Tissue is an issue in the search for biomarkers in idiopathic pulmonary fibrosis

Introduction

Biological markers, which are often referred to as biomarkers, are commonly defined
as objectively measured and evaluated indicators of physiological or pathological
processes or pharmacological responses to therapeutic intervention 1], although there are also several other definitions. During recent years, blood-originated
biomarkers from serum, plasma, or cells have been the most extensively reviewed with
respect to idiopathic pulmonary fibrosis (IPF) 2],3]. Biomarkers have been postulated to be useful in several ways, e.g., in making a
differential diagnosis between IPF and other interstitial lung diseases (ILDs), in
estimating prognosis and survival, in revealing the course of disease, and also for
monitoring drug efficacy. In addition, it is possible that biomarkers could be helpful
in distinguishing between various phenotypes of IPF.

Rationale for lung tissue biomarkers

It has been estimated that about one third of IPF patients do require a surgical lung
biopsy (SLB) in order to come to an ultimate diagnosis, and thus, it may be feasible
to obtain lung tissue samples from a relatively high proportion of patients 4]. One benefit for lung tissue biomarkers would be the fact that the tissue obtained
is probably the most appropriate source if one wishes to be able to link cell biological
phenomena to pathogenetic mechanisms of the disease. Many biomarkers can be presumably
located in several targets. Blood, sputum, and even broncho-alveolar lavage (BAL)
samples can be collected repeatedly, which is usually not possible for lung tissue
samples taken by surgical operation, because these procedures always carry a potential
risk of serious complications 4]. The novel less invasive method for obtaining lung tissue samples by the transbronchial
cryo-biopsy technique is expected to become more common in clinical practice; this
may mean that in the future, lung tissue samples could be obtained not only for diagnostics
but also for follow-up 5]. Some blood biomarkers have been investigated also in BAL and lung tissue, but there
are very few reports describing the simultaneous examination of blood and BAL or lung
tissue samples. The recent study of Seibold et al. combined multiple sources of materials
and showed that a polymorphism in the promoter of mucin-5 subtype B (MUC5B) was associated
with familial interstitial pneumonia (IP) and IPF 6].

This review article aims to focus on biomarkers in IPF, i.e., idiopathic usual interstitial
pneumonia (UIP), in lung tissue concentrating on studies with relevant clinical endpoints.
Studies focusing on IPF and UIP were included due to the changes in the classification,
which have taken place during the past decades, although all UIP cases do not necessarily
represent IPF. Publications comparing IPF with major types of ILDs like nonspecific
interstitial pneumonia (NSIP) and connective tissue disease-associated ILD (CTD-ILD),
which are the most common differential diagnostic dilemmas, were included. In addition,
studies conducted on lung tissue samples using modern large-scale transcriptomic and
proteomic technologies were included, although all of those had not used clinical
or radiological endpoints.

Studies on lung tissue samples with clinical endpoints

Studies of fibroblast focus

The specific aggregates of fibroblasts, myofibroblasts, and extracellular matrix (ECM)
proteins in fibrotic lung are called fibroblast foci (FF), and these structures are
more common in IPF than in other types of lung fibroses. Several studies have demonstrated
that a high amount of FF in lung tissue correlates with the shortened survival of
IPF patients 7]-12], as previously reviewed elsewhere 4]. At present, the number of the FF is the only histological biomarker that reproducibly
correlates with the prognosis of IPF.

Lung tissue biomarkers with clinical or radiological endpoints

The majority, 75.8%, of patients with IPF were found to be positive for protease-activated
receptor 2 (PAR-2) in the study of Park et al. (Table 1). Blood neutrophil counts were lower, whereas blood lymphocyte counts and honeycombing
scores in chest CT were higher in PAR-2-positive patients than in the PAR-2-negative
patients. All of the fatal cases belonged to the PAR-2-positive group, although this
difference between groups did not reach statistical significance 13]. In the study of Tzouvelikis and co-authors, lung tissue samples of IPF, cryptogenic
organizing pneumonia (COP), and NSIP patients were analyzed for epidermal growth factor
receptor (EGFR). It was observed that the EGFR mRNA levels negatively correlated with
forced vital capacity (FVC) and diffusion capacity (DLCO) 14].

Table 1. Compilation of studies using lung tissue biomarkers with clinical endpoints

Todd et al. studied IPF cases using both SLB and subsequent lung transplantation samples
from each patient, which made it possible to compare the histological features of
the early and late phases of the disease. It was revealed that numbers of lymphocytes
in lung tissue increased during progression since the amount of lymphocytes was higher
in the lung explants than in the SLB samples 15].

