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The combination of clinical parameters and immunophenotyping of intraepithelial lymphocytes allows to assess disease severity in refractory celiac disease

  • Federica Branchi
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Hindenburgdamm 30, 12203 Berlin, Germany

    Center for the Diagnosis and Prevention of Celiac Disease - Gastroenterology and Endoscopy Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, via Francesco Sforza 35, 20122 Milan, Italy
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  • Jakob Johann Wiese
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Hindenburgdamm 30, 12203 Berlin, Germany
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  • Claudia Heldt
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Hindenburgdamm 30, 12203 Berlin, Germany
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  • Subhakankha Manna
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Hindenburgdamm 30, 12203 Berlin, Germany
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  • Violaine Dony
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Hindenburgdamm 30, 12203 Berlin, Germany
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  • Christoph Loddenkemper
    Affiliations
    PathoTres Gemeinschaftspraxis für Pathologie und Neuropathologie, Teltowkanalstrasse 2, 12247 Berlin, Germany
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  • Christian Bojarski
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Hindenburgdamm 30, 12203 Berlin, Germany
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  • Britta Siegmund
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Hindenburgdamm 30, 12203 Berlin, Germany
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  • Thomas Schneider
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Hindenburgdamm 30, 12203 Berlin, Germany
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  • Severin Daum
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Hindenburgdamm 30, 12203 Berlin, Germany
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  • Michael Hummel
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institut für Pathologie, Molekularpathologie, Charitéplatz 1, 10117 Berlin, Germany
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  • Author Footnotes
    1 equal contributors.
    Verena Moos
    Footnotes
    1 equal contributors.
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Hindenburgdamm 30, 12203 Berlin, Germany
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  • Author Footnotes
    1 equal contributors.
    Michael Schumann
    Correspondence
    Corresponding author.
    Footnotes
    1 equal contributors.
    Affiliations
    Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Medizinische Klinik für Gastroenterologie, Infektiologie und Rheumatologie, Hindenburgdamm 30, 12203 Berlin, Germany
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  • Author Footnotes
    1 equal contributors.

      Abstract

      Background

      Flow cytometry of intestinal lymphocytes is discussed to be a stronger predictor of enteropathy-associated T-cell lymphoma development in refractory celiac disease than T-cell clonality analysis.

      Aims

      To investigate possible associations between clinical characteristics of refractory celiac disease patients and aberrant intraepithelial lymphocytes and to evaluate the accuracy of immunophenotyping for the identification of high-risk refractory celiac disease.

      Methods

      Flow cytometry of isolated lymphocytes from duodenal biopsies of controls and celiac disease patients was performed and results were compared to clinical data.

      Results

      Flow cytometry analysis was performed on 42 controls, 37 non-complicated celiac disease and 30 refractory celiac disease cases with or without T-cell receptor clonality. Elevated aberrant intraepithelial lymphocyte counts were significantly associated with severe malabsorption. A 15% cut-off (aberrant lymphocytes among all lymphocytes) had the best discriminatory ability to identify high-risk patients. However, this technique failed to identify some high-risk cases (sensitivity 63%, specificity 100%). The severity of malabsorption was added to the criteria for high-risk refractory celiac disease, improving the correct patients’ allocation (sensitivity 100%, specificity 96%).

      Conclusion

      Immunophenotyping of aberrant intraepithelial lymphocytes is a good predictor for high-risk refractory celiac disease. Furthermore, adding the evaluation of malabsorption to the diagnostic assessment of refractory celiac disease optimizes accuracy.

