Bilateral renal T-cell lymphoma with hepatic infiltration and secondary polycythemia in a dog: Utility of cytology slides

Abstract

This is a case of bilateral renal T-cell lymphoma associated with secondary erythrocytosis in a dog. This case is distinctive in using clonality combined with immunocytochemistry to support the diagnosis, thus emphasizing the utility of cytology slides when histology is unavailable. This combination may be a unique canine lymphoma entity.

Case description

An 8-year-old neutered male cross-bred Bernese mountain dog was presented to the emergency service of the Centre Hospitalier Universitaire Vétérinaire (CHUV) with a 2-week history of inappetence. Owners reported that the dog had chronic gastrointestinal signs. Nine days prior to presentation, a severe episode of profuse diarrhea had been symptomatically treated without major improvement. Upon presentation the dog was weak, bright, alert, responsive, and moderately dehydrated (6% to 7%). Abdominal examination caused discomfort and bilateral nephromegaly was suspected. The rest of the physical examination, including vital signs of the cardiopulmonary system (auscultation, heart and respiratory rates, mucous membrane color, femoral pulse) was unremarkable. The cardiopulmonary parameters remained unremarkable throughout the hospitalization. Initial diagnostic tests included blood work, complete urinalysis, and abdominal ultrasound followed by fine-needle aspirates of the kidneys, renal lymph nodes, and liver.

Routine hematology (Advia 120; Siemens Healthcare Diagnostics, Toronto, Ontario) revealed a moderate erythrocytosis [hematocrit (HCT): 0.65 L/L; reference interval (RI): 0.37 to 0.57 L/L; hemoglobin (HGB): 234 g/L; RI: 129 to 184 g/L; red blood cells (RBC): 9.6 × 10¹²/L; RI: 5.7 to 8.8 × 10¹²/L)]. Considering the observed dehydration, erythrocytosis was initially thought to be secondary to hemoconcentration. A mild leukocytosis [white blood cells (WBC): 14.97 × 10⁹/L; RI: 5.20 to 13.90 × 10⁹/L] composed of a mature mild neutrophilia (12.43 × 10⁹/L; RI: 3.9 to 8.0 × 10⁹/L) without toxic changes was attributed to a physiologic response, although an inflammatory process could not be ruled out.

A serum biochemistry profile (Synchron DXC 600; Beckman Coulter, Fullerton, California, USA) revealed a moderate azotemia [blood urea nitrogen (BUN): 11.33 mmol/L; RI: 2.09 to 7.91 mmol/L and creatinine 194 μmol/L; RI: 58 to 127 μmol/L] of renal origin because of concomitant isosthenuria (urine specific gravity, 1.012). Prerenal azotemia due to decreased glomerular filtration rate secondary to dehydration may also have partially contributed to the azotemia. Hypoproteinemia (52.30 g/L; RI: 56.6 to 74.8 g/L) was moderate considering the patient’s dehydration and was composed of a moderate hypoalbuminemia (25.80 g/L; RI: 29.10 to 39.70 g/L), probably secondary to a renal loss as a marked proteinuria was present (5 g/L, Chemstrip^®). This reagent strip reading was likely accurate and unaffected by either the pH (pH = 5.5) or the specific gravity (1.012), which are reported to falsely increase the protein value when the urine is concentrated and highly or moderately alkaline. A mild hyperphosphatemia was noted (2.63 mmol/L; RI: 0.75 to 1.70 mmol/L), most likely due to decreased renal excretion. Hepatic changes included a mild increase in alanine amino-transferase (ALT) activity (125 U/L; R: 4 to 62 U/L) and a marginal increase in alkaline phosphatase (ALP) activity (157 U/L; RI: 6 to 80 U/L). Moderate hyperbilirubinemia (20.80 μmol/L; RI: 0 to 8.6 μmol/L) along with slightly icteric serum and bilirubinuria (1+, Chemstrip^®) indicated that a mild icterus was present, most likely of hepatic origin as there was no evidence of hemolysis or signs of post hepatic biliary obstruction. Together these changes indicated mild hepatocellular damage and cholestasis. Electrolyte abnormalities consisted of a mild hypernatremia (158.3 mmol/L; RI: 143 to 154 mmol/L) and hyperchloremia (125.2 mmol/L; RI: 108 to 117 mmol/L), most likely secondary to pure water loss. Mild hypocalcemia (2.31 mmol/L; RI: 2.38 to 3.00 mmol/L) was attributed to hypoalbuminemia.

