Noninvasive, MultiOmic, and Multicompartmental Biomarkers of Reflux Disease: A Systematic Review

Abstract

Background and Aims

Gastroesophageal reflux disease (GERD) is a prevalent gastrointestinal disorder that may complicate conditions such as obstructive airway disease. Our group has identified predictive biomarkers of GERD in particulate exposed first responders with obstructive airway disease. In addition, GERD diagnosis and treatment is costly and invasive. In light of these clinical concerns, we aimed to systematically review studies identifying noninvasive, multiOmic, and multicompartmental biomarkers of GERD.

Methods

A systematic review of PubMed and Embase was performed using keywords focusing on reflux disease and biomarkers and registered with PROSPERO. We included original human studies in English, articles focusing on noninvasive biomarkers of GERD published after December 31, 2009. GERD subtypes (non-erosive reflux disease and erosive esophagitis) and related conditions (Barrett’s Esophagus [BE] and Esophageal Adenocarcinoma). Predictive measures were synthesized and risk of bias assessed (Newcastle-Ottawa Scale).

Results

Initial search identified n = 238 studies and n = 13 articles remained after applying inclusion/exclusion criteria. Salivary pepsin was the most studied biomarker with significant sensitivity and specificity for GERD. Serum assessment showed elevated levels of Tumor Necrosis Factor- alpha in both GERD and Barrett’s. Exhaled breath volatile sulfur compounds and acetic acid were associated with GERD. Oral Microbiome: Models with Lautropia, Streptococcus, and Bacteroidetes showed the greatest discrimination between BE and controls vs Lautropia; ROC_AUC 0.94 (95% confidence interval; 0.85–1.00).

Conclusion

Prior studies identified significant multiOmic, multicompartmental noninvasive biomarker risks for GERD and BE. However, studies have a high risk of bias and the reliability and accuracy of the biomarkers identified are greatly limited, which further highlights the need to discover and validate clinically relevant noninvasive biomarkers of GERD.

Background

Gastroesophageal reflux disease (GERD) is a highly prevalent disorder with an incidence of 5/1000 person-years and costs > $9–10 billion/year.1, 2, 3, 4, 5 GERD diminishes health-related quality of life (QoL), productivity, accounts for about 5% of outpatient visits, and is an independent risk factor of the metaplastic changes of Barrett’s Esophagus (BE).^3,6, 7, 8 Refractory reflux, which is defined as a partial or complete lack of response to twice daily proton pump inhibitors (PPIs) was associated with both anxiety and depression and once weekly episodes of GERD was detrimental to QoL.8, 9, 10, 11

Heterogeneous presentation of GERD, including cough, heartburn, and/or regurgitation, can make diagnosis challenging. Diagnosis can be made clinically as per the Montreal consensus or it can be definitive as per the Porto and Lyon consensus statements.12, 13, 14, 15, 16, 17, 18, 19, 20 In the recently published American College of Gastroenterology guidelines, GERD is objectively defined by the presence of characteristic mucosal injury seen and/or an abnormal reflux monitoring study.¹⁵ Conclusive evidence of GERD includes endoscopic findings of erosive esophagitis (ERD) Los Angeles grade C or D, a stricture or Barrett’s esophagus, and an esophageal acid exposure time > 6% on a pH or pH impedance study in at least one full day of recording.12, 13, 14, 19

However, the main methods of reflux testing are invasive and expensive. They include a wireless telemetry capsule attached to the esophageal mucosa during endoscopy and trans-nasal catheter-based testing.^21,22 Further invasive measures are required for GERD symptoms refractory to empiric PPI therapy, such as high resolution manometry and reflux monitoring.^15,23 Prior to use of these invasive diagnostic modalities, noncompliance to PPI should always be ruled out. Moreover, studies show that the correlation between symptoms, laryngoscopic findings, and other objective testing such as pH-impedance testing is low.^24,25

Several studies have recently focused on the utility and potentially improved diagnosis of GERD using noninvasive biomarkers such as pepsin.^26,27 In the context of these prior works and clinical need, we developed our systematic review which focuses on noninvasive biomarkers of reflux disease and severity.