      Keywords

      1. Introduction

      Refractory celiac disease (RCD) is a rare and complex clinical entity, defined as the persistence of clinical and histological signs of enteropathy after at least one year of strict gluten-free diet (GFD) [
      • Ludvigsson J.F.
      • Leffler D.A.
      • Bai J.C.
      • et al.
      The Oslo definitions for coeliac disease and related terms.
      ]. Patients with type II RCD (RCD II) have a poorer prognosis and a high risk of development of enteropathy-associated T-cell lymphoma (EATL) in the course of their disease [
      • Malamut G.
      • Cellier C.
      Complications of coeliac disease.
      ,
      • Cil T.
      • Altintaş A.
      • Işikdoğan A.
      • et al.
      Screening for Celiac disease in Hodgkin and non-Hodgkin lymphoma patients.
      ,
      • Daum S.
      • Cellier C.
      • Mulder C.J.
      Refractory coeliac disease.
      ]. It has been demonstrated that intestinal intraepithelial lymphocytes (IELs) of RCD II patients have undergone a transformation from T-cells to premalignant, highly proliferative clonal innate-like cells following an IL15- and NOTCH-dependent pathway [
      • Ettersperger J.
      • Montcuquet N.
      • Malamut G.
      • et al.
      Interleukin-15-dependent T-cell-like innate intraepithelial lymphocytes develop in the intestine and transform into lymphomas in celiac disease.
      ]. Therefore, RCD II and EATL are considered as part of the same clinical entity characterized by the presence of aberrant clones among intraepithelial lymphocytes, with RCD II often labelled as pre-lymphomatous condition. Widely used diagnostic tools to identify RCD II include PCR-based strategies to detect clonal rearrangements of the T-cell receptor (TCR) genes in the duodenal mucosa as well as the detection of an aberrant phenotype of intestinal IELs with immunohistochemistry [
      • Rubio-Tapia A.
      • Hill I.D.
      • Kelly C.P.
      • Calderwood A.H.
      • Murray J.A.
      • Gastroenterology ACo
      ACG clinical guidelines: diagnosis and management of celiac disease.
      ]. Multiparameter flow cytometry is a method for the analysis of cellular populations, introduced in recent years as an innovative technique for the identification of IELs with an aberrant phenotype from single-cell isolates of duodenal mucosa [
      • Verbeek W.H.
      • Goerres M.S.
      • von Blomberg B.M.
      • et al.
      Flow cytometric determination of aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more accurately than T-cell clonality analysis in Refractory Celiac Disease.
      ,
      • Verbeek W.H.
      • von Blomberg B.M.
      • Scholten P.E.
      • Kuik D.J.
      • Mulder C.J.
      • Schreurs M.W.
      The presence of small intestinal intraepithelial gamma/delta T-lymphocytes is inversely correlated with lymphoma development in refractory celiac disease.
      ,
      • Malamut G.
      • Afchain P.
      • Verkarre V.
      • et al.
      Presentation and long-term follow-up of refractory celiac disease: comparison of type I with type II.
      ]. Aberrant IELs in RCD display an altered expression of common T-cell markers and a distinct expression of TCR gene rearrangements [
      • van Wanrooij R.L.
      • Schreurs M.W.
      • Bouma G.
      • et al.
      Accurate classification of RCD requires flow cytometry.
      ,
      • Tack G.J.
      • van Wanrooij R.L.
      • Langerak A.W.
      • et al.
      Origin and immunophenotype of aberrant IEL in RCDII patients.
      . In the first studies, increased aberrant IELs proved to be a better predictor of EATL development than molecular-pathology assessment of T cell clonality [
      • Verbeek W.H.
      • Goerres M.S.
      • von Blomberg B.M.
      • et al.
      Flow cytometric determination of aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more accurately than T-cell clonality analysis in Refractory Celiac Disease.
      ]. Follow-up data suggested a possible role of aberrant IEL assessment also in the setting of treatment monitoring [
      • Tack G.J.
      • Verbeek W.H.
      • Al-Toma A.
      • et al.
      Evaluation of Cladribine treatment in refractory celiac disease type II.
      ]. However, as opposed to immunohistochemistry and TCR gene rearrangement studies, it is not a widely available technique and its application in routine clinical practice still needs validation.
      The aims of this study were to determine the presence of aberrant IELs in our cohort of non-celiac, celiac and RCD patients by means of immunophenotyping of intestinal lymphocytes isolated from duodenal biopsies; to verify the reproducibility of the previously proposed threshold for aberrant IELs in our cohort and thus validate this diagnostic technique; moreover, to evaluate whether the combination of IEL immunophenotyping with other parameters (clinical, biochemical, molecular pathological) contributes to accurately identify high-risk RCD patients (RCD II/pre-EATL).

      2. Methods

      2.1 Patients

      Adult subjects with and without CD, referred for gastroscopy as part of their clinical management, were consecutively enrolled in the study between 2014 and 2018. The following inclusion criteria allowed to assign patients to four groups:
      • 1.
        Patients with an established diagnosis of active CD in line with international guidelines [
        • Rubio-Tapia A.
        • Hill I.D.
        • Kelly C.P.
        • Calderwood A.H.
        • Murray J.A.
        • Gastroenterology ACo
        ACG clinical guidelines: diagnosis and management of celiac disease.
        ], with evidence of TTG-IgA positivity, Marsh duodenal lesions and an ongoing gluten containing diet;
      • 2.
        Patients with an established CD diagnosis and adhering to a GFD for longer than a year, without evidence of villous atrophy at duodenal histology at the time of enrolment, considered GFD responders;
      • 3.
        Patients with RCD, fulfilling the criteria for the diagnosis of refractory disease (established CD diagnosis, persistence of Marsh III duodenal lesions after longer than one year of GFD and after professional revision of dietary adherence, exclusion of differential diagnoses).
      • 4.
        Controls, who presented with unspecific gastrointestinal symptoms and had a normal gastroscopy including duodenal histology.
      Exclusion criteria were gastrointestinal bleeding at the time of endoscopy and refusal to participate in the study. Clinical and biochemical characteristics were collected and used to compare groups. As part of the diagnostic work-up for RCD, patients underwent endoscopic evaluation of the small bowel (capsule endoscopy, enteroscopy) as well as cross-sectional imaging (abdominal CT or magnetic resonance imaging) in order to assess the extent and severity of villous atrophy and to screen for malignancy. Follow-up data on the endpoints death and lymphoma development until 2021 were collected.

