Clinical Biomarkers in Otolaryngology—Head and Neck Surgery: Autoimmune Diseases

Abstract

Objective:

The purpose of this article is to review the literature and compile promising and clinically relevant biomarkers in autoimmune disease related to otolaryngology—head and neck surgery.

Study Design:

Narrative review.

Methods:

Pubmed and Google Scholar were queried using combined key words such as “biomarkers” and “otolaryngology.” Additional queries were made with combined key words such as “biomarkers” and a particular subspecialty such as “autoimmune” or “Meniere’s” to maximize yield of relevant titles. Subsequently, specific biomarkers identified, such as “anti-TPO-antibodies,” were used as key words. Relevant titles were reviewed and selected for abstract review. Applicable abstracts were then selected for review of the full text.

Results:

Biomarkers that are currently in clinical use for the management of autoimmune diseases within the field of otolaryngology were included in this review. The compiled biomarkers were then detailed individually regarding their molecular characteristics, function, and clinical significance.

Conclusions:

Based on this literature review, there are several biomarkers currently in clinical use within the field of otolaryngology relating to autoimmune diseases. The majority of these biomarkers are in the form of proteins such as Cogan peptide and c-ANCA. This survey may serve as a comprehensive resource on biomarkers for autoimmune diseases in clinical otolaryngology.

Introduction

Biomarkers are valuable tools that help diagnosis, guide treatment, and predict the trajectory of disease. They have become cornerstone of modern medicine and a foundation for the future of personalized medicine. A biomarker is defined as a quantifiable biological parameter that represents a clinical process or outcome that cannot be directly observed. Proteins, microRNA, DNA sequences, epigenetic modifications, cytokines, and cell receptors are just a few examples of the countless molecular entities that could serve as clinically relevant biomarkers. The application of biomarkers to guide clinical management has already been established in various fields, including endocrinology, immunology, and oncology. They can also be used to help clinicians diagnose disease, make predictions regarding clinical course, and monitor therapeutic effectiveness and adverse drug reactions. Although the importance of biomarkers is clear, many remain applicable predominantly within the realm of academia and research, especially in the field of otolaryngology. We set out to review the literature and create a comprehensive text identifying key clinically relevant biomarkers currently under investigation within otolaryngology. As a starting point, we specifically focus on autoimmune disorders relevant to the field, because it has the longest tradition of employing biomarkers and the area with which otolaryngologists most readily identify this assessment technique. In our companion article, we will consider novel biomarkers whose application is not limited to autoimmune disorders.

Autoimmune Inner Ear Disease

Autoimmune inner ear disease (AIED) was first described in the 1940s by Cogan in the context of Cogan syndrome, as a constellation of findings that included interstitial keratitis associated with vertigo, tinnitus, and usually profound deafness. ¹ Since that time, the understanding behind the pathophysiology of AIED has evolved and is now discussed within the context of molecular mechanisms that drive the autoimmune process. ^{1 -3} Currently, AIED is defined clinically as primary or secondary. ¹ Primary AIED occurs when pathology affects primarily the auditory system, whereas secondary AIED arises within the setting of a systemic autoimmune illness. ^1,2 Primary AIED is described as a bilateral sensorineural hearing loss (SNHL), particularly in the lower frequencies, progressing over weeks to months. ² Without treatment, hearing loss is typically progressive and decline occurs in at least 1 ear. ^2,3 The incidence of AIED is rare with less than 5 cases per 100 000. ¹ Cogan syndrome (CS) is an orphan disease with less than 300 cases reported so far. ⁴

The pathogenesis of AIED is related to an inflammatory attack on the proteins of the inner ear resulting in autoantibody formation and immune complex deposition. ^1,3 These immune complexes deposit onto the endothelial surfaces of the inner ear vasculature causing damage to the inner ear. ^1,3 Involvement of the stria vascularis, spiral ligament, and organ of Corti likely accounts for the SNHL identified in these patients. ¹ Unlike many autoimmune conditions in which single types of autoantibodies appear to predominate, there have not been any singular antibodies or molecules that define AIED. Although many seromarkers have been investigated, there has not been one particular biomarker with clinically validated sensitivity and specificity for the diagnosis, prognosis, or targeted treatment of AIED or for CS. ^1,3,4

