Baseline quantitative histology in therapeutics trials reveals villus atrophy in most patients with coeliac disease who appear well-controlled on gluten-free diet

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/YGH2.380 This article is protected by copyright. All rights reserved JUHA TAAVELA (Orcid ID : 0000-0003-3948-9555) KENNETH E TRUITT (Orcid ID : 0000-0001-9350-0212) DR. ROBERT PAUL ANDERSON (Orcid ID : 0000-0002-0764-7267) Article type : Original Article BASELINE QUANTITATIVE HISTOLOGY IN THERAPEUTICS TRIALS REVEALS VILLUS ATROPHY IN MOST PATIENTS WITH COELIAC DISEASE WHO APPEAR WELL-CONTROLLED ON GLUTEN-FREE DIET Short Title: Quantitative histology and treated coeliac disease Authors: Daveson, A James M.1; Popp, Alina2,3; Taavela, Juha2,4; Goldstein, Kaela E.5; Isola, Jorma6,7; Truitt, Kenneth E.5; Mäki, Markku2‡; Anderson, Robert P. 5 † ‡ on behalf of the RESET CeD Study Group†† Affiliations: 1University of Queensland, Brisbane, Queensland, Australia 2Tampere Centre for Child Health Research, Faculty of Medicine and Health Technology, Tampere University and Tampere University Hospital, Tampere, Finland 3University of Medicine and Pharmacy "Carol Davila" and National Institute for Mother and Child Health "Alessandrescu-Rusescu", Bucharest, Romania; 4Central Finland Central Hospital, Department of Internal Medicine, Jyväskylä, Finland 5ImmusanT, Inc., Cambridge, Massachusetts, USA 6Laboratory of Cancer Biology, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland; 7Jilab Inc, Tampere, Finland; Accepted Article This aT JUH A JU KEN K D R. RD A rtic A BAS EB V IL LV W E LW S hor t A u t hA J orm aJo C eD Affil iA 1 1 U ni vU 22 Ta mT U niv eUnive 33 U ni vU Heal tH 44 C en tC 55 I m mI 66 Lab oL Tam pT 77 J ila bJ This article is protected by copyright. All rights reserved ‡ Drs. Anderson and Maki contributed equally to this article as senior authors. † To whom correspondence should be addressed: Dr. Robert P. Anderson, ImmusanT Inc., One Kendall Square, Suite 7201, Cambridge, MA 02139, or at [email protected] Acknowledgments: ImmusanT, Inc., Cambridge, Massachusetts USA provided funding for the study. AP and MM were financially partly supported by the Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital, Grant No. 9X035. RPA, JI and MM designed the study; AJMD, KEG, KET and the RESET CeD Study Group conducted clinical studies; AP, JT, MM and JI performed histological assessments; RPA analysed the data. RPA and KEG prepared the tables and figures; RPA and MM wrote the manuscript. All authors reviewed and approved the manuscript, tables and figures. The authors made the decision to submit the manuscript for publication and vouch for the accuracy of data and analyses and for the fidelity of this report to the trial protocol. RPA had full access to all data in the study and had final responsibility for the decision to submit for publication. †† A. Adams (AB Clinical Trials, Las Vegas, NV, USA), J. Andrews (Royal Adelaide Hospital, Adelaide, SA, AUS), C. Behrend (Grand Teton Research Group, Idaho Falls, ID, USA), G. Brown, (Alfred Hospital, Melbourne, VC, AUS), S. Chen Yi Mei (Eastern Health-Box Hill Hospital, Box Hill, VC, AUS), A. Coates (Gastroenterology Associates of Western Michigan, PLC d.b.a. West Michigan Clinical Research Center, Wyoming, MI, USA), A.J. Daveson (Coral Sea Clinical Research Institute, North Mackay, QLD, AUS, and The Wesley Hospital - The Wesley Research Institute, Auchenflower, QLD, AUS), A. DiMarino (Thomas Jefferson University Hospital, Philadelphia, PA, USA), H. Ee (Department of Gastroenterology, Sir Charles Gairdner Hospital, Nedlands, WA, AUS), D. Elliott (University Of Iowa, Iowa City, IA, USA), R. Epstein (ActivMed Practices & Research, Inc., Portsmouth, NH, USA), B. Feyen, (PMG Research of McFarland Clinic, Ames, IA, USA), R. Fogel (Clinical Research Institute of Michigan, Chesterfield, MI, USA), K. Friedenberg (Great Lakes Gastroenterology Research, Mentor, OH, USA), R. Gearry (Department of Medicine, University of Otago, Christchurch, NZ), M. Gerdis (Drug Trials America, Hartsdale, NY, USA), M. Goldstein (Long Island Gastrointestinal Research Group, Great Neck, NY, USA), V. Gupta (Alliance Medical Accepted Article ‡ s †† To Ken dK Ack nA A P a nA Expe rE desig d A P, JA prep apr appr oap for p u t rial ptr t o su b † † A .