Lymphoma is a frequently diagnosed neoplasm originating from the lymphoid tissue (14,23). It is classified based on its anatomic location, histological characteristics, and the immunophenotype of the predominant lymphocytes (21). Each subtype exhibits distinct biological behavior. Generally, low-grade lymphomas predominantly comprised small, mature lymphocytes and exhibited slow progression. In contrast, high-grade lymphomas comprised intermediate-to-large lymphocytes, characterized by the aggressive progression that necessitated timely intervention (9,15).

When classified by immunophenotyping, B-cell lymphomas constituted approximately 70% of all canine lymphomas (13). Among these, diffuse large B-cell lymphoma (DLBCL) was the most frequently diagnosed histological subtype (14,19), typically showing aggressive behavior (1). In contrast, non-DLBCL B-cell lymphomas, such as marginal zone lymphoma (MZL), follicular, and small B-cell lymphocytic lymphoma, exhibited indolent behavior and slow growth (2). Here, we present a case of canine DSBCL, a non-DLBCL B-cell lymphoma, characterized by splenic infiltration and an aggressive clinical course following non-traumatic splenic rupture.

A 10-year-old neutered male, Shih Tzu, was presented with lethargy, fever, and generalized lymphadenopathy. Bilateral prescapular lymph node enlargement was first noted a month prior, followed by rapid growth of other superficial lymph nodes, including the bilateral submandibular, inguinal, and popliteal lymph nodes. Lethargy and fever developed acutely 2 weeks before presentation. On physical examination, the patient exhibited depression, hyperthermia (40.1°C), tachypnea (respiratory rate of 66 per minute), and tachycardia (heart rate of 156 beats per minute) which met the systemic inflammatory response syndrome (SIRS) criteria. Systolic blood pressure, measured using the Doppler method, was 150 mmHg. Abdominal palpation revealed a large, undefined intra-abdominal mass in the cranial abdomen. In addition, multiple petechiae and dark red nodules were observed in the left inguinal region, which were observed by the owner five days earlier.

We evaluated the lymph nodes for a primary complaint of generalized lymphadenopathy. The bilateral submandibular, prescapular, popliteal, and inguinal lymph nodes were severely enlarged (mean sum of the longest dimensions (16), 327 mm) without pain, which included a differential diagnosis of lymphoma, lymphadenitis, systemic infection, or metastatic neoplasia. Fine-needle aspiration cytology of the bilateral prescapular lymph nodes and the left popliteal lymph node revealed a homogeneous population of small to intermediate-sized lymphocytes with occasional neutrophils, eosinophils, plasma cells, and no detectable pathogens. We ruled out lymphadenitis based on the absence of an increase in the inflammatory cell population.

Flow cytometry immunophenotyping of the left popliteal lymph node aspirate was performed to characterize the immunophenotype of the predominant small to intermediate-sized lymphocytes. Its expression resulted in CD21+/CD45+/CD34–/CD3+/CD5–/CD4–/CD8– (Fig. 1). Among these, 85% of the cells expressed CD45 and CD21, while 99% expressed CD3. Because the predominant cells showed homogenous marker expression, lymphoma was identified as the primary differential diagnosis. T-zone lymphoma was excluded, as this subtype does not express CD45. These cells exhibited double positivity for both B-cell- and T-cell markers. Such a phenotype may indicate B-cell lymphoproliferative disease with TCR rearrangements (20) or an aberrant immunophenotype (4).

Figure 1. Flow cytometric features of the patient’s left popliteal lymph node. (A) The small lymphocytes show homogenous expression of CD21 and CD45, unlike TZL, which is negative for CD45. (B) The small lymphocytes show homogenous expression of CD3.

