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A Single-Tube, EuroClonality-Inspired, TRG Clonality Multiplex PCR Aids Management of Patients with Enteropathic Diseases, including from Formaldehyde-Fixed, Paraffin-Embedded Tissues

2018; Elsevier BV; Volume: 21; Issue: 1 Linguagem: Inglês

10.1016/j.jmoldx.2018.08.006

1943-7811

Coralie Derrieux, Amélie Trinquand, Julie Bruneau, Virginie Verkarre, Ludovic Lhermitte, Marion Alcantara, Patrick Villarèse, Bertrand Meresse, David Sibon, Olivier Hermine, Nicole Brousse, Thierry Jo Molina, Christophe Cellier, Nadine Cerf–Bensussan, Georgia Malamut, Elizabeth Macintyre,

Microscopic Colitis

Celiac disease is a chronic inflammation of the small intestine with villous atrophy that can become refractory to a gluten-free diet. Two categories of refractory celiac disease can be distinguished by the phenotype of intraepithelial lymphocytes and the status of TRG genes. Their distinction is important because 30% to 50% of type II but only 0% to 14% of type I evolve to an aggressive enteropathy-associated T-cell lymphoma and therefore require intensive treatment. Currently, differential diagnosis integrates immunohistochemistry, immunophenotyping, and TRG clonality analyses, but each has limitations. A single-tube multiplex TRG PCR (ECN) was prospectively compared to an in-house two-tube TRG PCR (N2T) in 73 samples, including 67 cryopreserved intestine tissues. Thirteen formalin-fixed, paraffin-embedded (FFPE) samples were also analyzed retrospectively. The ECN PCR had comparable efficiency to detect major clonal rearrangements in highly infiltrated tissues from T-cell lymphoproliferative disorders and type II refractory celiac disease and to detect the persistence of minor clones in type II refractory celiac disease follow-up samples. The ECN PCR abolished the risk of amplification of false-positive weak clonal rearrangements in cryopreserved specimens and allowed improved detection of clonal rearrangements in DNA from FFPE samples. The ECN PCR allows robust assessment of cryopreserved and FFPE digestive tissues at diagnosis and follow-up of enteropathies with villous atrophy, thus guiding therapeutic management. Celiac disease is a chronic inflammation of the small intestine with villous atrophy that can become refractory to a gluten-free diet. Two categories of refractory celiac disease can be distinguished by the phenotype of intraepithelial lymphocytes and the status of TRG genes. Their distinction is important because 30% to 50% of type II but only 0% to 14% of type I evolve to an aggressive enteropathy-associated T-cell lymphoma and therefore require intensive treatment. Currently, differential diagnosis integrates immunohistochemistry, immunophenotyping, and TRG clonality analyses, but each has limitations. A single-tube multiplex TRG PCR (ECN) was prospectively compared to an in-house two-tube TRG PCR (N2T) in 73 samples, including 67 cryopreserved intestine tissues. Thirteen formalin-fixed, paraffin-embedded (FFPE) samples were also analyzed retrospectively. The ECN PCR had comparable efficiency to detect major clonal rearrangements in highly infiltrated tissues from T-cell lymphoproliferative disorders and type II refractory celiac disease and to detect the persistence of minor clones in type II refractory celiac disease follow-up samples. The ECN PCR abolished the risk of amplification of false-positive weak clonal rearrangements in cryopreserved specimens and allowed improved detection of clonal rearrangements in DNA from FFPE samples. The ECN PCR allows robust assessment of cryopreserved and FFPE digestive tissues at diagnosis and follow-up of enteropathies with villous atrophy, thus guiding therapeutic management. The spectrum of intestinal disease with villous atrophy is heterogeneous, ranging from celiac disease (CD) and various other dysimmune enteropathies to aggressive T-cell lymphoma. Affecting nearly 1% of genetically predisposed individuals in Europe, CD is a chronic inflammation of the small intestine secondary to gluten ingestion and is associated with intestinal villous atrophy and malabsorption.1West J. Celiac disease and its complications: a time traveller’s perspective.Gastroenterology. 