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Large Granular Lymphocytic Leukemia: A Treatable Form of Refractory Celiac Disease
GASTROENTEROLOGY 2012;143:1470 –1472
BRIEF REPORTS
BRIEF REPORT Large Granular Lymphocytic Leukemia: A Treatable Form of Refractory Celiac Disease GEORGIA MALAMUT,*,‡,§ BERTRAND MERESSE,*,§ VIRGINIE VERKARRE,*,§,储 SOPHIE KALTENBACH,*,¶ NICOLAS MONTCUQUET,*,§ JEAN–PAUL DUONG VAN HUYEN,*,# CÉLINE CALLENS,*,** JULIEN LENGLET,*,‡ GABRIEL RAHMI,*,‡ ELIA SAMAHA,‡ BRIGITTE RANQUE,*,‡‡ ELIZABETH MACINTYRE,*,** ISABELLE RADFORD–WEISS,*,¶ OLIVIER HERMINE,*,§§ NADINE CERF–BENSUSSAN,*,§ and CHRISTOPHE CELLIER*,‡,§ *Université Paris Descartes, Paris; ‡Gastroenterology, #Pathology, ‡‡Internal Medecine, Hôpital Européen Georges Pompidou, APHP, Paris; §Inserm U989, Faculté de Médecine Paris Descartes, Paris; 储Pathology, ¶Cytogenetics, **Molecular Hematology, §§Hematology, Hôpital Necker-Enfants Malades, APHP, Paris, France
Large granular lymphocyte leukemia (LGL) is characterized by clonal expansion of CD3ⴙ T cells or CD3ⴚ natural killer cells and frequently is associated with autoimmune diseases. We describe 2 patients with celiac disease who no longer responded to gluten-free diets after they developed T-cell LGL, with intestinal localization of malignant lymphocytes. Flow cytometry phenotyping of isolated intestinal intraepithelial and lamina propria cells eliminated type II refractory celiac disease, identifying large-sized CD8ⴙCD57ⴙ T cells. Treatment with a combination of cyclosporine and methotrexate restored the patients’ sensitivity to gluten-free diets. LGL therefore might be a cause of refractory celiac disease that is sensitive to immunosuppressive therapy. Keywords: T-Cell Receptor; Immune Regulation; Lymphocyte Proliferation; Villous Atrophy.
M
uch insight has been gained over the past 10 years in the pathogenesis of refractory celiac disease (RCD). In a subset of patients, refractoriness is caused by intraepithelial clonal expansion of phenotypically abnormal T lymphocytes. In this condition, now called type II RCD (RCDII), malignant lymphocytes egress from gut epithelium and disseminate into the blood, where they can be identified by their phenotype, notably CD103⫹ and identical clonal T-cell receptor gamma chain (TCR␥) rearrangements.1,2 Herein, we report 2 cases of RCD with the same TCR␥ clone in intestine and blood. Identification by flow cytometry of CD3⫹CD8⫹CD57⫹ T large granular lymphocytic leukemia (T-LGL) in the blood and duodenum allowed a differential diagnosis from RCDII. Treatment of T-LGL induced spectacular clinical and histologic improvement.
Case 1 A 75-year-old woman with a 10-year history of celiac disease (CD) was referred to our hospital in 1999 for RCD. She presented with severe diarrhea, low serum albumin level, anemia, and weight loss of 12 kg since 1995. She was heterozygous for DQB1*0201. Celiac antibodies
were positive in 1995 but negative in 1999. Increased peripheral blood lymphocyte (PBL) counts (7000/mm3), with an increased percentage of CD3⫹CD8⫹ T cells, were noted since 1995. In 1999, 39% of PBLs and 25% of bone marrow cells were CD3⫹CD8⫹CD57⫹ and showed cytologic features of LGL. Duodenal biopsy showed severe villous atrophy, glandular apoptotic lesions, and increased numbers of intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). Flow cytometry detected 6% of CD3⫹CD8⫹CD57⫹TCR␣⫹ cells in LPL (vs ⬍0.5% in uncomplicated CD, n ⫽ 20). The same clonal TCR␥ rearrangement was found in duodenum, PBLs, and bone marrow. Steroids, combined with cyclophosphamide for the first 6 months and then as monotherapy for 2 years, reduced diarrhea but improved histology only marginally. The LGL population had decreased in bone marrow (3%), but remained unchanged in intestinal lamina propria (9.5% of LPLs) and blood (36% of PBLs). Methotrexate and budesonide for 1 year allowed total control of diarrhea, weight recovery, correction of nutritional deficiencies, and a decrease in the absolute peripheral LGL count. Because of the patient’s age, no control endoscopy was performed. At the latest clinical evaluation in 2009, the patient remained well.
