Asubgroup of patients with celiac disease develops

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1 GASTROENTEROLOGY 2009;136:81 90 CLINICAL Presentation and Long-Term Follow-up of Refractory Celiac Disease: Comparison of Type I With Type II GEORGIA MALAMUT,*,, PAULINE AFCHAIN, VIRGINIE VERKARRE,*, THIERRY LECOMTE, # AURÉLIEN AMIOT,** DIANE DAMOTTE,*, YORAM BOUHNIK,** JEAN FRÉDÉRIC COLOMBEL, JEAN CHARLES DELCHIER, MATTHIEU ALLEZ, JACQUES COSNES, ANNE LAVERGNE SLOVE, ## BERTRAND MERESSE,*, LUDOVIC TRINQUART,*, *** ELIZABETH MACINTYRE,*, ISABELLE RADFORD WEISS,*, OLIVIER HERMINE,*, NICOLE BROUSSE,*, NADINE CERF BENSUSSAN,*, and CHRISTOPHE CELLIER*,, *Université Paris Descartes, Paris; Assistance Publique des Hôpitaux de Paris, Gastroenterology, Hôpital Européen Georges Pompidou, Paris; INSERM Unité 793, Paris; Assistance Publique des Hôpitaux de Paris, Gastroenterology, Hôpital St Antoine, Paris; Assistance Publique des Hôpitaux de Paris, Pathology, Hôpital Necker-Enfants Malades, Paris; # CHU de Tours, Gastroenterology, Tours; **Assistance Publique des Hôpitaux de Paris, Gastroenterology, Hôpital Beaujon, Clichy; Assistance Publique des Hôpitaux de Paris, Pathology, Hôpital Hôtel Dieu, Paris; CHRU, Gastroenterology, Hôpital Huriez, Lille; Assistance Publique des Hôpitaux de Paris, Gastroenterology, Hôpital Henri-Mondor, Créteil; Assistance Publique des Hôpitaux de Paris, Gastroenterology, Hôpital St Louis, Paris; ## Assistance Publique des Hôpitaux de Paris, Pathology, Hôpital Lariboisière, Paris; ***Assistance Publique des Hôpitaux de Paris, Epidemiology and Clinical Research, Hôpital Européen Georges Pompidou, Paris; and Molecular Hematology, Cytogenetics, and Hematology, Assistance Publique des Hôpitaux de Paris, Hôpital Necker Enfants Malades, Paris, France See editorial on page 32. Background & Aims: Refractory celiac disease (RCD) was recently subdivided into 2 subtypes (RCD I and II) based on a normal or abnormal phenotype of intraepithelial lymphocytes (IELs), respectively. It is not clear, however, if these 2 entities differ in their presentation at diagnosis or long-term outcome. We compared the clinical and biological characteristics of RCD I and RCD II at diagnosis, the risk of developing an overt lymphoma, and the predictive factors of survival. Methods: Medical files of 14 patients with RCD I and 43 with RCD II were analyzed retrospectively. Predictive factors of overt lymphoma and survival were studied in univariate and multivariate analyses. Results: At diagnosis, malnutrition, ulcerative jejunitis, and lymphocytic gastritis were more common in patients with RCD II than RCD I (P <.05). Overt lymphomas occurred in 2 patients with RCD I and 16 with RCD II. In the univariate analysis, abnormal IEL phenotype and increased age at diagnosis of RCD were predictive factors for overt lymphoma. Abnormal IEL phenotype (P <.01), clonality (P.01), and overt lymphoma (P.001) predicted short survival time. Only abnormal IEL phenotype (P.03) and overt lymphoma (P.04) were predictive in the multivariate analysis. The 5-year survival rate was 93% in patients with RCD I and 44% with RCD II. Conclusions: RCD II has a much more severe presentation and prognosis than patients with RCD I; <44% of patients with RCD II survive 5 years after diagnosis. Abnormal IEL phenotype is a predictive factor but not a necessary condition for the development of overt lymphoma. Asubgroup of patients with celiac disease develops primary or secondary resistance to a gluten-free diet (GFD) with persistent symptoms of malabsorption and intestinal villous atrophy. Diagnosis of this condition, defined as refractory celiac disease (RCD), is made after exclusion of other small bowel diseases such as autoimmune enteropathy, tropical sprue, or common variable immunodeficiency. 1,2 We have shown that in a subset of patients, refractoriness to the diet was associated with the emergence of an abnormal population of intraepithelial lymphocytes (IELs) 3 characterized by the lack of surface expression of CD3/T-cell receptor (TCR) complexes and of CD8 but containing intracellular CD3 and a clonal rearrangement of the gamma chain of T-cell receptor (TCR ). 3 5 A second group of patients with clinical and histologic resistance to a GFD but with normal and polyclonal IELs was subsequently identified. Therefore, definition of these 2 recent entities is now based on the immunophenotyping of IELs: RCD of type I (RCD I) with normal IEL phenotype and RCD of type II (RCD II) with abnormal IEL phenotype. 6 Whether these 2 groups of patients can be differentiated by their clinical and biological presentations at diagnosis remains to be investigated and, in particular, whether they differ by the severity of the clinical symptoms and/or the severity of endoscopic or histologic features. Moreover, the long-term outcome and the respective risk of overt lymphoma in these 2 entities need to be Abbreviations used in this paper: CT, computed tomography; EC, epithelial cell; GFD, gluten-free diet; IEL, intraepithelial lymphocyte; RCD, refractory celiac disease; TCR, T-cell receptor by the AGA Institute /09/$36.00 doi: /j.gastro

