Biology and therapy of fibromyalgia. Evidence-based biomarkers for fibromyalgia syndrome

Researchers studying fibromyalgia strive to identify objective, measurable biomarkers that may identify susceptible individuals, may facilitate diagnosis, or that parallel activity of the disease. Candidate objective measures range from sophisticated functional neuroimaging to office-ready measures of the pressure pain threshold. A systematic literature review was completed to assess highly investigated, objective measures used in fibromyalgia studies. To date, only experimental pain testing has been shown to coincide with improvements in clinical status in a longitudinal study. Concerted efforts to systematically evaluate additional objective measures in research trials will be vital for ongoing progress in outcome research and translation into clinical practice.


Introduction
Fibromyalgia (FM) is a chronic condition characterized by widespread pain and tenderness on examination, along with symptoms of nonrestorative sleep, fatigue, and cognitive difficulties. Recent familial studies have suggested an underlying genetic susceptibility on which environmental factors trigger the expression of symptoms [1,2]. Despite the myalgias that patients experience, no abnormality in muscle has been reliably found [3]. Instead, aberrant pain and sensory processing probably caused by alterations in the central nervous system function are being consistently recognized in FM and related syndromes. Investigations into the autonomic nervous system and the hypothalamic-pituitary-adrenal axis also suggest a role of these stress-response systems in vulnerability to FM or in symptom expression in FM.
Our improved understanding of FM has stimulated the search for biomarkers to be used to identify individuals susceptible to the syndrome, for the diagnosis of FM, for objective measures of disease activity, or as surrogate endpoints of

Genetics
Increasing evidence supports a genetic predisposition to FM. First-degree relatives of individuals with FM display an eightfold greater risk of developing the syndrome than those in the general population [1]. As such, a genetic study using multicase families has been completed that identified an HLA linkage not yet replicated [4].
Polymorphisms in the serotonergic 5-hydroxy tryptamine 2A receptor (T/T phenotype), the serotonin transporter, the dopamine 4 receptor and the catecholamine o-methyl transferase enzyme have also been evaluated in patients with FM [5][6][7][8][9][10]. Notably, these polymorphisms all affect the metabolism or transport of monoamines, compounds that have a critical role in both sensory processing and the human stress response. With the exception of the catecholamine omethyl transferase finding and the dopamine-4-receptor gene polymorphism, however, which have not been replicated or refuted, the other findings initially noted were generally not found in subsequent studies [4][5][6][7][8][9][10]. In some cases, the findings in FM were found when all individuals with this disorder were studied, but not when individuals free of psychiatric comorbidities were studied, suggesting that some of the above findings may track more closely with psychiatric comorbidity than inherent features of FM. Other candidate genes evaluated but not shown to be associated with FM are presented in Table 1.

Evoked (experimental) pain measures
Even before the establishment of the American College of Rheumatology criteria for FM in 1990, which require both widespread pain and tenderness, investigators have used psychophysical pain testing to learn more about the nature of this condition. In fact, the early findings that the tenderness in FM was detectable throughout the body, rather than just confined to areas of tender points or muscle, was a hallmark finding that led investigators to believe this was a central nervous system pain amplification syndrome [11]. These measures are only relatively objective since they require patient self-report, but tender points do clearly measure a phenomenon that is independent from spontaneous, clinical pain.
Numerous experimental pain studies have evaluated methods of quantifying the sensory experience of pain. Various groups using an assortment of devices that produce several stimuli have assessed the pain threshold and have attempted to quantify the pain experience in FM. A review of the investigated modalities gives the greatest support for the use of the tender point intensity/index, pressure pain thresholds, or heat pain thresholds as objective measures of the degree of hyperalgesia (increased pain to normally painful stimuli) and allodynia (pain in response to normally nonpainful stimuli) of an individual. Another consistent finding has been an absence of descending endogenous analgesic activity in FM.