Case 2 A 48-year-old woman with a 10-year history of CD was referred to our hospital in February 2010 for suspicion of RCD. She presented with severe diarrhea, low serum albumin level, anemia, lymphopenia (200/mm3), neutropenia, duodenal ulcers at endoscopy, enlarged mesenteric lymph nodes, fever, and weight loss of 14 kg since 2005. She was homozygous for HLADQB1*0201. Celiac antibodies were positive in 2000 but negative in 2010. Abbreviations used in this paper: CD, celiac disease; IEL, intraepithelial lymphocytes; LGL, large granular leukemia; LPL, lamina propria lymphocyte; PBL, peripheral blood lymphocyte; RCD, refractory celiac disease; RCDII, type II refractory celiac disease; TCR, T-cell receptor. © 2012 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2012.08.028
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Steroids were ineffective and discontinued after 2 months. Cyclosporin, introduced at 4 mg/kg/day in June 2010, corrected the anemia and lymphopenia, controlled the diarrhea, and allowed discontinuation of parenteral nutrition and weight recovery. In January 2011, the proportion of LGLs remained high in the blood (45%) but duodenal biopsy specimens showed complete villous recovery and a drastic decrease of granzyme B⫹ intestinal lymphocytes and CD8⫹CD57⫹ IELs (15%) and LPLs (18%) (Figure 2). The patient remains cyclosporin-dependent and needing at least 1 mg/kg/day. LGL has been reported in 2 cases of CD, neither of which with proven intestinal involvement.3,4 We report 2 cases of LGL causing resistance to a gluten-free diet 5 years after diagnosis of initially responsive CD. The same clonal TCR␥ rearrangement was detected in intestine and blood, a situation reminiscent of RCDII with peripheral dissemination of abnormal IELs, which share cytologic features with LGLs2 (Supplementary Figure 2). Yet, the lymphocyte phenotype was not compatible with RCDII and flow cytometry allowed the diagnosis of CD3⫹
Figure 1. Duodenal biopsy (patient 2). Subtotal villous atrophy in 2010 (A, H&E ⫻100), glandular apoptotic lesions (B, H&E ⫻400, arrows). Infiltration by CD8⫹ (C, ⫻200), CD3⫹ (D, ⫻200), granzyme B⫹ (E, ⫻400) lymphocytes. (F) Expression of CD57 on CD8⫹ T cells freshly isolated from IELs, LPLs, and PBLs. Fluorescence-activated cell sorter analyses were gated on CD45⫹CD3⫹ cells.
Duodenal biopsy showed severe villous atrophy and glandular apoptotic lesions, a normal number of IELs, and a mild increase in LPLs (Figure 1). Flow cytometry showed that 70% of IELs, 54% of LPLs, and 35% of PBLs were CD3⫹CD8⫹CD57⫹TCR␣⫹ cells (Figure 1). CD94 was expressed in 90% of CD8⫹ IELs and LPLs and in 50% of CD8⫹ PBLs, whereas CD56, CD16, NKG2C, and NKp46 were less than 3%. Most CD8⫹CD57⫹ cells had an effector memory CD45RO⫹CD28⫺ phenotype. Numerous granzyme B⫹ LPLs and IELs were detected in situ. CD8⫹ or CD57⫹ cells also were observed in sinusoids of liver biopsy (Supplementary Figure 1). The same clonal TCR␥ rearrangement was detected in duodenal biopsy specimens, blood, and bone marrow. Genome-wide, array-based comparative genomic hybridization analysis performed on CD3⫹CD8⫹ CD57⫹ sorted cells and compared with matched genomic constitutional DNA did not show any acquired unbalanced abnormality.