†† A del aA (Alfr e(A Hill, VH Mich M I nstit uIn A uc hAuch U SA )U D. El D I nc., PIn Foge lF Gast rG Otag oO (Lon g(L This article is protected by copyright. All rights reserved Research LLC, Lighthouse Point, FL, USA), R. Holmes (PMG Research of Winston-Salem, LLC, Winston-Salem, NC, USA), G. Holtmann (The University of Queensland - Princess Alexandra Hospital, Woolloongabba, QLD, AUS), S. Idarraga (Allegiance Research Specialists, Wauwatosa, WI, USA), G. James (Digestive Health Research, LLC, Hermitage, TN, USA), T. King, (Auckland Clinical Studies Limited, Auckland, NZ), T. Klein (Heartland Research Associates, LLC, Wichita, KS, USA), S. Kupfer (The University of Chicago Medical Center, Chicago, IL, USA), B. Lebwohl (Celiac Disease Center at Columbia University, New York City, NY, USA), J. Lowe (Advanced Research Institute, Ogden, UT, USA), J. Murray (Mayo Clinic, Rochester, MN, USA), E. Newton (Omega Medical Research, Warwick, RI, USA), D. Quinn (P3 Research Limited, Wellington, NZ), D. Radin (Stamford Therapeutics Consortium, Stamford, CT, USA), T. Ritter (Texas Digestive Disease Consultants, Southlake, TX, USA), H. Stacey (Diablo Clinical Research, Inc., Walnut Creek, CA, USA), C. Strout (Coastal Carolina Research Center, Mount Pleasant, SC, USA), R. Stubbs (P3 Research Limited, Havelock North, NZ), S. Thackwray (Clinical Trials Centre - University of the Sunshine Coast, Sippy Downs, QLD, AUS), V. Trivedi (PMG Research, Inc., d/b/a PMG Research of Piedmont Healthcare, Statesville, NC, USA), J. Tye-Din (The Royal Melbourne Hospital - The Walter And Eliza Hall Institute of Medical Research, Parkville, VC, AUS), J. Weber (Center for Digestive Health, Troy, MI, USA), S. Wilson (Ocean State Clinical Research Partners, Lincoln, RI, USA) Accepted Article esea W ins W Hosp H W I, UW C lini cC KS, UK (Celi a(C Rese aR (Om e Radi nR C ons uC U SA )U Rese aR Suns h Pied mP A nd EA Heal tH This article is protected by copyright. All rights reserved ABSTRACT Background: The prevalence and severity of duodenal injury in coeliac disease patients controlled on a gluten-free diet is unclear. Aims: To use quantitative histology to assess duodenal injury in treated coeliac disease. Methods: Quantitative histology in pre-treatment duodenal biopsies collected in clinical trials assessing an investigational immunotherapy for coeliac disease were analysed. Morphometric readings were converted to Marsh classifications. Results: 93 patients had duodenal biopsies. For well-oriented sections of second part biopsies, six (6%) patients were classified as Marsh 0 or 1, 30 (33%) as Marsh 2, and 56 (60%) as Marsh 3a or 3b. In second part biopsies from 78 seronegative patients on gluten-free diet >2 years, 27 (35%) were Marsh 2 and 45 (58%) were Marsh 3a or 3b. Distal compared to proximal duodenal biopsies had significantly higher villus height to crypt depth ratios (median, third part: 2.1 versus bulb: 1.4; P<0.0001) and higher intraepithelial lymphocyte density (44 versus 30; P=0.0002). The sum of paired villus height and crypt depth measurements was correlated strongly with villus height (rs = 0.82, P<10-10). At least one biopsy was graded Marsh 3a or worse in all 26 patients who had serial biopsies from the bulb, first, second and third part, but was overlooked in 20 patients by subjective histology. Conclusions: Quantitative histology in well-oriented biopsy sections reveals villus atrophy in the majority of patients with coeliac disease who appear well-controlled on gluten-free diet. Standardisation of biopsy collection, processing and evaluation could substantially improve the value of follow-up biopsies in coeliac disease. Key words: Coeliac disease, transglutaminase IgA, serology, quantitative histology, gluten-free diet. Abbreviations: Human leukocyte antigen-DQ (HLA-DQ), transglutaminase 2 (TG2). Manuscript word count Introduction to Discussion: 3299 S Bac kB a glu ta Aim sA M e t hM asses sas r eadi nre ResuR (6%) I n se cIn Mars hM s igni fsi P<0. 