Indicators of Nonresponsive Celiac Disease
Indicators of Nonresponsive Celiac Disease
There exists an unmet diagnostic need for serological assays to monitor the recovery of CD patients on a GFD and identify those nonresponsive individuals that may require more aggressive therapy. Here, we have identified and validated dGP IgG assays as an effective serological assay to meet this need. Similarity between the best performing library-isolated peptides and known deamidated gliadin B- and T-cell epitopes led to the confirmation of dGP as the targeted antigen. In a cohort of 15 NRCD cases and 45 responsive controls, we observed 87% sensitivity and 89% specificity, which support the utility of IgG dGP testing in monitoring response to a GFD. Nevertheless, a more rigorous assessment of diagnostic performance will require a large prospective cohort of CD cases on a GFD to determine positive and negative predictive values. Our results demonstrate that bacterial display technology can provide both diagnostically useful reagents and an effective route to antigen discovery without prior knowledge of the cause or mechanisms of disease.
Refractory CD (RCD) is defined by persistent villous atrophy and CD-associated symptoms despite a year or more of strict GFD after CD diagnosis and no evidence of other small bowel disorders. Early detection of RCD is critical because of the increased risk of ulcerative jejunitis, enteropathy-associated T-cell lymphoma (EATL) and non-Hodgkin's lymphomas, epithelial neoplasms, oesophageal and pharyngeal squamous cell carcinomas, and concomitant autoimmune diseases. RCD diagnosis depends on the exclusion of other possible causes of villous atrophy and increased IELs. The only definitive test for RCD requires detection of an abnormal IEL phenotype specific to RCD type II. Our observations that EMA and TG2 did not detect persistent mucosal damage confirm previous results that neither test can substitute for intestinal biopsies for diagnosing NRCD or RCD. However, negative EMA and TG2 assays within our cohort supported strict GFD adherence. Overall, 96.7% and 91.7% of our patients were negative for EMA and TG2 respectively. These results are in agreement with a previous study wherein EMA and TG2 were negative in 97.5% and 97.2% of GFD-adherent patients respectively. Although occasional accidental gluten intake cannot be completely ruled out, the combination of the low anti-TG2 titre and comprehensive dietary assessment with dietitians is the gold standard for monitoring the strictness of GFDs.
IgG-class antibodies against dGP were significantly elevated in NRCD patients compared with responsive CD patients as detected by flow cytometry and ELISA. Only one misclassified responsive CD patient was on a GFD for more than 2 years. The titres of the remaining responsive patients could be expected to continue to decline (below the positive threshold) upon further follow-up. This observation is supported by the change in dGP antibody reactivity before and after 1 year of GFD, as the two patients with the highest titres at diagnosis also had the highest titres 1 year later. In this study, 14/15 NRCD patients were on a strict GFD for more than 3 years. Previous studies suggest that a period of 3 years of GFD is sufficient to allow titres to decline to baseline levels in responsive patients. Comparing the difference in dGP titre between Marsh 3c NRCD and fully recovered Marsh 0 patients, classification accuracy improves further to >90%. Given that there were no differences in serological or histological findings between RCD and NRCD patients, the definition of RCD could potentially be revised to include asymptomatic NRCD patients in cases where a strict diet is confirmed and other associated causes of refractory disease are ruled out.
Previous studies have proposed that the detection of antibodies specific for deamidated gliadin may be helpful in CD follow-up. One study reported that six of nine NRCD patients and 6/33 responsive CD patients maintained IgA dGP antibodies after 1 year of a strict GFD. Our assays using IgA antibodies had comparable NRCD sensitivity and specificity, but we observed that IgG-class dGP antibodies were significantly more sensitive and specific for NRCD when compared with IgA-class antibodies. Although IgA outperforms IgG in anti-TG2 assays, IgG anti-dGP ELISA has a greater diagnostic performance with untreated CD patients than its IgA counterpart. Another study reported that 10/15 NRCD patients on a GFD for 1 year had elevated anti-dGP IgA and IgG titres, but 7/10 positive cases were patients that had 'low compliance' with their GFD. Consequently, it was not possible to link dGP titre to NRCD. We did not include any patients with low or moderate adherence to GFD in our study. Thus, the present finding that anti-dGP IgG antibodies accurately discriminate NRCD from responsive CD provides compelling evidence that dGP testing may be useful to identify individuals with NRCD. Our study thus provides support for a prospective study in a large cohort of newly diagnosed CD patients on a GFD for identifying NRCD cases.
