Biomarkers for diagnosis, monitoring of progression, and treatment responses in ankylosing spondylitis and axial spondyloarthritis

Abstract

With the growing awareness of the impact of chronic back pain and axial spondyloarthritis and recent breakthroughs in genetics and the development of novel treatments which may impact best on early disease, the need for markers that can facilitate early diagnosis and profiling those individuals at the highest risk for a bad outcome has never been greater. The genetic basis of ankylosing spondylitis has been considerably advanced, and HLA-B27 testing has a role in the diagnosis. Knowledge is still incomplete of the rest of the genetic contribution to disease susceptibility, and it is likely premature to use extensive genetic testing (other than HLA-B27) for diagnosis. Serum and plasma biomarkers have been examined extensively in assessing disease activity, treatment response, and as predictors or radiographic severity. For assessing disease activity, other than C-reactive protein and erythrocyte sedimentation rate, the most work has been in examining cytokines (particularly interleukin 17 and 23), matrix metalloproteinase (MMP) markers (particularly MMP3). For assessing those at the highest risk for radiographic progression, biomarkers of bony metabolism, cartilage and connective tissue degradation products, and adipokines have been most extensively assessed. The problem is that no individual biomarkers has been reproducibly shown to assess disease activity or predict outcome, and this area still remains an unmet need, of relevance to industry stakeholders, to regulatory bodies, to the healthcare system, to academic investigators, and finally to patients and providers.

Introduction

Most recent data from NHANES 2009–2010 report a frequency of chronic back pain of nearly 20 % in those in the USA between the ages of 20 and 70 years [1]. Of these, up to one third have inflammatory back pain (IBP) [1, 2]. IBP is a hallmark of axial spondyloarthritis (AxSpA), which occurs in up to 25 % of those with IBP [3]. Other than by imaging, there is no direct test to diagnose AS. There is no serologic or blood marker specific for the disease.

Disease activity in AS has been measured by the Bath Ankylosing Spondylitis Disease Activity Index [4], which includes only patient-reported measures. Although used extensively since first described in 1994, it has noted limitations. It does not include the physician’s assessment of the disease and does not assess the impact of specific clinical factors. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) contains inherent redundancy in the questions and is not specific for true inflammation (as opposed to pain from other sources). The Assessment of SpondyloArthritis International Society (ASAS) developed a new AS disease activity score (ASDAS) that combines patient-reported assessments with acute phase reactants (C-reactive protein [CRP] or erythrocyte sedimentation rate [ESR]) [5]. ASDAS discriminates patients with different levels of disease activity and is sensitive over time, thus potentially useful in clinical trials. However, it too is based largely on subjective measures, and what is lacking is a comprehensive, objective biomarker of disease activity.

Disease severity is characteristically measured by metrology, functional impairment (the Bath Ankylosing Spondylitis Functional Index (BASFI)) [6], and radiographic severity, most commonly measured by the modified Stoke Ankylosing Spondylitis Severity Score (mSASSS) [7]. The presence of sacroiliitis on plain X-rays is required for the classification of ankylosing spondylitis [8] However, inflammation and pain precede radiographic sacroiliitis features on plain X-rays by several years [9]. In order to capture the disease earlier, the concept of AxSpA has been formulated, based on the evidence of active inflammation in the sacroiliac joints on magnetic resonance imaging (MRI) and/or a combination of other findings. Classification criteria have been put forth by the Assessment of SpondyloArthritis International Society (ASAS) to facilitate early diagnosis of AxSpA [10]. However, it would be especially useful if a biomarker could be found to identify those at the highest risk for progressing from AxSpA to AS or those at the highest risk for rapid radiographic progression to spinal fusion. AxSpA can progress to AS in a large proportion of patients and in some cause fusion and complete rigidity of the spine, which is irreversible. It may take up to 10 years before sacroiliitis can be clearly detected by conventional radiography [9]—critical delays before beginning novel and effective therapies. With observational data suggesting that early intervention in AS is most effective in slowing radiographic progression [11], early identification is important to prevent the incapacity caused by this disease. Moreover, novel therapies are expensive and fraught with side effects. The identification of those patients most likely to respond to these therapies would be a valuable addition to the current treatment armamentarium.