0P v illu s P<1 0P from fr C on cC m ajo rm Stan dS o f fo lof K ey wKey w A bbrA Man uM This article is protected by copyright. All rights reserved INTRODUCTION Persistent duodenal mucosal damage in patients with coeliac disease on gluten-free diet is associated with increased severe long-term complications (1). A recent meta-analysis found 31% of patients with coeliac disease had persistent villus atrophy on gluten-free diet, and only half of these patients had elevated transglutaminase 2 (TG2) IgA serology (2). A corollary of these findings is that about one in five patients apparently well controlled on gluten-free diet with normal TG2 serology has duodenal villus atrophy. These conclusions are, however, based on routine processing, and subjective or semiquantitative analysis of biopsies, usually from the distal duodenum. Quantitative histology provides accurate assessment of duodenal mucosal injury in coeliac disease by using highly reproducible measurements of villous height, crypt depth and intra-epithelial density in well-oriented distal duodenal biopsies (3, 4). With implementation of rigorous standard operating procedures, quantitative duodenal histology outperforms conventional qualitative histology using grouped classifications such as Marsh score (5-7), and is emerging as a preferred measure of efficacy in therapeutics trials for coeliac disease (5, 8-10). Perhaps surprisingly, quantitative histology has not yet, to our knowledge, been used to evaluate disease activity/duodenal injury in patients who appear “well-controlled” coeliac disease and are in serological remission. Quantitative histology in baseline duodenal biopsies collected in phase 1 and 2 clinical trials assessing an investigational immunotherapy, Nexvax2®, for coeliac disease provided the opportunity to assess duodenal injury in patients with “well-controlled” coeliac disease on gluten-free diet (11-13). The primary objective of this study was to use quantitative histology to assess duodenal mucosal injury in treated coeliac disease, and in particular those patients who are well established on gluten-free diet, and in serological remission. METHODS Study design The study analysed quantitative histology of duodenal biopsies collected during the pre-treatment periods of four clinical trials testing a novel investigational immunotherapy (Nexvax2®). Patients Accepted Article N Persi sPe with iw c oeli aco eleva el five pfi v illu sv quan tqu Qua nQ u sin gu wellow proc epr g rou p i n th ein yet, t oye “ wel l“ Qua nQ an in van duod edu prim aprima t reat etreate and i nan ME TM S tud yS The sT perio dp This article is protected by copyright. All rights reserved were recruited at sites in Australia, New Zealand, and the United States. In each study, a subgroup of patients had biopsies collected as a safety assessment before and after the treatment period. Details of the four studies are summarized in Table 1. In the three phase 1 studies, which have been reported in detail elsewhere (11, 12), patients with coeliac disease were maintaining their usual gluten-free diet had no study interventions before their pre-treatment biopsy. In the phase 2 (“RESET CeD”) trial, which has also been described elsewhere (13), patients with coeliac disease were maintaining their usual gluten-free diet, but had a single bolus gluten challenge (6 grams of gluten protein taken as 10 grams vital wheat gluten flour in water) three weeks before biopsy collection according to the protocol described by Tye-Din et al (14). Patients and interventions All patients gave written, informed consent prior to undergoing any trial-related procedures. Eligibility criteria for each clinical trial have been reported fully elsewhere (11-13). In brief, for all four studies, patients who had biopsies collected had met initial screening criteria, which included being aged between 18 to 70 years, having documented duodenal villus atrophy while consuming gluten, being positive for HLA-DQA1*05 and DQB1*02 (“HLA DQ2.5”), and having maintained a gluten-free diet for at least one year. Patients with uncontrolled complications of celiac disease including refractory coeliac disease were excluded. In phase 1 studies, additional study-specific eligibility criteria were intended to select for patients entering the treatment period who were strictly maintaining