2009; 136: 32-34Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar A small subset of patients become refractory to a gluten-free diet and are diagnosed as having refractory celiac disease (RCD), defined by the persistence of symptoms of malnutrition and intestinal villous atrophy for >12 months despite strict adherence to a gluten-free diet.2Meresse B. Malamut G. Cerf-Bensussan N. Celiac disease: an immunological jigsaw.Immunity. 2012; 36: 907-919Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar In the context of primary resistance, other causes of villous atrophy must be ruled out: autoimmune enteropathy or indolent T-cell lymphoproliferative disorders (T-LPDs) of the gastrointestinal (GI) tract.3Akram S. Murray J.A. Pardi D.S. Alexander G.L. Schaffner J.A. Russo P.A. Abraham S.C. Adult autoimmune enteropathy: Mayo Clinic Rochester experience.Clin Gastroenterol Hepatol. 2007; 5: 1282-1290Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 4Carbonnel F. d'Almagne H. Lavergne A. Matuchansky C. Brouet J.C. Sigaux F. Beaugerie L. Nemeth J. Coffin B. Cosnes J. Gendre J.P. Rambaud J.C. The clinicopathological features of extensive small intestinal CD4 T cell infiltration.Gut. 1999; 45: 662-667Crossref PubMed Scopus (58) Google Scholar, 5Perry A.M. Warnke R.A. Hu Q. Gaulard P. Copie-Bergman C. Alkan S. Wang H.Y. Cheng J.X. Bacon C.M. Delabie J. Ranheim E. Kucuk C. Hu X. Weisenburger D.D. Jaffe E.S. Chan W.C. Indolent T-cell lymphoproliferative disease of the gastrointestinal tract.Blood. 2013; 122: 3599-3606Crossref PubMed Scopus (120) Google Scholar Two types of RCD can be distinguished (RCD-I and RCD-II), based on the phenotype of intraepithelial lymphocytes by immunohistochemistry (IHC) in duodenal biopsy specimens or by multicolor flow cytometry on sorted intraepithelial lymphocytes and on molecular analysis of TRG gene rearrangements in intestinal DNA. In active CD and RCD-I, the number of intraepithelial lymphocytes is increased, but they are characterized by the expression of surface CD3, CD8, and CD103 and by the absence of clonal TRG gene rearrangements. RCD-II is defined by the accumulation of clonal and phenotypically abnormal CD103+ intraepithelial lymphocytes. An aberrant hybrid natural killer (NK)/T phenotype, lacking surface CD3 and CD8 but expressing cytoplasmic CD3 and NK markers (such as CD94, NKG2D, and NKp46), is observed.6Cellier C. Patey N. Mauvieux L. Jabri B. Delabesse E. Cervoni J.P. Burtin M.L. Guy-Grand D. Bouhnik Y. Modigliani R. Barbier J.P. Macintyre E. Brousse N. Cerf-Bensussan N. Abnormal intestinal intraepithelial lymphocytes in refractory sprue.Gastroenterology. 1998; 114: 471-481Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 7Cellier C. Delabesse E. Helmer C. Patey N. Matuchansky C. Jabri B. Macintyre E. Cerf-Bensussan N. Brousse N. Refractory sprue, coeliac disease, and enteropathy-associated T-cell lymphoma: French Coeliac Disease Study Group.Lancet. 2000; 356: 203-208Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar, 8Hüe S. Mention J.J. Monteiro R.C. Zhang S. Cellier C. Schmitz J. Verkarre V. Fodil N. Bahram S. Cerf-Bensussan N. Caillat-Zucman S. A direct role for NKG2D/MICA interaction in villous atrophy during celiac disease.Immunity. 