Figure 2. Duodenal biopsy (patient 2). Normal villi without glandular apoptosis in 2011 (A, HES ⫻100). Normal number of CD3⫹ (B, ⫻100) and CD8⫹ (C, ⫻200) IELs and decreased T CD3⫹, CD8⫹, and granzyme B⫹ (D, ⫻400) LPLs compared with 2010. (E) Decrease of LGLs in epithelium and LP, stability in blood.
BRIEF REPORTS
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CD8⫹TCR␣⫹CD57⫹ LGL cells. Both T-LGL and RCDII IELs can express a spectrum of natural killer markers5 but RCDII IELs were CD57⫺ in more than 40 cases analyzed by flow cytometry.1,2 Moreover, CD103, always present on circulating RCDII IELs, was not detected in peripheral CD8⫹CD57⫹LGL but only on intestinal lymphocytes, suggesting that LGL arise in the periphery and acquire CD103 after migration in the gut environment. In case 1, the much lower number of LGLs in intestine than in blood further points to their peripheral origin. Previous work has suggested that LGLs, similar to RCDII IELs, depend on interleukin-15 for their survival.5– 8 The presence of interleukin-15 in the gut of CD patients may promote local survival, but also the cytotoxicity of LGLs egressing from blood. Accordingly, a high proportion of granzyme B⫹ cells were observed in epithelium and LP in patient 2, which decreased after treatment in parallel with intestinal recovery. Distinction between RCDII and T-LGL–related CD is essential for appropriate treatment. There is as yet no curative treatment for RCDII; immunosuppressive drugs usually are ineffective or predispose to overt lymphoma. In contrast, intestinal T-LGLs can be controlled by cyclophosphamide, methotrexate, or cyclosporine, as shown herein.9
GASTROENTEROLOGY Vol. 143, No. 6
Gastroenterology at www.gastrojournal.org, and at http:// dx.doi.org/10.1053/j.gastro.2012.08.028. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
Cellier C, et al. Gastroenterology 1998;114:471– 481. Malamut G, et al. Gastroenterology 2009;136:81–90. Lopez P, et al. Am J Hematol 1993;43:116 –122. Molitor JL, et al. Rev Med Interne 1997;18:237–239. Meresse B, et al. J Exp Med 2006;203:1343–1355. Fehniger TA, et al. J Exp Med 2001;193:219 –231. Malamut G, et al. J Clin Invest 2010;120:2131–2143. Chen J, et al. Blood 2012;119:137–143. Lamy T, et al. Blood 2011;117:2764 –2774.
Received February 19, 2012. Accepted August 15, 2012. Reprint requests Address requests for reprints to: Georgia Malamut, MD, PhD, Hôpital Européen Georges Pompidou, 20 Rue Leblanc 75015, Paris, France. e-mail: [email protected]; fax: (33) 1-56-0935-29. Conflicts of interest The authors disclose no conflicts.
Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of
Funding Supported by Centre Expert National des Lymphomes Associés à la maladie Coeliaque, Institut National du Cancer.
