Application of a pattern‐based approach to histological diagnosis in very early onset IBD (VEO‐IBD) in a multicentric cohort of children with emphasis on monogenic disease with IBD‐like morphology

Very early‐onset inflammatory bowel disease (VEO‐IBD) is a clinical umbrella term referring to IBD‐like symptoms arising in children before the age of 6 years, encompassing both ‘pure’ IBD, such as ulcerative colitis (UC) and Crohn's disease (CD) and monogenic diseases (MDs), the latter often involving genes associated with primary immunodeficiencies. Moreover, histological features in gastrointestinal (GI) biopsies in MD can also have IBD‐like morphology, making differential diagnosis difficult. Correct diagnosis is fundamental, as MDs show a more severe clinical course and their inadequate/untimely recognition leads to inappropriate therapy.

Application of a pattern-based approach to histological diagnosis in very early onset IBD (VEO-IBD) in a multicentric cohort of children with emphasis on monogenic disease with IBD-like morphology Aims: Very early-onset inflammatory bowel disease (VEO-IBD) is a clinical umbrella term referring to IBD-like symptoms arising in children before the age of 6 years, encompassing both 'pure' IBD, such as ulcerative colitis (UC) and Crohn's disease (CD) and monogenic diseases (MDs), the latter often involving genes associated with primary immunodeficiencies.Moreover, histological features in gastrointestinal (GI) biopsies in MD can also have IBD-like morphology, making differential diagnosis difficult.Correct diagnosis is fundamental, as MDs show a more severe clinical course and their inadequate/untimely recognition leads to inappropriate therapy.
Methods and results: Biopsy samples from the lower and upper GI tract of 93 clinically diagnosed VEO-IBD children were retrospectively selected in a multicentre cohort and histologically re-evaluated by 10 pathologists blinded to clinical information.Each case was classified according to morphological patterns, including UC-like; CD-like; enterocolitis-like; apoptotic; eosinophil-rich; and IBD-unclassified (IBD-U).Nine (69%) MD children showed IBD-like morphology; only the IBD-U pattern correlated with MD diagnosis (P = 0.02) (available in 64 cases: 51 non-MD, true early-onset IBD/other; 13 MD cases).MD patients showed earlier GI symptom onset (18.7 versus

Introduction
Very early-onset inflammatory bowel disease (VEO-IBD) refers to a clinical presentation with IBD-like symptoms, such as chronic diarrhoea, vomiting, perianal lesions, haematochezia, abdominal pain and rectal bleeding, arising in children before the 6th year of life.These clinical features are found both in 'pure' IBD, such as Crohn's disease (CD) and ulcerative colitis (UC), and in causative monogenic or digenic defects, often involving genes associated with primary immunodeficiencies (PID). 1 'Pure' IBD represents the multifactorial result of a dysregulated immune response to environmental factors in a genetically susceptible host and, in older paediatric patients and adults, is most often polygenic, involving more than 200 risk loci spanning more than 300 genes.Conversely, monogenic disease (MD) has mendelian inherited transmission and involves genes crucial to the immune system network, although they can be classified into six main (and sometimes overlapping) immune dysregulation categories: (i) general immune dysregulation, (ii) T and B cell defects, (iii) phagocytic defects, (iv) hyper-and autoinflammatory conditions, (v) epithelial barrier dysfunction and (vi) other conditions. 2 For this reason, MD leads to a variety of conditions including gastrointestinal (GI) and extra-GI symptoms, such as pulmonary and/or upper airway and/or urinary recurrent infections starting in the first months of life and unresponsive to conventional therapy, early-onset tumours, endocrine dysfunction, hepatosplenomegaly, cytopenia, high ferritin levels (macrophage activation syndrome) and somatic defects in skin, hair, teeth and various other ectodermal elements.Non-immune syndromic aspects, such as facial malformations and delays in psychophysical development, are very common in MD, but they appear after 2-3 years of life. 3Moreover, GI symptoms in MD are characterised by a more severe clinical course, reduced responsiveness to conventional IBD therapy, intense treatment regimens involving extensive surgery, and more intensive medical treatment; most importantly, inadequate recognition of underlying MDs may lead to inappropriate therapy.Indeed, immunosuppressive therapy, used to treat IBD, can be fatal in children with MD/PID with IBDlike symptoms. 1With this in mind, consultation by an expert immunologist for in-depth immune deficiency evaluation and testing is recommended, especially in infants, before initiation of therapy.Moreover, as MDs follow mendelian transmission, patients and their families should be referred to geneticists for analysis of first-degree family members. 4][7][8][9][10] Erroneously labelling the process as 'IBD' in the histology report early in the disease course will prevent the physician from considering further conditions as well as initiating inappropriate clinical management and therapy. 11Moreover, lesions such as colitis without chronic damage, with eosinophilic-rich inflammation or apoptotic bodies, usually observed in infections, autoimmune disorders, allergic and/or food intolerance, have also been described in MDs. 12 In real clinical practice, the pathologist is often asked to report on histological findings before the diagnostic process is completed with no clinical or, sometimes, endoscopic details.The use of term 'VEO-IBD' in the histological report could be confounding and possibly lead to inappropriate and dangerous treatment. 11he aim of the study was to collect a large multicentric case series of lower GI (and any available upper GI) biopsy sets from children with a clinical diagnosis of VEO-IBD, with a review of histological aspects by a panel of experienced pathologists, using a morphological pattern-based approach, blinded to clinical information, and comparison of histology with the final multidisciplinary diagnosis (including true IBDs and MDs).