a gluten-free diet. In the first and second phase 1 studies, patients who had a known gluten exposure within two months prior to screening were excluded, and patients were discontinued if subjective assessment of Marsh class for any pretreatment biopsy was more severe than 1 (raised intra-epithelial lymphocytes with normal villous architecture). In the third phase 1 study, patients were excluded if both TG2 IgA and deamidated gliadin peptide IgG serology were elevated, or if their score in the Celiac Dietary Adherence Test suggested non-compliance to gluten-free diet. In the phase 2 study, patients in the biopsy subgroup had met the inclusion criteria that experienced worsening digestive symptoms on the day of screening gluten food challenge three weeks earlier. Quantitative duodenal histology Accepted Article we e patie npa t he f oth detai ld h ad nha whic hw u sual u g ram sgr proto Pati eP A ll p aA Eligi bE four s bein gb g lute ngl g lute n i nclu din eligi be m ain tm g lute ngl i f su bif sub i ntra -intrawere w s core sc 2 stu d2 diges di Qua nQ This article is protected by copyright. All rights reserved Biopsy collection, processing and quantitative histology methods in the first and second phase 1 studies have been described in detail elsewhere (3, 11, 12). Briefly, biopsies were collected from four sites: the duodenal bulb, and from the first, second, and third parts of the duodenum. Two biopsies were obtained at each site with a single biopsy per pass of the forceps, and placed in formalin fixative. Both biopsies from each site were processed together; they were embedded in paraffin, sectioned, and stained with haemotoxylin and eosin, and by anti-CD3 immunochemistry. The pathologist at the central laboratory made a subjective assessment of whether a patient had any biopsy consistent with a Marsh classification above 1. Subsequently the same histology slides as used to assess the initial Marsh score were batched and sent to the University of Tampere for quantitative histology. If sections from a biopsy were considered poorly oriented and prevented accurate assessment, the central pathology laboratory was requested to re-cut tissue sections. Two observers (JT, AP) analysed all slides independently and were unaware of the clinical data or laboratory findings of the patients. Replicate measurements of villus height, crypt depth and frequency of intraepithelial CD3+ lymphocytes per 100 villus enterocytes (IELs) with well-oriented villus-crypt units were determined according to published protocols (3). Final Marsh classifications for formalin-fixed biopsies were based on average villus height to crypt depth ratios according to the previously reported conversion scale: villus height to crypt depth ratio ≥2.8 for Marsh (M)0 if intraepithelial CD3+ lymphocyte density per 100 enterocytes <25, or M1 if ≥25; villus height to crypt depth ratio 2.0 - 2.7 for M2; villus height to crypt depth ratio 1.2 - 1.9 for M3a; villus height to crypt depth ratio 0.5 - 1.1 for M3b; and villus height to crypt depth ratio 0.0-0.4 for M3c (15). In the third phase 1 study and phase 2 study, biopsy collection was limited to the second part of the duodenum, PAXgene tissue fixative (QIAGEN, Hilden, Germany) was used, and biopsies were sent directly to Jilab Inc. (Tampere, Finland) for processing and quantitative histology. Biopsy collection and handling, and the protocol for quantitative histology are described elsewhere (3, 12, 16). Paxgene fixative facilitated a wider repertoire of immunohistochemical, immunofluorescence assessments, and genome-wide transcriptome analysis in the same tissue sample (16). In brief, after collection, biopsies were placed one per compartment of a multi-compartment tissue cassette. The cassette was immersed in PAXgene fixative for 1-4 hours, and then transferred to the proprietary storage solution in Accepted Article ops s tudi est s ites: si were w B oth B s tain est c entr ace Mars hM Mars h from fr path op s lide ssl Repl iR l ymp h acco rac base dba s cale : densi de v illu sv and van I n th eIn the duod eduode direc tdi and han fixati fi g eno mge were w i n P Ain This article is protected by copyright. All rights reserved PAXgene dual-chamber fixative