Pathogenesis

GSE is an immunologic disorder that occurs in genetically susceptible hosts. Environmental influences are important as well. The familial nature of the disease was originally established in a study of 17 probands and their families, who underwent biopsies of the small bowel.⁷ An increased incidence of GSE was found in first-degree relatives of symptomatic patients. Studies have documented that 10% to 20% of asymptomatic first-degree relatives have mild histologic changes, such as increased intraepithelial lymphocytes (IELs) without villous blunting, or have mild mucosal lesions.^7,19,20 In such a setting, the decision to institute a gluten-free diet depends on a variety of factors.

Genetic studies have established the strong association with major histocompatibility complex class II, with as many as 95% of patients carrying either the human leukocyte antigen HLA-DQ2 allele (the majority) or the HLA-DQ8 allele.^21–25 However, these genetic phenotypes do not explain the pathogenesis entirely, because 30% of the normal population carry the DQ2 haplotype without disease, and not all patients with GSE carry this specific phenotype.^21,23 Studies have identified at least seven non-HLA genes associated with an increased risk of GSE, including CCR3, and the interleukin locus IL2/IL21. Some of the identified genes are also common in type 1 diabetes and rheumatoid arthritis, suggesting that they impart a generalized increased risk of autoimmunity.^26–29 The reader is referred to several reviews on the genetics of GSE.^{26,27,30–32}

In the proper genetic environment, exposure to the prolamin fractions of gluten found in wheat, rye, and barley (gliadin, secalin, and hordein, respectively), results in a local immune response.^8,33 Additional environmental components may be related to previous infections.^34–36 For example, the E1b protein of adenovirus 12 has been shown to contain amino acid sequence homology with α-gliadin antibodies, and T cells that recognize these viral proteins cross-react with gliadin. The crucial role of intestinal tissue transglutaminase has also been elucidated. Tissue transglutaminase (tTG), a ubiquitous enzyme in human tissues, catalyzes the formation of cross-links between glutamine and lysine residues in substrate proteins.³⁷ tTG is believed to stabilize extracellular matrix molecules in the setting of mucosal injury and granulation tissue. In 1997, Dieterich and colleagues demonstrated that dietary gliadin, which is rich in the amino acid glutamine, serves as a substrate for tTG.³⁷ It is thought that the resulting cross-linked molecules (gliadin-gliadin or gliadin-tTG) become antigenic epitopes for a subsequent immune response. Of the more than 50 T cell–stimulating epitopes in gluten proteins, a 33-mer peptide that may be the primary initiator of the inflammatory response in GSE has been identified.³⁸ Investigations into the role of tTG in GSE have led to improved diagnostic tests (described later).³⁹

The immune response to dietary gluten involves both antibody- and cell-mediated injury. Antibody-induced injury may occur via antigen-antibody complex deposition with complement activation and also by the induction of cell-mediated cytotoxicity. Activated mucosal T cells have been shown to cause epithelial injury, probably through the release of cytokines, including IL15.^33,40,41

The clinical diagnosis of GSE depends on detection of the appropriate combination of symptoms, laboratory evidence of malabsorption, and the presence of serum autoantibodies. New and increasingly accurate serologic tests continue to be added to the “diagnostic tool kit” for GSE.⁴² These include the enzyme-linked immunosorbent assay for immunoglobulin A (IgA) anti-tTG antibodies, which has a high degree of sensitivity (77% to 100%) and specificity (91% to 100%). tTG antibody determination has become the serologic test of choice for GSE, largely replacing antigliadin and antiendomysial antibody tests.^43–45 The antiendomysial antibody (EMA) test is as sensitive and specific as the anti-tTG test with human recombinant protein, but the immunofluorescence EMA test is labor intensive and relies on a more subjective interpretation than the anti-tTG determination does.⁶ Today, the EMA is often used as a confirmatory test when tTG test results or clinical or biopsy findings are ambiguous.