One possible explanation of the persistence of antibodies against dGP despite complete removal of gluten from the diet is the presence of T-cell clones that have evolved antigen independence and continue to stimulate dGP antibody-secreting plasma cells. This phenomenon has been previously described, and pools of memory T- and B-cells can be maintained at constant levels for years even in the absence of the eliciting antigen. In addition, plasma cells can continuously secrete antibody even after the disappearance of memory cells. Further studies will be necessary to confirm the presence of dGP-specific memory B-cells or plasma cells in NRCD patients. Therefore, the mechanism responsible for the persistence of anti-dGP IgG antibodies remains to be elucidated.
We propose a revised diagnostic algorithm using non-invasive serological tests for the monitoring of CD patient recovery during a GFD (Figure 4). The first step in the follow-up of CD is to confirm compliance with a strict GFD. Although IgA anti-TG2 antibodies do not identify a majority of diet noncompliers (low positive predictive value (PPV)), our results suggest that a negative TG2 ELISA may be useful to assess strict adherence to GFD. However, consultation with an expert dietitian or nutritionist is still considered the gold standard. If trace gluten contamination is suspected, a modified diet including only fresh and unprocessed foods may be beneficial for a subset of NRCD patients. Once diet adherence is confirmed and other associated causes of villous atrophy have been excluded, an anti-dGP IgG ELISA test should be performed 1 year after the initial CD diagnosis. A negative result may reassure a physician that their patient is recovering and may help patients maintain compliance to a GFD. Such patients would not require serial follow-up biopsies unless their symptoms reoccur. A positive dGP test identifies probable nonresponders, and NRCD patients could seek alternative therapies (parenteral nutrition, corticosteroids and/or immunosuppressive drugs) and undergo a biopsy to test for RCD type II.
The antibody repertoire analysis method used here may have broad utility for the development of diagnostics. Here, we have isolated and characterised mimotope peptide sequences from bacterial display random peptide libraries that did not require a priori knowledge about refractory or nonresponsive CD. The use of convenient IgG dGP blood tests could aid the effective care of recovering CD patients on a GFD and reduce the need for costly and invasive endoscopy/biopsy procedures. Furthermore, the dGP assay can efficiently identify patients that require alternative treatment options to reduce the morbidity and mortality risks associated with NRCD. Our results strongly support the use of dGP serology in the clinical follow-up of individuals with CD.
Discussion
There exists an unmet diagnostic need for serological assays to monitor the recovery of CD patients on a GFD and identify those nonresponsive individuals that may require more aggressive therapy. Here, we have identified and validated dGP IgG assays as an effective serological assay to meet this need. Similarity between the best performing library-isolated peptides and known deamidated gliadin B- and T-cell epitopes led to the confirmation of dGP as the targeted antigen. In a cohort of 15 NRCD cases and 45 responsive controls, we observed 87% sensitivity and 89% specificity, which support the utility of IgG dGP testing in monitoring response to a GFD. Nevertheless, a more rigorous assessment of diagnostic performance will require a large prospective cohort of CD cases on a GFD to determine positive and negative predictive values. Our results demonstrate that bacterial display technology can provide both diagnostically useful reagents and an effective route to antigen discovery without prior knowledge of the cause or mechanisms of disease.
Refractory CD (RCD) is defined by persistent villous atrophy and CD-associated symptoms despite a year or more of strict GFD after CD diagnosis and no evidence of other small bowel disorders. Early detection of RCD is critical because of the increased risk of ulcerative jejunitis, enteropathy-associated T-cell lymphoma (EATL) and non-Hodgkin's lymphomas, epithelial neoplasms, oesophageal and pharyngeal squamous cell carcinomas, and concomitant autoimmune diseases. RCD diagnosis depends on the exclusion of other possible causes of villous atrophy and increased IELs. The only definitive test for RCD requires detection of an abnormal IEL phenotype specific to RCD type II. Our observations that EMA and TG2 did not detect persistent mucosal damage confirm previous results that neither test can substitute for intestinal biopsies for diagnosing NRCD or RCD. However, negative EMA and TG2 assays within our cohort supported strict GFD adherence. Overall, 96.7% and 91.7% of our patients were negative for EMA and TG2 respectively. These results are in agreement with a previous study wherein EMA and TG2 were negative in 97.5% and 97.2% of GFD-adherent patients respectively. Although occasional accidental gluten intake cannot be completely ruled out, the combination of the low anti-TG2 titre and comprehensive dietary assessment with dietitians is the gold standard for monitoring the strictness of GFDs.