Methods

Review Strategy

Our systematic review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines.^28,29 Our Population, Intervention, Control, Outcome question was “In adult patients with diagnosed GERD (P), we performed a systematic review to identify (I) the noninvasive multiOmic and multicompartmental biomarkers of reflux disease (O)". Given the design of our systematic review, no comparison control (C) was needed.

PubMed and Embase were searched on February 2, 2022 as per the protocol of our systematic review were registered on PROSPERO (2022-CRD42022301543) and can be accessed at https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=301543.

Search Terms

Databases were searched for the following: (Gastric Acid Reflux OR Gastric Acid Reflux Disease OR Gastro-Esophageal Reflux Disease OR Gastro Esophageal Reflux Disease OR Gastro-Esophageal Reflux Diseases OR Gastro-oesophageal Reflux OR Gastro oesophageal Reflux OR Gastroesophageal Reflux Disease OR GERD OR Reflux, Gastroesophageal OR Esophageal Reflux OR Gastro-Esophageal Reflux OR Gastro Esophageal Reflux) AND (Biological Marker OR Biologic Marker OR Biological Markers OR Biologic Markers OR Biomarker OR Immune Markers OR Immunologic Markers OR Immune Marker OR Immunologic Marker OR Serum Markers OR Serum Marker OR Surrogate Endpoints OR Surrogate End Point OR Surrogate End Points OR Surrogate Endpoint OR Clinical Markers OR Clinical Marker OR Viral Markers OR Viral Marker OR Biochemical Marker OR Biochemical Markers OR Laboratory Markers OR Laboratory Marker OR Surrogate Markers OR Surrogate Marker).

Reference-List Screening was also Used

For this review, we have defined reflux disease to include GERD and two of its main phenotypes: nonerosive reflux disease with no evidence of mucosal injury and ERD.³⁰ To maximize studies of noninvasive biomarkers of GERD, we included studies with GERD and/or GERD patients with BE and esophageal adenocarcinoma.

Study Criteria

Studies were included if they (1) were of noninvasive biomarkers of GERD in blood, serum, saliva, or exhaled breath in diagnosed adult (clinically or endoscopically) reflux patients; (2) evaluated diagnostic tests (assessed sensitivity, specificity, positive/negative predictive values, risk, and/or accuracy of these biomarkers); (3) were written in English; and (4) published after December 31, 2009.

Studies were excluded if they (1) were not original research; (2) not written in English; (3) published before January 1, 2010; (4) exclusively used any nonhuman subjects or in vitro studies; (5) were conducted in a pediatric population; (6) focused on biomarkers in biopsied specimens; or (7) involved invasive tissue sampling and immunohistochemistry.

Data Extraction

Articles were reviewed and data regarding study design, patient characteristics, sample size, tools used, severity, and prevalence of reflux disease were extracted. Results from each database search were filtered for human subjects, English language, publication date, and imported into (EndNote X9). The references were then screened for duplicates using RefWorks (ProQuest LLC). Original research papers were reviewed (title, abstract, and full text) to ascertain eligibility. We examined references cited in the relevant articles. All results were screened by M.S.F. and S.P. and further independently evaluated by A.N. Disagreements were resolved by consensus. Details as per 2020 PRISMA are found in Figure 1 and resultant manuscripts that meet these criteria are detailed (Supplemental Tables 1-6).³¹

Figure 1.

Study design. Flow diagram as per PRISMA guidelines.^28,29

Risk of Bias Assessment

Inherent biases (selection, detection, performance, and reporting) were addressed through the study design/search algorithm. Selection bias was addressed by having predetermined inclusion and exclusion criteria and distinct definitions. Detection and performance bias was addressed by having at least two rounds of screening individually performed by M.F. and S.P. Reporting bias was minimized by using PubMed and Embase search filters for peer-reviewed published articles of human subjects written in English and removing duplicates. The Newcastle-Ottawa Scale,³² a domain-based approach, was used to assess the degree of bias as in prior studies.³³ Low-risk studies reflected were concordant in all domains (green); studies with at least one unclear or high-risk domain were considered as unclear or high risk of bias studies (yellow or red), respectively (Supplemental Tables 7A-B).