2004; 21: 367-377Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar, 9Malamut G. Afchain P. Verkarre V. Lecomte T. Amiot A. Damotte D. Bouhnik Y. Colombel J.F. Delchier J.C. Allez M. Cosnes J. Lavergne-Slove A. Meresse B. Trinquart L. Macintyre E. Radford-Weiss I. Hermine O. Brousse N. Cerf-Bensussan N. Cellier C. Presentation and long-term follow-up of refractory celiac disease: comparison of type I with type II.Gastroenterology. 2009; 136: 81-90Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar Recently, new insights in the pathogenesis of RCD-II highlighted a novel differentiation pathway of innate intraepithelial lymphocytes via NOTCH and IL-15 signaling, leading to silencing of T-cell differentiation genes.10Ettersperger J. Montcuquet N. Malamut G. Guegan N. Lopez-Lastra S. Gayraud S. Reimann C. Vidal E. Cagnard N. Villarese P. Andre-Schmutz I. Gomes Domingues R. Godinho-Silva C. Veiga-Fernandes H. Lhermitte L. Asnafi V. Macintyre E. Cellier C. Beldjord K. Di Santo J.P. Cerf-Bensussan N. Meresse B. Interleukin-15-dependent T-cell-like innate intraepithelial lymphocytes develop in the intestine and transform into lymphomas in celiac disease.Immunity. 2016; 45: 610-625Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar RCD-II is a rare complication of CD associated with a poor prognosis, with 33% to 52% of patients evolving to an enteropathy-associated T-cell lymphoma (EATL) within 5 years of diagnosis.9Malamut G. Afchain P. Verkarre V. Lecomte T. Amiot A. Damotte D. Bouhnik Y. Colombel J.F. Delchier J.C. Allez M. Cosnes J. Lavergne-Slove A. Meresse B. Trinquart L. Macintyre E. Radford-Weiss I. Hermine O. Brousse N. Cerf-Bensussan N. Cellier C. Presentation and long-term follow-up of refractory celiac disease: comparison of type I with type II.Gastroenterology. 2009; 136: 81-90Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 11Al-Toma A. Verbeek W.H. Hadithi M. von Blomberg B.M. Mulder C.J. Survival in refractory coeliac disease and enteropathy-associated T-cell lymphoma: retrospective evaluation of single-centre experience.Gut. 2007; 56: 1373-1378Crossref PubMed Scopus (248) Google Scholar These are referred to as classic EATL to distinguish them from non–CD-associated EATL, recently renamed monomorphic epitheliotropic intestinal T-cell lymphoma.12Deleeuw R.J. Zettl A. Klinker E. Haralambieva E. Trottier M. Chari R. Ge Y. Gascoyne R.D. Chott A. Müller-Hermelink H.K. Lam W.L. Whole-genome analysis and HLA genotyping of enteropathy-type T-cell lymphoma reveals 2 distinct lymphoma subtypes.Gastroenterology. 2007; 132: 1902-1911Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 13Swerdlow S.H. Campo E. Pileri S.A. Harris N.L. Stein H. Siebert R. Advani R. Ghielmini M. Salles G.A. Zelenetz A.D. Jaffe E.S. The 2016 revision of the World Health Organization classification of lymphoid neoplasms.Blood. 2016; 127: 2375-2390Crossref PubMed Scopus (4469) Google Scholar To prevent the evolution of RCD-II to EATL, intensive treatments, such as high-dose chemotherapy, followed by autologous hematopoietic stem cell transplantation are required.9Malamut G. Afchain P. Verkarre V. Lecomte T. Amiot A. Damotte D. Bouhnik Y. Colombel J.F. Delchier J.C. Allez M. Cosnes J. Lavergne-Slove A. Meresse B. Trinquart L. Macintyre E. Radford-Weiss I. Hermine O. Brousse N. Cerf-Bensussan N. Cellier C. Presentation and long-term follow-up of refractory celiac disease: comparison of type I with type II.Gastroenterology. 2009; 136: 81-90Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 14Al-toma A. Visser O.J. van Roessel H.M. von Blomberg B.M. Verbeek W.H. Scholten P.E. Ossenkoppele G.J. Huijgens P.C. Mulder C.J. Autologous hematopoietic stem cell transplantation in refractory celiac disease with aberrant T cells.Blood. 