Laboratory analyses were conducted to assess potential underlying diseases and paraneoplastic syndromes. A complete blood count indicated moderate normocytic, normochromic regenerative anemia, as evidenced by a manual packed cell volume of 21% (reference range: 35-55%), a reticulocyte count of 153,300/μL (reference range: 10,000-110,000/μL), a mean corpuscular volume 64.7 fL (reference range: 61.6-73.5 fL), and a mean corpuscular hemoglobin concentration 35.1 g/dL (reference range: 32-37.9 g/dL). Additionally, moderate thrombocytopenia was observed, with a platelet count of 42,000/μL (reference range, 148,000-484,000/μL). No morphological abnormalities of red blood cells (RBCs) were noted in the blood smear, suggesting that hemorrhage could be considered a primary cause of regenerative anemia. Additionally, an increased number of band neutrophils (4,978 cells/μL; reference range, 0-1,000 cells/μL) and many nucleated RBCs (30 cells/100 nucleated cell counts) were found on the blood film. Serum biochemistry revealed hypoglycemia (47 mg/dL; reference range: 70-143 mg/dL). The activated partial thromboplastin time was delayed (146 seconds; reference range, 75-105 seconds), and thromboelastography confirmed a hypercoagulable state. Urinalysis of the cystocentesis-obtained urine did not reveal any infectious agents. Additionally, ehrlichiosis, anaplasmosis, and Lyme disease were excluded using the 4Dx SNAP kit (IDEXX Laboratories, Westbrook, ME, USA). In summary, the laboratory findings indicated regenerative anemia, thrombocytopenia, a neutrophilic left shift, hypoglycemia, and a hypercoagulable state.

An abdominal-focused assessment with sonography for trauma revealed generalized intra-abdominal lymphadenopathy and splenomegaly, accompanied by multifocal hyperechoic nodules distributed throughout the splenic parenchyma. Additionally, a well-defined, oval-shaped, encapsulated mass filled with echogenic fluid was identified in the tail region of the spleen (Fig. 2). Echogenic free fluid in the abdominal cavity was observed on ultrasound so we performed abdominocentesis. During abdominocentesis, the animal suddenly developed shock characterized by bradycardia (heart rate: 59 beats/min), hypotension (systolic blood pressure: 70 mmHg), and pale mucous membranes, necessitating resuscitative measures. However, the owner decided to euthanize the animal due to financial concerns.

Figure 2. The ultrasound image of well-defined, oval shaped, 5.25 × 2.13 cm sized mass in the tail region of the spleen. A hyperechoic substance is observed within the echogenic fluid.

The subsequent necropsy revealed a hemoabdomen within the abdominal cavity. The spleen was severely enlarged and displaced from its normal anatomical position. The splenic surface exhibited a white and reddish-black appearance with a friable texture. Additionally, the lateral margin of the splenic tail region, where it contacts the right lateral lobe of the liver, was ruptured (Fig. 3). No other hemorrhagic lesions were observed on the skin or subcutaneous fat, except in the inguinal region, and generalized lymphadenopathy was confirmed. Consequently, histopathological and immunohistochemical staining (IHC) was performed on the left prescapular lymph nodes and the spleen and punch biopsies of the dark red nodules in the inguinal cutaneous region.

Figure 3. White and reddish black splenic surface with friable texture and ruptured margin.

Histopathological examination of the left prescapular lymph node showed that it was markedly expanded due to a neoplastic population of small lymphocytes. These cells exhibited oval nuclei with stippled chromatin, a single central nucleolus, and a scant eosinophilic cytoplasm. The mitotic figures were an average of 1-2 per high-power field. Immunohistochemistry confirmed that the proliferating neoplastic lymphocytes were positive for Pax 5 (a B-cell marker) and only a small population of residual CD3 (a T-cell marker) positive cells are present, which indicated that the neoplastic population and the lymph node were of B-cell origin.

Histopathologically, the spleen was predominantly composed of proliferating small lymphocytes, similar to those in the lymph nodes. Moreover, multiple necrotic and hemorrhagic lesions were observed within the spleen. A neoplastic population of round cells was found extending from the superficial to the mid-dermis, with only a few other inflammatory cells present in the background of the skin biopsies. The individual cells were mildly pleomorphic and exhibited characteristics similar to those seen in the lymph nodes and spleen. The small lymphocytes infiltrated within the dermis are strongly positive for PAX 5, similar to those in the lymph node, indicating a cutaneous manifestation of lymphoma (Figs. 4, 5). Based on the histopathological findings and immunohistochemical staining, the dog was diagnosed with diffuse small B-cell lymphoma, which subsequently led to a non-traumatic splenic rupture due to the extensive infiltration of neoplastic cells into the spleen.