Results

Literature Search

A total of 238 studies were identified from PubMed, Embase, and reference-list screening (Figure 1). After application of selection criteria, 223 research arrticles were assessed for inclusion. Exclusion criteria were met by n = 208 (Supplemental Table 5). Finally, n = 13 original research articles were considered eligible. Data from screening and extraction are available (Supplemental Tables 1-6).34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48

Risk of bias using Newcastle-Ottawa Scale was performed in case-cohort (n = 5) and case-control (n = 8) studies. Among the case-cohort studies, n = 3 had a high risk and n = 2 had a low risk of bias. Similarly, for case-control studies, n = 7 had a high risk and n = 1 had a low risk of bias (Supplemental Table 7A-B).

Study Characteristics by Manuscript

The populations of patients studied include GERD (n = 13), BE (n = 5), and Esophageal Adenocarcinoma (EAC) (n = 2). The biomarkers were found in specimens such as saliva (n = 6), serum (n = 4), exhaled breath (n = 2), and exfoliated tongue cells (n = 1) (Table 1).

Table 1.

Study Characteristics (N = 13)

Studya		Country	Population/Design	Study size (N)	Specimen/Assay	Biomarker/End Points	Significant findings
1	Yuskel 201234	US	GERD, Prospective blinded case-control	GERD N = 58, controls N = 51	Saliva/ Lateral flow device	Pepsin/Positive peptest prevalence	Pepsin test with rapid LFD has acceptable test characteristics in patients with GERD and may replace the need for EGD and pH testing
2	Bor 201935	Turkey	GERD, observational case-cohort study	GERD N = 10	Saliva/ Lateral flow device	Pepsin/Positive peptest prevalence	Pepsin test showed a PPV of 69% to detect MII-pH reflux eventb
3	Wang 201936	China	GERD, multicenter case control study	Control N = 323, GERD N = 709, •NERD N = 488, •ERD N = 221	Saliva/ Lateral flow device	Pepsin/Predictive values	Results across all centers showed pepsin-positive sensitivity 85% Sensitivity of pepsin for NERD and ERD subgroups were 86% and 84%, respectively.
4	Guo 202027	China	ΦRefractory GERD, observational case-cohort study	Conclusive GERD N = 48; against GERD N = 24; inconclusive GERD N = 58	Saliva/ELISA	Pepsin/pepsin concentration	Pepsin test had a moderate diagnostic value for PPI refractory conclusive GERD patients
5	Buas 201740	US	GERD/BE, observational case-cohort study	SRD: GERD N = 100, BE N = 62	Serum/LC-MS	Multiple serum metabolites/FC serum metabolites	SRD: Nine metabolites were elevated in BE (FC > 1) (Creatine; homocysteine; 3-nitrotyrosine Hydroxyproline/amino levulinate; Arginine; tyrosine; Sorbitol; linoleic acid; ornithine)
6	Haider 201841	US	GERD/BE, WTC-FDNY cohort, observational prospective case-cohort study	Source cohort GERD N = 915, BE N = 106, controls N = 637 Biomarker cohort GERD N = 153, BE N = 20, controls N = 112	Serum- Luminex 200IS	TNF-α C-peptide Fractalkine IP-10 /Mean serum Biomarker level, odds ratio	C- peptide(pg/mL): GERD (791.2) vs control (550.7) TNF-α(pg/mL): GERD (4.7) vs BE (5.7) vs control (4.3) Fractalkine (pg/mL): BE (101.4) vs control (70.6) IP-10(pg/mL): BE (323.2) vs control (236.60);
7	Kim 201042	South Korea	NERD/ERD, observational case-cohort study	NERD N = 89, ERD N = 80	Exhaled breath/ Halimeter	Halitosis /VSC ppb levels	Erosive mucosal changes were strongly associated with VSC levels
8	Dryahina 201443	Czech Republic	GERD, case-control study	GERD N = 22, controls N = 24	Exhaled breath / SIFT-MS	Acetic acid /End expiratory conc.	Breath acetic acid is a useful biomarker of GERD and other conditions lowering pH of airway lining.
9	Snider 201844	US	BE (93.8% had GERD), prospective case-control	BE N = 32c, control N = 17	Saliva/ PCR	Oral microbiome /alpha and beta diversity	Oral microbiome in BE was markedly altered and distinguished BE
10	Yan 201645	China	GERD, case-control study	GERD N = 166, Control N = 72	Exfoliated tongue cell coatings/ rt-PCR	miR-203/miR-203 expression	miR-203 was significantly downregulated in GERD patients compared to healthy controls
11	Maddalo 201846	Italy	Phase III prospective case-control study	Case (N = 231) Controls N = 107)	Serum- ELISA	SCCA-IgM)	Patients with levels above cut off have a 33 times higher relative risk of developing BE or EAC.
12	Dosedelova 202047	Czech Republic	GERD, Case-control study	GERD N = 20, Control N = 12	Saliva/ capillary electrophoresis w/ capacitively coupled contactless conductivity detector	Salivary bicarbonate, phosphate, inorganic anions (CL-, NO2-, NO3-, SO4-, SCN-) organic anions (acetate, butyrate	Bicarbonate levels were significantly elevated in saliva of GERD patients
13	Thrift 201448	Australia	BE (models 1,2,3 included GERD frequency and duration in the questionnaire), case-control study	Definitive BE N = 141 Control N = 138	Serum/ electrochemiluminescence assay	Multibiomarker risk score, association with BE risk (odds ratio)	Multibiomarker score improved discrimination between BE and controls compared with using only GERD frequency and duration

