Gastroesophageal reflux disease (GERD) is caused by the reflux of gastric contents into the esophagus. GERD affects 3% to 4% of the population, and the incidence of GERD and its complications have increased in past decades. The annual cost of managing the disease is approximately $9 billion in the United States.1 Reflux esophagitis is the pathologic state resulting from GERD. Given the low pH of the gastric contents, duration and frequency of the exposure, and the strength of the mucosal defense mechanisms, the mucosa is variably damaged.
The diagnosis of GERD is based on the combination of clinical symptoms, endoscopic findings, pH monitoring, and histologic changes. However, GERD is a heterogeneous disease, and the findings in these tests do not always correspond. For example, some patients report typical symptoms but lack other positive tests, whereas other patients are asymptomatic yet exhibit typical endoscopic and histologic features of GERD. The sensitivity of reflux symptoms for diagnosing GERD is only 55%, if endoscopy is considered as the gold standard.2 In a systematic review of approximately 15 publications, the sensitivity of a “trial” of 1 to 4 weeks of proton pump inhibitor therapy for diagnosing GERD was 78% and the specificity was 54%.2 Long-term proton pump inhibitor therapy has been shown to normalize the histologic parameters such as dilated intercellular spaces (DIS), basal cell hyperplasia, and elongation of the papillae.3,4
Even though most patients have classic endoscopic findings (erythema, mucosal edema, erosions, or ulcers), many patients with typical reflux symptoms have normal or nearly normal endoscopic findings [nonerosive reflux disease (NERD)].2,5,6 The NERD spectrum includes patients with documented reflux, increased acid contact, and esophageal hypersensitivity.6,7 Although NERD is quite common, the endoscopy is 90% to 95% specific for GERD.2,5
Twenty-four–hour ambulatory pH monitoring is specific (85% to 90%), but has a low sensitivity (60%) for reflux.8,9 Similarly, histologic analysis has limited sensitivity, specificity, and reproducibility, each discussed in detail below. Moreover, each of these tests has significant limitations, and at present there is no gold standard method for the diagnosis of GERD.
Reflux disease is associated with a constellation of histologic features, representing changes from secondary to acid injury and mucosal healing. None of these features is pathognomonic and the final diagnosis remains a clinicopathologic correlation.10
The features most often used for diagnosis are related to epithelial proliferation after the initial injury: basal cell hyperplasia and elongation of the lamina propria papillae. These features were initially described by Ismail-Beigi (1970)11 and are still considered important histologic features of reflux disease today.
Basal Cell Hyperplasia
Basal Cell Hyperplasia is most frequently defined as a basal layer representing more than 15% of the total thickness of the epithelium (Fig. 1). The basal layer of the squamous epithelium comprises smaller cells with round nuclei and basophilic cytoplasm. It typically represents the reserve stratum from which the epithelium regenerates. Although pathologists easily recognize the basal layer, the upper limit of the basal layer can be difficult to define in a reproducible fashion, yet is critical to the determination of whether this compartment represents less than or greater than 15% of the total epithelial thickness. One useful definition of the uppermost limit of the basal zone is the point at which the majority of epithelial cell nuclei is separated by a distance of less than the diameter of 1 nucleus.12 Importantly, this assessment should not be made near the vascular papilla and requires reasonably well-oriented biopsies in which the entire epithelial thickness is visible.