2 82 MALAMUT ET AL GASTROENTEROLOGY Vol. 136, No. 1 ascertained to adapt follow-up and treatments. We and others have reported that patients with RCD II run a high risk of developing overt lymphoma. 5,7 In contrast, Al Toma et al observed that patients with RCD I had a relatively benign course 7 because none of the 43 patients with RCD I analyzed in their retrospective study developed an overt lymphoma over a mean follow-up of 5 years. In addition, no mortality related to RCD was observed in their patients with RCD I. 7 Similarly, no overt lymphoma was found in patients with RCD I in a recent German study screening endoscopic signs of RCD and enteropathy-associated T-cell lymphoma. 8 However, de novo lymphoma may develop directly from unknown celiac disease, 9 suggesting that the step of RCD II is not obligatory and thus indirectly that overt lymphoma may also arise in RCD I. Therefore, the goal of the present study was to compare the clinical and biological presentations at diagnosis of patients with RCD I and RCD II and to assess the onset of overt lymphoma and the predictive factors of survival in both types of RCD. Patients and Methods Patients The medical files of 83 consecutive patients with a diagnosis of RCD and referred to 6 large hospitals in France (Hospitals European Georges Pompidou, Lariboisière, Saint Louis, Henri Mondor, Claude Huriez, and Saint Antoine) between 1992 and 2007 were reviewed retrospectively. Eleven patients were excluded because of other diagnoses: enteropathy associated with primary hypogammaglobulinemia (n 4), collagenous sprue (n 1), autoimmune enteropathy (n 1), and lamina propria CD4 T-cell or CD8 small T-cell lymphomas (n 5). Also excluded were 9 other patients with indeterminate RCD due to unknown IEL phenotype, as well as 6 patients with celiac disease revealed by an overt T-cell lymphoma. Consequently, 57 patients were considered for the study and followed up from January 1992 to December 2007 to assess their responses to therapies and the onset of complications. Collection of Data Clinical data recorded for each patient included age, sex, symptoms (abdominal pain, diarrhea), autoimmune diseases, body mass index, and per day gluten intake calculated by a dietitian experienced in celiac disease. Blood tests included measurements of hemoglobin, folic acid, vitamin B 12, albumin, and transaminase levels. Serologic tests included the detection of serum immunoglobulin (Ig) A and IgG anti-gliadin antibodies, IgA class endomysial antibodies, and anti-human tissue transglutaminase IgA antibodies. HLA-DRB1 and -DQB1 genotyping was performed by means of hybridization with sequence-specific oligonucleotides following amplification by polymerase chain reaction, using the InnoLipa HLA genotyping test (Abbott, Rungis, France). 10 Endoscopic evaluation included upper gastrointestinal endoscopy or double balloon enteroscopy with gastric and small intestinal biopsies, colonoscopy with colonic biopsies, and video capsule endoscopy. Ulcerative jejunitis was defined as previously described. 5,11 For the histologic assessment, a minimum of 4 gastric, duodenal, and colonic biopsy specimens were fixed in 10% formalin and embedded in paraffin, and sections stained with H&E and Giemsa were reviewed by 2 expert pathologists (D.D. and V.V.). Villous atrophy was assessed in line with Oberhuber et al 12 modification of Marsh classification 13 and graded as absent, partial, subtotal, or total. The percentage of IELs (number of IELs per 100 epithelial cells [ECs]) was established on welloriented serial sections by counting at least 500 ECs. Lymphocytic gastritis was defined by the presence of more than 25 IELs per 100 gastric columnar ECs and lymphocytic colitis by the presence of more than 20 IELs per 100 colonic columnar cells. Immunohistochemistry was performed on sections from paraffin-embedded biopsy specimens using antibodies directed against CD3 (rabbit polyclonal antibody anti-human CD3; A0452), CD8 (mouse monoclonal anti-human CD8; C8/144B, M703) (Dako A/S, Glostrup, Denmark), CD4 (clone AB12; Labvision Corp, Fremont, CA), and CD30 (clone BER-H2; Dako A/S) and on acetone-fixed frozen tissue sections using antibodies directed against CD103 (clone 2G5.1, Coulter Immunotech, Marseille, France), F1 (8A3), and TCR 1 (5A6E9) (T Cell Sciences, Cambridge, MA) and a 3-stage indirect immunoperoxidase technique. 