Traditional antigliadin antibody testing no longer has a place in GSE diagnosis because of its lack of specificity. However, a new generation of antigliadin antibodies, known as deamidated gliadin peptides (DGP), are being used in a promising IgG-based test that has shown high concordance with both tTG and EMA tests, with possibly greater sensitivity in children and in early disease.^5,42,46–48 This test also has the advantage of detecting GSE in IgA-deficient patients.

HLA testing to detect susceptible HLA phenotypes (DQ2 and DQ8) has an increasing role in the diagnostic workup of GSE because the absence of DQ2 or DQ8 virtually excludes the diagnosis.^49,50 Finally, transglutaminase-specific IgA deposits can be found by immunofluorescence in frozen sections of biopsy specimens obtained from small-intestinal mucosa in patients with GSE, even those with normal to near-normal histology.^51–53 Although this technique is not currently in widespread use, it could become more prevalent in the future, especially in confirming histologically mild disease.⁵³ For a more detailed discussion of the current approach to clinical diagnosis and management of GSE, several excellent reviews are available.^{5,42,45,48–50,54,55}

Pathologic Features

Background

In Western countries before 1990, GSE was a diagnosis that was always made with ease and confidence on the basis of small bowel biopsies. The pathologic diagnosis required flat or nearly flat small bowel mucosa, usually accompanied by increased IELs and lamina propria inflammation in biopsy specimens obtained distal to the duodenal bulb. A diagnosis of untreated GSE was not normally considered if the biopsy specimen did not show significant loss of villous architecture.

Since the early 1990s, several changes in the understanding of GSE have altered the diagnostic algorithm for this disease and eliminated the gold standard status of small bowel biopsies.^5,42,56 These changes include (1) recognition that latent or occult (minimally symptomatic) GSE in adults represents a large, previously unappreciated disease population^13,14,56; (2) revision of histologic criteria to include minimal inflammatory changes with intact or only mildly abnormal architecture as a part of the histologic spectrum of GSE^14,56–58; and (3) the discovery that tTG is the target autoantigen of antibodies in patients with GSE and the subsequent development of a highly accurate diagnostic test for anti-tTG antibodies, which can now be used in combination with the tests described previously.^37,43,44,59

These developments have had a great impact on pathologists, who can now correlate minimal histologic findings with objective clinical and serologic evidence of GSE. They have also decreased the need for reliance on small bowel biopsy findings, which are often subject to interpretive challenges because of orientation and nonspecificity of changes. Today, the sophisticated pathologist and clinician must understand that evaluating patients for GSE may or may not include the need for mucosal biopsy.⁴² Small-intestinal mucosal biopsies can always be used to help fulfil diagnostic criteria, or they may be reserved for patients in whom results are inconclusive, the caveat being that histologic findings in the latter category of patients may also be inconclusive (Box 16.2).

Gross Pathology

In a landmark study, Rubin and associates described the appearance of normal duodenal mucosa on biopsy samples with the use of a hand lens and showed that the mucosa is characterized by numerous slender villi with a delicate capillary network that floats in formalin like “the tentacles of a sea anemone.”⁶⁰ In contrast, mucosa specimens from patients with GSE have a barren, stubby surface, without normal villi, containing widely spaced, irregular capillaries (Fig. 16.1). However, endoscopic findings in patients with GSE are subtle and often unreliable. A flat, scalloped appearance of the duodenal mucosa, most likely reflecting loss of villi, may be seen.⁶¹ One endoscopic study found that a reduction of folds, scalloping, mosaic pattern, and nodular mucosa were sensitive but not specific endoscopic findings in GSE, because they were also seen in some dyspeptic patients without GSE.⁶² A meta-analysis of studies testing the diagnostic sensitivity and specificity of video capsule endoscopy in GSE reported that the images seen were sufficient to correctly diagnose the condition.⁶³ However, there are insufficient data on the performance of this tool in the mild histologic forms that are commonly at issue today. Confocal laser microscopy shows promise as an endoscopic technique that can recognize villous irregularity and blunting, with the caveats that interobserver variability is high and the technique is not yet widely available.⁶⁴