Tender point count
The American College of Rheumatology criteria for FM require that an individual has a certain degree of tenderness. A tender point count is performed by applying 4 kg pressure manually to 18 predefined tender points, and then asking the patient whether these areas are tender. A positive response is considered a tender point; if an individual has 11 tender points or more, this element of the case definition is satisfied.
The apparent close link between tenderness and FM has been well studied in both clinical trials of new therapies and in mechanistic studies. In a number of longitudinal randomized, placebo-controlled trials, improvements in clinical pain have corresponded with a significant change in tender point counts or in the tender point index [12][13][14]. In contrast, other studies did not show a correspondence between improvements in clinical pain and tender point counts [15][16][17][18][19][20]. The discrepancies between studies could either be because the therapies did not improve tenderness or because tender points are not a good measure of tenderness. Both factors are likely to play a role since, in certain studies where multiple measures of the pain threshold were used, tender point counts did not significantly improve whereas other measures did [21,22]. Moreover, other studies have shown that tender points are not a pure measure of tenderness. For example, there is a strong correlation between tender point counts and measures of distress in population-based studies [23].
Tender points have also been demonstrated to be biased by cognitive and emotional aspects of pain perception, whereas other measures of tenderness are much less so (see below) [24]. Improvements in tender point counts in some previous FM trials therefore possibly occurred because of improvements in distress, rather than because of inherent improvements in pressure pain threshold. Finally, tender points are often not continuously distributed in samples; rather, most people have either very few or nearly 18 tender points. As such, many investigators do not feel that tender point counts are useful to assess tenderness, and have instead turned to psychophysically and statistically superior measures.

Pressure pain thresholds
Directly measuring pressure pain thresholds is an alternative method of documenting tenderness. Devices that measure pressure pain thresholds have been used to demonstrate a left-shift and lowered pressure pain thresholds in patients with FM compared with control individuals, and this finding is noted anywhere in the body, both at tender points and in areas previously considered control points (Table 2). These findings suggest to many investigators that the term control points should be abandoned, or replaced by a term such as high-threshold tender point, since FM patients are just as tender in these regions relative to healthy control individuals.
Many of these studies initially used commercial devices or dolorimeters to deliver continuously increasing pressure via blunt probes. These measures were found to be sensitive to psychophysical and psychological biases, however, slightly similar to tender point counts using digital palpation (reviewed in [25]). For instance, the rate of increase of stimulus pressure, controlled by the operator, and patient distress were both shown to influence the pain threshold [24,26]. To minimize the bias, more sophisticated paradigms using random delivery of pressures have been developed and investigated [27,28] (Table 3). Random delivery may be less sensitive to certain influences, but it is not free of bias. For instance, in a study by Petzke and colleagues, FM patients reported higher pain during random delivery than during ascending -possibly due to a perceived lack of control [28].
A recent longitudinal study compared the three different evoked measures -tender point counts, the dolorimeter (ascending pressure paradigm), and the multiple random staircase (random pressure paradigm) -with clinical reports of pain improvement [21]. Although both clinical pain measures improved during the course of the study involving acupuncture, only one of the evoked measures -the multiple random staircase measure, which presented stimuli to individuals in an unpredictable fashion -improved after treatment. These results suggest that, of the different methods, the random stimuli paradigm may be more likely to systematically change over time. Interpretation of the results is nonetheless limited and will need to be reproduced and examined using other treatment modalities.

Heat, cold, and electrical stimuli
In addition to the heightened sensitivity to pressure noted in FM, other types of painful stimuli also are judged more painful by these patients. A decreased heat pain threshold in FM patients as compared with control individuals has been shown by multiple groups [28][29][30] (Table 4). A reduced cold pain threshold has been reported by one group in two different studies [30,31]. Sensitivity to warmth and the ability to detect electrical stimuli do not appear to be discriminative measures at this time.

Diminished diffuse noxious inhibitory control
In the process of understanding altered evoked pain sensitivity present in FM, evaluation of the intrinsic analgesic systems has uncovered another potential biomarker: diminished diffuse noxious inhibitory control (DNIC). DNIC testing in both animals and humans involves testing the pain threshold at baseline, and then administering an acutely painful stimulus that leads to a systemic analgesic effect, presumably by activating endogenous analgesic systems.
Several studies by different groups, using different conditioning stimuli (the acute noxious stimulus) and test stimuli (the stimulus used to measure pain threshold at baseline and following the acute, noxious stimulus), have indicated a deficiency of DNIC in individuals with FM. Diminished DNIC was observed in four cross-sectional studies by different groups that used variable test and conditioning stimuli [31,[32][33][34] (Table 5). Diminished DNIC has also been noted in other types of chronic pain; that is, temporomandibular disorder and hip osteoarthritis [35,36]. The normalization of DNIC after hip osteoarthritis surgery suggests it may be an objective measure of chronic pain that can change over time with treatment [36].