14,15 The percentage of CD3 CD8 IELs was assessed as previously defined. 15 For flow cytometry phenotyping, IELs and lamina propria lymphocytes were isolated from duodenal biopsy specimens as described. 3 Peripheral blood lymphocytes were isolated on a Ficoll gradient according to standard procedures. Multicolor staining of lymphocytes was performed using PerCP-cyanin 5.5 labeled anti-cd45 (Becton Dickinson, Le Pont De Claix, France), phycoerythrinconjugated anti-cd3, anti-cd8 (Becton Dickinson), or anti-tcr antibodies (Coulter Immunotech, Marseille, France), fluorescein isothiocyanate conjugated anti-cd4, anti-cd3 (Becton Dickinson), and anti-tcr (Coulter Immunotech) antibodies and analyzed on a BDLSR I using CellQuest software (Becton Dickinson) as described. 3 Molecular detection of clonal TCR chain rearrangements was performed on DNA extracted from intestinal, cutaneous, and bronchopulmonary frozen biopsy specimens and from mononuclear cells isolated from peripheral blood or from pleural and/or peritoneal fluids by multiplex polymerase chain reaction as described. 5 Karyotypic studies were performed on interleukin-15 dependent lines derived from intestinal abnormal (CD103, scd3 ) IELs and/or from fresh peripheral lym-

3 January 2009 REFRACTORY CELIAC DISEASE PRESENTATION AND PROGNOSIS 83 phocytes in patients with RCD II with circulating abnormal IELs as described. 16 Diagnosis of RCD The diagnosis of RCD was based on clinical relapse (chronic diarrhea and/or severe abdominal pain) with persistent malabsorption syndrome (decreased serum levels of iron, folate, vitamin B 12, and/or calcium) and on histology showing persistent villous atrophy with increased numbers of IELs after 1 year of strict adherence to a GFD. RCD I was defined by normal IEL phenotype ( 25% CD103 or CD45 IELs lacking surface CD3 on flow cytometry or 50% CD3 CD8 IELs in formol-fixed sections) and the absence of detectable clonality in duodenal biopsy specimens. RCD II was defined by the following abnormal phenotype of IELs: more than 25% CD103 or CD45 IELs lacking surface CD3/TCR complexes on flow cytometry or 50% IELs expressing intracellular CD3 but no CD8 in formol-fixed sections and/or the presence of a detectable clonal TCR rearrangement in duodenal biopsy specimens. 5 Follow-up Evaluation was performed at diagnosis and at regular intervals (every 6 months) during follow-up. Computed tomography (CT) scan and positron emission tomography scan were performed at diagnosis to eliminate an overt lymphoma and then every 6 months or if justified by clinical symptoms. Clinical response to treatment was defined by the reduction of diarrhea with a decrease of 50% of the number of stools or of weight stools per day and/or the recovering of 50% of weight loss. Histologic response to treatment was defined as partial in case of villous architecture improvement by at least one grade and total if villous architecture was restored to normal in control biopsy specimens. Detection of abnormal IELs and clonality was performed as described previously. Follow-up extended from the diagnosis of RCD to the most recent events until December 1, 2007 (follow-up, years). Statistical Analysis Continuous data were summarized using mean (standard deviation) or median (25th percentile, 75th percentile), as appropriate. Continuous variables were compared between patients with RCD I and patients with RCD II using the nonparametric Mann Whitney test. Categorical variables were compared using Fisher exact test. Overt lymphoma-free survival and overall survival were analyzed using Kaplan Meier methodology. Cox regression models were used to assess the association between survival and covariates. Multivariate analyses were performed in a backward fashion, entering the variables associated with the outcome at the 0.20 level in univariate analysis. Statistical analyses were performed using SAS version 9.1 (SAS France, Brie Comte Robert, France) and GraphPad Prism software (GraphPad Software Inc, San Diego, CA). A P value of less than.05 was considered statistically significant. Table 1. Clinical Data at Diagnosis of RCD RCDI(n 14) RCD II (n 43) P Sex ratio (F/M) 11/3 25/18 NS Median age at diagnosis of celiac disease (y) (25th percentile, 75th percentile) 41.6 (36, 48.1) 49.1 (39.8, 58.3).08 Median age at diagnosis of RCD (y) (25th percentile, 75th percentile) 48.7 (43.7, 62.6) 52.7 (42, 58.3) NS No. of patients with diarrhea (%) 12/14 (85.7) 38/43 (88.4) NS No. of patients with abdominal pain (%) 9/14 (64.3) 23/43 (53.5) NS Body mass index, mean (kg/m 2 ) NS No. of patients with anemia (%) 6/12 (50) 32/41 (78).08 No. of