The urinary sediment revealed a few large phagocytic cells (Figure 1A) and rare small lymphocytes. Large cells contained variable amounts of hematoidin crystals but no obvious signs of erythrophagocytosis. Taking into account the predisposition of Bernese mountain dogs to histiocytic sarcoma (HS) along with the urinary sediment findings, an HS with renal/urinary involvement was included in the differential.

Figure 1.

A — Urinary sediment. A large binucleated round cell containing rhomboid crystals compatible with hematoidin crystals. Sedistain^®, bar = 25 μm. B — Right kidney. A cluster of cohesive epithelial cells arranged in a tubular fashion is observed with moderately abundant fine intracytoplasmic basophilic granules. This is consistent with an aspirate of a proximal tubule. Modified Wright’s-Giemsa, bar = 50 μm. C — Left kidney. The sample is highly hemodiluted with occasional cytoplasmic fragments in the background as well as numerous intermediate to large round cells and 1 atypical mitosis (asterisk). Modified Wright’s-Giemsa, bar = 50 μm. D — Left kidney. Neoplastic lymphocytes display strong membranous positivity for CD3 immunohistochemistry, bar = 50 μm. CD3 Antibody CA17.2A12 clone; chromogen: Vector NovaRed (Vector labs Burlingame California, USA).

Ultrasound examination by a board-certified radiologist confirmed a moderate bilateral nephromegaly (Figure 2) and showed a loss of cortico-medullary distinction with pelvic dilation. Other changes detected by ultrasound consisted of a slight hepatomegaly with multiple hyperechoeic nodules, moderate splenomegaly with no evidence of parenchymal changes and enlargement of the renal lymph nodes. Fine-needle aspirates of both kidneys, liver, and renal lymph nodes were obtained. Renal cytology smears were highly hemodiluted with overall adequate cellularity. The major population was composed of intermediate round cells with a high nucleus to cytoplasm ratio (Figure 1B). The cytoplasm was scant to moderate, pale basophilic and mostly localized on one side of the cell. A rounded to irregular indented nucleus was observed, containing an open to sometimes stippled chromatin associated with 1 or 2 discrete nucleoli. A few renal epithelial clusters mostly of tubular origin were present (Figure 1B). Low numbers of macrophages were observed. A moderate quantity of cytoplasmic fragments were scattered throughout the smear. Morphological appearance of the malignant population was diagnostic of a bilateral renal malignant round cell tumor that was most consistent with lymphoma. Cytology smears of the renal lymph nodes were highly hemodiluted with mainly ruptured cells. A final diagnosis could not be made although lymphoma was considered based on the numerous intermediate lymphocytes (Figure 1C). Liver cytology smears were composed of a few normal hepatocyte clusters on a highly hemodiluted background. Occasional intermediate lymphocytes were observed and lymphoma was suspected; however, a contribution of lymphocytes from the hemodiluted background could not be ruled out.

Figure 2.

Abdominal ultrasound examination. Marked enlargement of the right kidney with loss of cortico-medullary distinction and pelvic dilation. The distance between the 2 asterisks indicates the length of the kidney (9 cm).

Over the 72-hour hospitalization time, manual measurements of the hematocrit revealed a persistent erythrocytosis (values ranging between 0.57 and 0.65 L/L; upper reference limit: 0.57 L/L) despite fluid therapy composed of 2 to 3 times daily maintenance requirements [Plasmalyte A (Baxter, Mississauga, Ontario) supplemented with KCl]. Combined with the lack of evidence of cardiopulmonary abnormalities and diagnosis of renal neoplasia, inappropriate erythrocytosis was suspected. Measurement of erythropoietin (EPO) levels in the patient’s serum sample performed by radioimmunoassay (RIA) in a reference laboratory (NationWide Specialist Laboratories., Pampisford, Cambridge, UK) showed a moderately increased EPO (42 MIU/mL; RI: 8.4 to 28 MIU/mL). Further characterization of the neoplastic population was performed in a referral laboratory (Leukocytes Antigen Biology Laboratory, UC-Davis, Davis, California, USA). Immunostaining for CD3 yielded a positive reaction for CD3 in more than 75% of the cells on both kidney aspirates (Figure 1D), supporting a T-cell origin. Positive clonal gene rearrangements for T-cell receptor (TCR)gamma also indicated a T-cell origin. Clonality assay on the renal, liver, and nodal cytology smears revealed a similar positive TCRgamma rearrangement, supporting neoplastic involvement by a clonal T-cell population. A diagnosis of bilateral renal T-cell lymphoma with hepatic infiltration and secondary polycythemia was thus based on clinical findings, clinical pathology, imaging, clonality, and immunocytochemistry. The owners opted for euthanasia and declined necropsy.