Functional neural imaging
Functional neural imaging enables investigators to visualize how the brain processes the sensory experience of pain. The primary modes of functional imaging that have been used in FM include functional magnetic resonance imaging (fMRI), single-photon emission computed tomography (SPECT), and positron emission tomography.
fMRI studies evaluating pain processing have the strongest current evidence of the functional imaging studies, because local myalgia tolerance: fraction of masseter to serum serotonin levels masseter Continued overleaf they corroborate this left-shift in stimulus-response function (that is, hyperalgesia/allodynia) noted in FM. Specifically, several areas of the brain consistently show greater activation in FM patients than in control individuals given the same objective stimulus intensity -especially the secondary somatosensory cortex, insula and the anterior cingulate cortex. These findings have been noted in five cross-sectional studies by two different groups, using both pressure and heat stimuli [37,38] (Table 6). In the study by Giesecke and colleagues, the clinical pain intensity corresponded with an increase in the evoked regional cerebral blood flow [37]. The resting regional cerebral blood flow was evaluated by a third group in a longitudinal study using fMRI, and showed change after drug treatment [39]. These studies have also been useful in identifying differences in pain processing in individuals with and without psychological comorbidities, showing for example that depression does not seem to be influencing the magnitude of neuronal activation in sensory pain regions such as the secondary somatosensory cortex, whereas cognitive factors such as catastrophizing did influence the sensory intensity of pain [37,40].
Positron emission tomography imaging in FM has been reported in only a few studies with inconclusive results. The only positive study is a recent one showing there may be altered dopaminergic activity in FM [41].
SPECT imaging has been studied in four cross-sectional studies by different groups that consistently found reduced regional cerebral blood flow in the right thalamus of patients with FM (three of the four studies) [42][43][44][45]. No correlation between symptoms and findings were noted in the SPECT studies.
The consistent abnormalities seen in fMRI and SPECT studies suggest either of these methods might be useful to use as a biomarker, but longitudinal studies showing that improvements in symptoms coincide with normalization of functional imaging findings would be necessary to establish this role. The advantages of fMRI imaging over positron emission tomography and SPECT include the less invasive nature and the higher temporal and spatial resolutions of fMRI. Disadvantages of fMRI include the cost and prac- ticability as well as the inability to perform receptor-ligand studies that are possible with positron emission tomography and SPECT.

Event-related potentials
Cerebral potentials evoked by noninvasive stimulation provide a unique opportunity to investigate the functional integrity and magnitude of brain processing pathways. Expressing the ability of the human brain to discriminate, classify, and memorize the significance of exogenous stimuli, event-related potentials (ERPs) have been used as a marker of cognitive function in patients with psychiatric and neurological disorders. The electrical waveforms generated can be divided into late and early components, and the waveforms are designated by their polarity (P-positive, N-negative) and latency (timing of peak) after stimulus onset. Additionally, the amplitude -the size of the voltage difference between the component peak and a prestimulus baseline -is also quantified. Auditory, somatosensory, and visual ERPs have been evaluated in patients with FM in a few studies.
Among the ERPs evaluated to date, the P300 potential (most commonly generated by an auditory consciously attended stimuli) appears to be the most promising to differentiate FM patients from control individuals. The P300 wave is a late cortical neuropsychological event, the latency of which reflects the information processing speed and the amplitude of which expresses memory functions. A reduced P300 amplitude during an auditory discriminated-task paradigm has been significantly noted in FM patients as compared with control individuals in three cross-sectional studies by two different groups [46][47][48] (Table 7). All three studies also evaluated the P300 latency, but only the largest study by Alanoglu and colleagues noted an increase in P300 latency, a finding that may have not been found in the prior studies due to lack of power [46]. In the one of these three studies by Ozgocmen and colleagues that performed ERPs before and after treatment, 8 weeks of sertraline treatment led to an increase in the P300 magnitude [48].
These studies generally failed to show an association between the ERP findings and symptom severity, although    there was an association noted with the total myalgic score. Although the change in the P300 potential after sertraline treatment was attractive, the authors agreed that -given the corresponding significant clinical improvement in pain, fatigue, or depression -the mechanism for the change remained unclear, and they acknowledged it may represent regression to the mean. Larger studies by different groups with an attention to standardizing methods are essential prior to mainstream use of this marker.
In contrast to auditory potentials, there are few and varied studies evaluating somatosensory and visual ERPs. The assorted protocols used in the studies investigating somatosensory and visual ERPs may have contributed to the lack of consistently demonstrated differences in FM and normal individuals. The lack of an established standardized metho-dology makes direct comparison difficult and may limit the evidence of reproducibility.