      2.2 Flow cytometric analysis of duodenal biopsy specimens

      2.2.1 Acquisition of specimens and IELs isolation

      During the endoscopic examination, biopsy specimens were collected. In addition to formalin-fixed duodenal samples for histopathology investigation, another 4–8 biopsy specimens were obtained and placed in 10 ml NaCl 0.9% for IEL-isolation (for details about the isolation protocol see Supplementary Methods).

      2.2.2 Immunostaining

      Immunostaining of specimens was performed within a median of 3 days after fixation of IEL isolates in 4% paraformaldehyde (range 0–24 days). For full details about the immunostaining protocol see Supplementary Methods and Supplementary Table 1. Fluorescein-isothiocyanate (FITC), allophycocyanin (APC), peridinin chlorophyll protein (PerCP), phycoerythrin (PE), Pacific Blue, and Amcyan conjugated monoclonal antibodies directed to CD3, CD4, CD7, CD8, TCR-γδ, CD11b, CD14, CD19, CD20, CD56, CD103 were used.

      2.2.3 Flow cytometric analysis

      Flow cytometry was performed using a FACSCalibur with the CellQuest software or a FACSCanto II with the FACSDiva software (all BD Biosciences) and were further analyzed using the FlowJo software (Tree Star, Ashland, Oregon, USA). The minimum lymphocyte count analyzed by FACS was 104 lymphocytes as defined by their granulation (using Side Scatter, SSC) and particle size (using Forward Light Scatter, FSC). As regards the gating strategies for the identifications of aberrant IELs, further details are shown in the Supplementary Material. Particularly, we evaluated intracellular CD3 (cytCD3), surface-bound CD3 (surfCD3) as well as CD7 and CD103. In line with the previous work by Verbeek et al. [
      • Verbeek W.H.
      • Goerres M.S.
      • von Blomberg B.M.
      • et al.
      Flow cytometric determination of aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more accurately than T-cell clonality analysis in Refractory Celiac Disease.
      ], we defined aberrant IELs as CD7+ cytCD3+ surfCD3 among lineage- and TCRγδ-negative (lin TCRγδ) lymphocytes and expressed the results as% of total lymphocytes. This strategy differs slightly from the one initially proposed by Verbeek, since an initial gating for lin-TCRγδ- was performed before evaluating CD3 and CD7. Moreover, the initial selection for lymphocytes was not performed on the basis of CD45+ and low SSC, but in our case on low SSC and FSC. This strategy was evaluated and demonstrated to be comparable to the gating based on CD45-staining (Supplementary Figure 1 and Supplementary Table 2).

      2.3 Assessment of the optimal cut-off for aberrant IELs

      The initial publications on the role of aberrant IELs in RCD have proposed a cut-off of 25%, then 20%aberrant IELs to discriminate between RCD I and II [
      • Verbeek W.H.
      • Goerres M.S.
      • von Blomberg B.M.
      • et al.
      Flow cytometric determination of aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more accurately than T-cell clonality analysis in Refractory Celiac Disease.
      ,
      • Malamut G.
      • Afchain P.
      • Verkarre V.
      • et al.
      Presentation and long-term follow-up of refractory celiac disease: comparison of type I with type II.
      : However, a standard, validated cut-off is yet to be determined. In this study, considering the methodological differences and the slightly different gating strategy as compared to the original publication by Verbeek et al. [
      • Verbeek W.H.
      • Goerres M.S.
      • von Blomberg B.M.
      • et al.
      Flow cytometric determination of aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more accurately than T-cell clonality analysis in Refractory Celiac Disease.
      ], we set the goal to determine the cut-off for pathologically increased aberrant IELs in our cohort. The cut-off was initially developed as previously described, using the 95th percentile of aberrant IEL counts in controls and non-refractory CD patients within our cohort [
      • Verbeek W.H.
      • Goerres M.S.
      • von Blomberg B.M.
      • et al.
      Flow cytometric determination of aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more accurately than T-cell clonality analysis in Refractory Celiac Disease.
      ]. Successively, the diagnostic accuracy of the percentage of aberrant IELs to identify high-risk (pre-EATL and EATL) RCD patients was tested by means of a receiver operating characteristic (ROC) curve to predict the most accurate cut-off.

      2.4 Histopathology of duodenal biopsies

      Conventional histology was performed on duodenal biopsies. For this purpose, four to six formalin-fixed duodenal specimens were embedded in paraffin using conventional techniques, microtome-cut and stained using hematoxyline and eosine (H&E). The enteropathy, if present, was graded according to the Marsh-Oberhuber-Classification [
      • Marsh M.N.
      Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity ('celiac sprue').
      ,
      • Oberhuber G.
      • Granditsch G.
      • Vogelsang H.
      The histopathology of coeliac disease: time for a standardized report scheme for pathologists.
      . Immunohistochemical staining and evaluation of lamina propria infiltrates were performed in case of RCD and suspected EATL.