Figure 4. Histopathology of inguinal region skin. Neoplastic round cells expand the superficial to mid-dermis. H&E stain, ×20.

Figure 5. Histopathology of inguinal region skin. Immunohistochemical staining of Pax5. The small lymphocytes are diffusely immunoreactive with this marker. ×20.

Our case involved a canine patient presenting with generalized lymphadenopathy, subsequently diagnosed with diffuse small B-cell lymphoma based on cytology, immunophenotyping, and histopathology. The disease showed aggressive progression due to splenic infiltration and rupture. Lymphomas are histologically classified according to the Revised European American Lymphoma/World Health Organization (REAL/WHO) system. This classification system includes anatomical, morphological, and immunophenotypic criteria, dividing lymphomas into various subtypes, such as DLBCL, peripheral T-cell lymphoma not otherwise specified, T-zone lymphoma, and MZL (18). Immunophenotypically, the B-cell phenotype occurs more frequently than the T-cell phenotype in canine lymphomas (14). Among these, DLBCL is the most common subtype of canine B cell lymphoma (14,19). Histologically, DLBCL is characterized by predominantly intermediate to large cells with round nuclei and one or more nucleoli. The median survival time of DLBCL varies from 322 days (7) to 252 days (6).

In contrast, there are non-DLBCL subtypes of canine B-cell lymphoma such as MZL, follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt-type lymphoma, and small lymphocytic lymphoma (12). These subtypes are characterized by a predominance of small to intermediate-sized lymphocytes (7). MZL, FL, and MCL are considered indolent lymphomas (18), and canine small B-cell lymphoma is thought to be an indolent disease. However, a previous study reported a shorter median survival time (MST) for dogs with non-DLBCL (114 days) compared to those with DLBCL (325 days) after receiving a 19-week CHOP protocol. This study also found a higher objective response rate (ORR) to the 19-week CHOP chemotherapy in dogs with DLBCL (96.3%) compared to dogs with non-DLBCL (70%) (22).

A study published in 2021 introduced DSBCL, a condition that does not fit within the existing WHO criteria (8). In this study, B-cell lymphomas exhibiting a diffuse pattern of small-to intermediate-sized B cells, measuring 1 to 1.5 times the diameter of a red blood cell, with indistinct nucleoli, were categorized as DSBCL. The cells showed immunoreactivity for PAX 5. According to the study, they exhibited low forward light scatter properties (medium flow cytometry cell size, 379) on flow cytometry. Cytologically, the lymphocytes were small to intermediate in size, smaller than neutrophils. Neoplastic lymphocytes had a mitotic count ranging from 0 to 3 mitotic figures per five 500x magnification fields. The median overall survival of dogs with DSBCL was 140 days, ranging from 2 to 464 days, and the treatment protocols varied (8).

It has been confirmed that the characteristics of DSBCL described in a previous study are consistent with our case. In our case, the multiple lymph nodes contained a homogeneous population of small-to intermediate-sized lymphocytes with stippled chromatin, a single central nucleolus, and indistinct nucleoli on cytology. Immunophenotyping of these lymph nodes revealed that the small lymphocytes expressed B cell and T cell markers, CD21 and CD3, respectively. However, additional histopathological examination and immunohistochemical staining revealed diffuse small B cell lymphoma. Furthermore, most of the lymphocytes exhibited a forward scatter value of 500 in flow cytometry, corresponding to the criteria for DSBCL in previous studies (8,22).