Sleep and activity
In addition to pain, other symptoms very commonly seen in FM include disturbed sleep and poor function. Sleep logs and polysomnography have consistently confirmed patient reports of hypersomnolence [49,50]. Using polysomnography, investigators have correlated hypersomnolence with poor sleep quality by demonstration of fewer sleep spindles, an increase in the cyclic alternating pattern rate, or poor sleep efficiency [51][52][53]. Sleep abnormalities are rarely shown to correlate with symptoms in FM, however, and many investigators anecdotally feel as though even identifying and treating specific sleep disorders often seen in FM patients (for example, obstructive sleep apnea, upper airway resis-  [45] nucleus. FM patients with less depression had increased uptake in pons Kwiatek and 2000 17 22 HC SPECT Resting rCBF n/a Reduced rCBF in right thalamus colleagues [43] and potine tegmentum, no reduction in left thalamus, or caudate nucleus. No correlation between symptoms and findings Mountz and 1995 10 7 HC SPECT Resting rCBF n/a Reduced rCBF in bilateral colleagues [44] hemithalami and caudate nucleus correlated with low pain threshold No correlation between symptoms and findings ACC, anterior cingulate cortex; CLBP, chronic low back pain; fMRI, functional magnetic resonance imaging; HC, healthy control individuals; 5-HT-3, 5-hydroxytryptamine 3; IBS, irritable bowel syndrome; MDD, major depression disorder; MRS, multiple random staircase; n/a, not applicable; PET, positron emission tomography; QST, quantitative sensory testing; rCBF, regional cerebral blood flow; SI, somatosensory cortex I; SII, somatosensory cortex II; SPECT, single-photon emission computed tomography. tance, restless leg or periodic limb movement syndromes) does not necessarily lead to improvements in the core symptoms of FM.

Actigraphy
A method of motion assessment that infers sleep and wakefulness from the presence of limb movements, actigraphy is increasingly being used as a surrogate marker for both sleep and activity. The actigraph typically combines a movement detector and memory storage on a watch-like device. The device can be worn on the wrist or the ankle continuously for long periods of time. Sleep-pattern measures available via actigraphy analyses include sleep latency, the wake time after sleep onset, and the total sleep time; sleep architecture cannot be measured, as with polysomnography. Compared with polysomnography, however, actigraphy is less expensive, less invasive, and more conducive to repeated measures, resulting in extensive use in intervention studies [54].
Actigraphy is being increasingly used in FM studies and appears promising, but has not yet proven adequately sensitive to stand alone in clinical evaluation or treatment trials [50,55,56]. As a measure of sleep quality there have been inconsistent actigraphy results, with one group noting increased levels of activity at night in FM (also noted in patients with major depression) [55] and another group noting no difference [50]. Edinger and colleagues used actigraphy as an outcome measure in an intervention trial comparing cognitive behavior therapy intervention with sleep hygiene and usual care in the treatment of insomnia [57].
Deriving an actigraphic improvement criterion, the investigators showed a greater number of patients receiving cognitive behavior therapy had clinically significant improvement in the total wake time compared with sleep hygiene therapy. No statistical difference between cognitive behavior therapy and usual care was able to be demonstrated, even though a statistical difference between the groups was shown using sleep log data in the same study.
As an objective measure of functional status, actigraphy might hold more promise as a surrogate outcome measure, because it allows the direct recording of activity levels, rather than relying on patient self-report [58]. Kop [58]. The FM patients had much lower peak activity levels, however, suggesting that the problems in function that FM patients report might be more due to an inability to rise to the intermittent demands of day-to-day life than due to overall reduced function. Auditory Auditory P300 wave FM reduced P300 amplitude, but colleagues [47] discriminated no difference in potential latency. task paradigm P300 latency negatively correlated with total myalgic scores and the control point scores. P300 amplitude correlated with PPT and total myalgic scores. No correlation in amplitude or latency with depression or anxiety.
Ozgocmen and 2003 13 10 Auditory Auditory P300 wave FM reduced P300 amplitude, but colleagues [48] discriminated no difference in potential latency task paradigm ~at baseline. Sertraline treatment before and after resulted in increase in potential sertraline amplitude by 8 weeks without treatment change in latency. No correlation (8 weeks) between EP findings, fatigue and pain scores, but correlated to total myalgic scores EP, evoked potential; PPT, pain pressure thresholds.

