MOLECULAR DIAGNOSIS OF FELINE INTESTINAL T CELL LYMPHOMA: IMPLICATIONS FOR CASE MANAGEMENT Peter F. Moore, VM PMI, University of California, Davis, CA 95616 (firstname.lastname@example.org) Introduction Diagnosis of gastrointestinal lymphoma is currently made on the basis of clinical, histological and immunophenotypic criteria. Histological evidence of lymphoma includes the presence of dense cellular infiltration, a monomorphic appearance of infiltrating lymphocytes, cytological immaturity of the lymphoid infiltrate, and disruption, effacement and replacement of the normal structures of the involved tissue by infiltrating cells. Pathologists have relied on the presence of transmural invasion by cytologically atypical lymphocytes, in order to make a confident diagnosis of intestinal lymphoma. However, lymphoma manifests in the intestinal mucosa long before transmural progression, which may not even occur during the full duration of the disease process. Assessment of intestinal disease is challenging in endoscopic biopsy specimens, which consist of mucosal and scant submucosal tissue with random orientation. In this instance, evaluation of submucosal tissue is hampered, and assessment of transmural infiltration is impossible. However, an understanding of the mucosal immuno-architecture and lymphocyte trafficking patterns facilitates interpretation of transmural and endoscopic intestinal biopsy specimens. Mucosal Immunoarchitecture The diffuse, mucosal-associated lymphoid tissue (MALT) of the small intestine, which consists of lamina proprial (LPC) and intra-epithelial compartments (IEC), is populated largely by CD3+ T cells in normal cats (1). Less than 5% of villous lamina proprial lymphocytes (LPL) are B cells and even fewer are found in the IEC. Plasma cells are concentrated in the intestinal crypt lamina propria. The inductive environment for adaptive immune responses is distributed in the distal small intestine where Peyer’s patches are most numerous. Antigen-stimulated B cells are induced to class switch to IgA and home to the intestinal crypts, and T cells are induced by interactions with DC to express cell surface molecules that enable them to traffic to the diffuse MALT (2-4). The requirements for lymphocyte intestinal trafficking have been elucidated in humans and rodents (2-4). Beta-7) and specific chemokines and chemokine receptors (CCL25 and CCR9) induce lymphocytes to enter the intestinal lamina propria via the endothelium of small vessels. The endothelial cells express mucosal addressin cell adhesion molecule (MadCAM), which is the ligand for alpha-4 beta-7 expressed by mucosal homing lymphocytes. Once lymphocytes enter the lamina propria, epithelial derived TGF-β induces some to express the integrin alpha-E beta-7, which is highly expressed by intra-epithelial lymphocytes (IEL). Feline IEL have also been shown to express alpha-E beta-7, hence the principles of intestinal mucosal lymphocyte trafficking established for humans and rodents likely applies to cats (5). Molecular Clonality Assessment In Lymphoproliferative Disease Expansion of T cell populations occurs in diffuse MALT in feline inflammatory bowel disease (IBD) and feline intestinal lymphoma, which was most frequently a T cell lymphoma in our case series. The distinction of IBD and T cell lymphoma, by morphological criteria alone, can pose difficulties, especially when mucosal infiltrates are more purely lymphocytic and lack the cellular heterogeneity often present in lymphoplasmacytic IBD. Molecular assessment of the clonality status of T cell infiltrates in feline intestinal disease is a valuable adjunct to morphological assessment. The true frequency of intestinal T cell lymphoma has been seriously underestimated based on retrospective molecular clonality assessments performed on tissues from cats in which IBD was diagnosed (Moore and Rodriguez-Bertos, unpublished observations). T cell receptor (TCR) gene rearrangement analysis by polymerase chain reaction (PCR) is a methodology used to detect clonality in T cell populations. This methodology provides the advantage of allowing sensitive detection of T cell clonality in formalin-fixed paraffin embedded (FFPE) tissues (6, 7). This may be critical with intestinal lympho-proliferative disease in which a diagnosis is frequently made on small tissue fragments obtained by endoscopy. During T cell development in the thymus, T cells rearrange their antigen