      2.5 T-cell clonality analysis

      In line with the current routine clinical practice, patients with RCD were evaluated by molecular pathology in order to identify the presence of a clonal T-cell population. DNA was extracted from paraffin-embedded duodenal specimens (Maxwell, Promega) and amplified by multiplex PCR using fluorescence-labeled primer sets binding to the V- and J-gene segments of the rearranged TCR-γ (two primer pools) and TCR-β loci (three primer pools) employing the BIOMED-2/EuroClonality protocols [
      • van Dongen J.J.
      • Langerak A.W.
      • Brüggemann M.
      • et al.
      Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.
      ]. Assessment of the TCR rearrangement patterns was done via capillary electrophoresis (GeneScan; 3500 Genetic Analyzer, Thermo Fisher Scientific) of the fluorescence-labeled PCR products [
      • Daum S.
      • Weiss D.
      • Hummel M.
      • et al.
      Frequency of clonal intraepithelial T lymphocyte proliferations in enteropathy-type intestinal T cell lymphoma, coeliac disease, and refractory sprue.
      ]. All assays were performed as independent duplicates. The definition of clonality was carried out according to the criteria described by Langerak [
      • Langerak A.W.
      • Groenen P.J.
      • Brüggemann M.
      • et al.
      EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.
      ]. According to the result of this analysis, RCD were assigned to one of two correspondent groups. Since the currently recommended criteria to differentiate RCD type I from RCD type II are not univocal (T-cell clonality, CD8 staining in immunohistochemistry, flow cytometry of IELs), we named these groups “RCD without T-cell clonality” (RCDclon-) and “RCD with T-cell clonality” (RCDclon+). Of note, all patients in the RCDclon+ group had persistent clonality assessed in different biopsy specimens taken at least two different time points, in order to rule out isolated, unspecific clonality [
      • Hussein S.
      • Gindin T.
      • Lagana S.M.
      • et al.
      Clonal T cell receptor gene rearrangements in coeliac disease: implications for diagnosing refractory coeliac disease.
      ].

      2.6 Ethics

      The study was approved by the local Ethics Committee of Charité – Berlin (No. EA4/016/14) and was performed in accordance with the Helsinki Declaration. The examination with gastroscopy was performed only in presence of an appropriate clinical indication. All enrolled patients gave their informed consent to participation in the study.

      2.7 Statistical analysis

      Descriptive statistics (medians and interquartile ranges as well as 10th, 90th and 95th percentile) were calculated, Chi-Square or Fisher's exact test were used to compare proportions. Kruskall-Wallis test and Mann-Whitney's test were used to compare continuous variables unless specified otherwise. Correlation between continuous variables was evaluated by computing Spearman's ρ. Survival analyses were performed with the Log-rank (Mantel-Cox) test. The Graphpad Prism software (version 6.0, GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analysis.