One autoantibody identified in CS has been identified as a potential serum biomarker. This immunoglobulin G (IgG) antibody targets Cogan peptide, which has been reported as a component of inner ear epithelia, endothelial cells, and human cochlea components with features analogous to connexin 26. ^4,5 Additionally, injection of these antibodies in mice induced features of CS including ocular symptoms. ^4,5 This autoantibody has aided in characterizing CS as an autoimmune disease but has not been validated for clinical use as a clinical biomarker as of yet. Additionally, another peptide commonly discussed in the literature surrounding AIED and CS is the anti-70 kDa heat shock protein (HSP70) antibody as a serologic biomarker. ^{4 -6} This stems from a prospective study completed in 2007 by Bonaguri et al in which 88 individuals, 21 of whom were healthy controls, were evaluated for the presence of anti-HSP70 antibodies in circulation, which is an IgG antibody targeting the HSP70. ⁶ Results showed that anti-HSP70 antibodies were isolated in 52% of the study group patients and in 4% of the control group. In a follow-up study in 2014, Bonaguri et al studied 112 individuals, 19 of whom were controls. ⁷ The study group was divided into typical CS, atypical CS, and an autoimmune SNHL group. ⁷ Results showed 92.9% of the typical CS group demonstrated positivity of anti-HSP70 antibodies. Levels of the antibody were lowest in the control group (5.2%). ⁷ The test was positive in 52.7% of patients in the autoimmune SNHL group and 16.6% in the atypical CS group. ⁷ Another marker of interest identified in a study by Cadoni et al is antiendothelial cell antibody (AECA), which was detected at statistically significant higher levels in the serum of sudden patients with SNHL compared to healthy controls (P = .0004). ⁸ Additionally, they found that positivity was significantly associated with absent recovery of hearing loss (P = .0020). ⁸ They conclude that the cytotoxicity caused by these antibodies might play a role in damage to the stria vascularis seen in immune-mediated sudden sensorineural deafness. ⁸ Although this study does propose an interesting mechanism of action for strial damage in AIED, the inclusion criteria did not select for patients with AIED in particular, rather for patients with sudden SNHL. Therefore, the specificity of this marker of AEID may be in question. Nevertheless, the findings that these AECAs seem to be related to poorer hearing loss outcomes suggest that further investigation could help elucidate their role in disease monitoring and treatment selection. ⁸ Other antibodies currently under investigation that have shown some promise include anticochlin antibodies. Baek et al demonstrated significantly elevated cochlin-specific serum antibody titers in patients with autoimmune SNHL compared with both normal hearing age- and sex-matched controls and patients with noise- and/or age-related hearing loss (P < .05). ⁹ Despite the many studies investigating potential biomarkers for CS and AIED, there remains no specific easily collectible molecule currently in clinical use for the diagnosis, disease monitoring, or treatment management of these conditions. ^{1 -3}

