Hypercalcaemic Multicentric Lymphoma in a Dog Presenting as Clitoromegaly

'Hypercalcaemic multicentric lymphoma in a dog presenting as clitoromegaly', Clitoromegaly is a clinical manifestation of various local and systemic conditions in all species. The external genitalia are a very rare site of primary or metastatic lymphoma in canines, with only one previously-reported case in a dog and only sparse reports in the medical literature. Lymphoma is also very rare in dogs less than four years of age. This account reports on a T-cell multicentric lymphoma in a 16-month-old Basset hound presented primarily for clitoromegaly. The patient survived for 68 days with cyclophosphamide-vincristine-prednisolone therapy. The causes of clitoromegaly in all species, including humans, are tabulated with references.


Introduction
Lymphoma is a common malignancy in dogs.The clinical manifestations of this disease are wide and varied, ranging from clinical signs relating to lymphadenomegaly, organomegaly, paraneoplastic syndromes and signs relating to obstruction of the airways by enlarged tonsils.Pleural and abdominal effusions have also been described, but the extranodal forms are considered less common than multicentric, mesenteric and mediastinal lymphomas (Vonderhaar & Morrison 2002).Furthermore, there is less veterinary information on the causes of clitoromegaly than is available in the medical literature.Discussion instigated by this novel patient (with primary clitoral lymphoma and an associated hypercalcaemia of malignancy) is of value in highlighting the possibilities for diagnosis of previously undiagnosed conditions in veterinary patients.In addition, no review of all reported causes of medical or veterinary causes of clitoromegaly could be found.To the authors' knowledge, this is only the second report of clitoral lymphoma in a dog (or any domestic animal species).

Case history and diagnostic evaluation
A 16-month-old, 22.7 kg (body surface area, 0.8 m 2 ), spayed female Basset hound was presented to the Onderstepoort Veterinary Academic Hospital (OVAH) with a history of a swollen vulva, lethargy and polydipsia.Clinical examination revealed generalised lymphadenomegaly with marked enlargement of the vulva and clitoris (Figure 1).Apart from a specific gravity of 1.008, urine analysis (dipstick, refractometry and sediment analysis) was unremarkable.Haematology was within normal limits (Table 1) and clinical chemistry revealed a moderate hyperalbuminaemia, mildly elevated serum urea nitrogen and hypercalcaemia (Table 2).Fine needle aspirates of lymph nodes were consistent with a diagnosis of centroblastic lymphoma.Cells showed anaplastic changes, bizarre shapes and a high mitotic index (Figure 2), which is characteristic of a T-cell lymphoma (Fournel-Fleury et al. 1997;Raskin & Nipper 1992;Teske & van Heerde 1996;Vonderhaar & Morrison 2002).Impression smears and fine needle aspirates of the clitoris revealed the same criteria of malignancy as the lymph nodes.Thoracic radiographs taken to investigate involvement of the intrathoracic organs (lungs, lymph nodes and thymus) were negative for metastasis.Abdominal ultrasound was performed as part of the staging procedure.A mass of mixed echogenicity and irregular shape measuring 30 mm x 10 mm was seen in the region of the medial iliac lymph node, consistent with regional metastasis to or from the clitoral mass.It was not aspirated due to its proximity to vascular structures.
The patient was placed on an intravenous saline infusion to lower the blood calcium by sodium-induced calciuresis and, once the patient was rehydrated, incisional biopsies of the submandibular and popliteal lymph nodes were taken and submitted for histopathology and immunophenotyping.The paracortical areas of both lymph nodes were infiltrated by large numbers of pleomorphic, neoplastic CD3+ CD79a-lymphocytes with a high mitotic rate (Figure 3 and Figure 4).On the basis of this, stage IIIb T-cell centroblastic lymphoma with hypercalcaemia of malignancy was diagnosed.The patient was then placed on a standard protocol of weekly vincristine (ABIC-Vincristine, Pharmachemie, Irene, South Africa) at 0.5 mg/m 2 IV q7d, cyclophosphamide (Endoxan, Sanofi-Aventis, Midrand, South Africa) at 50 mg/m 2 PO every other day and prednisolone (Prednisolone tabs, Centaur, Isando, South Africa) at 40 mg/m 2 PO q24h.
Where necessary, nausea induced by treatment was controlled with oral metoclopramide syrup (Clopamon Syrup, Sanofi-Aventis, Midrand, South Africa) at 0.5 mg/kg PO, for three  12 µm days after each treatment.The clitoral mass (as well as the enlarged lymph node) showed a marked reduction in size within a week of initiation of treatment (Figure 5).
At week 3 of treatment (day 37), vincristine dosage was lowered by 25% because of gastrointestinal side effects.This alteration in dosage and metoclopramide treatment prevented further nausea.The patient was still normocalcaemic at this time, but all subsequent evaluations showed hypercalcaemia (Table 2).On day 37, the patient was severely neutropaenic (Table 1) and vincristine and cyclophosphamide treatment was delayed.
On day 66, the patient exhibited diarrhoea of two days' duration.On clinical evaluation the vulvar mucosa was reddened.The dog was euthanased on day 68 when clinical signs of nausea and vomition became more severe and the clitoral mass and lymphadenomegaly returned.
The duration of the first remission (66 days) was defined as the time between the start of treatment and the first relapse (recurrence of lymphadenomegaly, hypercalcaemia or clitoral mass as assessed by cytological examination).Overall survival time (OST), 68 days, was defined as the time between the start of treatment and the death of the dog.

