Juha Taavela

Validation of Morphometric Analyses of Small-Intestinal Biopsy Readouts in Celiac Disease

Background Assessment of the gluten-induced small-intestinal mucosal injury remains the cornerstone of celiac disease diagnosis. Usually the injury is evaluated using grouped classifications (e.g. Marsh groups), but this is often too imprecise and ignores minor but significant changes in the mucosa. Consequently, there is a need for validated continuous variables in everyday practice and in academic and pharmacological research. Methods We studied the performance of our standard operating procedure (SOP) on 93 selected biopsy specimens from adult celiac disease patients and non-celiac disease controls. The specimens, which comprised different grades of gluten-induced mucosal injury, were evaluated by morphometric measurements. Specimens with tangential cutting resulting from poorly oriented biopsies were included. Two accredited evaluators performed the measurements in blinded fashion. The intraobserver and interobserver variations for villus height and crypt depth ratio (VH:CrD) and densities of intraepithelial lymphocytes (IELs) were analyzed by the Bland-Altman method and intraclass correlation. Results Unevaluable biopsies according to our SOP were correctly identified. The intraobserver analysis of VH:CrD showed a mean difference of 0.087 with limits of agreement from −0.398 to 0.224; the standard deviation (SD) was 0.159. The mean difference in interobserver analysis was 0.070, limits of agreement −0.516 to 0.375, and SD 0.227. The intraclass correlation coefficient in intraobserver variation was 0.983 and that in interobserver variation 0.978. CD3+ IEL density countings in the paraffin-embedded and frozen biopsies showed SDs of 17.1% and 16.5%; the intraclass correlation coefficients were 0.961 and 0.956, respectively. Conclusions Using our SOP, quantitative, reliable and reproducible morphometric results can be obtained on duodenal biopsy specimens with different grades of gluten-induced injury. Clinically significant changes were defined according to the error margins (2SD) of the analyses in VH:CrD as 0.4 and in CD3+-stained IELs as 30%.

Degree of Damage to the Small Bowel and Serum Antibody Titers Correlate With Clinical Presentation of Patients With Celiac Disease

BACKGROUND & AIMS In patients with celiac disease, gluten-induced lesions of the small-bowel mucosa develop gradually. However, it is not clear whether clinical presentation correlates with the degree of mucosal damage based on histology analysis. We investigated whether the degree of mucosal damage to the small bowel correlates with clinical presentation and serum markers of celiac disease. METHODS We collected results from serology tests and mucosal biopsy samples from 638 consecutive patients with celiac disease and compared them with reported gastrointestinal symptoms, health-related quality-of-life scores, results from laboratory tests, and bone mineral densities of patients. We assessed mucosal injury based on the ratio of villous height to crypt depth, numbers of intraepithelial CD3(+) cells, and semiquantitative Marsh classification criteria. Correlations were established based on the Pearson or Spearman coefficients. RESULTS The ratio of the villous height to crypt depth correlated with the severity of gastrointestinal symptoms, quality-of-life scores, laboratory test results, numbers of intraepithelial CD3(+) cells, and serum levels of antibodies associated with celiac disease. There was no correlation between the ratio of villous height to crypt depth and bone mineral density. The number of intraepithelial CD3(+) cells was not associated with symptoms, whereas the Marsh classification and serum levels of antibodies associated with celiac disease correlated with gastrointestinal symptoms, laboratory test results, and numbers of intraepithelial CD3(+) cells. CONCLUSIONS The ratio of small-bowel villous height to crypt depth and results from serology tests correlate with reported symptoms and quality of life of patients with celiac disease. Patient-reported outcomes are therefore of value, in addition to histology findings, in assessing patients with celiac disease.