Systemic Lupus Erythematosus

Secondary AIED is distinguished from primary AIED in that the audiovestibular symptoms occur within the confines of an underlying known autoimmune condition, as opposed to primary AIED where the immune response acts directly against the inner ear. ¹⁰ Overall, secondary AEID is rare, accounting for less than 1% of acquired hearing loss. ¹⁰ Many mechanisms for the pathogenesis of secondary AIED are described, including immune complex deposition, antibody-dependent cell-mediated cytotoxicity, complement system involvement, or damage by cytotoxic T cells directly. ¹⁰ Although there are no specific markers in clinical use for the monitoring of ear-specific manifestations of systemic autoimmune disease, there are a vast number of circulating biomarkers in use for monitoring disease overall. For example, in systemic lupus erythematosus (SLE), anti-double-stranded DNA, anti-C1q, and anti-nucleosomes have been used clinically for diagnostic purposes. ¹¹ In SLE, the mechanism of audiovestibular dysfunction has been proposed to be secondary to a vasculitis following immune complex deposition resulting in cochlear and vestibular damage by reactive oxygen species due to reduced blood flow. Many markers of organ-specific involvement, such as lupus nephritis and neuropsychiatric lupus, have been identified for use in SLE. ¹¹ In antiphospholipid antibody syndrome (APAS), the disease is characterized by presence of antiphospholipid antibodies or anticardiolipin antibodies which serve as diagnostic biomarkers from patient serum. ^10,12 No biomarkers for monitoring of ear-specific involvement in SLE and APAS were able to be identified on review of the literature.

Meniere Disease

One unique etiology of hearing loss that is worth discussing within the context of autoimmune conditions is Meniere disease (MD). Although not entirely considered an autoimmune disorder due to lack of consensus on pathogenesis of MD, this disorder can often clinically appear similar to primary or secondary AIED. ¹⁰ Due to the wide spectrum of clinical signs in all of these conditions, such as hearing loss, vertigo, tinnitus, and fluctuating timing, a specific and easily collectible biomarker would certainly aid in differentiating these conditions. ^10,13,14 Meniere disease was first described in the 19th century by Prosper Meniere who recognized a syndrome of recurrent vertigo, tinnitus, and aural fullness with fluctuating loss of hearing. ¹³ Since then, the pathology of MD has been investigated revealing an underlying histopathologic finding of endolymphatic space dilatation or endolymphatic hydrops. ^13,14 No serologic biomarkers have been clinically validated for diagnosing or monitoring MD, and the diagnosis is largely clinical based on history and audiometric findings. ^13,14 Some proteomics studies have been conducted to identify serologic biomarkers that may characterize MD, however none have been clinically validated at this time. ¹³ A study by Chiarella et al compared serologic proteins in patients with MD to those without the disease and found that complement factor H and B, fibrinogen α and γ chains, β-actin, and pigment epithelium-derived factor are overexpressed, while the levels of β-2 glycoprotein 1, vitamin D binding protein, and apolipoprotein 1 are significantly decreased in the plasma of patients with MD. ¹⁴ Although the presence of characteristic molecules in patients with MD is promising, these proteins still require further investigation and validation before being implemented in clinical use.

Sjogren Syndrome

There is no shortage of biomarkers currently under investigation for the monitoring of Sjogren syndrome (SS). Sjogren syndrome is a systemic autoimmune condition with multiorgan involvement, particularly of the exocrine glands such as salivary and lacrimal glands resulting in xerostomia and dry eyes. ¹⁵ Diagnosis of SS already employs biomarkers in the form of serum positivity for anti-Ro/SSA and/or anti-La/SSB which are in current clinical use and in fact are diagnostic criteria. ^16,17 These are autoantibodies to the 3 antigens 52 kDa Ro, 60 kDa Ro, and La, (also designated SSA and SSB, respectively) which are thought to play a role in ribonucleoprotein complexes which function in cellular post-transcriptional regulation. ¹⁸ The assay with the highest sensitivity and specificity to detect anti-Ro/SSA and anti-La/SSB is an RNA precipitation assay and is therefore the gold standard. ^19,20 Antinuclear antibody and rheumatoid factor have also been employed for diagnosis of SS, however these are markers of other autoimmune disorders as well and are therefore not specific to SS. ^{18 -20} Definitive diagnosis can be accomplished with lip biopsy. ^{18 -20}