      3. Results

      3.1 Characteristics of patients

      A total of 130 flow cytometry analyses for IEL-immunophenotyping were performed on 109 patients, including 42 controls, 21 active CD, 16 CD responding to GFD and 30 RCD. In 13 cases of the latter group, more than one flow cytometry analysis was performed over time. The demographic and clinical characteristics of the patients are shown in Table 1. Of note, there were no significant age and sex differences between controls and CD patients, with the exception of RCD patients, who were older than subjects with uncomplicated CD as previously described in literature [
      • Malamut G.
      • Afchain P.
      • Verkarre V.
      • et al.
      Presentation and long-term follow-up of refractory celiac disease: comparison of type I with type II.
      ,
      • Rubio-Tapia A.
      • Kelly D.G.
      • Lahr B.D.
      • Dogan A.
      • Wu T.T.
      • Murray J.A.
      Clinical staging and survival in refractory celiac disease: a single center experience.
      .
      Table 1Characteristics of the subjects enrolled in the study.
      Controls (N = 42)Active CD (N = 21)CD on GFD (N = 16)RCD (N = 30)p
      Sex (F/M)28/1414/714/221/9Ns
      Age at diagnosis36 (8–80)44 (26–70)49 (2–76)0.01
      RCD vs. active CD, ^RCD vs. other groups.
      Age at enrolment43 (19–85)41 (21–81)53 (36–82)64 (36–80)<0.001^
      Autoimmune comorbidities
      - Hashimoto2 (5)2 (10)1 (6)4 (13)
      - DM type I0 (0)0 (0)1 (6)0 (0)
      - Micr. colitis0 (0)2 (10)1 (6)2 (7)
      - other0 (0)2 (10)3 (19)4 (13)
      Neoplastic comorbidities
      History of:
      -Colon Ca1 (2)1 (5)0 (0)0 (0)
      -Breast Ca0 (0)1 (5)0 (0)0 (0)
      -Small bowel Ca0 (0)0 (0)0 (0)2 (7)
      -other2 (2)0 (0)1 (6)1 (3)
      Symptoms/signs at enrolment
      -Anemia3 (7)9 (43)0 (0)10 (33)
      -Chr. diarrhea13 (31)13 (62)4 (25)14 (47)
      -Abd. pain30 (71)10 (48)8 (50)10 (33)
      -Weight loss8 (19)8 (38)2 (13)12 (40)
      CD, celiac disease; GFD, gluten free diet; RCD, refractory celiac disease; DM, diabetes mellitus; Ca, cancer.
      Data are expressed as median (range) or N (%).
      low asterisk RCD vs. active CD, ^RCD vs. other groups.
      Table 2 summarizes the characteristics of patients belonging to the RCDclon+ and RCDclon- groups (according to the presence of a clonality T-cell population at the molecular pathology analysis). Most RCDclon+ patients presented with mild to severe villous atrophy (two patients without villous atrophy were on budesonide treatment at the time of the endoscopy), while a considerable proportion of patients in the RCDclon- group showed only increased intraepithelial lymphocytes with or without crypt hyperplasia (7 patients, 4 of whom were on budesonide, and one on azathioprine).
      Table 2Characteristics of RCD patients stratified according to T-cell clonality status.
      RCDclon-(n = 13)RCDclon+(n = 17)P
      Sex (F/M)9/412/5ns
      Age at CD diagnosis41 (16–69)54 (2–76)0.04
      Age at RCD diagnosis54 (20–76)66 (35–81)0.01
      Age at enrolment62 (20–76)73 (35–82)0.01
      Autoimmune comorbidities
      Hashimoto3 (23)1 (6)ns
      DM type I0 (0)0 (0)ns
      Microscopic colitis2 (15)0 (0)ns
      RA2 (15)0 (0)ns
      Sjögren syndrome1 (8)0 (0)ns
      Polyarthritis1 (8)1 (6)ns
      Symptoms/signs at presentation
      Anemia1 (8)8 (62)0.04
      Chronic diarrhea7 (54)8 (62)ns
      Abdominal pain3 (23)7 (54)ns
      Weight loss4 (31)8 (62)ns
      Fever/Night sweats0 (0)4 (31)ns
      Malabsorption parameters
      Low levels of
       Iron2/135/17Ns
       Folate1/104/15ns
       B121/132/15ns
       Zink2/125/15ns
       Electrolytes0/136/170.02
       Albumin0/136/170.02
      Increased INR0/136/170.02
      Osteoporosis0/108/170.01
      Duodenal Histology
      first histology since enrolment.
       Marsh I31
       Marsh II41
       Marsh IIIa25ns
       Marsh IIIb33
       Marsh IIIc17
      DM, diabetes mellitus, CD, celiac disease, RCD, refractory celiac disease, RA, rheumatoid arthritis.
      Data are expressed as median (range) or N (%) unless specified otherwise.
      *low iron status defined as ferritin or transferrin saturation lower than normal level;.
      a first histology since enrolment.

      3.2 Flow cytometry results

      3.2.1 Assessment of aberrant IELs

      The results of flow cytometry analysis did not reveal any significant differences between RCDclon- patients and uncomplicated CD (as shown in Supplementary Table 3). In fact, RCDclon- patients had low%aberrant IELs and moderately elevated%TCRγδ, at a level comparable to other CD groups. Although no statistically significant difference was observed between RCDclon and RCDclon- patients regarding the number of aberrant IELs, the scatter in the RCDclon group was striking (Fig. 1, Panel A). This suggested that RCDclon+ was not a homogeneous group and that a subpopulation of patients with high aberrant IELs can be identified within the RCDclon+ group.
      Fig. 1
      Fig. 1Percentage of A) aberrant IELs and B) TCRγδ+ lymphocytes in controls and in CD groups as assessed by flow cytometry; C) classification of RCD patients according to presence or absence of elevated aberrant IELs (CD7+cytCD3+surfCD3) D) ROC curve: discriminatory ability of aberrant IELs for high-risk RCD.
      A higher percentage of TCRγδ+ IEL at flow cytometry analysis has been associated with better prognosis in RCD [
      • Verbeek W.H.
      • von Blomberg B.M.
      • Scholten P.E.
      • Kuik D.J.
      • Mulder C.J.
      • Schreurs M.W.
      The presence of small intestinal intraepithelial gamma/delta T-lymphocytes is inversely correlated with lymphoma development in refractory celiac disease.
      ], therefore we also assessed TCRγδ expression. In this cohort, no significant differences were observed in TCRγδ+ between uncomplicated CD and RCD. However, an inhomogeneous distribution of results in the RCDclon+ groups suggested that a subgroup of RCDclon+ patients reveal lower TCRγδ+ percentages, in line with previous observations showing a negative relation between TCRγδ+ and aberrant IELs (Fig. 1, panel B).