receptor genes TCRA, TCRB, TCRG and TCRD, and in the process create 2 lineages of T cells, alpha-beta and gamma- delta T cells. TCRG gene rearrangement occurs in the majority of T cells regardless of TCR lineage (8, 9). Recently, we characterised feline TCRG and developed PCR primers for determination of the clonality status of T cells in feline intestinal lymphoma. The assay is sensitive, reproducible, and discriminates between clonal, pseudoclonal and polyclonal TCRG gene rearrangements in DNA extracted from FFPE tissue (6). In a companion paper, we also characterised the feline immunoglobulin heavy chain locus (IGH), and established its usefulness in the diagnosis of B cell neoplasia (10). PCR-based lymphocyte antigen receptor clonality assays represent important adjunctive tools in the diagnosis of feline lymphoma, when interpreted in the light of clinical, morphological and immunophenotypic data from the feline patient. The role of immunophenotyping is critical for determination of cell lineage in lymphoma, since cross lineage rearrangement of antigen receptor genes limits its use for lineage determination. Armed with an understanding of intestinal mucosal trafficking patterns, as well as immunohistological and molecular diagnostic tools, it is now feasible to re-examine the mucosal architecture characteristic of feline gastrointestinal lymphomas, and to establish the distinguishing morphological features that correlate with clonal expansion of mucosal lymphocytes versus polyclonal expansion, which is characteristic of IBD. Feline Gastrointestinal Lymphoma Feline intestinal lymphoma in our case series was most frequently of T cell origin, and originated predominately in the diffuse MALT of the small intestine. Extension to the stomach and large bowel occurred with lower frequency; this was likely due to the different lymphocyte trafficking requirements in these sites. T cell lymphomas occurred as mucosal and transmural lesions. T cell lymphomas initially arose in the diffuse MALT of the intestine in the IEC and the LPC, and remained in the mucosal environment for extended periods. Progression to involvement of mesenteric lymph nodes occurred with or without transmural migration. The existence of epitheliotropic T cell lymphomas has recently been documented in cats. Lesions were most common in the duodenum and simultaneous involvement of the IEC and LPC occurred in all cats (11). We observed an additional type of epitheliotropic lymphoma, which involved the duodenum; neoplastic lymphocytes in this disease were T cells, which diffusely infiltrated the IEC in exceedingly large numbers (often >100 IEL per 100 enterocytes) with sparing or minimal involvement of the LPC. In all instances, the neoplastic T cells had a clonal (or oligoclonal) rearrangement of TCRG. Cytologically, most intestinal epitheliotropic lymphomas were composed of small- to intermediate-size lymphocytes (nuclear diameter < 2 red blood cell diameters). Intestinal T cell lymphomas, with and without marked epitheliotropism, involved the lamina propria either diffusely, or in discrete regions of increased lymphocyte density within the villous lamina propria. Alternatively, infiltration occurred as a band of increased lymphocyte density that spanned the crypt-villous junction. Invasion of the lamina propria at the base of the intestinal crypts, and the submucosa was evidence of progression. Transmural extension of T cell lymphoma occurred focally or multifocally. In early lesions, neoplastic lymphocytes traversed the muscular layers peri-vascularly, leading to a trabecular pattern of involvement. In advanced lesions, coalescence of the infiltrate obliterated the muscularis and spilled into the serosa and adjacent mesentery. This often resulted in mass formation, intestinal obstruction and even perforation with peritonitis. Intestinal segments proximal and distal to transmural foci almost invariably manifested with mucosal lymphoma. Cytologically, transmural T cell lymphoma consisted of small- to intermediate-sized lymphocytes, or more frequently large lymphocytes (nuclear diameter >2 red blood cell diameters). Transmural large cell lymphomas have a markedly worse prognosis than small cell lymphomas (12, 13). Lymphomas of granular lymphocytes (LGL), occurred mainly as transmural large cell lymphomas; these have the worst prognosis (14-17). In many instances, fine needle aspirate cytology was necessary to identify