The dog showed systemic signs, including anorexia, lethargy, and fever. Histopathological examination confirmed the involvement of the spleen and cutaneous manifestation of neoplastic lymphocytes, which were identical to those found in the lymph nodes. The condition was classified as clinical stage Va, according to the WHO clinical staging system for lymphoma (17). Shock, which developed during abdominocentesis, was diagnosed as a hypovolemic shock due to the hemoabdomen observed in the necropsy results. Previous studies have shown that non-traumatic hemoabdomen is associated with intra-abdominal neoplasia, coagulopathies, gastric dilatation and volvulus, liver lobe torsion, and splenic torsion (5,11). Malignant neoplasia is the most common cause of non-traumatic hemoabdomen, with haemangiosarcoma being the most frequently diagnosed neoplasm (11). In addition, hemoabdomen is a common clinical sign in dogs with splenic malignant neoplasia (5). In our case, necropsy revealed a rupture of the splenic tail, with no evidence of gastric dilation, volvulus, or liver or splenic torsion. As we confirmed the infiltration of small neoplastic lymphocytes in the spleen, the hemoabdomen may have resulted from the involvement of DSBCL in the spleen. This is significant because lymphoma as a cause of non-traumatic splenic rupture in dogs is rare, and there are few reports of splenic rupture secondary to MZL and only one report of DLBCL in dogs (3,10).

In this patient, delayed aPTT, and a hypercoagulable state on TEG were observed. Delayed aPTT reflects reduced activity in the intrinsic or common pathways of coagulation, as it measures the function of coagulation factors involved in secondary hemostasis. Therefore, it is an insensitive marker for assessing hypercoagulability. TEG, on the other hand, reflects not only the activity of coagulation factors but also that of platelets and fibrinogen. The hypercoagulable state observed in this patient suggests the possibility of increased cellular expression of tissue factor or platelet hyperactivity, which can occur in patients with neoplasia (17). The exact cause of the two conflicting coagulopathy patterns observed in this dog remains unclear, but it is suspected to be a type of paraneoplastic syndrome associated with atypical splenic lymphoma.

Although coagulation abnormalities were identified in this patient, they were considered unlikely to be the primary cause of the hemoabdomen. Coagulopathy-related hemoabdomen is typically suspected in dogs with severe coagulation factor deficiencies or platelet deficiencies, which can result in hemorrhage in the absence of other identifiable abdominal abnormalities (5). Necropsy in this patient clearly confirmed splenic rupture. While moderate thrombocytopenia was observed, which could potentially cause petechiae or ecchymoses, it was not considered severe enough to be the primary cause of the hemoperitoneum.

This report has a limitation in that further treatment, such as chemotherapy, was not pursued due to the onset of sudden hemorrhagic shock and the owner’s decision to euthanize the animal. As a result, we could not estimate the patient’s overall survival time. However, the aggressive nature of this case aligns with a previous study that reported a median overall survival time of 140 days for 22 dogs with DSBCL (8). Furthermore, our case confirmed metastasis to other organs, including the spleen and skin, through histological examination. The histological findings revealed splenic infiltration by neoplastic lymphocytes, which caused hemoabdomen and ultimately led to an aggressive outcome.

In summary, this report presents a case of DSBCL associated with a non-traumatic hemoabdomen secondary to splenic rupture. Hemoabdomen resulting from splenic rupture secondary to lymphoma is rare in dogs. This case suggests that DSBCL should be considered in the differential diagnosis of splenic rupture. In addition, small B-cell lymphoma should not be regarded as an indolent type, as our findings revealed infiltration of neoplastic small lymphocytes in other organs, which resulted in aggressive outcomes similar to those described in previous reports.

This research was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Companion Animal Life Cycle Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (322092-04-1-HD030).

Hayoung Ryu, ARom Cho, DoHyeon Yu, and Hyeona Bae contributed to the conception and design of the study. ARom Cho, Seongwon Heo, and Seulgi Hwang contributed to the formal analysis of data. Hayoung Ryu and ARom Cho wrote the first draft of the manuscript. DoHyeon Yu and Hyeona Bae wrote sections of the manuscript.

No conflicts of interest have been declared.