2007; 109: 2243-2249Crossref PubMed Scopus (133) Google Scholar, 15Tack G.J. Wondergem M.J. Al-Toma A. Verbeek W.H. Schmittel A. Machado M.V. Perri F. Ossenkoppele G.J. Huijgens P.C. Schreurs M.W. Mulder C.J. Visser O.J. Auto-SCT in refractory celiac disease type II patients unresponsive to cladribine therapy.Bone Marrow Transplant. 2011; 46: 840-846Crossref PubMed Scopus (56) Google Scholar, 16Malamut G. Chandesris O. Verkarre V. Meresse B. Callens C. Macintyre E. Bouhnik Y. Gornet J.M. Allez M. Jian R. Berger A. Châtellier G. Brousse N. Hermine O. Cerf-Bensussan N. Cellier C. Enteropathy associated T cell lymphoma in celiac disease: a large retrospective study.Dig Liver Dis. 2013; 45: 377-384Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar The discrimination between RCD-I and RCD-II is therefore crucial to appropriate management. Currently, the differential diagnosis integrates histologic, multiparametric immunophenotyping and molecular analysis of TRG gene rearrangements. However, reproducible multicenter distinction is difficult, particularly, but not only, when different TRG PCRs are used, with variability in terms of analytical sensitivity and interpretation of minor clonal populations as weak clonal rearrangements within irregular polyclonal repertoires. This can lead to significant differences in estimation of the incidence of RCD-II. In this context, a robust and reproducible system of assessment of TRG gene rearrangements is needed. A new single-tube TRG PCR, recently designed by the EuroClonality group and adapted in our laboratory, was prospectively compared to our reference in-house two-tube TRG PCR on fresh or cryopreserved samples from patients with intestinal disorders with villous atrophy. A comparison with the standardized BIOMED-2 two-tube TRG PCR was also performed for RCD samples.17van Dongen J.J. Langerak A.W. Brüggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. García-Sanz R. van Krieken J.H. Droese J. González D. Bastard C. White H.E. Spaargaren M. González M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. 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.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2497) Google Scholar As a secondary aim, the single-tube TRG PCR was evaluated in formalin-fixed, paraffin-embedded (FFPE) tissues. The results demonstrated superiority of the single-tube with respect to the loss of weak clonal rearrangements of unknown significance compared with the two-tube reference and BIOMED-2 PCR in cryopreserved tissues and the improved amplification of clonal rearrangements in FFPE specimens compared with the reference PCR. The diagnosis of RCD was based on well-defined clinical, histologic, immunophenotypic, and molecular criteria, as previously described.7Cellier C. Delabesse E. Helmer C. Patey N. Matuchansky C. Jabri B. Macintyre E. Cerf-Bensussan N. Brousse N. Refractory sprue, coeliac disease, and enteropathy-associated T-cell lymphoma: French Coeliac Disease Study Group.Lancet. 2000; 356: 203-208Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar Seventy-three fresh or cryopreserved samples included 67 digestive tissues from 28 patients with previously diagnosed or suspected enteropathy with villous atrophy were collected prospectively from the gastroenterology department of the European Georges Pompidou Hospital, Paris, France, through the national expert center coordinator of the Center Expert des Lymphomes Associés à la maladie Coeliaque network. The cryopreserved diagnostic samples of all follow-up RCD-II patients and 13 FFPE specimens were analyzed retrospectively. The 28 patients