cytoplasmic granules in LGL. LGL lymphomas routinely invaded mesenteric lymph nodes and spread rapidly to liver, spleen and kidney. LGL intestinal T cell lymphomas may invade peripheral blood; this may be sufficient to cause secondary leukaemia. Although welldescribed, the frequency of this event has not been quantified (17). None of the 10 cats with intestinal LGL lymphoma in our present case series had secondary leukaemia. Conclusion We have developed sensitive PCR-based assays for detection of clonal rearrangements of TCRG (T cells) and IGH (B cells) from small amounts of tissue, such as endoscopically-obtained biopsies. It is important that the assay is conducted in duplicate to avoid the potential pitfall of pseudoclonality. Molecular clonality assays should be conducted as adjuncts to clinical, morphological and immunophenotypic assessment, with immunophenotypic assessment taking precedence over molecular clonality results for the determination of lymphocyte lineage. Most importantly, molecular clonality analyses, used appropriately, have the potential to improve feline patient care by allowing earlier detection particularly of emerging T cell lymphoma in an inflammatory background such as that presented by chronic IBD. We have also presented the key morphologic correlates of clonal expansion of T cells within the intestinal mucosa to assist pathologists in the assessment of mucosal infiltrative processes when antigen receptor gene rearrangement analysis is not feasible. References. 1. Roccabianca P, Woo JC, Moore PF. Characterization of the diffuse mucosal associated lymphoid tissue of feline small intestine. Vet Immunol Immunopathol 2000;75(1-2):27-42. 2. Nagler-Anderson C. Man the barrier! Strategic defences in the intestinal mucosa. Nat Rev Immunol 2001;1(1):59-67. 3. Cheroutre H, Madakamutil L. Acquired and natural memory T cells join forces at the mucosal front line. Nat Rev Immunol 2004;4(4):290-300. 4. Miyasaka M, Tanaka T. Lymphocyte trafficking across high endothelial venules: dogmas and enigmas. Nat Rev Immunol 2004;4(5):360-70. 5. Woo JC, Roccabianca P, van Stijn A, Moore PF. Characterization of a feline homologue of the alphaE integrin subunit (CD103) reveals high specificity for intra-epithelial lymphocytes. Vet Immunol Immunopathol 2002;85(1-2):9-22. 6. Moore PF, Woo JC, Vernau W, Kosten S, Graham PS. Characterization of feline T cell receptor gamma (TCRG) variable region genes for the molecular diagnosis of feline intestinal T cell lymphoma. Vet Immunol Immunopathol 2005;106(3-4):167-78. 7. van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003;17(12):2257-317. 8. Thompson SD, Manzo AR, Pelkonen J, Larche M, Hurwitz JL. Developmental T cell receptor gene rearrangements: relatedness of the alpha/beta and gamma/delta T cell precursor. Eur J Immunol 1991;21(8):1939-50. 9. Theodorou I, Raphael M, Bigorgne C, Fourcade C, Lahet C, Cochet G, et al. Recombination pattern of the TCR gamma locus in human peripheral T-cell lymphomas. J Pathol 1994;174(4):23342. 10. Werner JA, Woo JC, Vernau W, Graham PS, Grahn RA, Lyons LA, et al. Characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of B-cell neoplasia. Vet Pathol 2005;42(5):596-607. 11. Carreras JK, Goldschmidt M, Lamb M, McLear RC, Drobatz KJ, Sorenmo KU. Feline epitheliotropic intestinal malignant lymphoma: 10 cases (1997-2000). J Vet Intern Med 2003;17(3):326-31. 12. Fondacaro JV, Richter KP, Carpenter JL, Hart JR, Hill SL, Fettman MJ. Feline gastrointestinal lymphomas: 67 cases (1988-1996). European Journal of Comparative Gastroenterology 1999;4(2):5-11. 13. Richter KP. Feline gastrointestinal lymphoma. Vet Clin North Am Small Anim Pract 2003;33(5):1083-98, vii. 14. Darbes J, Majzoub M, Breuer W, Hermanns W. Large granular lymphocyte leukemia/lymphoma in six cats. Vet Pathol 1998;35(5):370-9. 15. Endo Y, Cho KW, Nishigaki K, Momoi Y, Nishimura Y, Mizuno T, et al. Clinicopathological and immunological characteristics of six cats with granular lymphocyte tumors. Comp Immunol Microbiol Infect Dis 1998;21(1):27-42. 16. Kariya K, Konno A, Ishida T. Perforin-like immunoreactivity in four cases of lymphoma of large granular lymphocytes in the cat. Vet Pathol 1997;34(2):156-9. 17. Roccabianca P, Vernau W, Caniatti M, Moore PF. Feline large granular lymphocyte (LGL) lymphoma with secondary leukemia: primary intestinal origin with predominance of a CD3/CD8(alpha)(alpha) phenotype. Vet Pathol 2006;43(1):15-28.