MOLECULAR DIAGNOSIS OF FELINE INTESTINAL T CELL LYMPHOMA: IMPLICATIONS FOR
CASE MANAGEMENT
Peter F. Moore, VM PMI, University of California, Davis, CA 95616 (pfmoore@ucdavis.edu)
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.