Authors: Varsha Bhat (1Center for Integrative Genomics, School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, USA), Vivien A. Sheehan (2Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA, USA)
Categories: Article, Acute pain, sickle cell disease, vaso-occlusive crisis, pain biomarkers
Source: Expert review of hematology
Authors: Varsha Bhat, Vivien A. Sheehan
Acute pain episodes, also known as vaso-occlusive crises (VOC), are a major symptom of sickle cell disease (SCD) and lead to frequent hospitalizations. The diagnosis of VOC can be challenging, particularly in adults with SCD, 50% of whom have chronic pain. Several potential biomarkers have been proposed for identifying individuals with VOC, including elevation above the baseline of various vascular growth factors, cytokines, and other markers of inflammation. However, none have been validated to date.
We summarize prospective biomarkers for the diagnosis of acute pain in SCD, and how they may be involved in the pathophysiology of a VOC. Previous and current strategies for biomarker discovery, including the use of omics techniques, are discussed.
Implementing a multi-omics-based approach will facilitate the discovery of objective and validated biomarkers for acute pain.
1.1 Sickle cell disease (SCD) is an inherited red blood cell disorder caused by a mutation in the sixth amino acid residue of the beta-globin gene (HBB). The substitution of hydrophilic valine with hydrophobic glutamine causes the sickle hemoglobin (HbS) to polymerize under low oxygen, resulting in the formation of sticky rigid sickled red blood cells that obstruct blood flow, leading to complications such as acute chest syndrome, stroke, and chronic kidney disease[1]. SCD affects more than 8 million people worldwide, with the highest incidence of cases in sub-Saharan Africa[2,3]. A key clinical feature of SCD is acute pain episodes or vaso-occlusive crises (VOCs)., caused by obstruction of the microvasculature. VOCs are episodic and unpredictable bouts of pain that are triggered by the interaction of sickled erythrocytes with leukocytes and endothelial cells [4,5]. VOCs eventually elicit inflammation, tissue damage, and nociceptive pain; they are the most common reason for the hospitalization of individuals with SCD; however, up to 85% of VOCs are managed at home, so hospitalizations alone do not capture the burden of acute pain in SCD. Up to 50% of adults and 30% of children with SCD may develop chronic pain, which is pain that lasts for at least six months in a year[6]. Chronic pain may be exacerbated by anxiety and depression[6]. Both acute and chronic pain are usually treated with opioid analgesics in combination with non-steroidal anti-inflammatory drugs; unfortunately, treatment with opioids is associated with several adverse effects, including symptoms of opioid withdrawal, worsening renal function, constipation, and hyperalgesia[6–8]. Additionally, chronic pain is often poorly responsive to opioids. Our inability to separate chronic from acute pain in SCD is a barrier to effective treatment; we may exacerbate chronic pain through the overuse of opioids, and induce tolerance to opioid analgesic properties, reducing our ability to treat an acute pain episode in a patient with concomitant chronic pain.
1.2 The pathophysiology of pain in SCD, particularly chronic pain, is complex and poorly understood; it has been attributed to inflammatory, neuropathic, and nociceptive mechanisms[9]. Pain is subjective in nature with inter-individual variability, and there is an absence of quantitative measures for distinguishing between acute pain and chronic pain; this knowledge gap has greatly complicated the diagnosis and treatment of chronic pain[10]. The current practice for diagnosis involves the use of assessment tools for measuring pain intensity and impact; these tools are largely reliant on the patients’ account of pain experience and cannot be used for comparisons between patients since they exhibit considerable variability in pain tolerance[11]. Moreover, these measures are not descriptive of the patient’s pain phenotype, and all pain in an SCD patient is typically treated as a VOC in the emergency department setting. Several pain assessment tools that do not rely on patient-reported outcomes have been explored in the last decade, such as analgesic use, quantitative sensory testing, brain imaging, and electrophysiological studies; however, these methods have substantial limitations that currently prevent their widespread implementation[10]. Hence, quantitative and laboratory-based biomarkers for acute pain are vital for diagnosis, defining endpoints in clinical trials when testing potential therapeutics for SCD-linked acute pain and other outcomes, and guiding the design of appropriate treatment strategies. In this review, we discuss the two primary methodologies that have been used to identify objective biomarkers for acute single biomarker approach using targeted laboratory-based methods, and comprehensive, large-scale omics-based techniques such as genomics and transcriptomics.
