THE ROLE OF MOLECULAR SIGNATURES IN COMPLICATED CELIAC DISEASE

Introduction: Refractory celiac disease (RCD) is defined as the persistence of symptoms and villous atrophy despite adherence to a gluten-free diet (GFD). Based on their molecular signatures, aberrant intraepithelial lymphocytes (aIELs) and monoclonal TCRγ rearrangement, RCD patients are differentiated into RCD type 1 (RCD1) and RCD type 2 (RCD2). This distinction is challenging but essential, as RCD2 is associated with enteropathy-associated lymphoma (EATL) and a poor prognosis, with no effective treatment currently available. Flow cytometry (f-cyt) is the diagnostic modality of choice in RCD to characterize IELs phenotypes, however limited data are available on the role of f-cyt in differentiating RCD from other chronic enteropathies. Moreover, altered molecular signatures at diagnosis have a prognostic significance, but there is limited evidence on their role in monitoring RCD patients during follow-up. Aims: This study aimed at evaluating: 1. the presence of aIELs in a cohort of non-celiac, celiac and RCD patients by means of f-cyt of intestinal lymphocytes isolated from duodenal biopsies; 2. the association between clinical, endoscopic and biochemical characteristics of RCD patients and the presence of aIELs; 3. if f-cyt analysis of aIELs can accurately identify high-risk RCD patients in our cohort, either used alone or in combination with TCRγ rearrangement and duodenal atrophy; 4. the performance of γδ IELs in adult patients as a supporting tool and possible biomarker of celiac disease; 5. the treatment regimens for RCD and their clinical efficacy and effect on histopathologic and immunologic parameters. Methods: This single-center retrospective study included all adult patients who underwent f-cyt analysis between 2015 and 2023 at Fondazione Ca’ Granda IRCCS Policlinico di Milano (Milan, Italy). Patients were categorized based on the presence of aIELs and clinical diagnoses: RCD, non-complicated CD, non-celiac enteropathies (NCE). Clinical, endoscopic, histological data were analyzed to assess outcomes based on IELs phenotype at diagnosis and at follow-up. Treatment regimens for RCD and related outcomes were described and analyzed. Results: A total of 119 patients (38 RCD, 59 non-complicated CD, 22 NCE; 77 females; mean age 53.6 ± 14.7 years) underwent 168 f-cyt. Follow-up f-cyt data were available for 32 RCD patients (19 RCD1, 13 RCD2). Aberrant phenotype (aIELS>20%) was significantly associated with malabsorption, higher mortality (HR 3.78, 95% CI 1.30-11.01, p 0.02), and increased EATL occurrence (HR 9.00, 95% CI 2.10-38.61, p < 0.01) in RCD2. When patients were stratified based on the combination of TCR clonality and aberrant phenotype, there was no significant difference in outcomes among those with one or both markers altered. The presence of aIELs>20% demonstrated good diagnostic performance for RCD2 (AUC 0.95, 95% CI 0.8791-1.000; p < 0.01); however, there were six false positive cases in our cohort. During follow-up, there was no significant difference in outcomes between patients who achieved normalization of aIELs or TCR clonality and those who did not. In RCD patients, the correlation between molecular response and both histological response (κ = 0.12) and clinical response (κ = 0.27) was poor. Conclusion: Our study confirms the accuracy and prognostic role of aIELs in RCD, in line with previous research. Of note, an aberrant phenotype was observed in six NCEs (false positives RCD2), suggesting to further investigate f-cyt use in NCEs. Another novel finding from our study is that neither normalization of aIELs nor TCR clonality correlated well with histological or clinical outcomes, suggesting limited utility for these markers in assessing treatment response. Further studies are needed to confirm these findings, and to identify other possible biomarkers for monitoring disease progression.