      3.2.2 Discriminatory ability of aberrant IELs for the identification of high-risk RCD

      The 95th percentile of aberrant IEL counts in controls and non-refractory CD patients within our cohort was determined, resulting in a proposed cut-off of 11% for pathologically elevated aberrant IELs. Based on this cut-off, patients were classified into a low-risk subgroup (RCD I, n = 25) and into a high-risk subgroup (RCD II n = 5) as proposed by Verbeek et al. [
      • Verbeek W.H.
      • Goerres M.S.
      • von Blomberg B.M.
      • et al.
      Flow cytometric determination of aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more accurately than T-cell clonality analysis in Refractory Celiac Disease.
      ]. According to this new classification, the new RCD I group now included patients with and without T-cell clonality, but considered at low risk for malignant development due to the low counts of IELs.
      Clinical and prognostic data available due to long follow-up were used as reference standard in order to verify the accuracy of patient allocation to the RCD I and RCD II groups (i.e., low-risk versus high-risk groups) according to aberrant IELs (see Fig. 1 Panel C). Despite the introduction of a lower cut-off for detection of aberrant IELs than the ones proposed in literature (20%), the strategy still missed a small number (n = 4) of high-risk patients (for details see Supplementary Table 4).
      A ROC curve was built to predict the most accurate cut-off of the percentage of aberrant IELs for the identification of high-risk RCD patients. The area under the ROC curve (AUROC) showed a fair to good performance of the test (AUROC=0.81, 95%CI 0.65–0.98, p = 0.0009) and suggested that the best cut-off for the identification of high-risk RCD patients was 15%, with a 63% sensitivity (95%CI 30.8–89.1) and a 100% specificity (95%CI 96.3–100) (Fig. 1 Panel D).

      3.3 Association between clinical characteristics of RCD patients and aberrant IELs

      In view of the results of previous key studies suggesting the close association between aberrant IELs and EATL development [
      • Verbeek W.H.
      • Goerres M.S.
      • von Blomberg B.M.
      • et al.
      Flow cytometric determination of aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more accurately than T-cell clonality analysis in Refractory Celiac Disease.
      ,
      • Verbeek W.H.
      • von Blomberg B.M.
      • Coupe V.M.
      • Daum S.
      • Mulder C.J.
      • Schreurs M.W.
      Aberrant T-lymphocytes in refractory coeliac disease are not strictly confined to a small intestinal intraepithelial localization.
      , we evaluated if the presence of aberrant IELs in RCD patients was associated with specific clinical characteristics, in order to identify a possible “clinical profile” of high-risk RCD patients.

      3.3.1 Ulcerative jejunoileitis

      It is known that ulcerative jejunoileitis is per se considered an independent predictor of poor prognosis in RCD – therefore elevated aberrant IELs are expected to be found in this subgroup. In RCD patients with ulcerative jejunoileitis a significantly higher%aberrant IELs was observed as compared to patients without ulcerative jejunoileitis (p < 0.0001). Similarly,%TCRγδ+ (of all lymphocytes) were significantly lower in ulcerative jejunoileitis patients, with a median of 3% (range 1–36) versus 9% (range 1–61) in the other RCDclon+ (Fig. 2 Panel A-B).
      Fig. 2
      Fig. 2A) aberrant IELs and B) TCRγδ+ lymphocytes in RCD patients with and without ulcerative jejunoileitis (UJ); C) aberrant IELs in RCD patients with and without severe malabsorption; D) correlation between Malabsorption score and aberrant IELs in RCDclon+ patients; E) receiver operating characteristic (ROC) curve for the diagnostic performance of the malabsorption score.

      3.3.2 Malabsorption syndrome

      Malabsorption is a characteristic of RCD and already was observed more frequently in RCDclon+ and EATL patients, even in comparison to active CD [
      • Wierdsma N.J.
      • Nijeboer P.
      • de van der Schueren M.A.
      • Berkenpas M.
      • van Bodegraven A.A.
      • Mulder C.J.
      Refractory celiac disease and EATL patients show severe malnutrition and malabsorption at diagnosis.
      ]. Therefore, we evaluated several absorption parameters, including the iron status, folate, vitamin B12, zinc, as well as albumin, serum electrolytes, and vitamin K-dependent coagulation function (INR) as potential determinants of disease severity among RCDclon+ patients. No alteration in a single parameter was associated with the presence of elevated aberrant IELs or with poorer prognosis. We hypothesized that the presence of more than one altered parameter, delineating a relevant malabsorption syndrome, would show an association with aberrant IELs and/or poor prognosis. In order to quantify the severity of malabsorption among RCD patients we designed a score ranging from 0 to 8 (1 point for deficiency in each of the seven aforementioned blood parameters plus 1 point for presence of a diagnosis of osteoporosis). After observing a significant higher prevalence of aberrant IELs among RCD patients with severe malabsorption (Fig. 2 Panel C), we performed a correlation analysis between the malabsorption score and the%aberrant IELs in RCDclon+ patients (Fig. 2 Panel D), revealing a weak to moderate positive correlation (r = 0.38, 95% CI 0.02 to 0.65, p = 0.04) between the malabsorption score and the%aberrant IELs. Far from confirming a causal association, these results suggested that high-risk RCD patients are characterized by both an elevation in aberrant IELs and severe malabsorption. In order to verify the persistence of this correlation in a wider group, the same analysis was performed on all RCD patients (RCDclon+ and RCDclon-), where a weaker but still significant correlation was observed (r = 0.35 with p = 0.01).
      As we did for the flow cytometry results, clinical and prognostic data were used to test the diagnostic accuracy of the malabsorption score in identifying high-risk RCD patients. A ROC curve was built to evaluate the discriminatory ability of the score and identify the most accurate cut-off: the AUROC showed a good performance of the test (AUROC=0.90, 95%CI 0.81–0.98, p = 0.0008) and showed that with a cut-off of ≥3 the malabsorption score reached a 67% sensitivity (95%CI 41.0–86.7) and 97% specificity (95%CI 83.3–99.9) for the identification of high-risk RCD patients (Fig. 2 Panel E).