This review will focus on the role of markers, genetic, blood and synovial, in the management of AS and AxSpA, both in diagnosis, determining disease activity, and predicting those at risk for a worse outcome.

Genetic markers

HLA-B27

While HLA-B27 was not included in the modified New York classification criteria for AS [4], this HLA allele is an integral part of the new ASAS criteria for axial SpA [11]. The prevalence of HLA-B27 varies among different ethnicities. In a recent US national survey, its prevalence was 7.5 % among non-Hispanic whites while it was lower at 4.6 % in Mexican Americans and approximately 1.1 % among non-Hispanic blacks [12]. While 85–95 % of white AS patients have HLA-B27, only 7–8 % of HLA-B27 carriers in the general population develop AS. Thus, a positive test for HLA-B27 is not sufficient for the diagnosis of AS. Furthermore, the prevalence of HLA-B27 is substantially lower among African American AS patients at 50 % [5]. In terms of disease features, HLA-B27 is associated with younger age at disease onset, development of anterior uveitis, and positive family history of SpA among AS patients but is not associated with increased structural damage on radiographs.

HLA-B27 is particularly helpful in diagnosing non-radiographic AxSpA. In a longitudinal study with a mean follow-up time of 7.7 years, the combination of severe sacroiliitis on MRI with HLA-B27 positivity was an excellent predictor of future AS development [13]. The usefulness of HLA-B27 in determining who would benefit from rheumatologic evaluation in primary care setting was shown in a recent international study. Patients with chronic back pain (>3 months) and age of onset before 45 years who had either HLA-B27 positivity, inflammatory back pain, or sacroiliitis on MRI were referred to rheumatologists for further evaluation. Following this simple referral strategy, 35 % of patients were diagnosed with SpA [14]. Outside of HLA-B27, other MHC genes that have been implicated in disease susceptibility include other HLA-B alleles (B*40:01) and MICA.

Non-MHC genes

To date, over 41 genes have been identified in predisposition to AS. The overwhelming majority of these have genes are outside the MHC and include genes involved in Th17 mediated immunity (IL23R, TYK2, IL6R, IL7R?, IL7?, IL1R2/IL1R1, IL12B), peptide presentation (MHC class I proteins, ERAP1, UBE2E3, UBE2L3, NPEPPS), CD8 T cell function (RUNX3, EOMES, IL7R), microbial sensing (CARD9, NOS2) as well as genes involved in other or unclear immunologic function (ZMIZ1, FCGR2A, KIF21B, SH2B3, TNFRSF1A, GPR65, SULT1A1, GPR35, BACH2, ICOSLG, NKX2-3) [15]. These data beg the question: Can genetic testing be used to diagnose AS? Over 41 genes have been implicated in SpA risk; but other than HLA-B27 and other MHC genes (MICA, HLA-B40), the amount of risk imposed by individual non-MHC genes is very small [15] (Table 1). In fact, it is estimated that only about 25 % of the genetic contribution to overall disease susceptibility is currently known, the rest coming from such things, yet unknown, as rare genetic variants (that would not be detected from the standard GWAS approach), copy number variation of genes (including insertions and deletions), epigenetic factors, and the added variance provided by gene:gene interaction not currently recognized. This incomplete knowledge about the impact of genetic factors on disease susceptibility, common to most complex diseases, was one of the factors that led the US Food and Drug Administration to send genetic testing company “23andMe” a warning letter in November 2013 ordering it to “immediately discontinue marketing the PGS [Saliva Collection Kit and Personal Genome Service]” [16]. In addition, genetic factors have been identified that are associated with complications of AS such as acute anterior uveitis [17] and radiographic severity [18] (Table 2).

Table 1.