December 2012
Supplementary Materials and Methods Serum IgA, IgG antigliadin, IgA class endomysial, IgA antihuman tissue transglutaminase, and IgM antienterocyte antibodies were detected as described.2 HLA-DRB1 and DQB1 genotyping was performed by means of hybridization with sequence-specific oligonucleotides after amplification by polymerase chain reaction, using the InnoLipa HLA genotyping test (Inno-Lipa Innogenetics, Les Ulis, France).1 For histologic analysis gastrointestinal specimens were fixed in 10% formalin, embedded in paraffin, and sections were stained with H&E and Giemsa. Villous atrophy was assessed according to Oberhuber’s modification2 of the Marsh classification. Immunohistochemistry was performed on paraffin- and acetone-fixed frozen tissue sections using a 3-stage indirect immunoperoxidase technique.3 Flow-cytometry phenotyping of freshly isolated IELs, LPLs, and PBLs was performed as described.4 Molecular detection of clonal TCR␥ chain rearrangements was performed on DNA extracted from frozen specimens and from PBLs by multiplex polymerase chain reaction and confirmed by analysis of heteroduplex formation.5 Array-based comparative genomic hybridization was performed on genomic DNA from tumor-sorted cells (CD3,
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CD8, CD57⫹) and non–tumor-sorted cells (CD3⫹, CD8⫹, CD57⫺). Genomic DNA was isolated by standard proteinase K digestion and phenol-chloroform extraction. The array-based comparative genomic hybridization control was sex-matched DNA from pooled human individuals (Promega, Madison, WI). Samples were analyzed using 1 mol/L microarrays (Agilent Technologies, Palo Alto, CA). Array-based comparative genomic hybridization labeling (Roche, Mannheim, Germany) and hybridization were performed according to the manufacturer’s recommendations. Array slides were analyzed by the Agilent scanner (G2505C) with Feature Extraction software (v10.1.1.1). Data were analyzed with Agilent CytoGenomics software (v1.5.2.0). The study was approved by the Ethic committee Ilede–France II (Paris, France). Supplementary References 1. Jabado N, et al. Pediatrics 1996;98:420 – 428. 2. Oberhuber G, et al. Eur J Gastroenterol Hepatol 1999;11:1185– 1194. 3. Verkarre V, et al. Gut 2003;52:205–211. 4. Cellier C, et al. Lancet 2000;356:203–208. 5. Malamut G, et al. Gastroenterology 2009;136:81–90.
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Supplementary Figure 2. Aspect of LGLs in RCDII patient with abnormal circulating cells. Peripheral blood smear showing large lymphocytes with intracytoplasmic granules (arrows).
Supplementary Figure 1. Hepatic biopsy specimen (patient 2). Liver biopsy, performed for mild cytolysis and anicteric cholestasis, showed steatosis with LGLs in hepatic sinusoids CD3⫹ (A, ⫻200, arrows) and CD57⫹ (B, ⫻400, arrows).
BRIEF REPORTS
BRIEF REPORT Large Granular Lymphocytic Leukemia: A Treatable Form of Refractory Celiac Disease GEORGIA MALAMUT,*,‡,§ BERTRAND MERESSE,*,§ VIRGINIE VERKARRE,*,§,储 SOPHIE KALTENBACH,*,¶ NICOLAS MONTCUQUET,*,§ JEAN–PAUL DUONG VAN HUYEN,*,# CÉLINE CALLENS,*,** JULIEN LENGLET,*,‡ GABRIEL RAHMI,*,‡ ELIA SAMAHA,‡ BRIGITTE RANQUE,*,‡‡ ELIZABETH MACINTYRE,*,** ISABELLE RADFORD–WEISS,*,¶ OLIVIER HERMINE,*,§§ NADINE CERF–BENSUSSAN,*,§ and CHRISTOPHE CELLIER*,‡,§ *Université Paris Descartes, Paris; ‡Gastroenterology, #Pathology, ‡‡Internal Medecine, Hôpital Européen Georges Pompidou, APHP, Paris; §Inserm U989, Faculté de Médecine Paris Descartes, Paris; 储Pathology, ¶Cytogenetics, **Molecular Hematology, §§Hematology, Hôpital Necker-Enfants Malades, APHP, Paris, France
Large granular lymphocyte leukemia (LGL) is characterized by clonal expansion of CD3ⴙ T cells or CD3ⴚ natural killer cells and frequently is associated with autoimmune diseases. We describe 2 patients with celiac disease who no longer responded to gluten-free diets after they developed T-cell LGL, with intestinal localization of malignant lymphocytes. Flow cytometry phenotyping of isolated intestinal intraepithelial and lamina propria cells eliminated type II refractory celiac disease, identifying large-sized CD8ⴙCD57ⴙ T cells. Treatment with a combination of cyclosporine and methotrexate restored the patients’ sensitivity to gluten-free diets. LGL therefore might be a cause of refractory celiac disease that is sensitive to immunosuppressive therapy. Keywords: T-Cell Receptor; Immune Regulation; Lymphocyte Proliferation; Villous Atrophy.
