THE ROLE OF FLOW CYTOMETRY IN COMPLICATED CELIAC DISEASE
Creators: name: BRANCHI, FEDERICA
Publisher: name: Universit� degli Studi di Milano
Year: 2018

description: Background Refractory celiac disease (RCD) is defined as persistence or recurrence of clinical symptoms of malabsorption and histologic signs of villous atrophy despite at least 1 year of strict adherence to a gluten-free diet. Two different entities of RCD have been described: RCD I shows a polyclonal pattern of intraepithelial lymphocytes (IEL), while in RCD II a monoclonal transformation of IEL can be identified (usually detected by means of TCR clonality analysis). Patients with RCD II have a poorer prognosis and a high risk of development of enteropathy-associated T-cell lymphoma (EATL), so that it has recently been proposed to consider RCD II as a form of low-grade intraepithelial lymphoma (pre-EATL). Recently, flow cytometric analysis of isolated intestinal lymphocytes has been introduced as new diagnostic modality for the detection of aberrant intestinal lymphocytes (ILs) and as a stronger predictor of EATL development than gene clonality analysis. Aims The aims of this study were (i) to evaluate the presence of aberrant ILs in a cohort of non-celiac, celiac and RCD patients by means of flow cytometry, (ii) to verify whether there is an association between clinical characteristics of RCD patients and presence of aberrant ILs, (iii) to compare different ILs detection strategies with the purpose of validating a simpler strategy than the ones currently proposed and (iv) to evaluate whether flow cytometric analysis of aberrant ILs could accurately identify RCD II/pre-EATL patients in our cohort. Methods Flow cytometry analysis of intestinal lymphocytes isolated from duodenal biopsy specimens from RCD, uncomplicated CD patients and controls was performed. Several lymphocyte markers (CD3, CD4, CD8, CD7, CD103, TCR??) were applied in order to identify aberrant ILs, defined by means of several gating strategies including cytCD3+surfCD3-CD7+ and surfCD3-CD7+CD103+. Percentages of aberrant ILs as well as clinical characteristics in different patients groups were compared. Results A total of 130 flow cytometry assays were performed on 109 patients, including 42 controls, 21 active CD, 16 CD on GFD and 30 RCD. RCD patients were initially subgrouped according to the presence (TCRclon+, n=17) or absence (TCRclon-, n=13) of TCR clonality: the presence of elevated aberrant ILs was compared between the two subgroups, with elevated ILs detectable exclusively in the RCDclon+. Patients with elevated ILs also showed a significantly more severe malabsorption (assessed by a composite score). A cut-off of 11% aberrant ILs for the most reliable strategy allowed to identify 2 patients with EATL, as well as a small group of high-risk RCD (5/30) that were classified as RCDII/pre-EATL. According to low aberrant ILs levels, the other RCD patients (21/30) were classified as RCD I/low-risk RCD. However, this technique proved negative in 2 cases of overt EATL and was not able to correctly identify as ?high-risk RCD? one patient with ulcerative jejunitis (who later developed a EATL), as well as one patient in whom a diagnosis of gamma-delta T-cell lymphoma was made. In the setting of RCD, aberrant ILs assessment by means of flow cytometry showed a Specificity of 100% but a Sensitivity of 67% for the detection of pre-EATL/EATL Alternative, simpler gating strategies for aberrant ILs showed similar accuracy to the principal strategy, however these results need further validation. Conclusion In routine clinical practice, flow cytometry for the assessment of aberrant ILs could prove a simple and accurate predictor for high-risk RCD. However, its use as a diagnostic strategy to classify patients into RCD I (low risk) and RCD II/pre-EATL could lead to missing cases of RCD patients with elevated risk. In order to prevent the consequences of false negative results, a multifaceted diagnostic approach taking TCR clonality and clinical manifestations (i.e. malabsorption) into account could maximize accuracy.