IPF cases were investigated for the expression levels of alpha smooth muscle actin
(?-SMA), telomerase, interleukin 4 (IL-4), transforming growth factor-beta (TGF-?),
and beta fibroblast growth factor (?-FGF). It was noted that the levels of expressions
of myofibroblast ?-SMA and IL-4 were negatively associated with patient survival 16]. Calabrese et al. examined explanted lungs of IPF patients, of which patients with
high-grade dysplasia or carcinomas showed a greater increase in the levels of serpin
B3/B4 expression in metaplastic epithelial cells than the patients without these diseases.
The expression level of serpin B3/B4 was linearly and positively associated with age.
Furthermore, the patients with greater impairments in DLCO displayed significantly
higher expression of serpin B3/B4 17].

It was noted that the number of mast cells were increased in IPF, and in addition,
a high mast cell number also associated with a slower rate of decline in FVC in a
study of Cha and co-authors 18]. Nagata and others evaluated Krebs von den Lungen-6 antigen (KL-6) and surfactant
protein A (SP-A) in idiopathic interstitial pneumonia (IIP). In patients with IIPs
as a whole and also in those with UIP, the SP-A positive ratio was significantly lower
in those who died from the progression of disease in comparison to those patients
with another prognosis, i.e., stable, improved, and deteriorating but living 19].

Myllärniemi et al. investigated UIP and NSIP cases for gremlin and bone morphogenetic
protein 4 (BMP-4), revealing that the area of gremlin-positive staining correlated
negatively with FVC. The levels of gremlin mRNA correlated negatively with the specific
diffusion capacity corrected for alveolar volume (DLCO/VA), whereas BMP-4 mRNA correlated
positively with FVC and DLCO 20]. A negative correlation between gremlin mRNA levels and DLCO/VA was observed when
UIP and NSIP patients were analyzed. In contrast, a positive correlation was observed
between BMP-4 mRNA and FVC as well as between BMP-4 mRNA and DLCO.

Parra et al. revealed that the total density of inflammatory cells was significantly
increased in the patients with NSIP and diffuse alveolar damage (DAD) when compared
to those with UIP. In UIP, forced expiratory volume in 1 s (FEV1) and survival correlated
with the numbers of CD3-positive T lymphocytes (TL), the numbers of CD68-positive
cells correlated with FEV1, and the amounts of neutrophil elastase-positive cells
correlated with residual volume and residual volume/total lung capacity (TLC) and
carbon monoxide transfer factor. The most important predictor of survival in UIP/IPF
was CD3-positive TLs 21]. In another study it was found that in IPF, the numbers of CD8-positive TLs inversely
correlated with FVC% predicted, TLC% predicted, DLCO% predicted, and arterial oxygen
tension (PaO₂). Positive and statistically significant correlations were found between the numbers
of CD8-positive TLs and alveolar-arterial gradient (P(A-a)O₂) as well as the Medical Research Council (MRC) score. Furthermore, the CD8-positive
TLs displayed significant negative correlations with the FVC% predicted and the FEV1%
predicted 22].

Tsukamoto et al. observed that epithelial cells were positively stained for Epstein-Barr
virus-associated latent membrane protein 1 (LMP1) in 31% of the patients with IPF,
whereas none of the patients with systemic sclerosis (SSc)-associated ILD or the controls
showed this kind of positive staining. Death from respiratory failure was significantly
more common in LMP1-positive patients than in LMP1-negative patients. The use of systemic
steroids after lung biopsy was more frequent in the LMP-positive than in the LMP-negative
patients 23].

The amount of tenascin-C was analyzed in patients with UIP and also other types of
ILDs 24]. The mean survival of the patients with UIP with high scores of tenascin-C was significantly
shorter than that of patients with UIP with a lower tenascin-C sum score. Testing
of tenascin-C scores in different locations revealed that an increased accumulation
of tenascin-C underneath metaplastic bronchiolar-type epithelium was also associated
with a shorter survival.

Differential diagnostic biomarkers

Cipriani et al. investigated CTD-UIP and IPF to evaluate the count and area of both
FF and lymphocyte aggregates (LAs) (Table 2). They found that FF counts and areas were lower in patients with CTD-UIP, whereas
the LA counts and areas were greatest in the patients with CTD-UIP, although the differences
did not quite reach statistical significance. The only marked difference was observed
in NSIP features, which were more prevalent in CTD-UIP than in idiopathic UIP 25]. NSIP and IPF cases were evaluated for levels of chemokine receptors CXCR3 and CCR4.
It was found that the number of CXCR3-positive lymphocytes in NSIP patients was significantly
greater than the corresponding value in IPF patients. The number of CCR4-positive
lymphocytes in NSIP patients was significantly lower than that in IPF, and thus, the
CXCR3 to CCR4 ratio in the NSIP patients was significantly elevated 26].