Sjogren syndrome affects primarily the salivary and lacrimal glands and is an attractive candidate for pathology that may be monitored via salivary or lacrimal secretions. Saliva and tears are advantageous sites of collection due to accessibility, cost effectiveness, and low risk. ^15,16 There have been many studies devoted to analyzing the salivary proteome and identifying markers characteristic to various diseases, particularly SS. Saliva is a hypotonic fluid predominantly composed of water, ions, proteins, hormones, and countless other components. ^15,16 Production is via serous and mucous cells of the parotid, submandibular, sublingual, and minor salivary glands. ^15,16

Unfortunately, despite the significant advantages of salivary collection, there are some obstacles to genomic and proteomic analysis of salivary markers. Firstly, salivary products are more prone to degradation than molecules circulating in blood. ^15,16 This may prevent prolonged storage of samples. Additionally, presence of mucin (200 kDa), a normal component of saliva, may react with immunoassays and therefore requires removal during processing of the sample. ^{15 -17} Furthermore, the overabundance of nonspecific salivary proteins such as amylase (54 kDa) and albumin (66 kDa) may mask potential underlying biomarkers that are expressed to a lesser degree. ¹⁷

Studies have demonstrated the identification of inflammation-related markers such as cathepsin-D, α-enolase, cystatins, defensins, and Igγ-light chain in the saliva of patients with SS, however these lack specificity for SS. ^16,17 Presence of more specific biomarkers such as anti-transglutaminase, antihistone, anti-Ro/SSA, and anti-La/SSB has been identified in saliva of patients with SS, however these have yet to be clinically validated. ^{16 -19}

Patients with SS are at a significantly higher risk of developing lymphoma. ^16,17 Therefore, there is a role for identifying reliable biomarkers that could be used to identify risk of progression to malignancy. ^16,17 There are countless other promising biomarkers that have shown specificity to SS, ease of collection, and correlation to severity of disease, however none have become validated clinically because they have not proven superior to the already established biomarkers. ^16,17

Immunoglobulin G4-Related Disease

Immunoglobulin G4-related disease, also known as Mikulicz disease, is a distinct systemic inflammatory disorder, of likely autoimmune etiology, in which there is infiltration of IgG4-producing plasma cells within various tissue locations. ²¹ The systemic nature of the disease results in multisystem sequelae, including involvement of the head and neck. Immunoglobulin G4-related disease has been documented in the sinuses, larynx, ears, and salivary glands, among other locations within the head and neck. ^{21 -23} The disease is diagnosed partly by biopsy of the tissue, revealing infiltration of IgG4 positive plasmacytes. Additionally, serum levels of the antibody IgG4 can be used to aid the diagnosis and response to glucocorticoids. Detection of IgG4 yields a sensitivity of 87.2% and specificity of 82.6% for the diagnosis of IgG4-related disease. ²⁴ Interestingly, elevated IgG4 levels can also be markers of other hepatobiliary conditions and cancers, demonstrating limited specificity for antibody detection to diagnose IgG4-related disease, particularly with regard to head and neck involvement. ²⁴ Thus, tissue biopsy remains the gold standard for diagnosis of this condition. Other biomarkers of IgG4-related disease are under study and present potential adjuncts to IgG4. ²⁴ Immunoglobulin G2, another antibody, and soluble interleukin 2 receptor, a freely circulating interleukin 2 receptor, are both being investigated as additional biomarkers for diagnosis and treatment monitoring of IgG4-related disease. ²⁴