Discussion

Erythrocytosis is defined by an elevation of the erythrocyte count in the peripheral blood and is detected by an increase in the HCT, RBC count, or hemoglobin concentration. The first dichotomy for erythrocytosis involves the distinction between a relative process and an absolute elevation of the aforementioned erythroid parameters. The cause of a relative erythrocytosis is either hemoconcentration or a transient physiologic erythrocytosis (splenic contraction). In our reported case, the dog was moderately dehydrated when the blood was sampled and a relative erythrocytosis secondary to hemoconcentration was the initial diagnosis. Moderate dehydration was attributed to intestinal water loss from diarrhea and probable renal water loss. Renal disease was supported by moderate azotemia and isosthenuria. Although routinely recommended to further support relative erythrocytosis, assessment of the total protein concentration could not support hemoconcentration as obvious protein loss with marked proteinuria was present. Follow-up complete blood counts (CBCs) after normalization of hydration status is a way of confirming the relative nature of erythrocytosis. In our case, the dog’s hematocrit remained elevated during hospitalization despite appropriate fluid therapy, supporting a cause other than hemoconcentration or splenic contraction for erythrocytosis.

Once relative erythrocytosis is ruled out or made less likely, absolute erythrocytosis can be further characterized as primary or secondary. A primary absolute erythrocytosis is an autonomous and neoplastic production of the erythroid cells such as in polycythemia vera. Although bone marrow was not examined in this case, this condition was very unlikely. The term “secondary” indicates erythroid production in response to EPO stimulus as opposed to autonomous erythroid production found in polycythemia vera. Secondary absolute erythrocytosis can also be further described: it is “appropriate” when there is sustained hypoxia, documented with a PaO₂ on a blood gas while it is “inappropriate” if the EPO production is autonomous and independent of systemic hypoxia. The etiology for such process includes benign and malignant conditions with both involving either production of EPO or an EPO-like substance.

Shortly after documentation of the erythrocytosis in this case, a bilateral renal round-cell tumor was diagnosed on cytology. Canine renal tumors including carcinomas, sarcomas, and lymphomas have been associated with secondary absolute inappropriate erythrocytosis. Canine EPO measurement has been investigated by use of RIAs based on cross-reactivity of antibodies against human EPO, generating method-specific and laboratory-specific reference ranges. Here, the moderate elevation of EPO indicated a secondary absolute erythrocytosis. Human secondary erythrocytosis always displays increased EPO levels (1); however, in dogs normal to increased values can be expected (2) since there is a range overlap. It is highly likely that the reported absolute erythrocytosis was inappropriate due to a normal cardiovascular examination that didn’t support systemic hypoxia, although PaO₂ measurement would have been ideal to draw definitive conclusions. Unfortunately arterial blood gas measurement was not performed at any time. Histopathology and immunohistochemistry could have helped to determine if EPO was produced by neoplastic lymphocytes by assessing the pattern of EPO activity between normal remnant renal cells and neoplastic cells.

Over a 30-year period, 15 cases of canine renal lymphoma have been described (3–11). The present case is the 6th case of combined renal lymphoma and secondary erythrocytosis to have been reported in dogs (4–6,10). To the best of our knowledge, there is only a single recent case report of human renal lymphoma with EPO expressing neoplastic cells and secondary paraneoplastic erythrocytosis (12). This suggests that although rare, the association of erythrocytosis and renal lymphoma might be a more common lymphoproliferative entity in dogs than in humans. There have been 6 reports of human lymphoid tumors with concomitant non-primary absolute erythrocytosis, including a solid lymphoma in the lungs (13), 4 cases of lymphoid leukemias (14,15), and only 1 recent renal lymphoma case (12). This rare association is therefore not usually mentioned in the diagnostic approach to human erythrocytosis (2).