      3.4 Optimization of the diagnostic approach to identify high-risk RCD

      Considering the lower-than-expected discriminatory ability of aberrant IELs for the diagnosis of high-risk RCD on one hand, and the association between severe malabsorption and presence of aberrant clones at flow cytometry on the other hand, we moved on to establish a diagnostic approach which included both the evaluation of aberrant IELs and malabsorption. Applying the criteria of either “>15% aberrant IELs” or a “malabsorption score ≥3″ to our patients’ data, we were able to identify the high-risk RCD patients of our cohort (EATL, ulcerative jejunoileitis and patients with EATL diagnosis at follow up) with a good diagnostic accuracy, reaching an overall 100% sensitivity (95% CI 63.1–100) and 96% specificity (95% CI 80.4–99.9).
      Survival data observed by means of Kaplan-Meyer curves revealed that the new classification into RCD I and RCD II based on aberrant IELs and malabsorption was able to better identify RCD patients with poor prognosis than the former classification based on T-cell clonality analysis and, furthermore, better than the classification based solely on increased aberrant IELs (Fig. 3).
      Fig. 3
      Fig. 3Analysis of survival of RCD patients according to A) the traditional classification based on the presence (RCDclon+, formally RCDII) or absence (RCDclon-, formally RCD I) of TCR clonality; B) the proposed classification based on the presence or absence of aberrant IELs (RCD IEL+ or IEL-); C) the new classification taking both malabsorption and aberrant IELs into account. Panels D, E, F show survival curves with death or new cancer diagnosis as endpoint.