The relative impacts of genes associated with AS identified by genome-wide association studies on disease susceptibility^*

Locus	Gene	SNP	Odds ratio	Heritability explained (%)
1p36	RUNX3	rs6600247	1.164	0.138
1p31	IL23R	rs11209026	1.650	0.352
1p31	IL23R	rs12141575	1.147	0.100
1q21	IL6R	rs4129267	1.176	0.151
1q23	FCGR2A	rs1801274	1.123	0.080
1q23	FCGR2A	rs2039415	1.088	0.037
1q32	GPR25-KIF21B	rs41299637	1.201	0.162
1q32	HHAT	rs12758027	1.094	0.048
2p15	Intergenic	rs6759298	1.308	0.407
2q11	IL1R2-IL1R1	rs4851529	1.103	0.055
2q12	IL1R2-IL1R1	rs2192752	1.112	0.047
2q31	UBE2E3	rs12615545	1.109	0.062
2q37	GPR35	rs4676410	1.131	0.060
3p24	EOMES	rs13093489	1.119	0.067
5p13	PTGER4	rs12186979	1.093	0.047
5p13	IL7R	rs11742270	1.113	0.054
5q15	ERAP1	rs30187	1.318	0.411
5q15	ERAP1	rs1065407	1.171	0.136
5q15	ERAP2	rs2910686	1.171	0.147
5q33	IL12B	rs6871626	1.117	0.066
5q33	IL12B	rs6556416	1.107	0.054
6p21	HLA-B*27/MICA	rs116488202	60	20.089
6p21	HLA-A*02	rs2394250	1.214	0.220
6q15	BACH2	rs17765610	1.172	0.064
6q15	BACH2	rs639575	1.081	0.034
7q31	GPR37	rs2402752	1.108	0.052
9q34	CARD9	rs1128905	1.124	0.082
10q22	ZMIZ1	rs1250550	1.110	0.059
10q24	NKX2-3	rs11190133	1.181	0.136
12p13	LTBR-TNFRSF1A	rs1860545	1.131	0.086
12p13	LTBR-TNFRSF1A	rs7954567	1.113	0.062
12q24	SH2B3	rs11065898	1.129	0.060
14q31	GPR65	rs11624293	1.234	0.086
16p11	IL27-SULT1A1	imm_16_28525386	1.112	0.064
16p11	IL27-SULT1A1	rs35448675	1.236	0.007
17q11	NOS2	rs2531875	1.122	0.074
17q11	NOS2	rs2297518	1.129	0.055
17q21	NPEPPS-TBKBP1-TBX21	rs9901869	1.146	0.111
19p13	TYK2	rs35164067	1.155	0.080
19p13	TYK2	rs6511701	1.098	0.036
21q22	Intergenic	rs2836883	1.190	0.140
21q22	ICOSLG	rs7282490	1.100	0.052
22q11	UBE2L3	rs2283790	1.124	0.052

^*Modified from reference [15]

Table 2.