were divided into three categories (Table 1): a CD and dysimmune subgroup (n = 9), including six patients with CD on a gluten-free diet [of whom two had circulating large granular lymphocytes (LGLs)] and three patients with autoimmune/dysimmune enteropathies at diagnosis (one with autoimmune-like, one with Crohn-like, and one with non CD-enteropathy with hypogammaglobulinemia), a RCD subgroup (n = 12) composed of six patients with RCD-I at follow-up and six with RCD-II (one at diagnosis and five at follow-up), and a T-cell lymphoproliferative subgroup (n = 7) comprising five patients with classic EATL (four at diagnosis and one at follow-up) and three with indolent T-LPD of the GI tract (two at diagnosis and one at follow-up).Table 1Categories of Patients Included, Time of Assessment, and Type of Samples Analyzed Prospectively with the ECN and N2T PCRCategoryTotal, nCD and dysimmune subgroupRCD subgroupT-cell intestinal lymphoproliferative subgroupCDDysimmuneRCD-IRCD-IIClassic EATLIndolent T-LPDPatients, n28636643 At diagnosis8030131 At follow-up20606512Samples, n73127132786Type of samples Digestive biopsies67127132456Antrum16234511Fundus2000200Stomach1000100Duodenum29727913Jejunum6001131Ileum1000100Colon9211500Small intestine1000001Rectum2110000Others6000330 Bronchoalveolar lavage2000110 Pulmonary biopsy2000110 Skin biopsy1000100 Lymph node1000010CD, celiac disease; EATL, enteropathy-associated T-cell lymphoma; ECN, single-tube multiplex TRG PCR; N2T, in-house two-tube TRG PCR; RCD, refractory celiac disease; T-LPD, T-cell lymphoproliferative disorders. Open table in a new tab CD, celiac disease; EATL, enteropathy-associated T-cell lymphoma; ECN, single-tube multiplex TRG PCR; N2T, in-house two-tube TRG PCR; RCD, refractory celiac disease; T-LPD, T-cell lymphoproliferative disorders. For histologic assessment, intestinal biopsy specimens were fixed in 4% formalin and embedded in paraffin. Hematoxylin and eosin– and Giemsa-stained sections were reviewed by three expert pathologists (N.B., V.V., and J.B.). Villous atrophy was assessed with a Oberhuber et al18Oberhuber G. Granditsch G. Vogelsang H. The histopathology of coeliac disease: time for a standardized report scheme for pathologists.Eur J Gastroenterol Hepatol. 1999; 11: 1185-1194Crossref PubMed Scopus (1376) Google Scholar modification of the Marsh classification and graded as absent, partial, subtotal, or total.19Marsh M.N. Gluten, major histocompatibility complex, and the small intestine: a molecular and immunobiologic approach to the spectrum of gluten sensitivity ('celiac sprue’).Gastroenterology. 1992; 102: 330-354Abstract Full Text PDF PubMed Scopus (1904) Google Scholar, 20Cellier C. Cuillerier E. Patey-Mariaud de Serre N. Marteau P. Verkarre V. Brière J. Brousse N. Barbier J.P. Schmitz J. Landi B. Push enteroscopy in celiac sprue and refractory sprue.Gastrointest Endosc. 1999; 50: 613-617Abstract Full Text PDF PubMed Scopus (72) Google Scholar The percentages of intraepithelial lymphocytes and CD3+CD8− cells were determined, as previously described.7Cellier C. Delabesse E. Helmer C. Patey N. Matuchansky C. Jabri B. Macintyre E. Cerf-Bensussan N. Brousse N. Refractory sprue, coeliac disease, and enteropathy-associated T-cell lymphoma: French Coeliac Disease Study Group.Lancet. 2000; 356: 203-208Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar DNA was extracted with a Nucleon Kit (GE Health Care, Chicago, IL), after overnight incubation at 37°C in 200 μg/mL of proteinase K (Boehringer-Mannheim, Mannheim, Germany) for cryopreserved biopsy specimens. For FFPE tissues, DNA extraction and its quantity (measurement of DNA concentration) and quality (DNA size ladder) were performed as previously described.21Camilleri-Broët S. Devez F. Tissier F. Ducruit V. Le Tourneau A. Diebold J. Audouin J. Molina T. Quality control and sensitivity of polymerase chain reaction techniques for the assessment of immunoglobulin heavy chain gene rearrangements from fixed- and paraffin-embedded samples.Ann Diagn Pathol. 