Post-mortem findings
A post-mortem examination was performed and additional samples taken for histopathology.On gross pathology, there was diffuse lymphomatous infiltration of the bilaterally-and symmetrically-enlarged prescapular, popliteal, inguinal and sublumbar lymph nodes, as well as the severely-enlarged vulva and clitoris.On the cut surface, the infiltrated organs were diffusely whitish grey and uniformly rubbery in texture.

Histopathology
Pieces of enlarged lymph node, clitoris and vulva were trimmed into tissue cassettes after 48 hours in 10% neutral buffered formalin and then dehydrated in graded ethanol, cleared in xylene and embedded in paraffin wax.Duplicate paraffin sections of 3 mm were prepared, one series being stained with haematoxylin and eosin (H&E), whilst the second series was utilised for immunohistochemical (IHC) staining.
Histology of the lymph nodes revealed the presence of multiple small clusters of neoplastic lymphocytes within the thinned capsule, the extracapsular connective tissue and the intact peripheral sinus.The cortex was residual, featuring only a few germinal centres.In all sections there was a solidly cellular interfollicular proliferation of neoplastic lymphocytes admixed with normal small, cleaved lymphocytes.The same admixture of cells caused the marked expansion of medullary trabeculae and filled the medullary sinuses.Postcapillary venules in the deep cortex appeared moderately hyperplastic in the sections examined.
The histology of the vulva and clitoris revealed diffuse sheets and cords and nodular perivascular accumulations of fairly monomorphic neoplastic lymphocytes in the subepithelial stroma, with diffuse invasion of the stratified squamous epithelium at both sites (see Figure 6).At 1000x magnification, Note: Immunohistochemical staining for CD3 detection demonstrating weakly-to moderately-positive malignant lymphocytes (predominantly cytoplasmic membrane staining) intermingled with several strongly-positive normal small, cleaved lymphocytes and a few malignant cells that failed to express CD3.A modified streptavidin-peroxidasebiotin complex procedure was applied, using cross-reacting rabbit anti-human CD3 primary antibody (1:100, DAKO) and diaminobenzidine (DAB) as the chromogen, with a haematoxylin counterstain.quite significant nuclear pleomorphism was observed in the neoplastic cells.Nuclear shape was particularly varied; there were round, cleaved, reniform, angular, irregular and horseshoe-shaped nuclei.The nuclei were generally vesicular and measured approximately 1.5 red cells in diameter.The chromatin was commonly diffuse to quite finely granular with irregular juxtamembranous and perinucleolar stippling.The nuclear membranes were sharply delimited and unevenly thickened, usually with multiple irregular indentations (Figure 3).The prominent magenta nucleoli were typically quite large, single and centrally or paracentrally located (Figure 3).On average, there was one mitotic figure per field at 1000x magnification.The neoplastic cells had scant, irregularly-distributed, weakly -basophilic to -amphophilic cytoplasm and cellular boundaries were usually indistinct.A few normal small, cleaved lymphocytes and fewer plasma cells, eosinophils and neutrophils were scattered between the neoplastic lymphocytes.