Markers of disease activity and treatment response

Marker	Comment	Ref.
C-reactive protein (CRP)	Perhaps, it is the most widely used biomarker in clinical trials and practice. An elevated CRP is present in about 40–50 % of patients with AS.	[19–23]
Erythrocyte sedimentation rate (ESR)	It is also widely used in clinical trials and practice. An elevated ESR is present in only about 40–50 % of patients with AS.	[19–23]
Interleukin 6 (IL-6)	IL-6 levels elevated in the serum of SpA patients compared to controls and correlated with CRP and ESR levels in some but not all studies.	[25–29]
Interleukin 17 (IL-17)	Serum IL-17 levels elevated in SpA patients and, in one study, associated with enthesitis and peripheral joint involvement.	[27–34]
Interleukin 23 (IL-23)	Several studies reported significantly higher serum IL-23 levels in SpA patients compared with healthy subjects, with highest levels in one in synovial fluid—not seen in all studies.	[27–34]
Interleukin 33 (IL-33)	It was elevated in Chinese AS patients and correlated with clinical and serologic markers of disease activity.	[33–35]
Matrix metalloproteinase 3 (MMP3)	MMP3 levels were significantly higher in AS patients than in healthy controls and were associated with higher disease activity in most (but not all) studies, and they correlated significantly with greater structural damage.	[28, 36–45]
Matrix metalloproteinase 8 (MMP8)	It was associated with disease activity (BASDAI) and functional impairment (BASFI) in one study, but this needs further confirmation.	[38]
Matrix metalloproteinase 9 (MMP9)	It was associated with disease activity in one study from the UK, not replicated in a second from Taiwan.	[37, 38]
Chemokine, CXC motif, ligand 8 (CXCL8)	This correlated with disease activity in one study, but this needs further confirmation.	[38]
Osteoprotegerin (OPG)	OPG levels higher in some and lower in other in AS patients, although patients with active disease had significantly higher concentration of OPG compared with the inactive group. Other study showed that OPG correlated with markers of disease activity such as BASDAI and VAS spinal pain. Anti-TNF treatment did not lead to a reduction in OPG levels in three studies.	[29, 47–51]
Bone alkaline phosphatase (BAP)	In higher levels reported in patients with AS and SpA compared to controls, there were no clear-cut and consistent changes with biologic treatment.	[51, 59, 63, 66]
Aggrecan	Patients with AxSpA have compared with healthy subjects’ depressed levels total aggrecan. After anti-TNF treatment, levels changed towards normal in clinical responders.	[28]
Nitric oxide metabolites (NOx)	Higher levels seen in AxSpA patients versus controls, with levels lower in those on anti-TNF agents versus NSAIDs alone.	[51]
Cartilage oligomeric matrix protein (COMP)	Elevated levels of COMP have been reported in a variety of inflammatory joint diseases, although association with other clinical markers and other biomarkers is inconsistent.	[28]
YKL-40	AS patients had elevated YKL-40 levels compared with controls, and after treatment with anti-TNF agents, levels changed towards normal levels in clinical responders, with persistent reductions over 3 years.	[28]
Calprotectin	Serum levels of calprotectin significantly increased in AxSpA, especially in those with worsening radiographic severity. Treatment with anti-TNF agents decreased calprotectin levels.	[44, 52–55]

Markers of disease activity

The erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)

CRP and ESR are the two markers of disease activity most commonly utilized in clinical practice and in clinical trials. However, they have both low sensitivity and specificity [19] and do not comprehensively represent the disease process in AS [20]. An elevated CRP is included in the ASAS AxSpA classification criteria as well as in the ASDAS, a measure of disease activity [10]. However, an elevated CRP or ESR is present in only about 40–50 % of patients with AS [21]. Thus, a normal ESR or CRP does not rule out AS or comprehensively capture active disease. Levels of both acute phase reactants are higher in patients with AS than in non-radiographic axial SpA [22]. Elevated CRP is also associated with increased radiographic changes on spine X-rays [21] and signs of inflammation on sacroiliac MRI [23]. Furthermore, an elevation of CRP and ESR predict future radiographic progression in sacroiliac joints and spine in AS patients. A recent paper found hsCRP to correlate better than routine CRP with clinical disease activity parameters in patients with axial SpA [24].

Cytokines

Studies of cytokine levels have been highly variable. Interleukin 6 (IL-6) has been the one most extensively studied [25–28]. IL-6 is a classic proinflammatory cytokine produced by a variety of immune cells that induces production of a number of positive acute phase proteins such as serum amyloid A and C-reactive protein. IL-6 is involved in initiation and maintenance of inflammation by facilitating neutrophil trafficking to the inflammation site and regulates T lymphocytes activation and differentiation. Some studies have demonstrated associations of IL-6 levels with either disease activity or other inflammatory markers [25–27] whereas others have not [28, 29]. Interleukin 17 (IL-17) and IL-23 are key cytokines in the TH17 pathway. A number of studies have reported elevated IL-17 and IL-23 levels in the plasma and sera of AS patients [27, 29–35] and associated them with disease activity [31, 33], though the latter was not seen in all [30, 32]. Interleukin 33 (IL-33) is a member of the IL-1 family and has been recently implicated in several inflammatory and autoimmune diseases. IL-33 can be produced by various types of tissues and cells and induce gene expression of Th2-associated cytokines via binding to the orphan receptor ST2. Three independent centers in China [33–35] have reported elevated serum levels of IL-33 (and in one, also its receptor ST2 [33]) in patients with AS and correlated them with disease activity or other inflammatory markers.