2000; 4: 71-76Crossref PubMed Scopus (20) Google Scholar, 22Lenze D. Müller H.-H. Hummel M. Considerations for the use of formalin-fixed and paraffin-embedded tissue specimens for clonality analysis.J Hematopathol. 2012; : 27-34Crossref Scopus (16) Google Scholar Albumin gene real-time quantitative PCR was also performed on undiluted and 1/10 diluted DNA on a 7900 Taqman (Life Technologies, Austin, TX) to detect PCR inhibitors. FFPE samples were considered acceptable if the Ct of the diluted DNA was <3.3 Ct higher than a control undiluted DNA because dilution can improve PCR efficiency in the presence of inhibitors. The in-house reference TRG PCR (N2T) consists of a two-tube fluorescent multiplex PCR (PCR1 and 2), which is similar to the BIOMED-2 two-tube (B2T, PCRA and PCRB) multiplex but differs in the size of PCR products and inclusion of a distinctly NED-labeled JP/Jγ1.2 primer. It includes three Jγ fluorescent primers (JP/Jγ1.2, JP1/2 or Jγ1.1/2.1, and Jγ1/2 or Jγ1.3/2.3), which recognize all five known Jγ segments. The mixes were designed to allow partial determination of Vγ use based on PCR product size (Supplemental Figure S1). PCR were performed as previously described.23Delabesse E. Burtin M.L. Millien C. Madonik A. Arnulf B. Beldjord K. Valensi F. Macintyre E.A. Rapid, multifluorescent TCRG Vgamma and Jgamma typing: application to T cell acute lymphoblastic leukemia and to the detection of minor clonal populations.Leukemia. 2000; 14: 1143-1152Crossref PubMed Scopus (48) Google Scholar The test TRG PCR (ECN) corresponds to a single-tube fluorescent multiplex PCR adapted from the EuroClonality one-tube (OT) TRG PCR (data not shown) by addition of a NED-labeled JP/Jγ1.2 primer and by decreasing the primer annealing temperature from 60°C to 57°C. The four Vγ, the JP1/2, and the Jγ1/2 primer sequences were identical to the EuroClonality-OT2 (Table 2). The ECN PCR gave a slightly bimodal Gaussian distribution because the JP1/2 PCR products were larger than the Jγ1/2- Jγ1.3/2.3 products by approximately 12 bp. The JP/Jγ1.2 primer was intentionally placed downstream to generate larger, distinctly labeled PCR products, favoring distinction of canonical V9-JP TRG repertoires, which classically demonstrate a major peak at 205 bp. PCRs were performed using a 0.1-μg DNA template, 1 U of AmpliTaq gold (catalog number 4317742; Life Technology, Carlsbad, CA), and 0.25 μmol/L for each primer. Magnesium chloride and dNTP were used at final concentrations of 5 and 0.2 mmol/L, respectively. DNA was denatured at 94°C for 8 minutes, followed by 35 cycles that consisted of denaturation for 45 seconds, primer annealing for 1 minute at 57°C, and extension for 1 minute 30 seconds at 72°C. The two-tube B2T TRG PCR was performed as previously described.17van Dongen J.J. Langerak A.W. Brüggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. García-Sanz R. van Krieken J.H. Droese J. González D. Bastard C. White H.E. Spaargaren M. González M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. 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.