M
uch insight has been gained over the past 10 years in the pathogenesis of refractory celiac disease (RCD). In a subset of patients, refractoriness is caused by intraepithelial clonal expansion of phenotypically abnormal T lymphocytes. In this condition, now called type II RCD (RCDII), malignant lymphocytes egress from gut epithelium and disseminate into the blood, where they can be identified by their phenotype, notably CD103⫹ and identical clonal T-cell receptor gamma chain (TCR␥) rearrangements.1,2 Herein, we report 2 cases of RCD with the same TCR␥ clone in intestine and blood. Identification by flow cytometry of CD3⫹CD8⫹CD57⫹ T large granular lymphocytic leukemia (T-LGL) in the blood and duodenum allowed a differential diagnosis from RCDII. Treatment of T-LGL induced spectacular clinical and histologic improvement.
Case 1 A 75-year-old woman with a 10-year history of celiac disease (CD) was referred to our hospital in 1999 for RCD. She presented with severe diarrhea, low serum albumin level, anemia, and weight loss of 12 kg since 1995. She was heterozygous for DQB1*0201. Celiac antibodies
were positive in 1995 but negative in 1999. Increased peripheral blood lymphocyte (PBL) counts (7000/mm3), with an increased percentage of CD3⫹CD8⫹ T cells, were noted since 1995. In 1999, 39% of PBLs and 25% of bone marrow cells were CD3⫹CD8⫹CD57⫹ and showed cytologic features of LGL. Duodenal biopsy showed severe villous atrophy, glandular apoptotic lesions, and increased numbers of intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). Flow cytometry detected 6% of CD3⫹CD8⫹CD57⫹TCR␣⫹ cells in LPL (vs ⬍0.5% in uncomplicated CD, n ⫽ 20). The same clonal TCR␥ rearrangement was found in duodenum, PBLs, and bone marrow. Steroids, combined with cyclophosphamide for the first 6 months and then as monotherapy for 2 years, reduced diarrhea but improved histology only marginally. The LGL population had decreased in bone marrow (3%), but remained unchanged in intestinal lamina propria (9.5% of LPLs) and blood (36% of PBLs). Methotrexate and budesonide for 1 year allowed total control of diarrhea, weight recovery, correction of nutritional deficiencies, and a decrease in the absolute peripheral LGL count. Because of the patient’s age, no control endoscopy was performed. At the latest clinical evaluation in 2009, the patient remained well.
Case 2 A 48-year-old woman with a 10-year history of CD was referred to our hospital in February 2010 for suspicion of RCD. She presented with severe diarrhea, low serum albumin level, anemia, lymphopenia (200/mm3), neutropenia, duodenal ulcers at endoscopy, enlarged mesenteric lymph nodes, fever, and weight loss of 14 kg since 2005. She was homozygous for HLADQB1*0201. Celiac antibodies were positive in 2000 but negative in 2010. Abbreviations used in this paper: CD, celiac disease; IEL, intraepithelial lymphocytes; LGL, large granular leukemia; LPL, lamina propria lymphocyte; PBL, peripheral blood lymphocyte; RCD, refractory celiac disease; RCDII, type II refractory celiac disease; TCR, T-cell receptor. © 2012 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2012.08.028
LGL IN CELIAC DISEASE
1471
Steroids were ineffective and discontinued after 2 months. Cyclosporin, introduced at 4 mg/kg/day in June 2010, corrected the anemia and lymphopenia, controlled the diarrhea, and allowed discontinuation of parenteral nutrition and weight recovery. In January 2011, the proportion of LGLs remained high in the blood (45%) but duodenal biopsy specimens showed complete villous recovery and a drastic decrease of granzyme B⫹ intestinal lymphocytes and CD8⫹CD57⫹ IELs (15%) and LPLs (18%) (Figure 2). The patient remains cyclosporin-dependent and needing at least 1 mg/kg/day. LGL has been reported in 2 cases of CD, neither of which with proven intestinal involvement.3,4 We report 2 cases of LGL causing resistance to a gluten-free diet 5 years after diagnosis of initially responsive CD. The same clonal TCR␥ rearrangement was detected in intestine and blood, a situation reminiscent of RCDII with peripheral dissemination of abnormal IELs, which share cytologic features with LGLs2 (Supplementary Figure 2). Yet, the lymphocyte phenotype was not compatible with RCDII and flow cytometry allowed the diagnosis of CD3⫹
Figure 1. Duodenal biopsy (patient 2). Subtotal villous atrophy in 2010 (A, H&E ⫻100), glandular apoptotic lesions (B, H&E ⫻400, arrows). Infiltration by CD8⫹ (C, ⫻200), CD3⫹ (D, ⫻200), granzyme B⫹ (E, ⫻400) lymphocytes. (F) Expression of CD57 on CD8⫹ T cells freshly isolated from IELs, LPLs, and PBLs. Fluorescence-activated cell sorter analyses were gated on CD45⫹CD3⫹ cells.