Matrix metalloproteinases

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases which are involved in the degradation of extracellular matrix proteins, including the cleavage of cell surface receptors, the release of apoptotic ligands and chemokine/cytokine inactivation. MMPs are also thought to play a major role in cell behaviors such as cell proliferation, migration (adhesion/dispersion), differentiation, angiogenesis, apoptosis, and host defense. Several studies have examined serum levels of MMPs in AS, especially MMP3 [28, 36–44], MMP8 [37], MMP9 [38], TIMP-1[36, 37] and −2 [36]. Higher MMP3 levels have been shown to reflect disease activity and treatment response, though this has not been universally seen [38, 43, 44]. MMP3 has also been shown to be an independent predictor of structural damage progression in patients with ankylosing spondylitis [45]. Another recent study examined 30 potential biomarkers on a multiplex, bead-based assay on a suspension array system and examined by both principal component and hierarchal clustering analysis and found MMP8 and MMP9 (and not MMP3) to be better associated with disease activity [38].

Osteoprotegerin (OPG)

Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TNF) receptor superfamily. Activated T cells can directly trigger osteoclastogenesis through OPG ligand (RANKL). OPG levels have been described as lower in AS patients in some studies [46, 47] and higher in others [29, 48–51] and have been correlated with the BASDAI but not with treatment response [48, 51].

Human cartilage glycoprotein-39 (HC gp-39, otherwise known as YKL-40)

YKL-40 is one of the major secreted proteins of human articular chondrocytes and has been associated with disease activity in AS. However, longitudinal studies have not shown it to perform as well as other biomarkers (i.e., CRP, MMP3) [28].

Cartilage oligomeric matrix protein (COMP)

COMP is a pentameric extracellular matrix protein that catalyzes the assembly of collagens and promotes formation of well-defined fibrils. Elevated levels of COMP have been reported in a variety of inflammatory joint diseases, although association with other clinical markers and other biomarkers in AS is inconsistent [28].

Aggrecan

Aggrecan is a major component of cartilage extra-cellular matrix. The extent of glycosaminoglycan side chain substitution in aggrecan and resulting fixed charge density attracts counter-ions and water, resulting in swelling pressure that is crucial for the biomechanical properties of cartilage. Patients with AxSpA have compared with healthy subjects depressed levels of total aggrecan [28]. After anti-TNF treatment, the levels changed towards normal in clinical responders.

Calprotectin

Calprotectin is a heterodimeric protein complex composed of S100A8 and S100A9 subunits and is a major calcium- and zinc-binding protein in the cytosol of neutrophils, monocytes, and keratinocytes. It has become established as a marker of whole gut inflammation [52] and is one of the most upregulated proteins specifically expressed in lesional psoriatic skin [53]. The concept of calprotectin as a biomarker in SpA is not new. Over 20 years ago, Hammer et al. found peripheral SpA it to be a plasma marker of inflammation and treatment response in reactive arthritis [54]. Subsequently, it was found to be elevated in the feces of 41 % of patients with AS [51]. Serum levels of calprotectin are also highly significantly increased in AxSpA and that treatment with anti-TNF agent significantly decrease calprotectin levels [44]. In another study, baseline calprotectin serum levels were significantly higher in patients with mSASSS worsening versus those without [55].

Markers of radiographic severity and progression (Table 3)

Table 3.