Abbreviations: US, United States; UK, United Kingdom; GERD, Gastroesophageal Reflux Disease; NERD, Non-Erosive Reflux Disease; ERD, Erosive Reflux Disease; BE, Barrett’s Esophagus; EAC/EA, Esophageal Adenocarcinoma; WTC, World Trade Center; FDNY, Fire Department of New York; SRD, Study of Reflux Disease; ELISA, Enzyme-Linked Immunosorbent Assay; LC-MS, Liquid Chromatography Mass Spectrometry; SIFT-MS, Selected Ion Flow Tube Mass Spectrometry; rt-PCR, reverse transcriptase-Polymerase Chain Reaction; PPV, Positive Predictive Value; NPV, Negative Predictive Value; FC, Fold Change; VSC, Volatile Sulfur Compounds; ppb, parts per billion; mi-RNA, microRNA; SCCA-IgM, Squamous Cell Carcinoma Antigen-Immunoglobulin M Complex; ROC_AUC, Area Under the Receiver Operator Curve; TNF-α, Tumor Necrosis Factor-Alpha; IP-10, Interferon gamma-induced protein-10; IL, Interleukin; RR, Relative Risk; OR, Odds Ratio; CI, Confidence Interval.

Φ Conclusive GERD: LA, grade C and D erosive Esophagitis (n = 4), Long segment Barrett’s Mucosa (n = 1), Peptic strictures or Acid Exposure Time (AET) > 6% (n = 44). Evidence against GERD, group (n = 24): erosive esophagitis, AET < 4%, 40 reflux episodes, Reflux hypersensitivity (RH) (n = 10) or functional heart burn (FH) (n = 14).

Inconclusive GERD (n = 58): LA, grade A and B ERD (n = 42), AET, between 4% and 6% (n = 24), positive reflux symptom association (n = 22), reflux episode > 80 (n = 16), Low mean nocturnal baseline impedance (MNBI) (n = 33) and low postreflux swallow-induced peristaltic wave index (PSPWI) (n = 58).

^aFirst Author Year (Ref).

^bAt least one positive pepsin result was seen in 16 patients irrespective of GERD and LPR (80% both).

^cIncludes BE, without dysplasia N = 16, BE, with low grade dysplasia (LGD) N = 6, N = 5, Esophageal adenocarcinoma (EAC) N = 5.