2.1 A significant proportion of studies dedicated to discovering biomarkers for pain have used a candidate approach rooted in known SCD pathophysiology. One of the earliest acute pain biomarkers considered for acute pain was the vascular adhesion molecule-1 (VCAM-1). VCAM-1 is known to be involved in regulating the adhesion of the sickled RBC to the endothelium; higher serum VCAM-1 levels in plasma were observed during VOC when compared to steady state in cross-sectional studies[12–14]. Solovey et al. demonstrated that the levels of circulating endothelial cells are higher at the onset of VOC when compared to steady state[15]. Microparticles, which are extracellular vesicles primarily derived from RBCs, may induce adhesion of the RBCs to endothelial cells and potentiate inflammation; plasma levels of microparticles were shown to be elevated during pain crisis when compared to steady state[16–18]. While RBCs can adhere to the endothelium and contribute to vaso-occlusion, often the blockage is caused by leukocytes adhering to the endothelium, then the red cell adhering to the leukocyte. Oxygen dissociates from hemoglobin, the red cell sickles and ischemia may ensue. Okpala et al. explored the viability of leukocyte adhesion molecules as biomarkers for VOCs[19]. Patients in an acute pain episode had increased expression of L-selectin (CD62L) in monocytes. L-selectin is involved in initiating monocyte adhesion to the endothelial cells by binding to mucin ligands during monocyte rolling and migration, thus contributing to vaso-occlusion[20]. Conversely, plasma fibronectin, a modulator of cell adhesion and migration, was reduced during pain crises; fibronectin is likely being consumed during the adhesion of erythrocytes to endothelial cells[21,22].
2.2 In a study of 36 pediatric patients, relatively lower levels of L-arginine in plasma and exhaled nitric oxide were observed during a VOC compared to steady state. Therefore, L-arginine and nitric oxide are potential biomarkers and therapies, as they may promote vasodilation[23]. L-arginine is a substrate for nitric oxide, a potent vasodilator, and an inhibitor of vasoconstrictors such as endothelin-1[24,25]. Endothelin-1 and prostaglandin E2 are mediators of inflammation that are secreted by endothelial cells[26]. Plasma levels of endothelin-1 and prostaglandin E2 increased during pain crises compared to healthy controls and post-crisis[27]. Neutrophils in SCD are typically activated and release neutrophil extracellular traps (NETosis) composed of DNA-histone complexes and proteins, subsequently triggering inflammation[28]. Elevated levels of four neutrophil activation markers during VOC in comparison to steady state and healthy controls have been linked to an increased risk of acute peptidyl arginine deaminase 4 (PADI4), neutrophil elastase (ELANE), myeloperoxidase (MPO)[29], and pentraxin 3 (PTX-3, also released by endothelial cells)[29–31]. However, the changes between steady state and VOC for ELANE and MPO were not statistically significant after adjusting for white blood cell counts. Mast cell activation and elevation of tryptase has been implicated in pain crises in studies with transgenic SCD Berkeley mice models; mast cells are involved in the secretion of neuropeptides and immunomodulators, contributing to pain[32].
2.3 Systemic inflammation in SCD is a key driver of vaso-occlusion[33]. High sensitivity C-reactive protein (hs-CRP) is often elevated in individuals with SCD, particularly during VOC according to several studies[34,35]. Substance P, a neurotransmitter associated with neurogenic inflammation, was shown in two studies to be similarly elevated during acute pain events and is a proposed indicator of pain sensitization as well[36,37]. Vascular endothelial growth factors and angiogenic mediators, which can enhance endothelial cell growth and adhesivity, may also serve as biomarkers of acute pain[38,39]. Levels of vascular endothelial growth factor-A (VEGF), placental growth factor (PlGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) were demonstrated to be higher during VOCs as compared to the steady state; moreover, three genetic variants in the VEGF-A gene were shown to be associated with an increased risk of vaso-occlusive crises in a case-control study where the authors performed genotyping of the VEGF gene[38,40–43]. Using SCD murine models, Gu et al, demonstrated that the blockade of PlGF can reduce acute pain by inhibiting leukocyte recruitment and activation[44]. However, these candidates lack clinical utility as these associations have not been replicated across studies. Vaso-occlusion and tissue ischemia in SCD may lead to subsequent bone infarction[45]. The urinary concentration of deoxypyridinoline, a well-characterized marker of bone resorption, was comparatively higher in patients during pain crises compared to steady state. Complement activation via the alternative pathway has been implicated in vaso-occlusion and ischemia-reperfusion injury; studies with transgenic Townes HbSS mouse models have shown that complement-derived fragments such as C5a can drive vaso-occlusion by activating P-selectin on endothelial cells[46–48]. As pain crises are associated with a hypercoagulable state, patients during VOC may also exhibit increased levels of coagulation markers such as D-dimer and thrombin-antithrombin (TAT) complexes[49–51].