Markers of radiographic severity and progression

Marker	Comment	Ref.
C-reactive protein, ESR	Elevated CRP associated with radiographic severity on spine X-rays and elevation of CRP and ESR predict future radiographic progression in the sacroiliac joints and spine in AS patients.	[61]
Vascular endothelial growth factor (VEGF)	Mean baseline VEGF values were significantly higher in patients with mSASSS worsening by ≥2 units after 2 years than in those without progression and in patients with syndesmophyte formation as compared with those without new bone formation.	[56–58]
Matrix metalloproteinase 3 MMP3	Serum MMP is an independent predictor of structural damage progression in patients with ankylosing spondylitis.	[45]
C-terminal cross-linking telopeptide of type I (CTX-I) and type II (CTX-II) collagen	Baseline radiological damage correlated with CTX-II but not with CTX-I levels. There was a negative correlation between CTX-I and BMD of the trochanter. In multivariate analyses, CTX-II significantly and independently contributed to explaining variation in radiological damage and progression.	[59–61]
Circulating protein fragments of cartilage and connective tissue degradation (C2M and C3M)	Circulating protein fragments of cartilage and connective tissue degradation (C2M and C3M) were significantly higher in serum samples from AS patient than from healthy controls. A combination of C2M and C3M, dichotomized according to best cut-offs for individual markers, could correctly identify 80 % of the progressors and 61 % of the non-progressors. It needs further confirmation.	[62]
Degraded citrullinated vimentin fragments (VICM)	VICM levels were higher in AS patients than in healthy controls—those with highest levels of VICM levels had the highest mSASSS score. VICM levels associated with radiographic progression after 2 years.	[60]
Sclerostin	Serum levels of sclerostin were significantly lower in patients with AS than in healthy individuals. Of particular note, low serum sclerostin level in patients with AS was significantly associated with the formation of new syndesmophytes. It was not seen in other studies	[46, 50, 63–66]
Dickkop-1 (Dkk-1)	AS patients with no syndesmophyte formation show significantly higher functional Dkk1 levels suggesting that blunted Wnt signaling suppresses new bone formation and consequently syndesmophyte growth and spinal ankylosis.	[46, 50, 51, 66–69]
Osteoprotegerin (OPG)	OPG levels were higher in AS patients than in the controls, decreasing significantly after the 3-month anti-TNF-α therapy.	[50, 69]
Osteocalcin	Osteocalcin levels were higher in AS patients than in controls. Osteocalcin level increased after the 3-month anti-TNF-α therapy.	[61, 70–72]
Fetuin-A	Patients with syndesmophytes had significantly higher levels of fetuin-A compared with patients without syndesmophytes and controls. However, fetuin-A was not different between the patients without syndesmophytes and controls.	[65]
Adipokines	Serum levels of adipokines were inconsistently associated with disease activity. One study showed an association of leptins with syndesmophyte formation and another elevated levels of visfatin with radiographic progression in AS.	[73–75]

Vascular endothelial growth factor (VEGF)

Vascular endothelial growth factor is a heparin-binding growth factor specific for vascular endothelial cells that is able to induce angiogenesis in vivo. VEGF levels have been shown to be elevated in patients with AS and AxSpA [28, 33, 56–58], including in those with active disease [56] and to be higher in those patients with greater radiographic progression (increasing mSASSS scores) [56] compared to those with no worsening.

Circulating protein fragments of cartilage and connective tissue degradation

One study showed that the variation in radiological damage and progression correlated and was explained by type II collagen (CTX-II) levels but not with Type I (CTX-I) [58, 59]. CTX-II also correlated with serological markers of inflammation. Another found that levels of degraded citrullinated vimentin fragments (VICM) were significantly higher in AS patients than in healthy controls and that AS patients with the highest levels of VICM and a high baseline mSASSS had the highest risk of radiographic progression with progression occurring in 67 % of these patients [60]. In yet another examination looking at MRI scans in AxSpA patients [61], higher pretreatment MRI inflammation scores for SI joints and/or lumbar spine were associated with higher baseline CTX-II levels, but not with higher levels of other biomarkers of inflammation and bone turnover. Two neo-epitope biomarkers, C2M, which measures a matrix metalloproteinase (MMP)-generated neo-epitope of type II collagen and C3M, a marker of soft tissue turnover associated with inflammation which directly measure tissue remodeling, have been shown to be elevated in AS patients [62], and in one report, a combination of C2M and C3M was dichotomized according to the best cut-offs for individual markers to predict 80 % of the radiographic progressors and 61 % of the non-progressors.

Sclerostin

Sclerostin and noggin (NOG) are bone morphogenic protein (BMP) antagonists that modulate mitogenic activity through sequestering BMPs. Studies of sclerostin levels in patients with AS have been inconsistent. One report [63] showed that sclerostin expression by immunohistochemical analysis of osteocytes from German patients with AS was markedly reduced (compared to osteocytes of healthy individuals and RA patients) and that serum levels of sclerostin were significantly lower in patients with AS than in healthy individuals. Of particular note, low serum sclerostin levels in patients with AS in this study were significantly associated with the formation of new syndesmophytes. Similar findings were seen in two other cohorts of Swedish [50] and Brazilian AS patients [64]. Yet, other studies showed no differences or even demonstrated elevated levels of sclerostin comparing between AS patients and controls [46, 65, 66].