Duodenal biopsy showed severe villous atrophy and glandular apoptotic lesions, a normal number of IELs, and a mild increase in LPLs (Figure 1). Flow cytometry showed that 70% of IELs, 54% of LPLs, and 35% of PBLs were CD3⫹CD8⫹CD57⫹TCR␣⫹ cells (Figure 1). CD94 was expressed in 90% of CD8⫹ IELs and LPLs and in 50% of CD8⫹ PBLs, whereas CD56, CD16, NKG2C, and NKp46 were less than 3%. Most CD8⫹CD57⫹ cells had an effector memory CD45RO⫹CD28⫺ phenotype. Numerous granzyme B⫹ LPLs and IELs were detected in situ. CD8⫹ or CD57⫹ cells also were observed in sinusoids of liver biopsy (Supplementary Figure 1). The same clonal TCR␥ rearrangement was detected in duodenal biopsy specimens, blood, and bone marrow. Genome-wide, array-based comparative genomic hybridization analysis performed on CD3⫹CD8⫹ CD57⫹ sorted cells and compared with matched genomic constitutional DNA did not show any acquired unbalanced abnormality.
Figure 2. Duodenal biopsy (patient 2). Normal villi without glandular apoptosis in 2011 (A, HES ⫻100). Normal number of CD3⫹ (B, ⫻100) and CD8⫹ (C, ⫻200) IELs and decreased T CD3⫹, CD8⫹, and granzyme B⫹ (D, ⫻400) LPLs compared with 2010. (E) Decrease of LGLs in epithelium and LP, stability in blood.
BRIEF REPORTS
December 2012
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MALAMUT ET AL
BRIEF REPORTS
CD8⫹TCR␣⫹CD57⫹ LGL cells. Both T-LGL and RCDII IELs can express a spectrum of natural killer markers5 but RCDII IELs were CD57⫺ in more than 40 cases analyzed by flow cytometry.1,2 Moreover, CD103, always present on circulating RCDII IELs, was not detected in peripheral CD8⫹CD57⫹LGL but only on intestinal lymphocytes, suggesting that LGL arise in the periphery and acquire CD103 after migration in the gut environment. In case 1, the much lower number of LGLs in intestine than in blood further points to their peripheral origin. Previous work has suggested that LGLs, similar to RCDII IELs, depend on interleukin-15 for their survival.5– 8 The presence of interleukin-15 in the gut of CD patients may promote local survival, but also the cytotoxicity of LGLs egressing from blood. Accordingly, a high proportion of granzyme B⫹ cells were observed in epithelium and LP in patient 2, which decreased after treatment in parallel with intestinal recovery. Distinction between RCDII and T-LGL–related CD is essential for appropriate treatment. There is as yet no curative treatment for RCDII; immunosuppressive drugs usually are ineffective or predispose to overt lymphoma. In contrast, intestinal T-LGLs can be controlled by cyclophosphamide, methotrexate, or cyclosporine, as shown herein.9
GASTROENTEROLOGY Vol. 143, No. 6
Gastroenterology at www.gastrojournal.org, and at http:// dx.doi.org/10.1053/j.gastro.2012.08.028. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
Cellier C, et al. Gastroenterology 1998;114:471– 481. Malamut G, et al. Gastroenterology 2009;136:81–90. Lopez P, et al. Am J Hematol 1993;43:116 –122. Molitor JL, et al. Rev Med Interne 1997;18:237–239. Meresse B, et al. J Exp Med 2006;203:1343–1355. Fehniger TA, et al. J Exp Med 2001;193:219 –231. Malamut G, et al. J Clin Invest 2010;120:2131–2143. Chen J, et al. Blood 2012;119:137–143. Lamy T, et al. Blood 2011;117:2764 –2774.