patients with low albuminemia ( 35 g/l) (%) 6/11 (54.5) 38/41 (92.7).01 No. of patients positive for at least one celiac antibody under strict GFD (%) 4/14 (28.6) 10/36 (27.8) NS IgA anti-gliadin (%) 0/11 (0) 7/32 (21.9) NS IgG anti-gliadin (%) 4/14 (28.6) 6/33 (18.2) NS IgA class endomysial antibodies (%) 2/11 (18.2) 4/32 (12.5) NS IgA anti tissue transglutaminase (%) 3/8 (37.5) 2/15 (13.3) NS HLA (%) DQ2 10/11 (90.9) 26/26 (100) NS DQ2/DQ2 4/10 (40) 16/24 (66.7) NS DQ8 1/10 (10) 3/24 (12.5) NS DQ2/DQ8 0/10 (0) 3/24 (12.5) NS No. of patients with dermatis herpetiformis (%) 0/14 (0) 4/43 (9.3) NS No. of patients with autoimmune diseases (%) 2/14 (14.3) 13/43 (30.2) NS No. of patients with liver dysfunction (%) Chronic cytolysis 8/14 (57.1) 21/43 (48.8) NS Unexplained 4/8 (50) 13/21 (61.9) Viral hepatitis 3/8 (37.5) 4/21 (19) Other identified cause 1/8 (12.5) 4/21 (19)

4 84 MALAMUT ET AL GASTROENTEROLOGY Vol. 136, No. 1 Results Description of the Studied Population at Diagnosis Clinical and biological data. Fourteen patients were diagnosed with RCD I (11 female and 3 male; mean age, 49.3 years) and 43 with RCD II (25 female and 18 male; mean age, 52.3 years) (Table 1). Primary resistance to GFD was observed in 5 patients (35.7%) with RCD I and in 21 patients (48.8%) with RCD II. In the remaining patients, the onset of a secondary resistance to GFD was observed after a mean of 10.2 ( 7.6) years and 7.4 ( 4.3) years in patients with RCD I and RCD II, respectively. At diagnosis, all patients with RCD had diarrhea and/or abdominal pain. Chronic diarrhea and abdominal pain were observed in more than 85% of patients with RCD I and in 55% 65% of patients with RCD II. Low serum albumin level suggested that protein-losing enteropathy was more frequently observed in RCD II than in RCD I (P.01). Low body mass index and anemia tended to be more frequent in patients with RCD II than in patients with RCD I (not significant). Chronic elevated levels of transaminases were detected in approximately 50% of patients. The most frequent cause was viral hepatitis, with 3 cases of chronic viral hepatitis (1 B and 2 C) in patients with RCD I and 4 cases of chronic hepatitis C in patients with RCD II. All cases of hepatitis C were secondary to blood transfusion. All cases of chronic viral hepatitis were diagnosed before RCD. In addition, 1 patient with RCD I and 1 patient with RCD II had had acute hepatitis B, which healed before diagnosis of RCD. Sclerosing cholangitis was observed in 1 patient with RCD I and autoimmune hepatitis, macrophage activation syndrome, and liver lymphomatous infiltration in 3 patients with RCD II, respectively. In most cases, however, cytolysis remained unexplained. All patients were positive for at least 1 celiac antibody at the initial diagnosis of celiac disease. Celiac antibodies persistent despite the GFD were observed in approximately 30% of patients in both groups. The frequency of DQ2 homozygosity tended to be higher in patients with RCD II than in patients with RCD I (not significant). Endoscopic, histologic, phenotypic, and molecular features. Ulcerative jejunitis was more common in patients with RCD II than in patients with RCD I (P.04) (Table 2). Furthermore, large ulcerations with a diameter greater than 1 cm were only observed in patients with RCD II (Table 2). Small bowel villous atrophy was partial in 28.6% and 22% (not significant) and severe (total or subtotal) in 71.4% and 78% (not significant) of patients with RCD I and RCD II, respectively (Table 2). Median numbers of IELs quantified in duodenal sections tended to be higher in patients with RCD II (91.5/100 ECs) than in patients with RCD I (66.9/100 ECs) (not significant). Lymphocytic gastritis was more frequent in patients with RCD II than in patients with RCD I (P Table 2. Endoscopic and Histologic Characteristics at Diagnosis of RCD RCDI(n 14) RCD II (n 43) P Capsule endoscopy Villous atrophy 4/4 10/10 NS Erosions 1/4 9/10.04 Ulcerations ( 1 cm) 0/4 6/10.08 Strictures 0/4 3/10 NS Upper endoscopy proximal small bowel Ulcerative jejunitis 4/14 (28.6) 29/43 (67.4).01 Ulcerations ( 1 cm) 0/4 29/ Villous atrophy Partial villous 4/14 (28.6) 9/41 (22) NS atrophy Subtotal villous 5/14 (35.7) 18/41 (43.9) NS atrophy Total villous atrophy 5/14 (35.7) 14/41 (34.1) NS IELs (no./100 ECs) Mean Median NS TCR clonality 0/12 (0) 35/36 (97.2) a Trisomy 1q 15/16 (93.8) Lymphocytic gastritis 1/14 (7.1) 19/41 (46.3) b.01 Lymphocytic colitis 4/14 (28.6) 14/41 (34.1) b NS NOTE. All values are expressed as n/n (%) unless otherwise noted. a In 1 patient, TCR analysis revealed oligoclonality but no monoclonality; 69% (20/29) of patients with TCR clonality had biallelic TCR rearrangements. b Infiltration