2.4 Cytokines, particularly interleukins, have been extensively studied as prospective biomarkers for pain crises in SCD as they are often implicated in acute inflammation[52,53] (Table 1). However, the direction of change, that is, increase or decrease during pain crises, appears to be context-specific for cytokines. In the ELIPSIS at-home longitudinal study, the levels of various cytokines were examined every 3 weeks, during pain crises and after VOC resolution in 35 participants over six months[49]. The study included an electronic patient-reported outcome tool to help participants record VOC days, pain, fatigue, functionality, and medication. The authors identified several circulating biomarkers for VOCs, including interleukin-6 (IL-6) and interleukin-10 (IL-10), that all increased during VOC from the subject’s baseline. In addition, the authors observed higher levels of CRP and thrombin-antithrombin complexes during VOCs. However, in certain studies, a reduction in IL-10 and TNF- α was observed during VOCs[54,55]. Similarly, there have been contradictory findings about IL-1β[55,56]. Elevated serum levels of IL-8 and IL-4 during VOC compared to steady state have been associated with an increased risk of acute pain[57–59]. Table 1 summarizes the biomarkers proposed for VOC; a comprehensive literature search was performed with the keywords “pain”, “vaso-occlusive crisis”, and “sickle cell disease”, and the biomarkers were selected if statistically significant differences in the levels of the biomarkers between steady state and VOC were observed in patients in the studies. All studies defined VOC as an episode of acute pain in the abdomen, chest, back and extremities, requiring the administration of opioid analgesics or nonsteroidal anti-inflammatory drugs. In the studies by Graido-Gonzalez et al and Qari et al, patients with other conditions at the time of sampling were excluded[27,55].
3.1 The vast majority of previous efforts aimed at identifying acute pain biomarkers have been constrained by small sample sizes and the difficulty in investigating multiple candidates across individuals. Furthermore, candidate genes or proteins for analysis are typically chosen from prior knowledge of SCD pathophysiology, which limits our ability to identify and seek to validate novel biomarkers. Omics-based studies, particularly with large sample sizes and validation in a second cohort, or prospectively, present an unbiased approach to pain biomarker identification. Omics has the added advantage of being able to probe thousands of genes, proteins, or metabolites at once. Over the past decade, we have witnessed significant progress with respect to applying genomics and transcriptomics to understand the pathophysiology of SCD, including acute and chronic pain[67–70].
3.2 Genome-wide association and genotyping-based studies have found associations between the rate of acute pain crises and polymorphisms in the genes GCH1, MBL, PKLR, and upstream of KIAA1109[71–74]. Rampersaud et al. used whole-genome sequencing and discovered six genes associated with acute COMT, IL1A, FAAH, TBC1D1, NR3C1, and KCNJ6; these genes encode proteins with diverse functions, highlighting the complexity of pain pathophysiology in SCD[75]. Furthermore, the authors devised a 9-locus, 21-SNP polygenic risk score to stratify patients according to their risk of acute pain. Genetic variants implicated in fetal hemoglobin induction, such as those in the genes BCL11A, HBS1L-MYB, and HBG2, were associated with a reduced rate of pain crises; these associations persisted after controlling for fetal hemoglobin levels[76]. However, some concerns with the studies published so far are small sample sizes and lack of replication; to identify robust associations, genome-wide association studies require large sample sizes with adequate statistical power and the results must be replicated in independent cohorts[77].