Pepsin, A Classic Biomarker of Reflux Disease

N = 5 of the 13 studies used pepsin as a biomarker. Peptest, which detects salivary pepsin as low as 16 ng/mL using a lateral flow device, was used in n = 4 studies.34, 35, 36, 37 One study used Enzyme-Linked Immunosorbent Assay with a minimum detectable level of 0.93 ng/mL to quantify salivary pepsin (Table 1).³⁸

In a prospective blinded case-control study, a cut-off of 50 ng/mL of pepsin was evaluated using the area under receiver operating curve (ROC_AUC) analysis from 52 gastric samples and 54 sterile water samples.³⁴ Patients with GERD underwent endoscopy and wireless 48-hour pH monitoring. This study yielded a positive pepsin test prevalence of 22% (GERD) vs 12% (controls). There was a stepwise increase in the prevalence of positive pepsin with 24% positive with heartburn-only symptoms, 43% with abnormal pH, and 55% with endoscopic esophagitis. The positive and negative predictive values were calculated based on the disease definition of esophagitis and/or abnormal pH monitoring (Table 2) (Figure 2A).³⁴ Sensitivity ranged from 50% to 85%, with specificity ranging from 60% to 100%.34, 35, 36, 37^,49,50

Table 2.

Predictive Values of Biomarkers

Study	Biomarker	ROCAUC (95% CI)	Cutoffa	Sn %	Sp %	PPV %	NPV %	OR (95% CI)
Yuskel 201234	Salivary pepsin	-	50 ng/mL	50	92	85	68	-
Bor 201935	Salivary pepsin	-	16 ng/mL	75	-	78.6	-	-
Wang 201936	Salivary pepsin	-	75 ng/mL	85	60	82	65	-
Guo 202027	Salivary pepsin	0.76 (0.68–0.84)	4.21 ng/mL	76.36	63.41	-	-	-
Buas 201740	Multiple serum metabolites	SRD 0.64	-	-	-	-	-	-
Haider 201841	TNF-α C-peptide Fractalkine IP-10		C-peptide ≥ 360 pg/mL	-	-	-	-	GERD vs control: 2.08 (1.20–3.61)
			TNF-α ≥ 6 pg/mL					GERD vs control: 2.06 (1.15–3.70);
								BE vs control: 3.84 (1.23–12.03)
			Fractalkine ≥ 250 pg/mL					BE vs control: 3.42 (1.18–9.96)
			IP-10 ≥ 290 pg/mL					BE vs control: 4.47 (1.45–13.84)
Kim 201042	Halitosis	-	-	-	-	-	-
Dryahina 201443	Exhaled breath acetic acid	0.805	-	-	-	-	-
Snider 201844	Changes in oral microbiome	0.94 (0.81–1.00)		96.9	88.2	-	-
Yan 201645	miR-203	0.94 (0.90–0.7)	-	91.7	87.3	-	-
Maddalo 201846	Serum SCCA-IGM	0.799	56.6 AU/mL	91.5	75.4	85.8	84.4
Dosedelova 202047	Salivary bicarbonate, phosphate, inorganic anions (CL-, NO2-, NO3-, SO4-, SCN-) organic anions (acetate, butyrate)	-	-	-	-	-	-
Thrift 201448		Model 1 0.74 (0.69–0.80)	-	-	-	-	-
		Model 2 0.80 (0.75–0.85)	-	-	-	-	-	-
	IL-12p70, IL-6, IL-8, IL-10 leptin	Model 3 0.85 (0.80–0.89)	IL-12p70 0.77 pg/mL	-	-	-	-	Multibiomarker risk score	1	1.30 (0.51–3.27)
			IL-6 0.18 pg/mL
			IL-8 0.1 pg/mL
			IL-10 7.5 pg/mL						2	3.75 (1.44–9.78)
			Leptin 43 pg/mL						≥ 3	11.9 (4.06–34.9)

ABBREVIATIONS: SRD, Study of Reflux Disease; SN, Sensitivity; SP, Specificity; PPV, Positive Predictive Value; NPV, Negative Predictive Value; ppb, parts per billion; mi-miRNA, microRNA; SCCA-IgM, Squamous Cell Carcinoma Antigen-Immunoglobulin M Complex; ROC_AUC, Area Under the Receiver Operator Curve; TNF-α, Tumor Necrosis Factor-Alpha; IP-10, Interferon gamma-induced protein-10; IL, Interleukin; RR, Relative Risk; OR, Odds Ratio; CI, Confidence Interval.

^aCutoff determined as per individual manuscript methods.

Figure 2.