Basal-zone hyperplasia can be seen in squamous epithelial injury of many forms and thus is not a specific feature of reflux. The specificity can be as low as 45%.13
It is most frequently defined as papillae extending more than two-third of the distance to the epithelium surface (Fig. 2) and seems to correlate with reflux severity.14 As with basal cell hyperplasia, assessment of papilla elongation requires reasonably well-oriented specimens in which the entire epithelial thickness and the length of the papilla are visible for assessment. The sensitivity of biopsy varies with biopsy location but ranges from 30% to 60%; the reported specificity is 80%.13
Inflammatory Cell Infiltrates
Intraepithelial eosinophils, neutrophils, and lymphocytes are part of the inflammatory response triggered by reflux-induced injury. Before the 1970 description of basal-zone hyperplasia,11 inflammation was the most important criterion for the diagnosis of reflux esophagitis. Today, inflammation of any type (lymphocytes, eosinophils, and neutrophils) is more specific than sensitive for the diagnosis of reflux esophagitis.15 Few intraepithelial lymphocytes and scattered Langerhans cells can be seen in normal adults and are increased in some patients with reflux (Fig. 3), although they do not have any independent diagnostic significance.16,17 Most are cytotoxic T cells, sometimes exhibiting irregular nuclear contours when migrating through the epithelium.17,18
The accepted number of normal intraepithelial eosinophils in the esophagus has been debated. Frierson19 noted that the eosinophils are typically absent whereas others stated that under normal conditions scattered eosinophils can be seen, usually <5 per high power field.16 It is likely that part of the confusion lies in variable definitions of normal control subjects and GERD patients, and hopefully further clinical validation of this feature and others will shed further light on “normal” cut-off values. Increased intraepithelial eosinophils (Fig. 4) are a very useful criterion in cases of poor biopsy orientation but it is not sensitive, as only 20 to 40% of patients with reflux esophagitis will show this feature.13,20,21 In selected patient populations, eosinophils are a relatively specific feature of reflux esophagitis (90% specific).13 However, eosinophilic esophagitis (EE), infections, and pill-induced esophagitis typically present with the increased eosinophils. Indeed, EE constitutes an important and sometimes difficult differential diagnosis. Recent consensus recommendations on the diagnosis of EE recognize the clinical and histologic overlap between EE and GERD, emphasizing the clinicopathologic correlation of symptoms; high numbers of eosinophils (greater than 15 eosinophils per high power field), and exclusion of GERD by high-dose proton pump inhibitor therapy or pH monitoring.22
Neutrophils are thought to be the most specific inflammatory cell type among the group, although it is also the least sensitive, seen in only 10% to 40% of the cases (Fig. 5).16,20,23 Neutrophils, mucosal erosions (Fig. 6), and ulcers (Fig. 7) tend to be associated with severe GERD and usually some degree of mucosal erosion/ulceration are seen at endoscopy.13,23,24 Particularly in this subgroup of patients, other ulcerating conditions (infections, pill esophagitis) should be excluded.
Dilated Intercellular Spaces
DIS (Fig. 8) have been described in patients with erosive and nonerosive GERD.25–29 This feature was initially described by Hoopwood et al,29 using transmission electron microscopy and is defined as an increase in the spaces between squamous cells (>0.47 to 2.4 μm depending on the study), predominantly in the basal layer.5,25–28 On the basis of light microscopy studies, DIS is characterized by irregular intercellular spaces with an uneven separation of cell membranes and stretching or detachment of desmosomes.27 A more practical approach using light microscopy has transformed this new marker into a useful and promising criterion for early injury in GERD. This feature is related to loss of tight junctions between squamous cells, resulting in increased paracellular permeability that may facilitate leaking of the acid through the mucosa and direct contact with terminal dendritic processes of underlying sensory neurons in the epithelium.30–32 Slight acidification of intercellular spaces can trigger symptoms, potentially explaining the occurrence of typical GERD symptoms in the absence of an endoscopic lesion.33–35 The prevalence of DIS in GERD varies from 67% to 94% depending on the presence or absence of clinical symptoms, endoscopic lesions, and pH monitoring abnormalities.13
Although many histologic findings have been described in patients with GERD, the minimum criteria for diagnosis remain unknown. Does 1 abnormality suffice for a diagnosis of GERD? Must the finding be diffuse, or do focal abnormalities qualify? And how “abnormal” does that finding have to be (how much basal cell hyperplasia or papilla elongation? how many eosinophils?)? With increased recognition of the clinical heterogeneity of GERD (asymptomatic disease and frequency of NERD), the histologic criteria must be retested and validated in light of the most advanced clinical information. Rigorous clinical studies with well-characterized normal control subjects and study patients are required to establish minimum cut-offs for the histologic diagnosis of disease.
Zentilin et al13 have proposed a scoring system that takes multiple possible histologic abnormalities into account. Using a receiver operator characteristic curve analysis, a score of 2 was identified as the optimal cut-off value for separating GERD patients from controls.13 Interestingly, the authors noted that mild changes were often seen at the squamocolumnar junction (SCJ) in controls, and that the use of a composite score helped to distinguish these mild abnormalities from GERD.
Biopsy Site Location
It is frequently asserted that basal cell hyperplasia and papilla elongation represent normal findings when found in the distal most 2-cm of the esophagus, presumably related to “physiologic” episodes of reflux. However, some researchers have found biopsies from the SCJ to be more sensitive than more proximal biopsies, albeit less specific.13 It is worth noting that Zentilin et al13 used higher cut-off values for basal-zone hyperplasia and papilla elongation in biopsies obtained at the SCJ than for those obtained at 2 cm and 4 cm proximal to the junction (less than 20% vs. less than 15% for basal cell thickness, less than 66% vs. less than 50% for papilla elongation). These data call into question whether features of epithelial hyperplasia seen in biopsies from the SCJ represent normal findings or mild abnormalities that can contribute to a diagnosis of reflux when additional abnormalities are present, or if different normal cut-off values should be applied in biopsies from the SCJ.
