T. Marchal

Cytohistological and immunological classification of canine malignant lymphomas: comparison with human non-Hodgkin's lymphomas.

Non-Hodgkins lymphomas (NHLs) in man are on the increase. They are also common in dogs, which, as close companions of man, may constitute a useful experimental model. However, comparisons cannot be made without a reliable morphological and immunological classification of canine NHL. Canine NHLs (n = 134) were classified on the basis of fine-needle lymph-node aspirates according to the Kiel classification, and 92 were re-classified according to the Working Formulation and the updated Kiel classification, in a histological and immunological study. The immunophenotype was determined (1) in 92 cases by the use of the pan-T anti-CD3 polyclonal antibody and the pan-B anti-mb1 monoclonal antibody on paraffin wax-embedded tissue sections, and (2) in 47 cases by the use of a panel of polyclonal and monoclonal antibodies on fresh preparations and frozen tissue. Cytological analysis showed a predominance of high-grade lymphomas (73.9%) over low-grade lymphomas (26.1%); it also demonstrated forms not reported in other species (small-cell variants, lymphomas with macronucleolated medium-sized cells [MMCs], and polymorphic lymphomas with a centroblastic component). Histological examination revealed the rarity of follicular lymphomas (2.2% of cases), an appearance suggestive of T-cell neoplasia (8.7% of cases), and evidence that some MMC lymphomas originated in the marginal perifollicular zones. Some (26%) of the lymphomas were of the T-cell phenotype: the majority of these consisted of small-cell, low-grade lymphomas and mycosis fungoides, the rest being either high-grade pleomorphic lymphomas (mixed or large-cell) or, rarely, high-grade, small noncleaved-cell, plasmacytoid lymphomas. No lymphoma expressed a double (T and B) phenotype. This study revealed similarities with, but also notable differences from, human NHL. In particular, the MMC lymphomas may constitute an interesting equivalent of human marginal zone B-cell lymphomas.

Immunophenotype of the canine transmissible venereal tumour

The canine transmissible venereal tumour is a naturally occurring contagious round-cell neoplasia which is primarily located in the mucous membrane of the external genitalia in dogs of either sex. In order to specify the controversial cytogenetic origin of this round-cell tumour, 14 cases of canine transmissible venereal tumour, formalin- or Bouin-fixed and paraffin-embedded, were subjected to extensive immunophenotypic analysis using reagents specific to a variety of cytoplasmic or surface antigens: lysozyme, ACM1 antigen, vimentin, neuron-specific enolase, glial fibrillary acidic protein, desmin, alpha smooth muscle actin, CD3, IgG, kappa and lambda light chains, and keratin. Lysozyme immunoreactivity was detected in all cases, ACM1 antigen in 11 of 14, neuron-specific enolase in 11 of 14, vimentin in 10 of 14, glial fibrillary acidic protein in 4 of 14 and desmin in 1 of 14. All the sections were negative to keratins, alpha smooth muscle actin and CD3, whereas in five cases, perivascular tumour cells contained Ig G, kappa and lambda light chains. The immunoreactivity to lysozyme and ACM1 antigen supports the hypothesis of a histiocytic immunophenotype for the canine transmissible venereal tumour.

Growth fractions in canine non-Hodgkin's lymphomas as determined in situ by the expression of the Ki-67 antigen.

The proportion of proliferating cells in non-Hodgkins lymphomas (NHLs) as determined in situ by the expression of the Ki-67 antigen, has prognostic value in human oncology, and is strongly related to the different grades of malignancy. The evaluation of the Ki-67 index in canine NHLs may be useful in assessing the individual variability of the growth fraction in the different sub-types of lymphoma, and also the validity of the classification in terms of grade of malignancy. The growth fraction was evaluated in 92 canine NHLs, previously classified according to the Kiel classification (as adapted to the canine species), by determining the expression of the Ki-67 antigen with the MIB1 antibody on (1) paraffin-wax tissue sections in all 92 cases, and (2) fine-needle aspirates or tumour imprints in 30 cases. The labelling appeared satisfactory in 88% of the cases, with good concordance between the histological and cytological data. A highly significant correlation (P < 0.001) was established between the proportion of Ki-67+ cells and the classification into low-grade (Ki-67 index < 21%) and high-grade malignancy (Ki-67 index > 21% and usually > 29%). In the low-grade lymphoma group, a macronucleolated medium-sized-cell lymphoma not found in man had the lowest proliferation index. In the high-grade malignancy group, the number of Ki-67+ cells seemed to be proportional to cell size, whatever the phenotype, with the rare exceptions of some unclassifiable small-cell Burkitt-type or plasmacytoid lymphomas, which were highly proliferating. The classification of lymphomas into low-grade and high-grade appears to correlate well with their proliferative index. The existence of individual variations, within given categories of canine NHL, suggests that, as in human medicine, prognosis may be assisted by determining the growth fraction at initial diagnosis, and by fine-needle aspiration at relapses.