by abnormal IELs (CD3 CD8 phenotype on paraffin sections and/or presence of the same TCR clonal rearrangement as in duodenal biopsy specimens)..01), while lymphocytic colitis was detected in one third of patients in both groups. In all 14 patients with RCD I, immunohistochemistry on paraffin duodenal biopsy specimens indicated that less than 40% IELs (mean, 22.9%; range, 14% 31%) were positive with anti-cd3 and negative with anti-cd8 antibodies. In 12 patients with RCD I, the normal phenotype of IELs was confirmed using multicolor fluorescence-activated cell sorter analysis (mean number of IEL CD3 CD8, 6.75% [0.5% 25%]). In all 43 patients with RCD II studied, duodenal immunohistochemistry indicated that more than 50% IELs (mean, 87.1%; range, 56% 100%) were positive with anti- CD3 but negative with anti-cd8 antibodies. In 26 patients with RCD II, the abnormal phenotype of IELs was confirmed using fluorescence-activated cell sorter analysis, which showed that 74.8% (range, 25% 99.8%) of IELs isolated from biopsy specimens were CD45 and CD103 but lacked surface expression of CD3 and of a TCR or. CD8 expression was observed in 3 patients but at a low level undetectable by immunohistochemistry in tissue sections. None of the patients with RCD I exhibited clonality of the TCR or TCR chain. In contrast, clonality of the TCR chain was detected in the duodenal biopsy specimens in 33 of 36 tested patients with RCD II. In 2

5 January 2009 REFRACTORY CELIAC DISEASE PRESENTATION AND PROGNOSIS 85 patients with no detectable rearrangement of the TCR chain, a clonal rearrangement of the TCR chain was present. Notably, in the remaining patient, analysis of the TCR chain showed a restricted oligoclonal repertoire (the TCR chain was not studied). Characteristics and diffusion of abnormal IELs in patients with RCD II. Cytogenetic analysis, performed in the cell lines derived from the IELs of 16 patients with RCD II, detected a 1q trisomy in 15 patients (93.8%) (Table 2). In 3 patients who had a substantial proportion ( 20%) of circulating abnormal CD103 lymphocytes, the 1q trisomy was also detected in freshly isolated peripheral lymphocytes. Gastric and colonic lymphocytic infiltrations by abnormal IELs (CD103, CD3i, CD8 ) were observed in 19 (46.3%) and 14 (34.1%) patients, respectively, and the presence of abnormal cells was confirmed by the detection of the clonal TCR chain in the corresponding biopsy specimens. Lymphocytes with the same abnormal phenotype as IELs (CD103 lacking surface CD3/TCR complexes and CD8 but containing CD3 ) were detected in extraintestinal sites in 26 patients (60.5%) with RCD II (Table 3). The most common extraintestinal locations were blood, bone marrow, and skin, where diffusion of abnormal IELs was confirmed by the detection of the same clonal TCR rearrangement as in duodenal biopsy specimens. Liver biopsies, performed in 2 patients with cytolysis and cholestasis, suggested diffusion of abnormal IELs (CD3, Table 3. Extraintestinal Locations of Abnormal IELs in Patients With RCD II Location Blood CD103 CD3s cells a 5% and clonality 18/35 (51.4%) Bone marrow CD103 CD3s cells a 5% and/or 11/26 (42.3%) clonality Skin CD103 CD3i CD8 cells a and/or 7/41 (17.1%) clonality Liver CD3i CD8 CD4 cells a in sinusoids n 2 Lung CT scan 9/41 (21.9%) CD3i CD8 CD4 cells in bronchial biopsy 4/4 specimens or CD103 CD3s cells a 5% in bronchoalveolar lavage, pleural effusion, and/or of clonality Mesenteric lymph nodes CT scan ( 1 cm) 18/30 (60%) Head CT scan (maxillary sinus) n 6 NOTE. Total 26/43 (60.5%). a CD3s, absence of surface CD3 as determined by flow cytometry. The latter cells contained intracellular CD3 detectable by intracellular staining (not shown) as well as by immunohistochemistry in tissue sections (CD3i). Table 4. Treatments RCDI(n 14) RCD II (n 43) P GFD 14/14 (100) 42/43 (97.7) NS Parenteral nutrition 5/14 (35.7) 29/42 (69).03 Corticosteroids 10/14 (71.4) 32/43 (74.4) NS Azathioprine 1/14 (7.1) 5/43 (11.6) NS Methotrexate 0/14 (0) 8/43 (18.6) NS Anti tumor necrosis 1/14 (7.1) 3/43 (7) NS factor Cyclosporine 0/14 (0) 2/43 (4.6) NS Cladribine 0/14 (0) 2/43 (4.6) NS Anti-CD52 0/14 (0) 2/43 (4.6) NS Surgery 3/14 (21.4) 12/43 (27.9) NS Perforation 4/12 Hemorrhage 1/12 Obstruction 2/3 2/12 Exploration 1/3 5/12 NOTE. All values are expressed as n/n (%) unless otherwise noted. CD4, CD8 ) to the liver sinusoids. Lung involvement was suspected on CT examination in 9 patients and confirmed in 4 patients; abnormal CD103 lymphocytes lacking surface CD3 and CD8 were detected by flow cytometry in the bronchoalveolar lavage of 3 patients who underwent bronchoscopy and in the pleural effusion of a fourth patient and the presence of clonal cells confirmed by