Materials and methods
Histological slides from gastrointestinal biopsy sets of clinically diagnosed VEO-IBDs in children younger than 6 years were retrospectively selected from January 2015 to July 2022 from the archives of the Pathology Units of Fondazione IRCCS Casa Sollievo della Sofferenza (San Giovanni Rotondo), Ospedale San Camillo Forlanini (Rome), Azienda Ospedaliera-Universitaria Meyer (Firenze), Universit a degli Studi Federico II (Napoli), Universit a degli studi di Padova (Padova), IRCCS Ospedale Gaslini (Genova), Ospedale Pousillipon (Napoli), Universit a degli Studi di Pavia (Pavia), Ospedale Pediatrico Bambino Ges u (Rome).Ten pathologists with experience in gastrointestinal paediatric and adult pathology jointly re-evaluated all cases during monthly meetings and blinded to clinical information.All available original haematoxylin and eosin-stained slides were reassessed and categorised according to the histological pattern in four previously described VEO-IBD categories: [5][6][7] (a) UC-like pattern, characterised by a continuous marked inflammatory infiltrate with eosinophils, monocytes, plasma cells and lymphocytes with or without cryptitis, Paneth cell metaplasia, ulcerations and architectural gland atrophy/distortion (Figure 1A, B     Lower GI sampling protocols were complete with samples from the ileum and different sites of the colon in 72% of cases (67 of 93), while 11% (10 cases) had no ileal sample, 15% (14 cases) of samples were limited to the left colon and rectum and in 2% (two cases), only one left-sided site was sampled.Most cases (81%, 75 cases) had complete endoscopic reports attached and the majority (76%, 57 of the 75 cases) of these showed various endoscopic alterations.Upper GI sampling was performed in 63% (59 cases) with variable biopsy sets including both stomach and duodenum samples (46%), with the addition of oesophageal samples in 25%, and other combinations of just one site in a third of cases (29%).
Histologically, the most frequent pattern was the UC-like colitis pattern seen in 39%, while the least was the apoptotic pattern and IBD-U pattern both seen in 4%; see Table 1.Mixed patterns were present in nine cases and histology was unremarkable in seven cases; in one case, no agreement was reached in the assignation of a histological pattern (five pathologists for CD-like pattern and five pathologists for an enterocolitis-like pattern).
In 64 patients (69%), follow-up and final outcome were available (see Table 2) and 51 patients were diagnosed with UC (33), CD (eight) or other causes (10) (non-MD group), while in 13 patients a final diagnosis of MD was made.No differences were seen in the male : female ratio between MD and non-MD; however, MD patients tended to show earlier onset of GI symptoms (18.7 versus 26.9 months) and were sent to endoscopy earlier (22 versus 37 months), and these differences were statistically significant (P < 0.05).
No significant differences in histological patterns were seen between groups except for the IBD-U pattern, which was significantly correlated with MD cause (P = 0.02).No significant differences in the topographic distribution of lesions were seen between non-MD and MD cases.