Immunohistochemical Detection of Leishmania Infantum in Formalin-Fixed, Paraffin-Embedded Sections of Canine Skin and Lymph Nodes

Canine leishmaniasis is a protozoal infection caused by Leishmania infantum which multiplies through the process of binary fission in macrophages of the skin and the reticulolymphoid organs (lymph nodes, bone marrow, etc.). The disease is transmitted to humans and animals by bloodsucking sandflies. Outside of America, 2 canine leishmaniasis is endemic in several countries around the Mediterranean basin, with an average prevalence 7 of from 20% to 40%; the growing international displacement of humans and their pets and the viral (acquired immunodeficiency syndrome: AIDS) and iatrogenic factors of immunodepression 1 have helped to increase the frequency and the distribution of the zoonosis. Clinical signs of the illness are numerous and varied: 6 generalized lymphadenopathy, cachexia, exfoliative dermatitis with scaling, hyperkeratosis, alopecia, and ulcers. The disease is extremely polymorphic, so observation of the organism in appropriate tissue samples and demonstration of serum anti-Leishmania antibodies are necessary to confirm the diagnosis. The differential diagnosis includes several immunologic skin diseases (pemphigus foliaceus, systemic lupus) and other scaling dermatoses of the dog (zinc-responsive dermatoses, necrolytic migratory erythema, etc.); hence, the veterinarian often performs a skin or lymph node biopsy. Unfortunately, skin biopsy results vary considerably: typical features 4,7 are inflammatory infiltrate of macrophages and lymphocytes, granulomatous perifolliculitis, and perivascular and diffuse dermatitis. Parasitic organisms are frequently not easily recognized and the examination of the hematoxylin and eosin (HE) sections is sometimes inconclusive. The purpose of this study was to evaluate two immunohistochemical techniques for the demonstration of Leishmania in skin and lymph node biopsies. Material consisted of formalin-fixed biopsies from 26 dogs examined in the Dermatology Department of the Veterinary School of Lyon (4 dogs) and the surgeries of veterinarians located in an area where leishmaniasis is endemic (22 dogs). The diagnosis of leishmaniasis was confirmed by serologic immunofluorescent assay. a Thirty-four skin biopsies were obtained from selected areas of the bodies of 26 dogs in apparently healthy (11 biopsies) or visibly abnormal areas (23 biopsies). Fifteen lymph nodes were obtained from 9 dogs that were euthanatized for medical reasons. All the samples were fixed in 10% neutral formalin and then embedded in paraffin using routine procedures. One section from each sample was stained with standard HE staining to identify the parasites in the infected cells, since it is a common pathological technique that is able to define subtle cellular differences if there are pathologic changes.

Lymphocyte subset abnormalities in German shepherd dog pyoderma (GSP).

Peripheral blood lymphocyte subpopulations were studied in 12 German shepherd dogs suffering from deep pyoderma (GSP). Twelve other healthy but matched dogs were used as controls. GSP was found to be associated with an imbalance in the CD4 and CD8 subsets (respectively 37.3 +/- 8.7% and 28.6 +/- 6.6%, as compared to 47.5 +/- 8.8% and 19.3 +/- 4.0% in the controls). The activation markers were not affected by GSP. Moreover, analysis of the B-cell populations showed a striking decrease in the level of CD21 cells (5.5 +/- 3.3% of CD21+ lymphocytes, compared to 12.2 +/- 6.0 in the controls). This study suggests that the immunological imbalance observed in GSP may be associated with defective helper cells, and provides further evidence that dogs suffering from GSP are not immunologically normal reactors.

High-Grade Canine T-cell Lymphoma/Leukemia with Plasmacytoid Morphology: A Clinical Pathological Study of Nine Cases:

The aim of this study is to determine the clinical, morphological, and immunophenotypical presentation of 9 cases of a particular type of canine T-cell lymphoma/leukemia. The morphological presentation was a diffuse infiltration of small, medium-sized, or large blast cells with eccentric nuclei, hyperbasophilic cytoplasm, and a juxtanuclear, pale cytoplasmic area, giving a plasmacytoid appearance and suggesting a B-cell morphology. Surprisingly, all 9 cases were of T-cell phenotype (CD3+). Among the 7 immunophenotyped cases, 4 were CD4–/CD8+, 2 CD8+/CD4+, and 1 CD4+/CD8–. The median Ki-67 index was 65.7%, which placed this lymphoma in the high-grade group. This type of lymphoma/leukemia was found in dogs between 1 and 11 years of age, with a median age of 5.8. The male-female ratio was 0.8 for a reference population of 1.04. The most significant clinical findings were lymphadenopathy either generalized or localized in all cases, a mediastinal mass in 4 cases, bone marrow involvement in 7 cases, hypercalcemia in 4 cases, along with an aggressive clinical course and a poor response to chemotherapy in all cases, with a median disease-free survival time of 3 months.