Not only does there seem to be a variation in the features at and proximal to the SCJ, but there also seems to be circumferential variation in the severity of reflux-related abnormalities. In SCJ biopsies from patients with NERD, basal cell thickness, and DIS were more prominent at the 3 o'clock quadrant of the distal esophagus than the 9 o'clock position.36 Further, in biopsies from the SCJ, NERD patients and controls differed only in DIS at the 3 o'clock position, and endoscopic findings were also more frequent at the 3 o'clock position. However, biopsies taken at 1 to 2 cm proximal to the SCJ demonstrated significant differences with respect to basal cell thickness, papilla elongation, and DIS among patients and controls, suggesting that these more proximal biopsies have the greatest potential to discriminate patients with and without GERD.36
Are Pathologists Reproducible in the Diagnosis?
It is difficult to assess how reproducible pathologists are in the diagnosis of GERD or the assessment of its component histologic features. This is, in part, due to the scarcity of publications on sufficiently large patient populations assessing interobserver agreement since the entry of κ statistics into the medical literature.9,24,37–39 Zentilin et al13 in 2005 published an interesting study in over 100 patients and their interobserver agreement (among the 2 experienced pathologists from the same department) was 90.7% (κ=0.89), considering 3 histologic criteria: basal layer thickness, papilla elongation, and DIS. As a field, we are probably far from this level of agreement.
After reviewing the extensive literature in this topic, we can conclude that GERD is a common, yet heterogeneous disease. Diagnosis is based on a combination of symptoms, endoscopy, pH monitoring, and histologic findings. However, although many diagnostic tools are applied, the correlation between these tests is variable, and there is no gold standard for diagnosis. Hence, a combination of data from multiple tests seems to be the most useful approach to accurate diagnosis. It is vital for pathologists to understand the spectrum of the disease and the strengths, weaknesses, and potential pitfalls of each diagnostic test to maximize the diagnostic yield of esophageal mucosal biopsies.
1. Sandler RS, Everhart JE, Donowitz M, et al. The burden of selected digestive diseases in the United States. Gastroenterology. 2002;122:1500–1511.
2. Moayyedi P, Talley NJ. Gastro-esophageal reflux disease. Lancet. 2006;367:2086–2100.
3. Amstrong D, Vieth M, Fiocca R, et al. Esomeprazole therapy and esophageal histology in endoscopy-negative reflux disease (ERND) – The CHEER study. Gastroenterology. 2004;124(suppl 1):A416.
4. Kiesslich R, Kausler S, Vieth M, et al. Minimal change esophagitis: prospective comparison of endoscopic and histological markers between patients with non-erosive reflux disease and normal controls using magnifying endoscopy. Dig Dis. 2004;22:221–227.
5. Dent J. Microscopic esophageal mucosa injury in nonerosive reflux disease. Clin Gastroenterol Hepatol. 2007;5:4–16.
6. Long JD, Orlando RC. Nonerosive reflux disease. Minerva Gastroenterol Dietol. 2007;53:127–141.
7. Fass R. Erosive esophagitis and nonerosive reflux disease (NERD). Comparison of epidemiologic, physiologic, and therapeutic characteristics. J Clin Gastroenterol. 2007;41:131–137.
8. Kahrilas PJ, Quigley EMM. Clinical esophageal pH recording: a technical review for practice guidelines development. Gastroenterology. 1996;110:1982–1996.
9. Schindlek NE, Wiebecke B, Klauser AG, et al. Diagnostic value of histology in non-erosive gastro-esophageal reflux disease. Gut. 1996;39:151–154.
10. Takubo K, Honma N, Aryal G, et al. Is there a set of histologic changes that are invariably reflux associated? Arch Pathol Lab Med. 2005;129:159–163.
11. Ismail-Beigi F, Horton P, Pope CE. Histological consequences of gastroesophageal reflux in man. Gastroenterology. 1970;58:163–174.
12. Mitros FA. Atlas of Gastrointestinal Pathology. Philadelphia: J. B. Lippincott; 1988:1.2–1.12.
13. Zentilin P, Savarino V, Mastracci L, et al. Reassessment of the diagnostic value of histology in patients with GERD, using multiple biopsy sites and an appropriate control group. Am J Gastroenterol. 2005;100:2299–2306.
14. Jonson LF, Demeester TR, Haggitt RC. Esophageal epithelial response to gastroesophageal reflux. A quantitative study. Am J Dig Dis. 1978;23:498–509.
15. Vieth M, Peitz U, Labenz J, et al. What parameters are relevant for the histological diagnosis of gastroesophageal reflux disease without Barrett's mucosa? Dig Dis. 2004;22:196–201.