Granulomatosis With Polyangiitis

Granulomatosis with polyangiitis (GPA), formerly known as Wegener granulomatosis, is a systemic autoimmune vasculitis that results in necrotizing inflammation of the small and medium-sized vessels. ²⁰ Histopathology shows inflammatory, noncaseating granulomatous lesions primarily affecting the respiratory tract and the kidneys. ²⁰ The disease is heterogeneous and manifests in multiple organ systems, including the head and neck. ²⁰ In otolaryngology, GPA affects primarily the nasal cavities, sinuses, nasopharynx, larynx, and ears. ^25,26 The biomarker most closely associated with the disease is c-ANCA(antineutrophil cytoplasmic antibodies, also designated PR3-ANCA), making it a member of a collection of ANCA-associated vasculitides which also includes eosinophilic GPA and microscopic polyangiitis. ^27,28 The direct role of ANCA in pathogenesis of disease has been disputed, however it has been shown to bind to antigens in neutrophils and monocytes which results in release of proinflammatory cytokines and lytic enzymes. ²⁸ ANCA binding to neutrophils also appears to induce adhesion to endothelial cells promoting cytotoxicity. ²⁸ Detection of ANCAs is a well-known and widely utilized diagnostic test for the diagnosis of GPA and other associated vasculitides. Despite its use in clinical practice, the use of ANCA, particularly for disease monitoring and predicting disease relapse, remains controversial. There remain many issues with the use of ANCA as a prognostic tool. Part of this stems from the varied prevalence of ANCA reported in GPA which can range from 50% to 95% depending on disease stage and activity, therapy at the time of sampling, and the detection assay used. ²⁹ The assay primarily used for screening purposes is indirect immunofluorescence technique (IFT). Positive results are then followed by antigen-specific assays, such as enzyme-linked immunosorbent assay, that detect PR3-ANCA, which creates the c-ANCA pattern on IFT and is mainly associated with GPA. ²⁹ When combining these methods, c-ANCA (specifically PR3-ANCA) is detectable in almost all patients with GPA, however it is only detectable in 60% of patients who have limited or local disease. ²⁹ All in all, although c-ANCA is currently clinically validated for use in diagnosis of GPA, its use for disease monitoring and prediction of relapse is controversial. Histologic diagnosis remains the gold standard. ²⁹

Hashimoto Disease

Autoantibodies have also been studied extensively as biomarkers for the diagnosis and disease monitoring of autoimmune thyroid diseases, such as Hashimoto thyroiditis and Graves disease. Thyroid peroxidase (TPO) is a 100 kDa dimeric transmembrane glycoprotein enzyme found almost exclusively in the thyroid and functions to oxidize iodide ions. ³⁰ Autoantibodies against this enzyme have been identified as indicators of autoimmune thyroiditis. Anti-TPO antibodies (anti-TPO-Ab) have been identified in various autoimmune thyroid conditions including postpartum thyroiditis and Graves disease but are most closely associated with the diagnosis of Hashimoto thyroiditis, with reports of up to 90% to 95% of patients with Hashimoto thyroiditis testing positive for anti-TPO-Ab. ³⁰ Various types of immunometric assays exist for detection of anti-TPO-Ab, which yield different sensitivities and specificities for the diagnosis of Hashimoto thyroiditis. ³¹ Engler et al conducted a clinical validity study to assess one particular radioimmunoassay for the detection of anti-TPO-Ab in patient serum. Their study demonstrated a sensitivity of 96% in the diagnosis of Hashimoto thyroiditis with a specificity of 100% when compared with previously validated assays, such as hemagglutination methods. ³² Although anti-TPO-Ab detection has been primarily used as a diagnostic biomarker for Hashimoto thyroiditis, anti-TPO-Ab also has clinical implications as a marker for screening other conditions of thyroid dysfunction, such as patients undergoing treatment with medications known to trigger thyroid autoimmunity (amiodarone, lithium, etc). ³¹ Its clinical utility, therefore, remains widespread, and future studies are ongoing in assessing wider applicability of anti-TPO-Ab.