Immunophenotypic and Ultrastructural Evidence of the Langerhans Cell Origin of the Canine Cutaneous Histiocytoma

Canine cutaneous histiocytoma (CCH), a histiocytic benign, dermal, self-healing tumor in the young dog, and epidermal Langerhans cells (LC) are thought to be related. In this study, we used immunohistochemical staining and transmission electron microscopy for 5 fresh CCH and 17 fixed tumors, to examine if, on the basis of their immunophenotype and their ultrastructural morphology, these tumor cells originate as LCs. The immunophenotype of CCH: canine CD11a, 11c, 18, 45, MHC II positive and ACM1, human CD14 negative, was different from canine macrophage immunophenotype but very similar to the canine LC phenotype. Furthermore, we have described ultrastructural markers in CCH cells for the first time: these consist of coated vesicles, regularly laminated bodies, pleiomorphic inclusions, paracrystalline structures, and deep invaginations of the plasma membrane, usually observed in congenital self-healing histiocytosis, a human LC tumor, or occasionally observed in human LC. The occurrence of such immunophenotype and ultrastructural markers confirmed the common lineage of LCs and CCH cells.

Cellular immune response of lymph nodes from dogs following the intradermal injection of a recombinant antigen corresponding to a 66 kDa protein of Echinococcus granulosus

A recombinant polypeptide (referred to as EgA31), which represents a 66kDa protein, was prepared from an Echinococcus granulosus cDNA library. In order to assess its potential to induce cellular immune responses, dog popliteal and prescapular lymph nodes were sensitized with this recombinant polypeptide. Subpopulations of lymphocytes were then analyzed by flow cytometry and immunohistochemistry on lymph node sections. Five days after the sensitization, the paracortical areas of the lymph nodes appeared hypertrophic, the number of CD3+, CD4+, CD8+ and CD5+ cells increased, the number of B-cells began to augment and some secondary follicles occurred, and a number of CD4+ cells appeared in germinal centers. Many large secondary follicles and a significantly augmented number of CD5+ cells in cords of medullae were observed 10 days after the sensitization. These active cellular responses strengthen the interest for further studies on the development of a vaccine with this recombinant polypeptide.

Quantitative assessment of feline epidermal Langerhans cells

The densities of feline epidermal dendritic cells expressing CD18. MHC class II and CD1a antigens were determined for four anatomical locations in 19 cats of European breed in blind conditions. The densities (±SD) of CD1a+ Langerhans cells in the skin of the abdominal wall (269±68 cells/mm2), the back (363±19), the internal side of the ear (572±30) and the external side of the ear (502±32) were significantly different, with young and old animals displaying less stained cells than adults. No significant differences in the mean densities were found with regard to sex, colour or antibody used.

Aggressive large granular lymphocyte lymphomas in five dogs: a clinical cytohistological and immunological study

Among 276 canine lymphomas referred to the Haematology–Cytology–Immunology laboratory of the Lyon Veterinary School between 1997 and 2003, there were five aggressive large granular lymphocyte (LGL) malignancies. The five dogs were clinically examined and followed up. Cytological and histological analyses of the liver, spleen, lymph nodes, intestine and bone marrow were performed. The immunophenotype and proliferation index were established. The most significant clinical finding was that of an aggressive clinical course, the presence of hepatomegaly and/or splenomegaly, an abdominal lymphadenopathy, anaemia in four cases and blood and bone-marrow involvement in two cases. The cytological presentation was a diffuse infiltration of atypical, large granular lymphoid cells. Two cases were of the null type (CD3−, CD79a−, CD4−, CD8−), and three were of the T-cell type (CD3+, CD79a−, CD4−, CD8+). The proliferation index was high in all cases, with a median of 54.4%. The histological presentation of the null-type cases was an infiltration of the liver’s portal triads and the spleen’s red pulp. The T CD8+ cases showed two different patterns, characterised by infiltration: in the first, of all the intestine’s layers, the liver’s portal triads, the spleen’s red pulp and the lymph nodes and, in the second, infiltration of the liver’s sinusoids, the spleen’s red pulp. Although these aggressive LGL lymphomas are still poorly known, they may be compared to three types of human lymphoma: aggressive NK cell lymphoma/leukemia, hepatosplenic T-cell lymphoma and enteropathy-type T-cell lymphoma.