3.3 Transcriptomics and proteomics enable the examination of gene and protein expression under various clinical conditions and can provide additional insights into disease mechanisms. In a microarray-based study by Abdulwahab et al. the gene PLSCR4 was demonstrated to be upregulated during VOCs[78]. Creary et al performed bulk RNA-Seq profiling of ten SCD patients during baseline and VOCs, and showed that the genes CD177, CASP5, SOCS3, and ANXA3 levels increased during pain crises[79]. In another bulk transcriptomic study with CD45+ cells, four genes—IL-1β, MS4A4A, FAM20A, and SERPINB2—were revealed to be upregulated at VOC in most of the longitudinal samples[80]. Das et al., identified six genes that were elevated during pain MS4A4A, FCAR, CKAP4, SLC1A3, ICA1 and ABCA1[81]. Proteomic profiling of monocytes in ten SCD patients revealed proteins linked to five-year VOC rate[82]. Far upstream element-binding protein, alpha actinin 1 or alpha actinin 4 levels are positively correlated with VOC rate, while heat shock 70 kDa protein cognate 4, transketolase, and coronin levels are negatively correlated[83].
Due to high individual variability in pain severity among patients with SCD, there is a pressing need for objective measures to identify patients at risk of acute pain; this would significantly advance pain diagnosis, treatment, and clinical research. Numerous promising biomarkers for acute pain have been discovered with single candidate approaches as well as genomic and transcriptomic methods; these have been linked to various aspects of pain pathophysiology and encompass adhesion molecules, vascular growth factors, angiogenic factors, markers of inflammation, and neutrophil activation, and cytokines. However, none of these biomarkers have been validated thus far. Further studies are necessary to establish the utility of these biomarkers in clinical settings.
5.1 Many candidate biomarkers for acute pain in SCD have been proposed and tested in small cohorts. So far, most studies have attempted to identify a single biomarker or a small set of biomarkers that can be used to stratify patients at risk of acute pain. Transgenic sickle mouse models such as Townes mice and Berkeley mice have been used to discover biomarkers by exposure of the mice to cold or systemic administration of TNF-alpha or heme to induce VOC. However, these findings have not been sufficiently replicated across different cohorts. There is also a stepwise progression to validating a biomarker; cross-sectional or retrospective studies may be helpful for candidate biomarker generation, but given the variability among individuals with SCD, may lead to false positive or false negative associations. Better support is generated by longitudinal data, comparing an individual’s baseline levels to their levels when in VOC. These results should then be verified in a separate cohort, and the predictive value of the biomarker assessed prospectively. Although many of the candidate biomarkers discussed above have been replicated in cross-sectional studies, they have either not been evaluated in longitudinal analyses, or their predictive and prognostic value has not been assessed to further promote their validation[84].
5.2 Even with rigorous validation and verification, the candidate biomarker strategy is limited and abrogates the possibility of true unbiased discovery. We propose a multi-omics approach to identifying pain biomarkers, rather than a candidate approach based on known SCD pathophysiology. An unbiased approach, measuring biomarkers across different omics modalities—genome, transcriptome, metabolome, proteome—will allow head-to-head comparison of many potential biomarkers and their magnitudes of effect; in conjunction with hemorheological studies, they may reveal new molecular mechanisms underlying pain in SCD[85]. Integration of multi-omics data collected from the same patients via statistical techniques such as regression-based and network-based methods will enable the discovery of molecular patterns and regulatory networks, subsequently aiding the identification of robust biomarkers[86]. We can leverage technical advances in single-cell sequencing to dissect the heterogeneity of gene expression across cell types. Investigating changes in the plasma and red blood cell proteome presents some technical hurdles, such as insufficient sample purification and extensive validation; addressing these challenges is imperative to make headway in the discovery of acute pain biomarkers[87,88].
5.3 Pain biomarker study designs must incorporate longitudinal analyses where the biomarkers are assessed at steady state, VOC and after VOC resolution in large cohorts comprising patients with acute or chronic pain. To ensure that the biomarkers are generalizable across populations, study cohorts must include adult and pediatric patients of different genetic ancestries. Prospective biomarkers must be validated in behavioral studies with transgenic mouse models that express sickle hemoglobin to define endpoints for future clinical trials. Furthermore, validating the biomarkers in multi-center studies will ensure their reproducibility and generalizability in different populations. Implementing these changes will help in identifying accurate and reliable biomarkers for acute pain as well as accelerate the development of safe and effective therapeutics for treating pain in SCD.