CYP3A4-Catalyzed Simvastatin Metabolism as a Non-Invasive Marker of Small Intestinal Health in Celiac Disease

OBJECTIVES:Histological examination of duodenal biopsies is the gold standard for assessing intestinal damage in celiac disease (CD). A noninvasive marker of disease status is necessary, because obtaining duodenal biopsies is invasive and not suitable for routine monitoring of CD patients. As the small intestine is a major site of cytochrome P450 3A4 (CYP3A4) activity and also the location of the celiac lesion, we investigated whether patients with active CD display abnormal pharmacokinetics of an orally administered CYP3A4 substrate, simvastatin (SV), which could potentially be used for noninvasive assessment of their small intestinal health.METHODS:Preclinical experiments were performed in CYP3A4-humanized mice to examine the feasibility of the test. Subsequently, a clinical trial was undertaken with 11 healthy volunteers, 18 newly diagnosed patients with CD, and 25 celiac patients who had followed a gluten-free diet (GFD) for more than 1 year. The maximum concentration (Cmax) of orally administered SV plus its major non-CYP3A4-derived metabolite SV acid (SV equivalent (SVeq)) was measured, and compared with clinical, histological, and serological parameters.RESULTS:In CYP3A4-humanized mice, a marked decrease in SV metabolism was observed in response to enteropathy. In the clinical setting, untreated celiac patients displayed a significantly higher SVeq Cmax (46±24 nM) compared with treated patients (21±16 nM, P<0.001) or healthy subjects (19±11 nM, P<0.005). SVeq Cmax correctly predicted the diagnosis in 16/18 untreated celiac patients, and also the recovery status of all follow-up patients that exhibited normal or near-normal biopsies (Marsh 0-2). All patients with abnormal SVeq Cmax showed a reduction in the value after 1 year of following a GFD.CONCLUSIONS:SVeq Cmax is a promising noninvasive marker for assessment of small intestinal health. Further studies are warranted to establish its clinical utility for assessing gut status of patients with CD.

Epitope-specific immunotherapy targeting CD4-positive T cells in coeliac disease: two randomised, double-blind, placebo-controlled phase 1 studies