the study of TCR rearrangement. Enlarged mesenteric lymph nodes with a diameter greater than 1 cm were observed in 60% of patients (18/30). Clinical and radiologic (CT scan) examination suggested a location to the maxillary sinus in 6 patients. Outcome Treatments. Parenteral nutrition was more often necessary in patients with RCD II than in patients with RCD I. Twenty-one percent of patients with RCD I and 28% of patients with RCD II had surgery, either to eliminate an overt lymphoma or to treat complications. Obstruction was observed in both patients with RCD I and patients with RCD II, whereas small bowel perforation and bleeding was only observed in patients with RCD II (Table 4). Corticosteroids were the first-line immunosuppressive treatment in patients of either group. A clinical response was obtained in 90% and 76.7% of patients with RCD I and RCD II, respectively (Table 5). Corticosteroid dependence was, however, observed in 80% (8/10) of patients with RCD I and in 74.2% (23/31) of patients with RCD II. Other immunosuppressive drugs were used in corticosteroid-refractory or -dependent patients (Table 4). A histologic response was observed in some of the few patients with RCD II treated with methotrexate or anti tumor necrosis factor but not with azathioprine or cyclosporine (Table 5). However, none of the treatments attempted in patients with RCD II had any significant and/or durable effect on the number of abnormal IELs. Similarly, some patients with RCD II were treated over a few weeks with fludarabine (n 1), imatinib (n 1), or

6 86 MALAMUT ET AL GASTROENTEROLOGY Vol. 136, No. 1 Table 5. Response to Treatments Treatment No. of patients treated Mean duration Clinical response Histologic response GFD RCD I mo RCD II mo Parenteral nutrition RCD I mo 5/5 (100%) 0/5 (0%) RCD II mo 29/29 (100%) 0/29 (0%) Corticosteroids RCD I mo 9/10 (90%) 4/10 (40%); P:0; T:4 RCD II mo 23/30 (76.7%) 10/30 (33.3%); P:7; T:3 Azathioprine RCD I 1 35 mo 1/1 (100%) 0/1 (0%) RCD II mo 2/4 (50%) 0/4 (0%) Methotrexate RCD I 0 RCD II mo 5/7 (71.4%) 2/7 (28.6%); P:2; T:0 Anti tumor necrosis factor RCD I 1 19 cycles 1/1 (100%) 0/1 (0%) RCD II cycles 2/3 (66.7%) 1/3 (33.3%); P:1; T:0 Cyclosporine RCD I 0 RCD II mo 1/2 (50%) 0/2 (0%) Cladribine RCD I 0 RCD II cycles 1/2 (50%) 1/2 (50%) a ; P:1; T:0 Anti-CD52 RCD I 0 RCD II wk 2/2 (100%) 2/2 (100%) a ; P:1; T:1 P, partial; T, total. a Treatments stopped because of onset of overt lymphoma in 2 patients treated with cladribine and in 1 patient treated with anti-cd52. by the association cyclophosphamide-doxorubicin-etoposide-steroids (n 1), but none of these drugs provided any therapeutic effect. Mechlorethamine (n 1) and bexarotene (n 1) were also used in a patient with cutaneous location of abnormal IELs but to no effect. Cladribine (n 2) and alemtuzumab (anti-cd52 monoclonal antibody) (n 2) improved clinical symptoms and restored villous atrophy partially or almost completely. However, the onset of an overt lymphoma was observed in the 2 patients treated with cladribine, just after the third cycle and at 2 months, and in 1 patient receiving anti-cd52 during the fifth week of treatment. In the latter patient, full villous recovery was observed at the last endoscopic evaluation 15 days before surgery for peritonitis disclosing the lymphoma. Overt lymphoma. Overt lymphoma developed in 2 patients with RCD I and in 16 patients with RCD II during follow-up (Table 6). The rate of overt lymphoma during the first 5 years after diagnosis was 14.3% and 32.6% in patients with RCD I and RCD II, respectively (Figure 1A). In univariate analysis, an abnormal IEL phenotype and an increase in age at diagnosis of RCD were predictive risk factors for an overt lymphoma (Figure 1B). The 2 patients with RCD I and 6 of the 16 patients with RCD II had not received any immunosuppressive drugs before the onset of overt lymphoma. Small bowel and skin were the most common initial locations of overt lymphoma, which consisted usually of large cells often positive with the CD30 marker. In cutaneous overt lymphoma, transformation always developed from a preexisting cutaneous location of RCD II. One patient treated Table 6. Staging and Phenotype of Overt Lymphoma Complicating RCD RCDI(n 14) RCD II (n 43) Overt lymphoma 2/14 (14.3) 16/43 (37.2) Initial location Small bowel 2/2 (100) 10/16 (62.5) Skin 3/16 (18.8) Lung 1/16 (6.3) Lymph nodes 3/16 (18.8) Size of cells Medium 0/2 (0) 2/16 (12.5) Large 2/2 (100) 14/16 (87.5) Phenotype CD3 2/2 (100) 16/16 (100) CD8 1/2 (50) 0/11 (0) CD30 0/2 (0) 6/10 (60) CD56 0/2 (0) 0/11 (0) CD103 1/1 (100) 6/8 (75) Staging Ie 0 2/16 (12.5) IIe 1/2 (50) 5/16 (31.3) IV 1/2 (50) 9/16 (56.3) NOTE. All values are expressed as n/n (%).