Synthesis of biomarker outcomes. (A) Predictive values of pepsin. (B) Area under the receiver operator characteristic curve (ROC_AUC) of biomarkers. (C) Odds ratio (OR) of biomarkers.

In another study, GERD had a similar positive Peptest prevalence of 80%.³⁵ A multicenter case-control study enrolled 1032 participants with GERD who received endoscopy and a Peptest.³⁶ The overall sensitivity of Peptest was 85% and 60% specificity. The authors acknowledge that there was poor selection of controls in some centers resulting in specificity as low as 37%–40% and suggest room for improvement by investigating the impact of smoking history.

Furthermore, Guo et al assayed salivary pepsin levels in patients with refractory GERD, using Enzyme-Linked Immunosorbent Assay.³⁸ Subjects were categorized into three groups (conclusive, inconclusive, and evidence against GERD) as per the Lyon consensus based on upper endoscopic findings, 24-h multichannel intraluminal impedance-pH monitoring (24-h pH-MII), and high resolution manometry. This study noted that salivary pepsin concentrations were significantly different among patients with conclusive GERD (8.2 ng/ml), inconclusive GERD (4.0 ng/ml), and evidence against GERD (2.4 ng/ml) (P < .001) (Table 1, Table 2 and Figure 2A).²⁷

Serum metabolites and Other Biomarkers of GERD, BE, and Esophageal Adenocarcinoma

To predict the risk of EA in patients with GERD, BE, an observational case-cohort study explored the metabolome in serum. In the study of reflux disease (n = 162), Table 1, nine metabolites were elevated in the BE vs GERD comparison group (study of reflux disease) (ROC_AUC 0.64; P < .05) (Table 2 and Figure 2B).⁴⁰

Similarly, in a study on the World Trade Center (WTC) particulate-exposed firefighters with obstructive airway disease (OAD), serum biomarkers were assessed. The study showed that serum TNF-α ≥ 6 pg/mL predicted both GERD and BE, C-peptide ≥ 360 pg/mL predicted GERD, Fractalkine ≥ 250 pg/mL, and IP-10 ≥ 290 pg/mL predicted BE. These biomarkers sampled prior to disease presentation showed strong predictive abilities (Table 2 and Figure 2C).⁴¹

Exhaled Breath Biomarkers to Distinguish Subtypes of GERD

In a case-cohort study, a Halimeter was used to measure volatile sulfur compounds in exhaled breath. Erosive mucosal changes were strongly associated with volatile sulfur compounds levels with mean Halimeter ppb levels of 191.85 vs 136.43 (P = .042) in ERD vs nonerosive reflux disease (Table 1).

Another case-control study compared the end expiratory concentrations of acetic acid in the exhaled breath condensate (EBC) of GERD patients with controls. Median acetic acid concentration was significantly higher in GERD compared to controls (85 ppbv vs 48 ppbv; p = 6 × 10^-5) (Table 1). Acetic acid as a diagnostic biomarker for GERD had an ROC_AUC of 0.805 (Table 2 and Figure 2B).⁴³

Oral Microbiome: A Screening Biomarker of BE

Reflux-related conditions including BE alter the esophageal microbiome.^51,52 The oral microbiome was compared in a case-control study in BE patients (n = 32) and controls (n = 17). Among controls, n = 10 (59%) had GERD and n = 6 (35%) were on PPI. Alpha diversity was no different from BE patients to controls (mean Shannon index: BE 2.73 vs controls 2.89; P = .10). At the phylum level, there was a significant increase in the relative abundance of Firmicutes (27.1 vs 14.6 %; P = .005) and a significantly decreased Proteobacteria (23.8 vs 34.5%; P = .02) in BE vs controls, respectively. Other notable differences included an increased relative abundance of Streptococcus, Veillonella, and Enterobacteriaceae in BE and several taxa (Neisseria, Lautropia, and Corynebacterium) in controls. Models with Lautropia, Streptococcus, and Bacteroidetes showed the greatest discrimination between BE and controls vs Lautropia alone; ROC_AUC 0.94 (95% confidence interval [CI] 0.85–1.00; P = .04) (Figure 2B and Table 2).⁴⁴

Additional Biomarkers

One study determined microRNA (miRNA) expression (miR-143, -145, -192, -194, -203, and -205) on exfoliated tongue cells across a discovery cohort (GERD n = 24, control n = 24).⁴⁵ Validated results showed significantly downregulated miR-203 in GERD (n = 142) as compared to healthy controls (n = 48), P < .0001; ROC_AUC 0.94 (95% CI: 0.90–0.97), with a sensitivity of 91.7% and a specificity of 87.3% (Figure 2B and Table 2).