BACKGROUND A gluten-free diet is the only means to manage coeliac disease, a permanent immune intolerance to gluten. We developed a therapeutic vaccine, Nexvax2, designed to treat coeliac disease. Nexvax2 is an adjuvant-free mix of three peptides that include immunodominant epitopes for gluten-specific CD4-positive T cells. The vaccine is intended to engage and render gluten-specific CD4-positive T cells unresponsive to further antigenic stimulation. We assessed the safety and pharmacodynamics of the vaccine in patients with coeliac disease on a gluten-free diet. METHODS We did two randomised, double-blind, placebo-controlled, phase 1 studies at 12 community sites in Australia, New Zealand, and the USA, in HLA-DQ2·5-positive patients aged 18-70 years who had coeliac disease and were on a gluten-free diet. In the screening period for ascending dose cohorts, participants were randomly assigned (1:1) by central randomisation with a simple block method to a double-blind crossover, placebo-controlled oral gluten challenge. Participants with a negative interferon γ release assay to Nexvax2 peptides after the screening oral gluten challenge were discontinued before dosing. For the biopsy cohorts, the screening period included an endoscopy, and participants with duodenal histology who had a Marsh score of greater than 1 were discontinued before dosing. Participants were subsequently randomly assigned to either Nexvax2 or placebo in ascending dose cohorts (2:1) and in biopsy cohorts (1:1) by central randomisation with a simple block method. In the three-dose study, participants received either Nexvax2 60 μg, 90 μg, or 150 μg weekly, or placebo over 15 days; in a fourth biopsy cohort, patients received either Nexvax2 at the maximum tolerated dose (MTD) or placebo. In the 16-dose study, participants received Nexvax2 150 μg or 300 μg or placebo twice weekly over 53 days; in a third biopsy cohort, patients also received either Nexvax2 at the MTD or placebo. In the 4-week post-treatment period, ascending dose cohorts underwent a further double-blind crossover, placebo-controlled oral gluten challenge, which had a fixed sequence, and biopsy cohorts had a gastroscopy with duodenal biopsies and quantitative histology within 2 weeks without oral gluten challenge. Participants, investigators, and study staff were masked to the treatment assignment, except for the study pharmacist. The primary endpoint was the number and percentage of adverse events in the treatment period in an intention-to-treat analysis. Both trials were completed and closed before data analysis. Trials were registered with the Australian New Zealand Clinical Trials Registry, numbers ACTRN12612000355875 and ACTRN12613001331729. FINDINGS Participants were enrolled from Nov 28, 2012, to Aug 14, 2014, in the three-dose study, and from Aug 3, 2012, to Sept 10, 2013, in the 16-dose study. Overall, 62 (57%) of 108 participants were randomly assigned after oral gluten challenge and 20 (71%) of 28 participants were randomly assigned after endoscopy. In the three-dose study, nine participants were randomly allocated to Nexvax2 60 μg and three to placebo (first cohort), nine were allocated to Nexvax2 90 μg and four to placebo (second cohort), eight were allocated to Nexvax2 150 μg and four to placebo (third cohort), and three were allocated to Nexvax2 150 μg and three to placebo (biopsy cohort). In the 16-dose study, eight participants were randomly allocated to Nexvax2 150 μg and four to placebo (first cohort), ten were allocated to Nexvax2 300 μg and three to placebo (second cohort), and seven were allocated to Nexvax2 150 μg and seven to placebo (biopsy cohort). The MTD for Nexvax2 was 150 μg because of transient, acute gastrointestinal adverse events with onset 2-5 h after initial doses of the vaccine, similar to those caused by gluten ingestion. In the ascending dose cohorts in the three-dose study, six (55%) of 11 placebo recipients, five (56%) of nine who received Nexvax2 60 μg, seven (78%) of nine who received Nexvax2 90 μg, and five (63%) of eight who received Nexvax2 150 μg had at least one treatment-emergent adverse event, as did all three (100%) placebo recipients and one (33%) of three Nexvax2 150 μg recipients in the biopsy cohort. In the ascending dose cohorts of the 16-dose study, five (71%) of seven placebo-treated participants, six (75%) of eight who received Nexvax2 150 μg, and all ten (100%) who received Nexvax2 300 μg had at least one treatment-emergent adverse event, as did six (86%) of seven placebo recipients and five (71%) of seven Nexvax2 150 μg recipients in the biopsy cohort. Vomiting, nausea, and headache were the only treatment-emergent adverse events that occurred in at least 5% of participants in either study. Among participants given the MTD, eight gastrointestinal treatment-emergent adverse events occurred in four (50%) of eight participants in the third cohort and none (0%) of three participants in the biopsy cohort in the three-dose study, and five events occurred in five (63%) of eight participants in the first cohort and three events in two (29%) of seven participants in the biopsy cohort of the 16-dose study. Median villous height to crypt depth ratio in distal duodenal biopsies was not significantly different between those who received the vaccine at the MTD on either schedule and those who received placebo. Of the participants who completed the post-treatment oral gluten challenge per protocol, interferon γ release assay to Nexvax2 peptides was negative (responders to treatment) in two (22%) of nine placebo-treated participants in the three-dose study versus two (33%) of six who received Nexvax2 60 μg, five (63%) of eight who received Nexvax2 90 μg, and six (100%) of six who received Nexvax2 150 μg (p=0·007); in the 16-dose study, none (0%) of five placebo-treated participants had a negative assay versus six (75%) of eight who received Nexvax2 150 μg (p=0·021). INTERPRETATION The MTD of Nexvax2 was 150 μg for twice weekly intradermal administration over 8 weeks, which modified immune responsiveness to Nexvax2 peptides without deterioration in duodenal histology. The gastrointestinal symptoms that followed the first intradermal administration of the vaccine resembled those associated with oral gluten challenge. These findings support continued clinical development of this potential therapeutic vaccine for coeliac disease. FUNDING ImmusanT.

A Prospective Study on the Usefulness of Duodenal Bulb Biopsies in Celiac Disease Diagnosis in Children: Urging Caution.