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2497) Google Scholar Each PCR experiment included a control reaction without DNA, a polyclonal peripheral blood lymphocyte DNA control, and mixes of clonal T-cell line DNA controls (SUDHL1, HSB2, KARPAS, JURKAT, and PEER cell lines). PCR products were sized by high-resolution analysis on an ABI PRISM 3130 GeneScan (Perkin-Elmer Applied Biosystems, Warrington, UK).Table 2Primers Sequences of the Single-Tube Multiplex TRG PCRNameSequenceVγI family5′-GGTTGTGTTGGAATCAGGAGTCA-3′Vγ95′-CGGCACTGTCAGAAAGGAATC-3′Vγ105′-AGCATGGGTAAGACAAGCAA-3′Vγ115′-TTGCTCAGGTGGGAAGACTA-3′Jγ1.3/2.3fluo5′-FAM-GTGTTGTTCCACTGCCAAAGAG-3′Jγ1.1/2.1fluo5′-FAM-AGTTACTATGAGCYTAGTCCCTT-3′JγP/1.2fluo5′-NED-ACTTACCTGTAATGATAAGCTTT-3′ Open table in a new tab Between February 2013 and February 2014, 73 fresh or cryopreserved samples from 28 patients previously diagnosed with or suspected of having enteropathy with villous atrophy were prospectively analyzed with the N2T and ECN TRG PCR. The diagnosis, time of assessment, and type of specimens are reported in Table 1. Supplemental Figure S1 shows polyclonal rearrangements obtained from healthy donor peripheral blood lymphocyte for the N2T, ECN, and B2T PCR. The limit of detection of the ECN PCR, determined by dilution of clonal positive controls in peripheral blood lymphocytes, was approximatively 1% to 5%, similar to the N2T PCR (Supplemental Figure S2). Overall, of the 67 cryopreserved digestive tissue biopsy specimens analyzed, 51 (76%) had similar results with the N2T and ECN PCR, interpreted according to the EuroClonality guidelines.24Langerak A.W. Groenen P.J. Brüggemann M. Beldjord K. Bellan C. Bonello L. Boone E. Carter G.I. Catherwood M. Davi F. Delfau-Larue M.H. Diss T. Evans P.A. Gameiro P. Garcia Sanz R. Gonzalez D. Grand D. Håkansson A. Hummel M. Liu H. Lombardia L. Macintyre E.A. Milner B.J. Montes-Moreno S. Schuuring E. Spaargaren M. Hodges E. van Dongen J.J. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.Leukemia. 2012; 26: 2159-2171Crossref PubMed Scopus (319) Google Scholar No JP/Jγ1.2 rearrangements were detected. Discordant cases were heterogeneously distributed (Table 3), and predominated in RCD-I samples, as detailed below.Table 3Concordance Rate and Discrepancies between TRG PCR in Cryopreserved SamplesSubgroups of patientsN2T and ECN, total n/n (%)B2T, N2T, and ECN, total n/n (%)ConcordantDiscordant (N2T and ECN)ConcordantDiscordantT-cell lymphoproliferative EATL6/8 (75)2 (weak clonal different from tumor ad polyclonal) Indolent T-LPD5/6 (83)1 (clonal similar to diagnosis and irregular polyclonal)RCD RCD-II diagnosis6/6 (100)∗Prospective RCD-II DNA./5/5 (100)†Retrospective RCD-II DNA. RCD-II follow-up19/21 (90)2 (weak clonal and strong clonal)11/14 (79)3 (B2T, ECN, and N2T‡See The ECN PCR Is at Least as Sensitive as the N2T PCR for the Follow-Up of RCD-II for details.) RCD-I follow-up6/13 (46)7 (weak clonal and polyclonal)5/13 (38)8 (minor clones B2T plus N2T and polyclonal ECN)CD and dysimmune15/19 (79)4 (3 weak clonal and 1 oligoclonal products and polyclonal)B2T, BIOMED-2 two-tube; CD, celiac disease; EATL, enteropathy-associated T cell lymphoma; ECN, single-tube multiplex TRG PCR; N2T, in-house two-tube TRG PCR; RCD, refractory celiac disease; T-LPD, T-cell lymphoproliferative disorder.∗ Prospective RCD-II DNA.† Retrospective RCD-II DNA.‡ See The ECN PCR Is at Least as Sensitive as the N2T PCR for the Follow-Up of RCD-II for details. Open table in a new tab B2T, BIOMED-2 two-tube; CD, celiac disease; EATL, enteropathy-associated T cell lymphoma; ECN, single-tube multiplex TRG PCR; N2T, in-house two-tube TRG PCR; RCD, refractory celiac disease; T-LPD, T-cell lymphoproliferative disorder. Major clonal rearrangements were easily detected, with concordant results between the N2T and ECN PCR, in 15 of 16 infiltrated specimens from four patients with classic EATL (two associated with an RCD-II and two developed on CD without underlying RCD-II, n = 5), three patients with indolent T-LPD of the GI