Immunohistochemistry
For immunophenotyping, the 3 mm-thick tissue sections were mounted on Superfrost Plus glass slides and dried overnight in an oven at 58 °C to enhance tissue adhesion.Routine dewaxing and rehydration were performed in xylene and graded ethanol and the sections were washed in distilled water.Antigen unmasking was performed by immersing the sections to be stained with the pan-T cell anti-CD3 polyclonal antibody in a plastic container with 0.01 M citrate buffer (pH 6).The sections to be stained with the pan-B cell anti-CD79a monoclonal antibody were immersed in a separate container with 0.01 M Tris buffer and 0.001 M EDTA (pH 9).Both containers were microwaved at high power for 15 min.After cooling for 15 min at room temperature, the buffer was decanted and the sections were washed in distilled water and then for 5 min in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) (pH 7.6).
The tissue sections were further treated according to a streptavidin-immunoperoxidase staining system (specifically the LSAB+ Kit, HRP from Dako, K0690).As the first stage in the LSAB+ Kit, endogenous peroxidase activity was quenched by incubating the tissue sections with 3% hydrogen peroxide (provided in the kit) for 5 min, followed by rinsing in PBS-BSA buffer (pH 7.6) for 5 min.
Thereafter, selected slides were incubated with a monoclonal mouse anti-human CD79a antibody (Dako, M7051) for 1 h at a dilution of 1:75.Slides for CD3 staining were incubated with the polyclonal rabbit anti-human CD3 antibody from Dako, A0452 (dilution 1:100) for 30 min.Both of these antibodies have been shown to be cross-reactive to canine B-and T-lymphocytes respectively (Jacobs, Messick & Valli 2002;Milner et al. 1996).The incubation of slides with the primary antibody was followed by sequential incubations with biotinylated link antibody and peroxidase-labelled streptavidin according to specific kit directions.Staining was completed after incubation of the sections at room temperature with the 3,3'-diaminobenzidine substratechromogen solution provided.Sections were then rinsed in distilled water, counterstained with Mayer's haematoxylin, washed in PBS-BSA buffer (pH 7.6) for 5 min, then dehydrated and mounted.Positive immunohistochemical controls for the CD3 and CD79a antibodies included normal canine lymph node.Negative controls were done on duplicate sections of canine lymph node that were treated identically to the other sections, except that the primary antibody was replaced with PBS-BSA buffer (pH 7.6) in each case.Samples were subsequently reviewed by a pathologist.
Cells were considered to be CD3 or CD79a positive if there was clear perinuclear, cytoplasmic and/or cell membrane staining (Day 1995;Jacobs et al. 2002).In this particular case, 80% -100% of the neoplastic cells in the sections of lymph node, clitoris and vulva were consistently CD3+ (pale to moderate cytoplasmic and cytoplasmic membrane staining) and CD79a-.They were therefore considered to be of the T-cell phenotype (Figure 4).The normal small, cleaved lymphocytes that were intermingled with the neoplastic T-lymphocytes in the sections of lymph node, vulva and clitoris were consistently strongly CD3+ (Figure 4).The observation that some of the neoplastic cells were not positive for the CD3 molecule can, in all probability, be ascribed to a loss of expression of the CD3 surface molecule in these cells (Day 1995).It is unlikely that tissue treatment and antigen retrieval methods played a part in the variable staining of the tumour cells in this case, since the positive control sections of normal canine lymph node revealed strong CD3 and CD79a expression and the normal T-lymphocytes in all of the sections of tumour were strongly CD3-positive.In addition, the lymphatic nodules within the residual cortex of several neoplastic lymph nodes in this case contained numerous strongly CD79a-positive B-lymphocytes.

Discussion
Clitoral neoplasia is very rare, with one source reporting that only eight cases (the last in 1987) had been described in the medical literature up to the time of the literature search in 2004 (Alvarez & Varner 1987), although an extensive literature search revealed more case reports (Table 3) but no reviews of all reported cases.In the veterinary literature, only one report exists of a clitoral lymphoma and this seems unlikely to have been primary (Ladds, Strafuss & Clifford 1969).
Although treatment was initiated in the patient described in this report, OST was relatively short (68 days vs 153 days) by the standards of other T-cell lymphomas (Vonderhaar & Morrison 2002).There are insufficient cases described to enable any predictions about outcomes of clitoral lymphoma.Nonetheless, this case was remarkable for its poor response to treatment despite an initial remission, a characteristic shared by many multicentric T-cell lymphomas.Also, unusually, the cytological and immunohistochemistry were not in complete agreement, although both predicted a highgrade lymphoma (Fournel-Fleury et al. 1997;Milner et al. 1996;Raskin & Nipper 1992).In humans, there are only a few reports of clitoral involvement in non-Hodgkin's lymphoma (Ferrando-Marco et al. 1992;Kosari et al. 2005;Ludwig, Heinrich & Brandeis 1987;Minderhoud-Bassie, Chadha-Ajwani & Huikeshoven 1992).One report described an adult patient with primary, isolated vulvar pseudolymphoma, a condition difficult to distinguish from lymphoma and of uncertain aetiopathogenesis.It represents a reactive, nonneoplastic accumulation of lymphocytes in the skin (usually the nipples or face), containing an admixture of B-and T-cell types.Other names for pseudolymphoma are Spiegler-Fendt sarcoid, lymphocytoma cutis or cutaneous lymphoid hyperplasia (Minderhoud-Bassie et al. 1992).To the authors' knowledge, these entities have not been reported in dogs.The report by Ludwig and coworkers described a two-year-old girl admitted for clitoromegaly, who was subsequently found to have renal, mediastinal, central nervous and multicentric involvement.After treatment with chemoradiation, the patient was still in full remission six years later (Ludwig et al. 1987).
A review of 186 cases of lymphoma of the human female genital tract described only one primary lymphoplasmacytic lymphoma, which occurred on the clitoris (Kosari et al. 2005).Nonetheless, Table 3 illustrates the wide array of benign and malignant neoplastic and non-neoplastic growths that may occur in this organ or even metastasise or extend into it.The importance of fine needle aspiration cytology, incisional biopsy and proper evaluation of the draining lymphatics and adnexa become obvious when the malignant nature of some of the neoplasms listed is considered (Fournel-Fleury et al. 1997;Raskin & Nipper 1992;Teske & Van Heerde 1996).
A trait of around 20% of canine lymphomas is hypercalcaemia of malignancy (Vonderhaar & Morrison 2002).This normally results from production of parathyroid hormone-related peptides (PTHrP) by the lymphomatous cells, although local factors related to the paracrine effects of TNF-α, IL-1 and other cytokines secreted by marrow lymphoma cells may also be involved.Unfortunately, a bone marrow aspirate or biopsy that might have detected myelophthisis was not performed on the patient in this report.Nevertheless, the patient did not have a leukaemic presentation and the hypercalcaemia responded to saline diuresis, although recalcitrant hypercalcaemias of malignancy may require additional treatment with one or more of the following drugs: prednisolone, furosemide, mithramycin, salmon calcitonin or pamidronate (Vonderhaar & Morrison 2002).Although the patient was hypercalcaemic and hyperalbuminaemic at initial presentation, there is evidence that correction of calcium levels for alterations in albumin status is superfluous (Schenck & Chew 2005).In any event, both ionised and total calcium were markedly raised, both initially and in the terminal stages of therapy (Table 2).