Objectives:Several recent celiac disease guidelines recommend the acquisition of duodenal bulb biopsies for diagnostics. This is in conflict with previously reported evidence and routine practice from the 1960s onward. We reopened the issue in a prospective multicenter study and used morphometric variables in evaluating the usefulness of bulb biopsies in children. We further sought to establish whether deposits of IgA targeting bulb transglutaminase 2 (TG2) could be of diagnostic help.Methods:Diagnoses of celiac disease were based on clinic and distal duodenal histopathology statements. Centralized reading of villous height (VH) to crypt depth (CrD) ratios and IgA deposits was performed on anatomical duodenal bulb specimens. All children participating also underwent routine investigations for other diseases.Results:Twenty-two children had celiac disease, and another 22 served as non-celiac disease controls. The quality of the anatomical bulb specimens was unsatisfactory for reliable morphometric measurements in 20 out of 44 (45%) patients even after recuttings. All celiac disease patients had VH:CrD<2.0 (mean 0.2) but also 10 out of 13 (77%) non-celiac control patients had an injured bulb mucosal lining (mean 1.3) even up to false-positive “flat lesion”. Bulb IgA deposits were able to separate celiac disease from disease controls.Conclusions:Morphological injury is common in the anatomical bulb even without celiac disease, increasing the risk of false-positive diagnoses. Premature conclusions might have been drawn on current care guidelines as to celiac disease diagnosis based solely on anatomical bulb specimens. Bulb mucosal IgA targeting TG2 in poor quality biopsy specimens is a powerful clinical tool in finding celiac disease patients.

Novel diagnostic techniques for celiac disease

ABSTRACT The diagnosis of celiac disease has long been based on the demonstration of gluten-induced small-bowel mucosal damage. However, due to the constantly increasing disease prevalence and limitations in the histology-based criteria there is a pressure towards more serology-based diagnostics. The serological tools are being improved and new non-invasive methods are being developed, but the constantly refined endoscopic and histologic techniques may still prove helpful. Moreover, growing understanding of the disease pathogenesis has led researchers to suggest completely novel approaches to celiac disease diagnostics regardless of disease activity. In this review, we will elucidate the most recent development and possible future innovations in the diagnostic techniques for celiac disease.

Outcome measures in coeliac disease trials: the Tampere recommendations

Objective A gluten-free diet is the only treatment option of coeliac disease, but recently an increasing number of trials have begun to explore alternative treatment strategies. We aimed to review the literature on coeliac disease therapeutic trials and issue recommendations for outcome measures. Design Based on a literature review of 10 062 references, we (17 researchers and 2 patient representatives from 10 countries) reviewed the use and suitability of both clinical and non-clinical outcome measures. We then made expert-based recommendations for use of these outcomes in coeliac disease trials and identified areas where research is needed. Results We comment on the use of histology, serology, clinical outcome assessment (including patient-reported outcomes), quality of life and immunological tools including gluten immunogenic peptides for trials in coeliac disease. Conclusion Careful evaluation and reporting of outcome measures will increase transparency and comparability of coeliac disease therapeutic trials, and will benefit patients, healthcare and the pharmaceutical industry.

Response to Kurien et al.

of Brunner’s glands, lymphoid follicles, and gastric acid ( 1,3 ). In our study, many of the disease control patients had an injured bulb mucosa even up to a “fl at lesion”, whereas the IgA deposit result was negative in all ( 1 ). Hence, bulb biopsy should defi nitively be in a diff erent pot than distal duodenal specimens as otherwise wrong positive statements are possible ( 2,4 ). When the specimens are in one pot, it is impossible to know for sure which sample is from bulb as Brunner’s glands have been demonstrated not to be a histologic feature exclusive of the duodenal bulb ( 5 ). On the basis of our results, there is an evident need to standardize the defi nition of pathologic features of bulb samples in routine practice and research ( 1 ). We believe that such standardization would improve celiac disease diagnostics. We want to emphasize that, whenever a sectioning is evaluated, orientation of the specimen should be inspected as a part of routine. Marsh class or morphometry result may not be given from tangential cuttings wherein only cross-sections of crypts are seen ( 1 ). Special care should always be taken when establishing celiac disease diagnosis in subjects with negative or low-celiac disease antibody titers and an isolated lesion in the anatomical duodenal bulb.