The role of immunocomplexed squamous cell carcinoma antigen (SCCA-IgM) as a screening biomarker of BE and EAC, was determined in a phase III cancer screening biomarker development study (n = 213).⁴⁶ Median serum SCCA-IgM levels were higher in BE 90 U/mL (95% CI: 89.31–131.55; P < .0001) and EAC 76 U/mL (95% CI: 56.63–178.87; P < .0001) as compared to controls which included GERD patients 36.6 U/mL (95% CI: 40.32–68.28) and blood donors 41.5 U/mL (95% CI: 49.37–91.33) with a relative risk 33 (95% CI: 12.66–89.46). BE patients with long segment or dysplastic BE had SCCA-IgM levels significantly higher than those with short nondysplastic BE (P = .035) (Table 2).

Salivary anion levels were measured in GERD (n = 20) vs healthy controls (n = 12) using background electrolyte with capillary electrophoresis.⁴⁷ They found a significant difference in mean bicarbonate concentration in GERD vs controls (8.1 mM vs 5.7 mM; P = .004). No difference was reported in the mean concentrations of phosphate (6.4 mM vs 5.7 mM; P = .272) (Table 1).

Multibiomarker Model of BE Risk

A case-control study of 279 subjects compared the accuracy of three risk prediction models that used demographic and clinical variables (Table 1).⁴⁸ Model 1 included GERD frequency and duration; Model 2 included GERD frequency, duration, age, sex, race, waist-to-hip ratio, and H-pylori status; and Model 3 included all variables in model 2 and used multiple serum biomarkers (IL-12p70, IL6, IL8, IL10, and leptin). The addition of risk scores associated with BE and multibiomarker risk scores improved the AUC compared to Model 1 significantly (ROC_AUC of 0.85 [95% CI 0.80–0.89; P = .01]).

Discussion

GERD a highly prevalent disease which affects 20% of the US population⁵³ and has a heterogeneous biomarker profile. GERD diminishes QoL, productivity, and accounts for about 5% of outpatient visits. Furthermore, refractory reflux is associated with anxiety, depression, and even once weekly episodes of GERD were detrimental to QoL.8, 9, 10

Therefore, GERD diagnosis and management is highly relevant to the health and wellbeing of a significant portion of our patient population. While GERD can be a clinical diagnosis (ie, symptomatic heartburn and regurgitation) as per the Montreal consensus statement both invasive and noninvasive biomarkers are often used.⁵⁴ GERD detection with endoscopy, ambulatory pH testing, and other invasive testing poses a rare but potential risk and contributes to a higher economic burden.⁴ Sensitivity of endoscopy may be limited.⁵⁵ Conversely, those with endoscopic evidence of reflux may be completely asymptomatic.^56,57 Invasive adjunctive testing to aid in disease detection pose rare but real medical risk, contribute to a higher economic burden, but little benefit is gained as the methods are plagued with poor sensitivity.⁴ Sensitivity of endoscopy is limited because many patients with GERD do not have mucosal injury.⁵⁵ Conversely, those with endoscopic evidence of reflux may be completely asymptomatic.^56,57 Therefore, optimizing biomarkers and specifically enriching for noninvasive biomarkers of GERD were the focus of our review.