Of interest is the exclusive T-cell phenotype that has been identified in canine renal lymphoma, particularly when secondary erythrocytosis is present. To our knowledge, no primary canine renal B-cell lymphoma has been reported when immunohistochemistry was performed. By fully documenting a 4th confirmed T-cell lymphoma with secondary erythrocytosis in dogs, we provide further evidence that this association is not isolated and that it may rely on a particular underlying pathological pathway of T-lymphocytes. In veterinary medicine, paraneoplastic EPO production seems to be the main stimulus for erythrocytosis in cases of lymphoma. This is supported by elevated plasma EPO (4,5,15), evidence of active EPO transcription (16) and cytoplasmic EPO expression by the neoplastic lymphocyte population (5,16). Further research with systematic phenotyping of canine renal lymphoma, EPO measurement and identification of EPO activity in neoplastic T-cells is warranted.

Lymphoma is a common canine neoplasm with an updated classification (17) based on the human World Health Organization classification of tumors of hematopoietic and lymphoid tissues. It is based on the recognition of entities that meet unique morphological, immunophenotypical, genetic, molecular, and clinical criteria in order to facilitate diagnosis and provide practical data for the oncologist. Some entities are not fully understood, thus justifying provisional groups, such as the heterogeneous Peripheral T-Cell Lymphoma Not Otherwise Specified (PTCL-NOS). Updates or clarifications are regularly proposed following new discoveries including better understanding of the normal T-cell counterpart (18). Canine T-cell lymphomas represent only up to a third of all canine lymphomas (17) including the heterogeneous PTCL-NOS group and other subtypes. Recent studies, including molecular profiling (19) and subtyping of T-Cell lymphoma (20) will likely help establish subgroups or entities with prognostic significance. In the case of renal lymphoma, the first matter would be to determine if it is of primary renal origin as opposed to a secondary site. In this reported case, the main clinical signs were associated with renal disease; however, there was extrarenal lymphomatous involvement. It is unknown if the neoplasm disseminated rapidly from a primary renal origin, or if renal involvement was an extension from an adjacent site. Unfortunately further description and classification of our case was not possible since clear evidence of the origin, complete grading of the tumor and histopathology were lacking. In the future, systematic characterization of renal lymphoma, including clinical data, histopathology, immunophenotyping and subtyping as well as molecular profiling could be rewarding in determining if renal T-cell lymphoma can be considered a unique entity in dogs.

In dogs, cytology is a sensitive and specific diagnostic test for lymphoma. In our case, the breed predisposition to HS, the occasional irregular indented nuclei among renal atypical round cells and the presence of large phagocytic atypical cells in the urine raised suspicion for HS, although lymphoma remained the primary rule out. Equivocal cases can benefit from a polymerase chain reaction (PCR) clonality assay to support the presence of a clonal lymphoid population (21) as in the present case. Sensitivity for TCRgamma rearrangement has recently been reported to be 100% (22) while specificity is > 95% (23). The number of neoplastic cells or their intact DNA content required for the test is low (21), thus making cytology specimens including stained slides a convenient choice. This is especially relevant for critically ill patients. Confirmation of a clonal lymphoid expansion can be quickly obtained without further invasive procedures such as surgery and biopsies. Immunocytochemistry is often recommended to confirm the lineage of neoplastic cells in lymphoma and leukemia. In this particular case, CD3 was strongly positive, further supporting the T-cell origin. Nonetheless, histology and immunophenotyping remain essential for a more precise diagnosis by providing architecture assessment and subtyping.

In conclusion, we report a 6th canine case of combined renal lymphoma and secondary erythrocytosis. The diagnosis was based on clinical findings, clinical pathology, imaging, clonality, and immunocytochemistry. To our knowledge this case is unique in its use of cytology smears to confirm a diagnosis using clonal proliferation of T-lymphocytes with the TCRgamma rearrangement clonality assay and immunocytochemistry, thus emphasizing the utility of cytology slides when histology is unavailable. Renal lymphoma of T-cell lineage with secondary erythrocytosis may constitute a unique canine entity among the heterogeneous PTCL-NOS lymphoma group. Further systematic study is recommended to investigate this hypothesis when such cases present.