Microscopic Pathology: Biopsy Strategy

GSE is a disease of the proximal small intestine in most patients, with severity usually greatest in the duodenum and proximal jejunum. The ileum may be involved in severe cases, but it is not a reliable site for biopsy diagnosis.^61,65–67 It is now widely recognized that many patients with GSE do not have diffusely abnormal duodenal histology, but instead show mild and patchy involvement in duodenal mucosal biopsies. The duodenal bulb, once strictly avoided in the evaluation of GSE because of its susceptibility to peptic injury and prominent Brunner glands, is now a recommended biopsy site because it is reliably involved in patients with patchy disease.^68,69 In a pediatric study, 16% of children had patchy villous atrophy, but all of them had involvement of the duodenal bulb, and in four patients the bulb was the only site of abnormality.⁶⁸ Duodenal bulb biopsies may also be more sensitive in patients following a low-gluten diet.⁷⁰ Further evidence supporting the validity of the duodenal bulb as a diagnostic site is found in the study by Walker and co-workers (2010), in which paired IEL counts were identical in the duodenal bulb and in the descending (second) duodenum in GSE patients.⁷¹ Based on studies in both adults and children, it appears that the most reliable biopsy protocol to detect all cases of GSE is a three- (or preferably five-) biopsy regimen that includes the duodenal bulb, the proximal duodenum, and the distal duodenum.^68,69,72,73

Once the small bowel biopsy specimen is received in the laboratory, accurate evaluation in cases of suspected GSE requires proper orientation in tissue blocks to assess mucosal architecture and to avoid overinterpretation of short villi (which are invariably seen in poorly oriented sections) as abnormal. Although specimens are rarely perfectly oriented, an attempt should be made to find three or four well-oriented villi in a row to assess architecture. Similarly, now that duodenal bulb biopsies are requested in this setting, care must be taken to distinguish peptic injury (neutrophils, gastric surface metaplasia, and surface injury) and villous blunting resulting from mucosal Brunner glands from GSE. IEL counts should be helpful in making this distinction.

Histology of Untreated Disease

In normal small bowel mucosa, the ratio of villus height to crypt depth is between 3 : 1 and 5 : 1, depending on the anatomic location (Fig. 16.2). Epithelial cells lining the villi contain basally located nuclei with abundant mature cytoplasm and a preponderance of absorptive cells admixed with goblet cells. A faint density, representing the microvillus brush border, can be appreciated on hematoxylin and eosin (H&E)-stained tissue sections. Studies have confirmed that there are approximately 20 lymphocytes per 100 epithelial cells in healthy people, with minor variations.^74–78 The lamina propria normally contains a mixture of plasma cells, lymphocytes, and occasional eosinophils. Each of the three mucosal components (i.e., architecture, epithelium, and lamina propria) should be carefully examined in cases of suspected GSE.

The pathology of GSE is described here in conjunction with a grading scheme that can be used in pathology reports (Table 16.1). At the time of this writing, the Marsh-Oberhuber classification is being used regularly in Europe but has not gained wide acceptance in the United States for routine use in pathology reports. Inclusion of the Marsh-Oberhuber subtype requires prior knowledge (e.g., by serology) that the patient has GSE, and biopsy requisition forms often do not provide that information.^56,58 In addition, differences between types 0, 1, and 2 in the Marsh scheme are subtle, so there is a high degree of inter-observer variability among pathologists. To avoid these issues, it is reasonable to use the term mucosal lesion or villous blunting,⁷⁹ as detailed in the following discussion, with a modifier (i.e., mild, moderate, or severe) to reflect the degree of villous blunting.

B, Mild mucosal lesion (or mild villous blunting), see note.
Note: The findings are nonspecific but may represent GSE in the proper clinical setting. Correlation with serologic studies is indicated. (Include differential diagnosis as appropriate.)