Stress-response systems and sex hormones
The theoretical link between stress-response systems and symptom expression is supported by studies demonstrating alterations of the hypothalamic-pituitary-adrenal axis and the autonomic nervous system in FM. Probing different aspects of the stress systems is underway to uncover objective ways to identify persons at risk or to identify reproducible abnormalities. One group clearly with increased susceptibility is women. Investigators hypothesize a potential effect of sex hormones on the stress response to partly explain the female predominance seen in FM, but this connection has not yet been specifically examined in FM patients [59].

Hypothalamic-pituitary-adrenal axis
In basal and diurnal cortisol studies, the most consistently found measure is a flattened diurnal plasma cortisol level with an elevated trough, found in three of four crosssectional studies by two out of three groups [60][61][62] ( Table 8). Studies evaluating basal plasma cortisol levels, salivary basal and diurnal cortisol levels, and urinary cortisol levels have shown inconsistent results, but they generally demonstrate normal to reduced basal levels. Since atypical depression can show a reduced cortisol level, biopsychological factors that influence cortisol levels may be contributing to the inconsistent results currently found in the literature [63]. These factors need to be better elucidated and accounted for in future studies. Nonetheless, a flattened diurnal cortisol level is a promising objective measure. . Depression also typically follows a pattern of resistance to the dexamethasone test, and therefore is a confounding factor in a large number of these evaluations.
Studies have also been completed to assess the cortisol response to exogenous corticotropin-releasing hormone or endogenous activators of corticotropin-releasing hormone (that is, hypoglycemia, IL-6) in FM. Investigators found normal to reduced cortisol levels in patients with FM after an increase in corticotropin-releasing hormone, but these results were not reproduced in other similar studies. Further investigation taking into account psychological factors as well as doses of different drugs will be prudent.

Autonomic reactivity
Tilt table testing and heart rate variability have been evaluated in patients with FM. The consistent and reproducible finding of lower heart rate variability in FM patients compared with control individuals (in three cross-sectional studies by two different groups) makes it a more useful measure than tilt table testing [71][72][73]. An abnormal drop in blood pressure or an excessive rate of syncope during tilt table testing has been noted in two out of three cross-sectional studies completed by three different groups [74][75][76]

Serologic and biochemical abnormalities
Physicians from multiple disciplines have used simple blood tests to diagnose and evaluate treatment for various diseases. Scientists have similarly evaluated a number of compounds in the serum and cerebrospinal fluid of patients with FM to find a comparable marker of disease or disease activity. Despite the effort to find easily accessible measures, no clinically suitable tests have yet been appropriately validated for FM.

Autoantibodies
The search for representative autoantibodies is a predictable step for a disease like FM, often evaluated by rheumatologists and coexisting with autoimmune diseases. Antiserotonin antibody, antiganglioside antibody, and antiphospholipid antibody have been shown to be different in FM patients and control individuals, but the applicability of these findings is not yet clear [81] (  The nonspecific increase in antibodies to a number of antigens may be a nonspecific finding that arises from a subtle shift in immune function in this spectrum of illness. In the closely related chronic fatigue syndrome, investigators have noted a shift from a T1 to a T2 immune response, which would be expected to lead to increased production of nonspecific antibodies. Any antibody or autoantibody proposed as either a diagnostic test for FM or a biomarker of FM must therefore be carefully tested using various control individuals to ensure its authenticity.

Muscle abnormalities
Despite the interest and investigation for objective peripheral muscle abnormalities, the results have remained variable and have not yet been reproduced by different groups. Additionally, there is great heterogeneity in the methods evaluating for objective muscle abnormalities that render a complete review of the data beyond the scope of the present study. To dissect out possible useful objective measures, further investigations are necessary, preferably utilizing noninvasive procedures. Table 9 Autoantibodies and fibromyalgia (FM) Year

Conclusion
Except for psychophysical pain testing, no objective measure has been appropriately evaluated and shown to improve with improvements in clinical status in a longitudinal study, and thus to qualify as a biomarker (see Table 10 for summary). These tests are not, however, entirely objective. Of the objective tests, those that hold the most promise as biomarkers are probably tests that directly assess elements of neural function, such as functional neuroimaging, ERPs, and DNIC. An effort by different groups to systematically evaluate these measures in research trials to obtain useful, comparable results will be vital for ongoing progress in outcome research. There will be an ongoing need to identify biomarkers for future studies that have reproducibility and predictive value, practicability, and biological and temporal relevance in FM.

Additional files
The following Additional files for this article are available online: Additional file 1 is an Excel file containing a