Table 2. Examples of studies focusing on differential diagnostics between IPF and NSIP or CTD-ILD

The levels of epimorphin protein and mRNA expression in NSIP were significantly higher
than those in the patients with UIP in the study of Terasaki and co-authors 27]. Nakashima et al. evaluated IPF and NSIP cases for signaling molecules associated
with tumor protein p53-mediated apoptosis 28]. Western blotting revealed that the expression of p53, phosphorylated p53, and mouse
double minute 2 homolog (Mdm2) protein was significantly higher in IPF and NSIP than
in the controls. The numbers of cells positive for the p53, phosphorylated p53, Mdm2,
and apoptosis regulator Bax proteins as well as the number of TUNEL-positive cells
were higher in IPF than in NSIP. Suga et al. investigated cases with IPF, NSIP, and
bronchiolitis obliterans organizing pneumonia (BOOP) for various matrix metalloproteinases
(MMP) and the specific tissue inhibitors of metalloproteinases (TIMP) 29]. The intense expression of MMP-9 in metaplastic epithelial cells was a special characteristic
of UIP.

Studies focusing on omics techniques

Zuo et al. conducted a microarray analysis of lung specimens from the patients with
IPF and CTD-UIP (Table 3). A marked increase in the expression of genes that encode for muscle proteins was
observed. The expression of genes that encode for proteins associated with cell contraction
and actin filament organization was increased as well as that of genes encoding for
collagens I, III and VI, tenascin-C, osteopontin, and fibronectin 30]. Selman et al. investigated lung tissues from the patients with IPF, hypersensitivity
pneumonitis (HP), and NSIP by microarray 31]. IPF cases were enriched for genes involved in development, extracellular matrix
structure and turnover, and cellular growth and differentiation. The levels of several
epithelium-related genes were also upregulated in IPF lungs.

Table 3. Omic studies using lung tissue in IPF research

Yang et al. profiled lung tissue from the patients with sporadic pulmonary fibrosis
and patients with familial pulmonary fibrosis. The genes involved in ECM turnover,
ECM structural constituents, proteins involved in ECM degradation, and cell adhesion
molecules were increased. Most of the genes that were differentially expressed in
the familial IIP belonged to the same functional categories as those that distinguished
IIP from control samples, but they were over- or under-expressed to a greater extent
in familial IIP than in all cases of IIP 32].

Konishi et al. evaluated lungs from the patients with stable IPF and patients with
acute exacerbation of IPF (IPF-AEx) by microarrays. When compared with control samples,
the global gene expression patterns of IPF-AEx were almost identical to those of stable
IPF. In the direct comparison of IPF-AEx and stable IPF, the differentially expressed
genes included those related to stress responses such as heat shock proteins and ?-defensins
as well as mitosis-related genes including histones and cyclin-A2 protein (CCNA2)
33]. The study of Boon and co-authors compared stable or slowly progressing IPF patients
with those suffering from progressive IPF. It was found that about a 100 of transcripts
were upregulated in the progressive group 34].

Yang et al. conducted a microarray analysis of 119 IPF cases and 50 controls. There
was elevated expression of the cilium genes associated with microscopic honeycombing
as well as higher expression of MUC5B and MMP-7. Two novel subtypes of IPF/UIP could
be defined by the expression of cilium-associated genes 35]. Patients with high cilium gene expression demonstrated more microscopic honeycombing,
but not FF, and displayed elevated tissue expression of MUC5B and MMP7.

A proteome analysis of explanted lungs from IPF patients identified that many proteins
upregulated in the IPF fell into the related categories of unfolded protein response
(UPR), endoplasmic reticulum (ER) stress, proteasome, degradation, and general cell
stress response 36]. Subsequently, the same researchers evaluated IPF and fibrotic NSIP patients with
proteomics and noted that the majority of the proteins which were upregulated in IPF
and NSIP fell into the related categories such as chaperone/protein folding, protein
processing, energy generation/glycolysis, and antioxidant function 37].

Patel et al. studied IPF patients with pulmonary hypertension (PH-IPF) and IPF patients
without PH (NPH-IPF). The comparison of PH-IPF arteriole with NPH-IPF arteriole results
achieved no separation between the two groups. When gene expression of the combined
IPF samples was compared to the controls, a total of 255 genes were differentially
expressed in IPF arterioles 38]. In a gene expression microarray study of DePianto et al., microscopic pathological
heterogeneity in IPF lung tissue corresponded to patterns related to bronchiolization
and lymphoid aggregates 39]. Recently, researchers were able to identify 2,130 differentially methylated regions
in IPF, of which 738 were associated with significant changes in gene expression 40].