7 January 2009 REFRACTORY CELIAC DISEASE PRESENTATION AND PROGNOSIS 87 Figure 1. (A) Kaplan Meier curve of overt lymphoma free survival according to the type of RCD. The dashed and solid curves represent the overt lymphoma free survival in patients with RCD I and patients with RCD II, respectively. The 5-year rate of overt lymphoma was 14.3% and 32.6% in patients with RCD I and II, respectively. (B) Predictive factors of onset of overt lymphoma. with anti-cd52 antibody had concomitant cutaneous and intestinal overt lymphoma. Loss of the intraepithelial marker CD103 was observed in the overt lymphoma of 2 patients with RCD II. Of the 18 patients with overt lymphoma, 16 (RCD I, n 1; RCD II, n 15) died due to tumoral progression after a median of 11.5 months (range, 0 48 months). Mortality. Three patients with RCD I and 26 patients with RCD II died during follow-up. The 5-year survival rate was 92.9% and 43.9% in patients with RCD I and patients with RCD II, respectively (Figure 2A). On univariate analysis, abnormal IEL phenotype (P.01), clonality (P.01), and onset of overt lymphoma (P.001) were predictive factors of short survival in patients with RCD (Figure 2B). On multivariate analysis, only abnormal IEL phenotype (P.03) and onset of overt lymphoma (P.04) were predictive of short survival. In patients with RCD I, death was related to overt lymphoma progression with sepsis and malnutrition in 1 case and to malnutrition alone in the 2 other cases. In patients with RCD II and overt lymphoma, the first cause of death was tumoral progression combined with malnutrition (n 9), infections (n 4), intestinal hemor-

8 88 MALAMUT ET AL GASTROENTEROLOGY Vol. 136, No. 1 Figure 2. (A) Kaplan Meier curve of survival according to the type of RCD. The dashed and solid curves represent the overall survival in patients with RCD I and RCD II, respectively. The 5-year survival rate was 92.9% in RCD I versus 43.9% in RCD II. (B) Predictive factors of survival. rhage (n 3), or lethal thrombosis (n 2). One patient died from esophageal bleeding related to portal hypertension and hepatitis C after a 4-year remission. Another patient died from pulmonary embolism at the end of the first year of remission. In patients with RCD II but no lymphoma, causes of death were malnutrition (n 8), sepsis (n 4), and thrombosis (n 1). Discussion The present study of 57 patients with RCD confirms that they can be divided into 2 subtypes, I and II, according to the normal or aberrant phenotype of intestinal IELs. At diagnosis, malnutrition was more severe and lymphocytic gastritis and ulcerative jejunitis were more frequent in patients with RCD II than in patients with RCD I. The outcome of RCD II was much more severe, with a high mortality rate mainly due to the development of overt lymphoma. Our work confirms beyond doubt that RCD II is a major risk factor for overt lymphoma but also shows for the first time that overt lymphomas can develop in patients with RCD I. RCD is defined by villous atrophy resistant to a strict GFD for more than 1 year. 1,2 If inaugural, other causes of villous atrophy, notably autoimmune enteropathy and

9 January 2009 REFRACTORY CELIAC DISEASE PRESENTATION AND PROGNOSIS 89 common variable immunodeficiency, need to be eliminated. Because RCD I had no specific features, the confirmation of refractoriness relies only on a very precise evaluation of the diet. The persistent positive serology observed in approximately 30% of our patients with RCD I may lead to question their strict adherence to the GFD. Anti-gliadin IgG persisted in some patients, but this test lacks specificity. 17 Anti-gliadin IgA antibodies, however, disappeared, and only EAM and IgA anti tissue transglutaminase antibodies persisted in patients with RCD I. Disappearance of anti-gliadin IgA antibodies may be the indication of a strict GFD. Conversely, persistent tissue damage in RCD I may be sustained, independently of gluten exposure, with transglutaminase up-regulation and thereby the production of autoantibodies. Consistent with this hypothesis, TG2 up-regulation is induced by various stimuli. 18 In contrast, diagnosis of RCD II has now become easier. Two characteristic endoscopic features were large and numerous ulcerations and strictures. This severe ulcerative jejunitis may explain the significantly more severe protein loss in RCD II than in RCD I. The diagnosis of RCD II was sustained by the detection of IELs expressing CD3 but no or low CD8 on paraffin sections. Nevertheless, it must be stressed that TCR IELs, which represent a substantial fraction of normal IELs in patients with uncomplicated celiac disease, 14 can express CD3 but not CD8 and may be confused with abnormal IELs when the latter cells are present in a moderate percentage. Conversely, a recent histopathologic study reported that some clonal RCD may have positive expression of CD8 IELs on paraffin sections. 19 However, the percentage of CD3 CD8 IELs was not determined, precluding a firm conclusion. The abnormal phenotype of IELs must be ascertained using immunohistochemistry on frozen tissue sections or by flow cytometry to show that IELs identified by the expression of the pan-leukocyte CD45 marker or by the IEL-specific integrin CD103 express neither a TCR nor surface CD3. The diagnosis of RCD II must finally be ascertained by showing the presence of a clonal rearrangement of the TCR chain in intestinal biopsy specimens and if negative by the presence of a clonal rearrangement of the TCR chain. Flow cytometry was particularly useful to assess precisely the percentage of abnormal IELs, particularly in patients with low percentages of aberrant IELs ( 40%), and to show the diffusion of abnormal CD103 IELs lacking surface CD3 to other compartments such as blood, bone marrow, and bronchioloalveolar fluid. Prognosis of RCD II was severe with a 5-year survival rate of less than 45%, a prognosis even worse than in the cohort previously recorded by Al Toma et al, who reported a 5-year survival rate of 58%. 