Pepsin was the most commonly studied noninvasive biomarker of GERD in our systematic review. Recent American College of Gastroenterology guidelines have raised concerns about pepsin testing due to poor diagnostic reliability and its inability to distinguish between patients with extraesophageal symptoms.^15,22 However, based on the finding of papers highlighted in our manuscript, pepsin likely has some diagnostic utility that needs to be further studied in targeted subpopulations, additional compartments such as EBC, or in a multivariate biomarker model.⁵⁸

EBC is a window into the aerodigestive compartment and is composed of droplets of airway lining fluid. It has emerged as a target for noninvasive biomarkers of GERD. Specifically, it has the potential to address unmet medical needs by expanding the portfolio of noninvasive assays to diagnose erosive changes and multiple coexisting pathological mechanisms of GERD.⁵⁹ Compounds identified in EBC include volatile sulfur compounds, histamine, adenosine, ammonia, and leukotrienes.⁶⁰ The identified compounds are of biologically plausible since histamine stimulates cells in the stomach lining to produce hydrochloric acid. H₂-blockers a common treatment of GERD competes with histamine for H₂-receptors on the stomach’s parietal cells and thereby depresses the production of hydrochloric acid.⁶¹ Similarly, adenosine has been found to regulate acetylcholine release, which stimulates the proton pumps.⁶²

The host-gut microbiome interaction has been an area of active investigation in several gastrointestinal disorders and is particularly of interest in light of the clinical relevance of EBC highlighted above.⁶³ Gut microbiota are linked to the regulation of the innate immune system and have been linked to markers found in EBC.^59,64, 65, 66, 67 Alteration in the microbiota and bacterial products can result in the activation of pathways involved in inflammation. Changes in esophageal microbiota can lead to the production of large amounts of bacterial components like lipopolysaccharide which can delay gastric emptying via COX1/2 and predispose to GERD. The role of Lautropia (which was one of the organisms identified as predictive of BE) is analogous to that of Clostridia in the colon and a decrease in bacterial load leads to the proliferation of other proinflammatory bacteria. In another study, a decrease in the concentration of Lautropia was seen in patients with periodontitis and successful treatment resulted in a subsequent increase in lautropia.⁶⁸ This taxonomic difference was used by Snider et al. using oral swabs and 16S rRNA gene sequencing in BE patients with GERD.^44,69

Biomarkers of GERD and BE in the FDNY WTC Exposed Cohort

A prime example of the importance of the gut/lung axis was found in the WTC-exposed first responder cohort.⁷⁰ WTC-particulate matter exposure is associated with OAD, GERD, and BE.71, 72, 73 WTC-exposed firefighters with OAD had a three times higher risk of developing GERD.⁷⁴ Approximately 44% of WTC responders developed GERD symptoms by 2005, which is 8.2 times its pre-9/11 prevalence.⁷⁵ We identified serum biomarkers of GERD and BE in a cohort of nonsmoking firefighters with WTC exposure.^41,76 Greater odds of developing GERD were associated with elevated TNF-α and C-peptide, whereas BE was associated with TNF-α, Fractalkine, and IP-10 (Table 2).⁴¹

Systematic Reviews by their Very Nature are Subject to Limitations and Inherent Biases

The heterogeneity of baseline characteristics, diagnostic criteria, standards for comparison of MII-pH testing, manometry, 24-hour reflux monitoring, questionnaires, endoscopy, and lack of validation limits the interpretability. This underlines the importance of developing diagnostic biomarkers of GERD and Barrett’s and invites future studies for developing standardized methods of diagnosis. Although several studies that were a focus of our review used Pepsin as a biomarker, current guidelines do not recommend pepsin testing for evaluation of patients with reflux and limit further clinical interpretation.¹⁵ While our review focused on noninvasive biomarkers of reflux disease, we understand that optimal diagnostic testing would be an integration of invasive and noninvasive biomarkers. The studies included in this review did not distinguish between refractory GERD and refractory GERD symptoms.⁵⁷ Finally, risk of bias was high in most studies included in this review. This adds additional importance to all future work, in that the clinically relevant noninvasive biomarkers of GERD and associated conditions are needed.

Conclusion

Studies identified include multiOmic, multicompartmental noninvasive biomarkers of GERD. This further highlights the fact that several pathways are biologically active in GERD. However, due to study limitations and variable controls, further validation studies are warranted to ascertain the reliability and accuracy of these biomarkers.

Future Plans

Our future work will focus on validating the previously discovered biomarkers of WTC-aerodigestive disease in longitudinally phenotyped WTC-exposed cohorts. We will also develop novel, noninvasive disease phenotyping of premalignant diseases such as BE and identify potential targeted therapeutics to improve care; ClinicalTrials.gov #NCT05216133.