containers (Qiagen #765112, Venlo, Netherlands). Samples in fixative were shipped at ambient temperature and then stored at +4°C until processing. Each biopsy was embedded in a separate paraffin block under a dissection microscope and aimed for a cutting plane perpendicular to the mucosal lumen surface to orientate the specimens correctly. As for the earlier studies, the slides were stained with haematoxylin and eosin and by anti-CD3 immunochemistry. De-identified slides were scanned as whole-slide images using a SlideStrider scanner at a resolution of 0.28 μm per pixel (Jilab Inc., Tampere, Finland). Images were stored as JPEG2000 files in the image server and viewed over the Internet with web-based client software developed for this study (Celiac Slide Analyzer). The same two observers (JT, AP) analysed all slides independently and were unaware of the clinical data or laboratory findings of the patients. Final Marsh classifications for PAXgene-fixed biopsies were based on average villus height to crypt depth ratios according to the previously reported conversion scale: villus height to crypt depth ratio ≥2.3 for Marsh (M)0 if intraepithelial CD3+ lymphocyte density per 100 enterocytes <25, or M1 if ≥25; villus height to crypt depth ratio 1.8 - 2.3 for M2; villus height to crypt depth ratio 1.1 - 1.7 for M3a; villus height to crypt depth ratio 0.5 - 1.0 for M3b; and villus height to crypt depth ratio 0.0-0.4 for M3c (16). Statistical analyses No formal power calculation was undertaken for these exploratory, post hoc analyses. Non-parametric tests were used to assess the significance of differences in paired (Wilcoxon signed rank test) or unpaired (Mann-Whitney test) observations, and to assess correlations (Spearman’s coefficient). All statistical tests were 2-tailed. Summary and significance statistics were computed using Graphpad Prism V7.0d. RESULTS Patient characteristics Table 1 shows the characteristics of the 93 patients who had pre-treatment duodenal biopsies collected across three phase 1 trials and one phase 2 study investigating Nexvax2 in patients with coeliac disease. Disposition of patients in the phase 1 studies who had biopsies has been reported previously, Accepted Article g fixati fix was ew plan ep earli eea i mm um sc an nsc J PE GJP deve l i nde pin Mars hM r atio sa Mars hM h eig h h eig hh ( 16 ) . (1 S tati sSt No f oN t ests wte u npa iun s tatis tstatist Pris mPrism R E SUR Pati eP Tabl eT acros ac disea sd This article is protected by copyright. All rights reserved (11, 12) and is shown in Figure S1 for patients in the phase 2 study. Patient characteristics were similar for the four studies. The median period since diagnosis of coeliac disease was six years. Three (4%) of the 85 patients who had serology evaluated had elevated levels of transglutaminase IgA. At screening, 30 (75%) patients in phase 1 studies and 48 (91%) patients in the phase 2 study had been diagnosed and managed with a gluten-free diet for at least two years, and were confirmed seronegative for TG2-specific IgA. Quantitative histology in second part duodenal biopsies Overall, among all 93 patients assessed, Table 2 shows six (6%) had second part duodenal biopsies classified as Marsh class 0 or 1, 30 (33%) had Marsh 2, and 56 (60%) had Marsh 3a or 3b. The proportion of patients with second part duodenal histology graded as Marsh 3 was similar for each study. There were non-significant trends for Marsh 3a and 3b classification to be less prevalent in patients confirmed as seronegative for transglutaminase 2 IgA who had been diagnosed for at least two years (58%) compared to other patients (78%), and more prevalent in patients subjectively assessed during screening in phase 1 studies as having Marsh class worse than 1 (84%) than those subjectively assessed as Marsh 0 or 1 (68%). Table 3 shows that there was no difference in the distribution of Marsh grades between patients recruited in Australia, New Zealand, and the United States. Proximal to distal changes in duodenal quantitative histology For all 26 patients having pre-treatment biopsies in the first and second phase 1 Nexvax2 clinical trials, two biopsies were collected from each of the bulb, first, second and