7 Consistent with results from the same investigators, the prognosis of RCD I was much better with a comparable 5-year survival rate of approximately 95%. The difference in survival of patients with RCD I and patients with RCD II is largely explained by the much more frequent incidence of overt lymphomas in patients with RCD II than in patients with RCD I, the first predictive factor for overt lymphoma being the presence of aberrant IELs. However, while neither Al Toma et al 7 nor Daum et al 8 observed any overt lymphoma in RCD I, in the present study, 2 of the 14 patients with RCD I developed an overt lymphoma, stressing the necessity to screen these patients for lymphoma. We also may propose small bowel CT scan or magnetic resonance imaging and wireless video capsule endoscopy at diagnosis and during the follow-up of patients with RCD I. A second predictive factor of overt lymphoma was an increase in age at diagnosis of RCD. Because the rate of primary resistance to GFD was elevated, particularly in patients with RCD II (48%), this age factor may indirectly represent the impact of the lifelong gluten intake on the onset of overt lymphoma. Progression of overt lymphoma, worsened by malnutrition, was the first cause of mortality. Thrombotic events, favored by inflammation and hypoalbuminemia, were also frequently involved in the mortality of patients with RCD II. It has not yet been possible to design an effective treatment for patients with RCD I or II. Corticosteroids improved clinical symptoms in most patients with either type of RCD, yet a histologic response was observed only in 30% 40% of cases. The bad prognosis of RCD II led to more aggressive treatments. Unfortunately, commonly used immunosuppressive drugs had only a poor effect on the histologic response and, as could be expected, had no impact on the abnormal clonal IEL population. Nucleoside analogues such as pentostatin or cladribine and anti- CD52 have previously shown clinical, histologic, and hematologic efficacies In our study, cladribine and anti-cd52 induced a histologic response. However, explosive onset of overt lymphoma was observed in 3 of 4 patients treated with one of these 2 drugs within 3 8 weeks after treatment initiation, precluding further use of these drugs inasmuch as enhanced risk of transformation into overt lymphoma has been previously observed in a series of 17 patients with RCD II treated with cladribine. 22 One possible alternative strategy suggested by a recent study is the use of autologous hematopoietic stem cell transplantation, which induced not only a significant clinical and histologic response but also reduced significantly the percentage of abnormal IELs in 2 of the 7 treated patients. 23 It is, however, noticeable that this heavy regimen did not reduce the proportion of abnormal IELs in the majority of the patients, suggesting a risk of relapse and overt lymphomas and stressing the necessity to develop complementary strategies. Identification of the mechanisms underlying the onset of RCD II may help find potential therapeutic targets. We have provided evidence that interleukin-15 may play a key role in the pathogenesis of RCD through an antiapoptotic effect that promotes the expansion of clonal IELs. 24 Blocking IL-15 may thus help to reduce the numbers of clonal IELs and prevent epithelial damage. The recent development of a humanized anti

10 90 MALAMUT ET AL GASTROENTEROLOGY Vol. 136, No. 1 interleukin-15 antibody that has already been used without any major side effects in a phase I II trial in rheumatoid arthritis suggests the feasibility of this therapeutic approach. 25 Finally, it is interesting that we confirmed our previous finding 16 and showed that IELs in RCD II of 15 out of 16 patients tested bore a 1q22-44 trisomy. This finding is even more interesting given the recent observation of an association between celiac disease and the 1q31 locus that encodes RGS-1, a regulator of G protein electively expressed in IELs. 26 Further studies should help to delineate the possible role of this molecule in the loss of homeostasis of IELs in celiac disease and RCD, particularly of type II, and perhaps provide a novel clue to therapy. Although all except one of the patients with RCD I improved clinically with corticosteroids, their inconstant histologic response and the risk of severe bone demineralization and fractures point to the need for additional strategies. Azathioprine and anti tumor necrosis factor used in 2 corticosteroid-dependent patients failed to improve histology and may perhaps increase the risk of overt lymphoma, which is, as shown in our study, present in these patients. In conclusion, patients with RCD II have much more severe presentation and prognosis than patients with RCD I who, nevertheless, are also at risk for developing overt lymphoma. Understanding the mechanisms that perpetuate antiapoptotic mechanisms in RCD II and intestinal inflammation in RCD I will be necessary to design appropriate therapeutic strategies. References 1. Trier JS, Falchuk ZM, Carey MC, et al. Celiac sprue and refractory sprue. Gastroenterology 1978;75: Green PH, Cellier C. Celiac disease. 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Enhanced detection of receptor constitutive activity in the presence of regulators of G protein signaling: applications to the detection and analysis of inverse agonists and low-efficacy partial agonists. Mol Pharmacol 2002;61: Received May 24, Accepted September 18, Address requests for reprints to: Christophe Cellier, MD, PhD, Hôpital Européen Georges Pompidou, 20 rue Leblanc Paris, France. christophe.cellier@egp.aphp.fr; fax: (33) N.C.-B. and C.C. contributed equally to this work. The authors disclose the following: Supported by Lymphocoeliaque, Institut National du Cancer.