16. Haggitt RC. Histopathology of reflux-induced esophageal and supraesophageal injuries. Am J Med. 2000;108:109S–111S.
17. Wang HH, Mangano MM, Antonioli DA. Evaluation of T-lymphocytes in esophageal mucosal biopsies. Mod Pathol. 1994;7:55–58.
18. Mangano MM, Antonioli DA, Schnitt SJ, et al. Nature and significance of cells with irregular nuclear contours in esophageal mucosal biopsies. Mod Pathol. 1992;5:191–196.
19. Frierson HF Jr. Histology in the diagnosis of reflux esophagus. Gastroenterol Clin North Am. 1990;19:631-644.
20. Tummala V, Barwick KW, Sontag SJ, et al. The signficance of intraepithelial eosinophils in the histologic diagnosis of gastroesophageal reflux. Am J Clin Pathol. 1987;87:43–48.
21. Brown LF, Goldman H, Antonioli DA. Intraepithelial eosinophils in endoscopic biopsies of adults with reflux esophagitis. Am J Surg Pathol. 1984;8:899–905.
22. Furuta GT, Liacouras CA, Collins MH, et al. Eosinophilic esophagitis in children and adults: a systematic review and consensus recommendations for diagnosis and treatment. Gastroenterology. 2007;133:1342–1363.
23. Frierson HF. Histological criteria for the diagnosis of reflux esophagitis. Pathol Annu. 1992;27(Pt 1):87–104.
24. Collins BJ, Elliott H, Sloan JM, et al. Oesophageal histology in reflux oesophagitis. J Clin Pathol. 1985;38:1265–1272.
25. De Hertogh G, Ectors N, Van Eyken P, et al. Review article: the nature of esopharyngeal injury in gastro-esophageal reflux disease. Aliment Pharmacol Ther. 2006;24:17–26.
26. Van Malenstein H, Farre R, Sifrim D. Esophageal dilated intercellular spaces (DIS) and nonerosive reflux disease. Am J Gastroenterol. 2008;103:1021–1028.
27. Solcia E, Villani L, Luinetti O, et al. Altered intercellular glycoconjugates and dilated intercellular spaces of esophageal epithelium in reflux disease. Virchows Arch. 2000;436:207–216.
28. Villanacci V, Grigolato PG, Cestari R, et al. Dilated intercellular spaces as marker of esophageal reflux: histology, semiquantitative score and morphometry upon light microscopy. Digestion. 2001;64:1–8.
29. Hoopwood D, Milne G, Logan KR. Electron microscopic changes in human oesphageal epithelium in oesophagitis. J Pathol. 1979;129:161–167.
30. Orlando RC, Powell DW, Carney CN. Pathophysiology of acute acid injury in rabbit esophageal epithelium. J Clin Invest. 1981;68:286–293.
31. Tobey NA, Carson JL, Alkiek RA, et al. Dilated intercellular spaces: a morphological feature of acid reflux-damaged human esophageal epithelium. Gastroenterology. 1996;111:1200–1205.
32. Rodrigo J, Hernandez DJ, Vidal MA, et al. Vegetative innervation of the esophagus III. Intraepithelial endings. Acta Anatomica. 1975;92:242–258.
33. Ravelli AM, Villanacci V, Ruzzenent N, et al. Dilated intercellular spaces: a major morphological features of reflux. J Pediatr Gastroenterol Nutr. 2006;42:510–515.
34. Kollarik M, Undem BJ. Mechanisms of acid-induced activation of airway afferent nerve fibers in guinea pig. J Physiol. 2002;543:591–600.
35. Steen KH, Reeh PW. Sustained graded pain and hyperalgesia from harmless experimental tissue acidosis in humans. Neurosci Lett. 1993;154:113–116.
36. Edebo A, Vieth M, Tam W, et al. Circumferential and axial distribution of esophageal mucosal damage in reflux disease. Dis Esophagus. 2007;20:232–238.
37. Seefeld U, Krejs GJ, Siebenmann RE, et al. Esophageal histology in gastroesophageal reflux. Morphometric findings in suction biopsies. Dig Dis Sci. 1977;22:956–964.
38. Kasapidis P, Xynos E, Mantides A, et al. Differences in manometry and 24-h ambulatory pH-metry between patients with and without endoscopic or histological esophagitis in gastroesophageal reflux disease. Am J Gastroenterol. 1993;88:1893–1899.
39. Narayasi RI, Burton MO, Young GS. Utility of esophageal biopsy in the diagnosis of non-erosive reflux disease. Dis Esophagus. 2003;16:187–192.