Conclusion
Clitoromegaly as a presentation of lymphoma is extremely unusual, although it should be included on differential lists for this presenting complaint, together with endocrinopathies and non-endocrine causes (Table 3).It could conceivably be primary (which would require demonstration of any other sites of involvement) or secondary (most likely and suspected in this patient) in which case it may represent a form of non-epitheliotropic lymphoma.Patients presenting with clitoromegaly, with or without obvious concurrent lymphadenomegaly, should be examined thoroughly.
The minimum database should include fine needle aspiration cytology for most masses where overt evidence of simultaneous endocrinopathy does not exist or where the history does not indicate conditions such as androgen administration or intersexuality (Table 3).In all cases, abdominal ultrasound examination should be performed, paying particular attention to the superficial inguinal, medial iliac and hypogastric lymph nodes, adrenal glands, ovaries and internal genitalia.In selected cases, ACTH-stimulation tests with measurement of cortisol, 17-hydroxyprogesterone and androstenedione, positive contrast retrograde vaginourethrocystography or intravenous pyelography, exploratory laparotomy, karyotyping and human chorionic C li to ri s gonadotrophin stimulation tests in apparently sterilised animals, should be performed to elucidate the chromosomal, hormonal and anatomical sexual status of the patient.
In humans, primary neoplastic clitoromegaly can be managed by surgery, including cosmetic reconstruction techniques, radiotherapy, or combinations thereof (Björses et al. 1997;Brown & Warne 2005;Chan et al. 2004;Houk & Lee 2005;Hyun & Kolon 2004;Jones & Matthews 1999;McNeely 1992;Oyama et al. 2004;Sur 1983;Warne, Grover & Zajac 2005).In this patient, adequate medium-term control was obtained by the use of chemotherapy alone, although surgical clitorectomy is reported in the veterinary literature (Soderberg 1986).It is possible that chemoradiation of the clitoris and draining lymphatics could play a role in future cases of isolated or regional clitoral lymphoma (and other animal neoplasms in this area), although it is difficult to advise this with any degree of certitude.The presence of a paraneoplastic syndrome with this lymphoma complicated management and contributed to the patient's demise.Nonetheless, both the neoplasm and paraneoplastic syndrome responded well to initial management.

FIGURE 2 :
FIGURE 2: Fine needle aspirate cytology of the clitoral mass (shown in Figure 1).

Note:
Malignant lymphocytes possess vesicular nuclei the approximate size of 1.5 red cells in diameter.Nuclei irregularly indented and nuclear membranes, though distinct, are unevenly thickened.The chromatin pattern is diffuse to finely granular and juxtamembranous and peri-nucleolar stippling is prominent.Cells are distinctly nucleolated.The large, commonly single magenta nucleoli are typically central or paracentral in location.The pale basophilic to amphophilic cytoplasm is scant and cell boundaries usually poorly defined.H&E.

FIGURE 7 :
FIGURE 7: Schematic illustration of lymphatic drainage of the canine clitoris.
Note: The presence of large, bizarrely-shaped cells with multiple nucleoli is indicative of centroblastic morphology.

TABLE 3 :
Reported causes of clitoromegaly in the veterinary and medical literature.