U P P E R G I H I S T O L O G I C A L F I N D I N G S
Upper GI findings, in part, reflect the variability in sampling protocols (see above).Most cases showed no histological lesions, and 72% (13 of 18), 64% (32 of 50) and 70% (33 of 47) of cases from the oesophagus, stomach and duodenum, respectively, were diagnosed as normal.Histological lesions in the oesophagus, stomach and duodenum are shown in Table 1 and Figure 3.No statistically significant differences in histological features were seen between non-MD causes and MDs.IBD-like histological patterns in VEO-IBD 445 presented with a UC-like pattern, two cases with a CD-like pattern and three cases with an IBD-U pattern, while four cases showed non-IBD-like morphology (three cases with an enterocolitis-like pattern, one case with an apoptotic pattern).Although, at first endoscopy, no clinical information/suspicion was given, the case (case 9) with an apoptotic pattern was identified as suspicious for MD in the pathology report, and a recommendation for in-depth clinical study and genetic evaluation on the basis of the peculiar morphology, including numerous apoptotic bodies, dramatic, diffuse glandular atrophy, Paneth cell metaplasia (Figure 1E,F) and mild inflammation, in the whole intestinal tract.

D E S C R I P T I O N O F M D C A S E S
A further three cases presented as an IBD-U histological pattern showing mild and focal glandular architectural damage; mild, occasionally discontinuous, lymphomonocytic inflammation; and ulcers and granulation tissue.One child had also undergone upper GI endoscopy with diffuse duodenal blunting with increased CD3 intra-epithelial lymphocytes (coeliac disease-like in the absence of serological and genetic tests positive for coeliac disease), isolated apoptotic bodies and focally enhanced gastritis.