third part of the duodenum. Figure 1 shows there were no significant differences in quantitative histology between the bulb and first part, but there were differences in villus height to crypt depth ratio and intra-epithelial lymphocyte density between proximal and distal sites that were progressive and statistically significant. Median villus height to crypt depth ratio increased from 1.4 (Marsh 3a) in the bulb to 2.1 (Marsh 2) in the third part, and median intra-epithelial lymphocyte density increased modestly from 30 in the bulb to 44 in the third part (normal <25). Intra-epithelial lymphocyte density in second part biopsies was significantly correlated with the bulb (Spearman rs = 0.70, P = 0.0001), first part (rs = Accepted Article (, s imil asi (4%) (4 s cree nsc diag ndi n ega tn Qua nQ Over a c lass icl prop opr s tud yst patie np t wo y asses sa s ubje su distri bd S tate sSt Prox iP For a Fo t rials ,trials, Figu rFigur first pfir l ymp hly s igni fsi (Mar s(M 30 in 30 biop sb This article is protected by copyright. All rights reserved 18. Haere P, Hoie O, Schulz T, Schonhardt I, Raki M, Lundin KE. Long-term mucosal recovery and healing in celiac disease is the rule - not the exception. Scand J Gastroenterol. 2016;51(12):143946. 19. Tuire I, Marja-Leena L, Teea S, Katri H, Jukka P, Paivi S, et al. Persistent duodenal intraepithelial lymphocytosis despite a long-term strict gluten-free diet in celiac disease. Am J Gastroenterol. 2012;107(10):1563-9. 20. Newnham ED, Shepherd SJ, Strauss BJ, Hosking P, Gibson PR. Adherence to the gluten-free diet can achieve the therapeutic goals in almost all patients with coeliac disease: A 5-year longitudinal study from diagnosis. J Gastroenterol Hepatol. 2016;31(2):342-9. 21. Burbure N, Lebwohl B, Arguelles-Grande C, Green PH, Bhagat G, Lagana S. Olmesartanassociated sprue-like enteropathy: a systematic review with emphasis on histopathology. Hum Pathol. 2016;50:127-34. 22. Mahadev S, Murray JA, Wu TT, Chandan VS, Torbenson MS, Kelly CP, et al. Factors associated with villus atrophy in symptomatic coeliac disease patients on a gluten-free diet. Aliment Pharmacol Ther. 2017;45(8):1084-93. Accepted Article 8. and han 4 6 .4 19. 1 i ntra ein Gast rG 20. 2 diet cdi s tud y 21. 2 asso cas 2016 ;2 22. 22 asso c Phar mP This article is protected by copyright. All rights reserved Figure Legends Figure 1. A shows villous height to crypt depth ratios, and B shows intraepithelial lymphocyte (IEL) densities in formalin-fixed biopsies from the proximal and distal duodenum. Two biopsies were collected from the bulb, 1st, 2nd and 3rd part of the duodenum in 26 patients enrolled in the first and second phase 1 studies of Nexvax2. Medians and interquartile ranges are shown; p values by Wilcoxon test. Data for biopsies with well-oriented villus-crypt units is shown; data for biopsies from the bulb for two patients are missing as neither had any well-oriented villus-crypt units observed. Figure 2: A-F a series of hematoxylin eosin-stained cuttings of the same endoscopy forceps biopsy specimen fixed in PAXgene and embedded in paraffin. A shows the cutting that was graded as Marsh 1 because of tall normal looking villi and an increased density of intraepithelial lymphocytes. The hallmark for tangential cutting is evident, no crypts are cut longitudinally, only cross sections of crypts are seen. Morphometry cannot be performed, and neither can a Marsh class can be given. B shows a section from the same biopsy block re-cut after tilting. C-F show four further sections resulting from further tilting between each re-cut. Crypt hyperplasia is evident and measurable villuscrypt units in the 6 sections give a global mean villus height crypt depth ratio of 0.85, i.e Marsh class 3b according to Taavela et al (16) Figure 3. Relationship between measurements of duodenal villus height and crypt depth, and their ratio or sum for the pooled set of 169 biopsy assessments from 93 patients. A shows villus height and crypt depth are significantly, but weakly correlated, (Spearman correlation coefficient and associated P-value). B shows the individual sums of paired values of villus height and crypt depth measurements in each clinical trial for each site sampled. The