Received February 19, 2012. Accepted August 15, 2012. Reprint requests Address requests for reprints to: Georgia Malamut, MD, PhD, Hôpital Européen Georges Pompidou, 20 Rue Leblanc 75015, Paris, France. e-mail: [email protected]; fax: (33) 1-56-0935-29. Conflicts of interest The authors disclose no conflicts.
Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of
Funding Supported by Centre Expert National des Lymphomes Associés à la maladie Coeliaque, Institut National du Cancer.
December 2012
Supplementary Materials and Methods Serum IgA, IgG antigliadin, IgA class endomysial, IgA antihuman tissue transglutaminase, and IgM antienterocyte antibodies were detected as described.2 HLA-DRB1 and DQB1 genotyping was performed by means of hybridization with sequence-specific oligonucleotides after amplification by polymerase chain reaction, using the InnoLipa HLA genotyping test (Inno-Lipa Innogenetics, Les Ulis, France).1 For histologic analysis gastrointestinal specimens were fixed in 10% formalin, embedded in paraffin, and sections were stained with H&E and Giemsa. Villous atrophy was assessed according to Oberhuber’s modification2 of the Marsh classification. Immunohistochemistry was performed on paraffin- and acetone-fixed frozen tissue sections using a 3-stage indirect immunoperoxidase technique.3 Flow-cytometry phenotyping of freshly isolated IELs, LPLs, and PBLs was performed as described.4 Molecular detection of clonal TCR␥ chain rearrangements was performed on DNA extracted from frozen specimens and from PBLs by multiplex polymerase chain reaction and confirmed by analysis of heteroduplex formation.5 Array-based comparative genomic hybridization was performed on genomic DNA from tumor-sorted cells (CD3,
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CD8, CD57⫹) and non–tumor-sorted cells (CD3⫹, CD8⫹, CD57⫺). Genomic DNA was isolated by standard proteinase K digestion and phenol-chloroform extraction. The array-based comparative genomic hybridization control was sex-matched DNA from pooled human individuals (Promega, Madison, WI). Samples were analyzed using 1 mol/L microarrays (Agilent Technologies, Palo Alto, CA). Array-based comparative genomic hybridization labeling (Roche, Mannheim, Germany) and hybridization were performed according to the manufacturer’s recommendations. Array slides were analyzed by the Agilent scanner (G2505C) with Feature Extraction software (v10.1.1.1). Data were analyzed with Agilent CytoGenomics software (v1.5.2.0). The study was approved by the Ethic committee Ilede–France II (Paris, France). Supplementary References 1. Jabado N, et al. Pediatrics 1996;98:420 – 428. 2. Oberhuber G, et al. Eur J Gastroenterol Hepatol 1999;11:1185– 1194. 3. Verkarre V, et al. Gut 2003;52:205–211. 4. Cellier C, et al. Lancet 2000;356:203–208. 5. Malamut G, et al. Gastroenterology 2009;136:81–90.
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Supplementary Figure 2. Aspect of LGLs in RCDII patient with abnormal circulating cells. Peripheral blood smear showing large lymphocytes with intracytoplasmic granules (arrows).
Supplementary Figure 1. Hepatic biopsy specimen (patient 2). Liver biopsy, performed for mild cytolysis and anicteric cholestasis, showed steatosis with LGLs in hepatic sinusoids CD3⫹ (A, ⫻200, arrows) and CD57⫹ (B, ⫻400, arrows).