for
hybridization defects and quality control procedures were applied, as
recommended by the manufacturer of the arrays. For array readout we used
Agilent Feature Extraction Software. Data files were imported into a microarray
database and linked with updated gene annotations using SOURCE
(http://genome-www5.stanford.edu/cgi-bin/SMD/source/sourceSearch) and then
normalized using cyclic LOESS[14]. Differentially expressed genes were
identified using Significant Analysis of Microarrays (SAM)[15]. Probes
corresponding to the genes that encode the 49 protein markers were identified
through their gene symbols or Entrez gene ids. Expression levels for the probes
that corresponded to these markers were extracted. In the case of multiple
redundant probes we selected the highest expressing probe with the lowest Qvalue.
Pulmonary function testing (PFT): Measurements were made using standard
equipment according to American Thoracic Society recommendations [12]
(SensorMedics, Yorba Linda, CA). Forced expiratory volume in one second
(FEV1), forced vital capacity (FVC), and diffusion capacity (DLCO) were
expressed as percentages of predicted values.
Data handling and statistical analysis
Protein concentrations obtained from 5-PL curve fitting were exported
from the Luminex software to tab-delimited text files. Concentrations with
fluorescence below background were set to zero. Data visualization and
clustering were performed using Genomica
(http://genomica.weizmann.ac.il/index.html), previously described[16] and
Spotfire Decision Site 9 (TIBCO, Palo-Alto, CA).
A protein was considered differentially expressed when there was a
change of at least 25% in concentration and statistical signifcance at p-value <
0.05 corrected for multiple testing. Data are reported as mean + standard
deviation. The Wilcoxon rank-sum test was used to identify potential biomarkers
that univariately distinguish IPF samples from controls. Statistical analysis was
performed using the R language and environment for statistical computing ([17];
http://www.r-project.org). Univariate comparisons were performed using the
Wilcoxon rank-sum test; for multiple testing, the familywise error rate (FWER)
was controlled at 0.05 using the Bonferroni method. Fold ratios are expressed
as ratios of medians. Descriptive statistics and box plots were used to examine
univariate distributions of protein concentrations in IPF and control subsets.
Classification trees were estimated with the rpart package for recursive
partitioning, an implementation of the classification and regression trees (CART)
algorithm. Classification performance was assessed using the ROCR package
(http://rocr.bioinf.mpi-sb.mpg.de). Briefly, the CART algorithm begins by
searching the entire data set for a protein and a binary split on that protein, to
minimize the misclassification of samples in the resulting “nodes” of the split.
Each potential biomarker is compared to a threshold, and each sample is
classified as IPF or Control depending on whether the marker is above or below
the threshold. After the first split is chosen, the algorithm is applied recursively to
each resulting node, until the resulting nodes are too small to split further. The
usual procedure is to overfit classification trees to the data and prune back the
trees using optimal cost-complexity pruning. For oligonucleotide array data
analysis we applied SAM and significance was controlled at FDR = 5% (SAM Q
value = 5)[15].
To investigate the ability of the MMP7 and MMP1 to jointly distinguish IPF from
HP, we plotted the values MMP1 (x-axis) and MMP7 (y-axis) in all patients (manuscript,
Figure 4D). Closed circles indicate patients with IPF and open circles indicate patients
with HP. Corners represent points in which the trade-off between positive predictive
value (PPV) and negative predictive value (NPV) are optimal for ruling out IPF (blue) or
ruling in IPF (red) based on MMP1 and MMP7 concentrations. As can be seen in Figure
4D the performance of the combination of MMP7 and MMP1 concentrations is actually
quite impressive, in terms of the predictive value of a positive or a negative test result.
For example, a serum level of MMP7>2.6ng/ml and a serum level of MMP1>8.9ng/ml
had a 91% positive predictive value (PPV) for IPF and a serum level of MMP7<2.9 ng/ml
and MMP1 >3.5ng/ml has a 96% negative predictive value (NPV) for IPF (figure S1). To
derive the corners on Figure 4D we calculated optimal concentration combinations in the
trade-off of PPV and NPV by plotting the PPV vs. NPV for all combinations (Figure S1).
Each “corner point” demarcated in Figure S1 has a corresponding line of the same color
in Figure 4D of the manuscript.
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Supplementary Figure Legend
Figure S1. PPV and NPV for Figure 4D. Red and blue Corners in manuscript
Figure 4D were derived by computing PPV and NPV at all observed
combinations of MMP-7 and MMP-1 concentrations. The points delineated by
corners in this figure correspond to optimal trade-off points between PPV and
NPV and also to corners of the same line color in manuscript Figure 4D.
66%
80% 85% 91%
100
Negative Predictive Value (%)
96%
90
80
77%
75%
70
70%
60
50
40
40
50
60
70
80
90
Positive Predictive Value (%)
100