median (interquartile range) for all 169 samples was 533 μm (482.5 - 575.5). No significant differences were found between biopsies from the 2nd part compared to biopsies from other sites in Study 1 and 2 (formalin-fixed), or 2nd part biopsies in Study 3 and 4 (PAXgene-fixed). C shows the expected strong correlations between villus height to crypt depth ratio and villus height, and to crypt depth. D shows villus height increases more about fourAccepted Article gu F igu rF densi d c olle cco s eco nse W ilc oW t he b uth F igu rF s peci msp 1 bec 1 h all mh c rypt s s how sh r esul tre c rypt 3b ac 3b F igu rF r atio ra c rypt crypt P-val P-valu i n ea cin 5335 μ c om pco 3 an d3 dept hde This article is protected by copyright. All rights reserved times more rapidly than crypt depth decreases as the sum of villus height and crypt depth increases (VH+CrD = 0.81*VH+273, and CrD = -019*VH+273). Figure 4. Schematic representation of the relationship between villus height and crypt depth measurements in the duodenal mucosa of patients with treated coeliac disease in the present study. Accepted Article tes ( VH +(V F igu rF m eas um This article is protected by copyright. All rights reserved Table 1. Study details and patient characteristics Study: 1 2 3 4 Total Study details Nexvax2 trial 1st Phase 1 2nd Phase 1 3rd Phase 1 Phase 2 Biopsy locations in duodenum Bulb, 1st-3rd Bulb, 1st-3rd 2nd 2 nd Biopsies per site 2 2 4 6 Start date 8Apr13 17Apr14 4Jan16 7Sept18 End date 10Sept13 31Jul14 20Sept16 25Mar19 Patient characteristics, number (%) of patients unless indicated Number of patients having biopsies 16 10 14 53 93 Females 12 (75) 7 (70) 7 (50) 40 (75) 66 (71) Age, years † 44 (33-57) 40 (25-55) 44 (33-57) 39 (33-51) 40 (32-51) Years after diagnosis CeD † 6 (5-7) 3 (2-7) 6 (3-10) 7 (4-11) 6 (3-11) <2 y after diagnosis CeD 1 (6) 0 1 (7) 3 (6) 5 (5) 2 copies HLA-DQB1*02 7 (44) 4 (40) 5 (36) 9 (17) 25 (27) TG2 IgA‡ serology elevated 1 (6) 0 0 2 (4) 3 (3) TG2 IgA‡ serology not done 2 (13) 4 (40) 2 (14) 0 8 (8) Screening histology Marsh 0 or 1 †† 14 (88) 6 (60) ND ND TG2 IgA‡ negative & diagnosis ≥2 y 13 (81) 6 (60) 11 (79) 48 (91) 78 (84) † Median (interquartile range). ‡TG2 IgA, INOVA rh transglutaminase 2 IgA. †† Modified Marsh score assessed by local pathologist on initial cuts of biopsies before re-cutting and quantitative histology was performed. GFD, gluten-free diet. ND, not done. Accepted Article Ta b l eT S tudy St S tudy St N e x v ax 2N B iopsy lBi B iopsie sBi S tart da tSt En d d at e Patie nP Numbe rNu F e mal esFe A ge, ye aAg Y ears a f <2 y aft e<2 2 copie s2 c TG2 Ig ATG TG2 Ig AT Screeni nSc TG2 Ig ATG †† Medi aM biopsie s l c e t This article is protected by copyright. All rights reserved Table 2. Number (%) of patients with well-controlled coeliac disease (1O)†, and other coeliac disease patients, with indicated Marsh (M) grading based on quantitative histology of 2nd part duodenal biopsies. Study: 1 & 2 3 4 Total Patients: 1O Other All 1OOther All 1OOther All 1OOther All M0 0 0 0 1 (9) 0 1 (7) 0 0 0 1 (1) 0 1 (1) M1 1 (5) 0 1 (4) 1 (9) 0 1 (7) 3 (6) 0 3 (6) 5 (6) 0 5 (5) M2 5 (26) 1 (14) 6 (23) 3 (27) 0 3 (21) 19 (40) 2 (40) 21 (40) 27 (35) 3 (20) 30 (32) M3a 12 (63) 6 (86) 18 (69) 6 (55) 2 (67) 8 (57) 25 (52) 2 (40) 27 (51) 43 (55) 10 (67) 53 (57) M3b 1 (5) 0 1 (4) 0 1 (33) 1 (7) 1 (2) 1 (20) 2 (4) 2 (3) 2 (13) 4 (4) M3c 000 0 00 000000 Total 19 7 26 11 3 14 48 5 53 78 15 93 †Patients with TG2 IgA serology in the normal range and on gluten-free diet for >2 years are in the primary analysis population indicated by 1O, patients who had positive TG2 serology or it was positive, or were on GFD for <2 years are not in the primary analysis group are indicated by “Other”. Accepted Article Ta b l eT c oeli aco h isto lh S tudy St Pati enP M 0M M1 M M 2M M 3 a M 3b M 3cM T o tal To †† Patien tP i ndicate analysi sn c i A This article is protected by copyright. All rights reserved Table 3. Number (%) of patients† with indicated Marsh (M) grading based on quantitative histology of 2nd part duodenal biopsies according to country of recruitment. Location: Australia New Zealand USA Study: 1&2 3 4 Total 1&2 4 Total 4 Total M0 01 (9) 01 (3) 00 00 1 (1) M1 01 (9) 2 (11) 3 (8) 1 (10) 1 (13) 2 (11) 0 5 (6) M2 3 (33) 3 (27) 7 (37) 13 (33) 2 (20) 2 (25) 4 (22) 10 (48) 27 (35) M3a 6 (67) 6 (55) 9 (47) 21 (54) 6 (60) 5 (63) 11 (61) 11 (52) 43 (55) M3b 0 0 1 (5) 1 (3) 1 (10) 01 (6) 0 2 (3) M3c 000 000 000 Total 9111939 10 8 18 21 78 †Limited to patients in the primary analysis population: TG2 IgA serology in the normal range and on gluten-free diet for >2 years Accepted Article Tabl eT of 2o nd Loc at iL S tudy S M 0M M1 M M 2M M 3 a M M 3bM M 3cM T o tal To †† Limit e l i This article is protected by copyright. All rights reserved Bulb 1st 2nd 3rd Lowest 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Villus height to crypt depth ratio M1 M2 M3a M3b M3c P = 0.0002 P < 0.0001 P = 0.003 P = 0.03 1.4 (1.1-1.5) 1.5 (1.1-1.7) 1.7 (1.4-2.0) 2.1 (1.7-2.2) 1.2 (0.9–1.4) Median (25th-75th%) Bulb 1st 2nd 3rd Highest 0 20 40 60 80 100 CD3+ IELs (per 100 enterocytes) P = 0.006 P = 0.0002 P = 0.006 P = 0.04 30 (18-47) 34 (24-49) 38 (30-48) 42 (31-57) 45 (34-58) Figure 1. A shows villous height to crypt depth ratios, and B shows intraepithelial lymphocyte (IEL) densities in formalin-fixed biopsies from the proximal and distal duodenum. Two biopsies were collected from the bulb, 1st, 2nd and 3rd part of the duodenum in 26 patients enrolled in the first and second phase 1 studies of Nexvax2. Medians and interquartile ranges are shown; p values by Wilcoxon test. Data for biopsies with well-oriented villus-crypt units is shown; data for biopsies from the bulb for two patients are missing as neither had any well-oriented villus-crypt units observed. Accepted Article Article F igu rF l ymp hy duod edu duod ed Medi M with ww patie npa Figure 2: A-F a series of hematoxylin eosin-stained cuttings of the same endoscopy forceps biopsy specimen fixed in PAXgene and embedded in paraffin. A shows the cutting that was graded as Marsh 1 because of tall normal looking villi and an increased density of intraepithelial lymphocytes. The hallmark for tangential cutting is evident, no crypts are cut longitudinally, only cross sections of crypts are seen. Morphometry cannot be performed, and neither can a Marsh class can be given. B shows a section from the Accepted Article F i g for c c ut t de n c ry p be p same biopsy block re-cut after tilting. C-F show four further sections resulting from further tilting between each re-cut. Crypt hyperplasia is evident and measurable villuscrypt units in the 6 sections give a global mean villus height crypt depth ratio of 0.85, i.e Marsh class 3b according to Taavela et al. (16). This article is protected by copyright. All rights reserved 0 100 200 300 400 500 600 0 100 200 300 400 500 Villus height (VH), μm Crypt depth (CrD), μm rs = -0.22, P = 0.004 0 100 200 300 400 500 600 0.0 0.5 1.0 1.5 2.0 2.5 3.0 μ m VH:CrD ratio VH, rs = 0.78, P < 10-10 CrD, rs = -0.74, P < 10-10 Bulb 1st 2nd 3rd 2nd 2nd 0 200 400 600 800 Sum (VH + CrD), μm Study: 1 & 2 3 4 Site: Median & interquatile range 0 100 200 300 400 500 600 0 200 400 600 800 Villus height (VH), μm μm Sum (VH + CrD), rs = 0.82, P < 10-10 CrD AB CD Accepted Article This article is protected by copyright. All rights reserved Figure 3. Relationship between measurements of duodenal villus height and crypt depth, and their ratio or sum for the pooled set of 169 biopsy assessments from 93 patients. A shows villus height and crypt depth are significantly, but weakly correlated, (Spearman correlation coefficient and associated P-value). B shows the individual sums of paired values of villus height and crypt depth measurements in each clinical trial for each site sampled. The median (interquartile range) for all 169 samples was 533 μm (482.5 - 575.5). No significant differences were found between biopsies from the 2nd part compared to biopsies from other sites in Study 1 and 2 (formalin-fixed), or 2nd part biopsies in Study 3 and 4 (PAXgene-fixed). C shows the expected strong correlations between villus height to crypt depth ratio and villus height, and to crypt depth. D shows villus height increases more about four-times more rapidly than crypt depth decreases as the sum of villus height and crypt depth increases (VH+CrD = 0.81*VH+273, and CrD = -019*VH+273). Accepted Article