Wnt proteins

Wnt proteins are extracellular signaling molecules involved in the control of embryonic development. They may also contribute to neoplastic processes. There are at least two families of secreted inhibitors of Wnt signaling: the secreted frizzled-related protein family, all of which have an N-terminal cysteine-rich domain and are transmembrane receptors, and the Dickkopf (German for “big head” or “stubborn”) family, which includes Dkk1. In general, Dkk1 levels have been described as being lower in most [50, 67–69] but not all studies [46, 51]. Furthermore, AS patients with no syndesmophyte formation have been shown to have significantly higher functional Dkk1 levels, suggesting that blunted Wnt signaling suppresses new bone formation and consequently syndesmophyte growth and spinal ankylosis [66, 67], although they were not affected by 3-month anti-TNF-α therapy [68]. In yet another study [50], patients with AS had significantly higher serum levels of Wnt-3a compared with the controls.

Osteocalcin

Bone gamma-carboxyglutamic acid (Gla) protein (BGLAP, or BGP, otherwise known as osteocalcin) is a small, highly conserved molecule associated with the mineralized matrix of bone. Initial studies showed osteocalcin levels to be lower [70], but in more recent reports higher in AS patients than in controls, especially in those who would develop new syndesmophytes [61, 69, 71, 72] and increased bone mineral density [71]. Osteocalcin level increased after the 3-month anti-TNF-α therapy.

Bone-specific alkaline phosphatase (BAP)

Bone-specific alkaline phosphatase (bone ALP) is a marker of active bone formation. Higher levels of bone-specific alkaline phosphatase have been reported, albeit inconsistently, in patients with patients with AS and AxSpA, although no clear-cut changes with treatment have been observed [51, 59, 63, 66].

Fetuin-A

The fetuin family is part of the cystatin superfamily and encompasses a series of tightly related proteins that have been implicated in several diverse functions, including osteogenesis and bone resorption, regulation of the insulin and hepatocyte growth factor receptors, and response to systemic inflammation. AS patients with syndesmophytes have been shown to have significantly higher levels of fetuin-A compared with patients without syndesmophytes and controls [65]. However, fetuin-A levels were not different between patients without syndesmophytes and controls, and on regression analysis, fetuin-A was an independent, significant predictor of syndesmophyte formation.

Adipokines

Adipokines are cytokines (cell signaling proteins) secreted by adipose tissue that mediate energy homeostasis, immune responses, and regulate bone mass. In relevance to AS, they include adiponectin, resistin, leptin, visfatin, and apelin. The result is an increase in cortical bone growth through either inhibition of osteoclasts or stimulation of osteoblasts. Studies of leptin levels in AS have been inconsistent, with some showing association with disease activity and others not [73]. Likewise, no consistent association has been identified with treatment response. Serum levels of leptin have been associated with syndesmophyte formation in male AS patients [74]. Serum resistin and visfatin levels were recently found to be elevated in AS patients and elevated visfatin levels at baseline were in a recent study associated with subsequent progression of radiographic damage in AS patients [75].

To whom are these questions of potential interest?

These questions are of no little importance. To patients and providers, this offers the possibility to improve diagnosis, treatments, and predictions of prognosis of AS. To the healthcare system, this can inform judicious use of resource-intense preventive care in the highest risk patients and minimize cost. To academic investigators, valuable resource for biomarker research into pathogenesis of AS and related SpA diseases can emerge. To regulatory bodies, such as the FDA, this can result in the development of biomarkers for prognostic, predictive, pharmacodynamic, and safety markers in trials of AS. And finally, for industry stakeholders, especially those who are manufacturers of AS or SpA-related therapeutics, this can lead to smaller and shorter trials, better patient selection and stratification. In other words, for all, this can reduce costs.