IgG-class antibodies against dGP were significantly elevated in NRCD patients compared with responsive CD patients as detected by flow cytometry and ELISA. Only one misclassified responsive CD patient was on a GFD for more than 2 years. The titres of the remaining responsive patients could be expected to continue to decline (below the positive threshold) upon further follow-up. This observation is supported by the change in dGP antibody reactivity before and after 1 year of GFD, as the two patients with the highest titres at diagnosis also had the highest titres 1 year later. In this study, 14/15 NRCD patients were on a strict GFD for more than 3 years. Previous studies suggest that a period of 3 years of GFD is sufficient to allow titres to decline to baseline levels in responsive patients. Comparing the difference in dGP titre between Marsh 3c NRCD and fully recovered Marsh 0 patients, classification accuracy improves further to >90%. Given that there were no differences in serological or histological findings between RCD and NRCD patients, the definition of RCD could potentially be revised to include asymptomatic NRCD patients in cases where a strict diet is confirmed and other associated causes of refractory disease are ruled out.
Previous studies have proposed that the detection of antibodies specific for deamidated gliadin may be helpful in CD follow-up. One study reported that six of nine NRCD patients and 6/33 responsive CD patients maintained IgA dGP antibodies after 1 year of a strict GFD. Our assays using IgA antibodies had comparable NRCD sensitivity and specificity, but we observed that IgG-class dGP antibodies were significantly more sensitive and specific for NRCD when compared with IgA-class antibodies. Although IgA outperforms IgG in anti-TG2 assays, IgG anti-dGP ELISA has a greater diagnostic performance with untreated CD patients than its IgA counterpart. Another study reported that 10/15 NRCD patients on a GFD for 1 year had elevated anti-dGP IgA and IgG titres, but 7/10 positive cases were patients that had 'low compliance' with their GFD. Consequently, it was not possible to link dGP titre to NRCD. We did not include any patients with low or moderate adherence to GFD in our study. Thus, the present finding that anti-dGP IgG antibodies accurately discriminate NRCD from responsive CD provides compelling evidence that dGP testing may be useful to identify individuals with NRCD. Our study thus provides support for a prospective study in a large cohort of newly diagnosed CD patients on a GFD for identifying NRCD cases.
One possible explanation of the persistence of antibodies against dGP despite complete removal of gluten from the diet is the presence of T-cell clones that have evolved antigen independence and continue to stimulate dGP antibody-secreting plasma cells. This phenomenon has been previously described, and pools of memory T- and B-cells can be maintained at constant levels for years even in the absence of the eliciting antigen. In addition, plasma cells can continuously secrete antibody even after the disappearance of memory cells. Further studies will be necessary to confirm the presence of dGP-specific memory B-cells or plasma cells in NRCD patients. Therefore, the mechanism responsible for the persistence of anti-dGP IgG antibodies remains to be elucidated.
We propose a revised diagnostic algorithm using non-invasive serological tests for the monitoring of CD patient recovery during a GFD (Figure 4). The first step in the follow-up of CD is to confirm compliance with a strict GFD. Although IgA anti-TG2 antibodies do not identify a majority of diet noncompliers (low positive predictive value (PPV)), our results suggest that a negative TG2 ELISA may be useful to assess strict adherence to GFD. However, consultation with an expert dietitian or nutritionist is still considered the gold standard. If trace gluten contamination is suspected, a modified diet including only fresh and unprocessed foods may be beneficial for a subset of NRCD patients. Once diet adherence is confirmed and other associated causes of villous atrophy have been excluded, an anti-dGP IgG ELISA test should be performed 1 year after the initial CD diagnosis. A negative result may reassure a physician that their patient is recovering and may help patients maintain compliance to a GFD. Such patients would not require serial follow-up biopsies unless their symptoms reoccur. A positive dGP test identifies probable nonresponders, and NRCD patients could seek alternative therapies (parenteral nutrition, corticosteroids and/or immunosuppressive drugs) and undergo a biopsy to test for RCD type II.
The antibody repertoire analysis method used here may have broad utility for the development of diagnostics. Here, we have isolated and characterised mimotope peptide sequences from bacterial display random peptide libraries that did not require a priori knowledge about refractory or nonresponsive CD. The use of convenient IgG dGP blood tests could aid the effective care of recovering CD patients on a GFD and reduce the need for costly and invasive endoscopy/biopsy procedures. Furthermore, the dGP assay can efficiently identify patients that require alternative treatment options to reduce the morbidity and mortality risks associated with NRCD. Our results strongly support the use of dGP serology in the clinical follow-up of individuals with CD.
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