Graves Disease

For the diagnosis of Graves disease, antibodies to the thyroid stimulating hormone receptor (TSH-R-Ab) have been studied extensively, and validated assays are now in widespread clinical use. TSH-R-Ab positivity is detected in 80% of patients with Graves disease and has been strongly implicated in Graves ophthalmopathy. ³³ The TSH receptor is a G-protein coupled transmembrane receptor, which enacts its function via a cyclic adenosine monophosphate cascade. ³³ TSH-R-Abs can be activating, thereby overstimulating this cascade, which results in hyperthyroidism, or blocking, which results in hypothyroidism. ³³ Both activating and blocking antibodies may be found in the same patient. In most recent years, third generation immunometric assays have become the most clinically used due to their high sensitivity and specificity, both over 98%. ³⁴

Conclusion

The diagnosis and monitoring of various autoimmune diseases within otolaryngology are aided immensely by the use of biomarkers. There is no shortage of biomarkers being investigated in the field, however the process by which a biomarker enters routine clinical use can be a long and challenging one. The ideal biomarker should be easy to collect, have high specificity to the disease in question, correlate well to the disease process, cost-effective, and clinically validated. Many key biomarkers such as IgG4 antibodies and anti-TPO-Ab have achieved this status, while others, such as Cogan peptide autoantibody, remain under scrutiny. The clinically relevant biomarkers in use within autoimmune disorders related to otolaryngology have been described in this text and summarized in Table 1. There is no doubt, however, that further study into this topic will yield valuable information for diagnosis and monitoring of disease.

Table 1.

Clinical Biomarkers in Autoimmune Disorders Within Otolaryngology.

Disorder	Biomarker	Molecular characteristics	Use	Sensitivity/specificity	Ease of collection	Limitations	In clinical use	Reference	Level of evidence 34
Cogan’s	Anti-Cogan peptide antibody	IgG binds a peptide sequence homologous to connexin 26	Diagnosis	NA	Blood	Not clinically validated	No	Shamriz et al (2018)	V
	Anti-HSP70	IgG against heat shock protein	Disease monitoring	NA	Blood	Not clinically validated	No	Bonaguri et al (2014)	III
Systemic lupus erythematosus	Anti-dsDNA	IgG against double-stranded DNA	Diagnosis	Method-dependent (EIA, Farr, PEG)	Blood	Limited sensitivity	Yes	Wu et al (2017)	V
Sjogren’s	Anti-Ro/SSA Anti-La/SSB	IgG to antigens 52 kDa Ro, 60 kDa Ro and La which comprise ribonucleoprotein (RNP) complexes	Diagnosis, disease monitoring	RNA precipitation (gold standard) ELISA: sensitivity 72%-specificity 95% CIE: sensitivity 89%-specificity 100%	Blood	Limited sensitivity	Yes	Chen et al (2015)	V
IgG4-related disease	IgG4	IgG4	Diagnosis	Sensitivity 87.2% and specificity of 82.6%	Blood	Low specificity	Yes	Tang et al (2019)	V
	sIL2	Circulating serum IL2 receptor	Diagnosis and disease monitoring	NA	Blood	Not clinically validated	No	Tang et al (2019)	V
Granulomatosis with polyangiitis	c-ANCA	Anti-cytoplasmic IgG against proteinase 3	Diagnosis, disease monitoring	Method-dependent (IFT, ELISA)	Blood	Inconsistencies in testing	Yes	Gadó et al (2013)	V
Hashimoto’s thyroiditis	anti-TPO-Ab	Anti-thyroid peroxidase IgG	Diagnosis, disease monitoring, treatment response	Sensitivity >96% Specificity >100%	Blood	Interassay variability	Yes	Engler et al (1994)	III
Graves’ disease	TSH-R-Ab	Anti-TSH-receptor IgG	Diagnosis, disease monitoring, treatment response	Sensitivity >98% Specificity >98%	Blood	Interassay variability	Yes	Roti et al (1998)	V

Abbreviations: Ab, antibody; dsDNA, double-stranded DNA; EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay; HSP70, 70 kDa heat shock protein; IFT, immunofluorescence technique; IgG, immunoglobulin G; NA, not available; PEG, polyethylene glycol; sIL-2; soluble interleukin 2; TPO, thyroid peroxidase; TSH-R-Ab, thyroid stimulating hormone receptor antibody.