Discussion
VEO-IBD is a clinical umbrella term used to describe IBD-like symptoms in children aged fewer than 6 years and comprises a heterogeneous spectrum, both phenotypically and genetically, of diseases with GI and extra-GI symptoms. 1Numerous studies concerning the clinical manifestations of VEO-IBD identified up to 15% of patients suffering from MD, often involving genes associated with PID. 1,2,13ue to the wide phenotypical and genetic heterogeneity of these conditions and the lack of specific clinical, endoscopic or histological findings, it is often difficult to reach a final diagnosis. 14In this context, very few studies, mostly case reports, have described IBD-like histological features in MD and, more specifically, in PID.
6][7] In our study, differences in disease onset (MD children tend to be sent to endoscopy earlier, probably due to earlier onset of symptoms and more severe disease) were found between non-MD and MD cases in our cohort; however, no significant differences in histological patterns were found.Our findings are similar to the study by Conrad et al., who evaluated a mono-institutional retrospective case series comprising 57 cases, and found no statistical differences in histological features between 'pure' IBD (46 patients) and MD (11 patients). 15In particular, the authors analysed 11 histological features [active inflammation (neutrophil-predominant cryptitis and crypt micro-abscesses), chronic architectural changes, apoptoses, granulomas, lymphoid aggregates, intraepithelial lymphocytes, plasma cell infiltrates in the lamina propria, eosinophils in the lamina propria or crypts or surface epithelium, mucus and goblet cell depletion, small bowel villous blunting and colonic villous surface transformation] in all cases, and   similar histological features were found between the two groups. 15n our cohort, a final diagnosis of MD was made in cases showing all morphological features, including IBD-like features (UC, CD, IBD-unspecified) as well as enterocolitis-like and apoptotic patterns, with no significant statistical differences, even though other studies in the literature have proposed some diagnostic clues to a correct MD diagnosis. 12,15These include increased frequency of apoptoses, severe and diffuse gland loss in the absence of inflammation (as seen in our case 9), villous atrophy (cases 6 and 13), diffuse eosinophilic infiltrates and lack of plasma cells in the mucosa.Of note, a poorly defined IBD-U pattern of injury in our cohort was statistically correlated with MD, meaning that ill-defined aspects of IBD-like damage should prompt suspicion.Furthermore, while upper GI findings are often normal, some features such as focally enhanced gastritis 17 and the aforementioned villous blunting/atrophy should again be looked for and mentioned in the pathology report.
We did not find some patterns in our cohort which have been described, such as the lymphocytic (with lymphocytosis and nodular lymphoid hyperplasia) or the granulomatous patterns, 15 and this may be due to the low number of diagnosed MD cases.The rarity of this disease entity is highlighted by the fact that in 7 years and more than five tertiary paediatric gastroenterology centres, 93 children were biopsied and only 13 definitive cases of MDs were found, making it difficult to accrue sufficient numbers for statistically sound interpretation.
]11 First, a 'definitive' diagnosis of UC/CD/IBD-U should be avoided in a clinically suspected VEO-IBD, without appropriate multidisciplinary work-up.Secondly, as morphological lesions can be found in endoscopically healthy mucosa, biopsy sampling should always be performed at all intestinal sites (according to IBD guidelines 16 ).Thirdly, upper GI histological findings can sometimes lead to unexpected diagnoses.Indeed, three cases of congenital tufting enteropathy were documented in clinically suspected VEO-IBD, two cases showing a UC-like pattern in intestinal biopsies.In this context, it would be useful to always acquire a whole upper GI biopsy set [including oesophageal, gastric (both body and antrum) and duodenal (I and II portion) biopsies]. 6Finally, clinical information should always be made available to the pathologist, and discussion of cases in a multidisciplinary context is imperative.
Our study has some limits, such as its retrospective nature and the absence of a definitive diagnosis for some children (molecular profiling to investigate MD was not available in all cases).A further limitation is that we did not perform a concordance study for pattern recognition between the participating pathologists due to the need for standardisation of reporting at the start of the study.A future concordance analysis will be important to help in identifying key issues with this pattern-based approach, but will require the collection of a large international series of cases.
In conclusion, our results show that IBD-like morphology is frequent in MD (69%), and that no single pathological feature is able to predict the true nature of VEO-IBD; a pattern-based approach is very much advised, so that the multidisciplinary team can perform more in-depth clinical, laboratory or genetic investigations to correctly diagnose these young patients.
); (b) CD-like pattern, characterised by a discontinuous inflammatory infiltrate with eosinophils, neutrophils and plasma cells, transmural inflammation, deep ulceration of mucosa, non-necrotising granulomas, pronounced lymphoid hyperplasia, aphthous lesions over lymphoid aggregates and, in the small bowel, villous atrophy (Figure 1C,D); (c) enterocolitis-like pattern, characterised by focal detachment of colonic

Table 1 .
Clinicopathological findings in the total cohort and in the non-monogenic disease and monogenic disease groups (total 93 cases; in 64 cases a definitive diagnosis was reached, including 51 cases of non-MD causes and 13 cases of MD) Ó 2023 The Authors.Histopathology published by John Wiley & Sons Ltd., Histopathology, 84, 440-450.IBD-like histological patterns in VEO-IBD 443 epithelium, extensive, widespread continuous marked inflammatory infiltrate with eosinophils, monocytes, plasma cells and lymphocytes with minimal architectural distortion in the absence of chronic damage (Figure 1E,F); (d) apoptotic pattern,

Table 3
describes in detail the 13 cases of diagnosed MDs in our series, showing that IBD-like morphology is frequent in MD cases (69%).Indeed, four cases