      4. Discussion

      This study was aimed at evaluating flow cytometric analysis of aberrant intestinal lymphocytes in a cohort of patients affected by uncomplicated CD and RCD, in order to identify relevant differences among RCD patients and to discriminate high-risk RCD patients with better accuracy than T-cell clonality assessment.
      In our cohort, the determination of aberrant IELs by means of flow cytometry proved a good yet imperfect diagnostic tool for the identification of high-risk RCD patients. First, the comparison of aberrant IEL counts between different CD groups and controls allowed to confirm that increased aberrant IELs were detected in a subgroup of RCDclon+ patients (traditionally defined RCD II as a whole), while these were never found in patients within the RCDclon- group. These results corroborate our experience of a good negative likelihood of molecular analysis for the detection of clonal/aberrant T-cell populations according to validated protocols: in our cohort, in fact, the absence of T-cell clonality was per se sufficient to classify RCD patients as “low-risk”. RCDclon- patients also showed overall less signs of malabsorption as compared to RCDclon+, thus further delineating them as a low-risk population. These observations differed from those published by another group, who reported that a small proportion of RCDclon- patients may be at risk of EATL development [
      • Verbeek W.H.
      • Goerres M.S.
      • von Blomberg B.M.
      • et al.
      Flow cytometric determination of aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more accurately than T-cell clonality analysis in Refractory Celiac Disease.
      ]. This discrepancy might be due to a higher resolution of the T-cell clonality analysis performed at our center, to the restriction of published data to TCR-gamma gene rearrangement analysis or secondary to limitations regarding negative predictions that arise in clinical studies that include only a moderate number of study participants.
      We tested the discriminatory ability of elevated aberrant IELs in RCD patients to identify the subset of high-risk RCD (pre-EATL and EATL) and realized that in our cohort this diagnostic strategy missed four RCD patients that deserved classification as high-risk RCD, resulting in a specificity of 100% but a sensitivity of only 63% for the detection of pre-EATL/EATL with clinical follow-up as reference standard.
      Of note, the lower cut-off of 15% we identified by means of the ROC curve analysis as compared to the previously proposed 20% [
      • Verbeek W.H.
      • Goerres M.S.
      • von Blomberg B.M.
      • et al.
      Flow cytometric determination of aberrant intra-epithelial lymphocytes predicts T-cell lymphoma development more accurately than T-cell clonality analysis in Refractory Celiac Disease.
      ] should be further validated. Importantly, it did not result in higher rates of false-positive results in our cohort.
      Immunophenotyping data of our cohort prompted us to question whether the flow cytometric assessment of aberrant IELs is sufficiently accurate to discriminate between RCD I and RCD II/pre-EATL without the aid of further diagnostic tools. Possible reasons for false negative results at flow cytometry may be due, among others, to a sampling error of duodenal biopsies: in fact, previous data have shown that villous atrophy in celiac disease is distributed non-uniformly in the duodenum [
      • McCarty T.R.
      • O'Brien C.R.
      • Gremida A.
      • Ling C.
      • Rustagi T.
      Efficacy of duodenal bulb biopsy for diagnosis of celiac disease: a systematic review and meta-analysis.
      ,
      • Hopper A.D.
      • Cross S.S.
      • Sanders D.S.
      Patchy villous atrophy in adult patients with suspected gluten-sensitive enteropathy: is a multiple duodenal biopsy strategy appropriate?.
      and this may also apply to the distribution of aberrant lymphocytes and thus influence a single flow cytometric assessment. Moreover, in routine clinical practice some patients with diagnosis of RCD may undergo the first flow-cytometric analysis when already in treatment. Although this option does not apply to the false-negative cases in our cohort, budesonide or other immunomodulators may lead to underestimation of the amount of aberrant IELs.
      In view of the evidence that a relevant malabsorption syndrome, defined as deficiency of three or more of relevant vitamins or micronutrients, is associated with high-risk RCD (data corroborated by a recent paper [
      • Wierdsma N.J.
      • Nijeboer P.
      • de van der Schueren M.A.
      • Berkenpas M.
      • van Bodegraven A.A.
      • Mulder C.J.
      Refractory celiac disease and EATL patients show severe malnutrition and malabsorption at diagnosis.
      ]), we allocated patients in the low-risk (RCD I) group based on the absence of both aberrant IELs at flow cytometry and a severe malabsorption. Cases with elevated aberrant IELs or severe malabsorption need to be classified as a high-risk (RCD II) group. Short-term follow-up with repeated immunophenotyping of IELs is advised in case of severe malabsorption without elevated aberrant IELs at first sampling. This further diagnostic step resulted in a higher accuracy that allowed for correct identification of high-risk RCD patients without missing those in which aberrant IELs could not (yet) be detected.
      All in all, considering the confirmatory data from our cohort showing that high-risk RCD are found exclusively within the RCDclon+ subgroups, and the evidence that a relevant malabsorption syndrome is associated with high-risk RCD, we hereby propose a possible diagnostic algorithm for the accurate classification of patients into RCD I or low-risk and RCD II or high-risk (Fig. 4). Interestingly, recent literature data suggest that other biomarkers such as beta2-microglobulin and chromogranin A are good predictors of a refractory course of CD and of EATL, although their discriminatory ability between high-risk and low-risk RCD has still to be evaluated [
      • Lenti M.V.
      • Aronico N.
      • Giuffrida P.
      • et al.
      Serum markers of refractoriness and enteropathy-associated T-cell lymphoma in coeliac disease.
      ]; in this study, those parameter were not routinely assessed, therefore we were not able to determine if there was an association with aberrant IELs in our cohort, but further confirmatory data may allow to include them in the diagnostic algorithm.
      Fig. 4
      Fig. 4Proposed diagnostic algorithm for the identification of low-risk/RCD I and high-risk/RCD II patients. Malabsorption parameters are routinely checked in celiac patients, and a more thorough evaluation is currently recommended in case of RCD, so that including them into the evaluation of RCD is practical and feasible. The initial performance of a TCR clonality study on histopathology samples in all patients with persistent villous atrophy still makes sense due to its high sensitivity despite the lack of specificity. Immunophenotyping by means of Flow cytometry is a simple method that can be easily performed in the setting of follow-up gastroscopy and will need to be re-assessed at follow-up in unclear cases.
      In conclusion, in view of the clinical implications involved with a diagnosis of RCD II and the possible advantages of a prompt treatment start, a correct and reliable classification of patients is of utmost importance. We would still support the T-cell clonality analysis on histopathology samples as initial diagnostic step in all patients with persistent villous atrophy, due to its role as negative predictor. The combination of IEL-immunophenotyping and the malabsorption score we developed is promising as a positive predictor for high-risk RCD and may play a key role in future diagnostic strategies but needs further internal and external validation.

      Funding

      Dr. Schumann is recipient of funding by Dr. Schär, by the Deutsche Zöliakie Gesellschaft and by the Deutsche Forschungsgemeinschaft (DFG GRK2318 and TRR241)

      Appendix. Supplementary materials

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