tract (n = 4), and one with RCD-II at diagnosis (n = 6) (Figure 1, A–D ). All 15 concordant samples were highly infiltrated by IHC. In the only discordant infiltrated duodenal case from a T-LPD of the patient with GI disease, a clonal product similar in size to the contemporary jejunal clone was detected with the N2T PCR, whereas the ECN PCR gave an irregular polyclonal profile. This corresponded to the presence of a clonal product close to the peak of the polyclonal background in a sample with a heterogeneous IHC infiltration, compatible with a false-negative result with the ECN PCR. Four digestive noninvolved specimens (three from a classic EATL and one from an indolent T-LPD of the GI tract) were also analyzed at diagnosis. The N2T PCR demonstrated weak clonal rearrangements, different from the pathological clones in 3 of 4 samples, whereas these minor peaks, of unknown significance, were lost in the polyclonal background with the ECN PCR for two of these three samples (Table 3). These results suggest that major clonal rearrangements are appropriately amplified with the ECN PCR in highly infiltrated tissues from classic EATL, indolent T-LPD of the GI tract, and diagnostic RCD-II, with a lower risk of nonspecific positivity. Twenty-one samples from five patients with RCD-II at follow-up were collected prospectively as part of the monitoring workup or before autologous hematopoietic stem cell transplantation. RCD-II diagnostic duodenal or jejunal DNA from each patient was analyzed retrospectively with both PCRs to establish clonal relatedness. Follow-up samples demonstrated a concordance rate of 90% (n = 19 of 21), with polyclonal profiles detected with both PCRs in 13 samples, clonal rearrangements in four samples, and oligoclonal profiles in two samples (Table 3). All but two clones of unknown significance in follow-up samples (one colonic and one antral) corresponded to diagnostic clonal peaks (data not shown). Diagnostic and follow-up duodenal specimens from a patient with RCD-II are illustrated in Figure 2, A–C. The N2T and ECN PCR both detected clonal rearrangements (two VγIf-Jγ1/two in the N2T PCR) within a polyclonal background in the follow-up sample (Figure 2E), which were identical to the diagnostic clones (Figure 2D). Two RCD-II samples from a single patient had minor discrepancies because they demonstrated major clonal rearrangements with the ECN PCR, corresponding in size to diagnostic clonal rearrangements, which were weak in the N2T PCR. However, the global interpretation was similar for the two PCRs, and these findings were not considered to be significant discrepancies. All duodenal/jejunal immunohistologic study results were consistent with the PCR results, with the persistence of abnormal intraepithelial lymphocytes without invasive lymphoma and clonal/oligoclonal profiles in seven of eight samples and with a slight increase of normal intraepithelial lymphocytes and a polyclonal profile in one of eight specimens. In conclusion, the ECN PCR is at least as sensitive as the N2T PCR for surveillance of clonal evolution in RCD-II samples, including for minor clones. For 13 RCD-I follow-up samples taken as part of the reevaluation workup, no major clonal rearrangements were detected by either PCR. However, weak clonal rearrangements within a polyclonal background were observed with the N2T PCR in most cases (7 of 13 or 54%, including two duodenal, one jejunal, three gastric, and one colonic biopsy specimens). In striking contrast, no significant clonal rearrangements, even of weak intensity, were detected with the ECN PCR in any sample, which were all considered to be polyclonal (9 of 13) or irregular polyclonal (4 of 13). A normal relief to partia