[Retropharyngeal manifestation of T-cell-rich B-cell lymphoma in a 7-year-old Icelandic gelding]. / Retropharyngeale Manifestation eines T-Zell-reichen B-Zell-Lymphoms bei einem 7-jährigen Isländerwallach.

A 7-year-old Icelandic gelding was presented with acute severe dyspnea of one day duration and purulent nasal discharge that had been present for 6 weeks. Clinically, the initial examination focused on severe enlargement of the mandibular and retropharyngeal lymph nodes as well as a mixed dyspnea.The diagnosis of a malignant lymphoma was evident following laboratory diagnostics, endoscopy, and cytological examination of a fine needle aspiration of a mandibular lymph node. The gelding was euthanized due to the poor prognosis and a significantly disturbed general condition. Pathohistological examination revealed a multicentric T-cell-rich B-cell lymphoma.

Gene expression profiles associated with early relapse during first remission induction in canine multicentric high-grade B-cell lymphoma.

Although chemotherapy using CHOP-based protocol induces remission in most cases of canine multicentric high-grade B-cell lymphoma (mhBCL), some cases develop early relapse during the first induction protocol. In this study, we examined the gene expression profiles of canine mhBCL before chemotherapy and investigated their associations with early relapse during the first whole CHOP-based protocol. Twenty-five cases of mhBCL treated with CHOP-based protocol as first induction chemotherapy were included in this study. Sixteen cases completed the first whole CHOP-based protocol without relapse (S-group), and nine developed relapse during the chemotherapy (R-group). RNA-seq was performed on samples from neoplastic lymph nodes. Differentially expressed genes (DEGs) were extracted by the comparison of gene expression profiles between S- and R-groups, and the differences in the expression levels of these genes were validated by RT-qPCR. Extracted 179 DEGs included the genes related to chemokine CC motif ligand, T-cell receptor signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway. We focused on chemokine CC motif ligand, and CCL4 was confirmed to be significantly downregulated in the R-group (P=0.039). We also focused on the genes related to T-cell signaling pathway, and CD3E (P=0.039), ITK (P=0.023), and LAT (P=0.023) genes were confirmed to be significantly upregulated in the R-group. The current results suggest that both changes in tumor cells and the interactions between tumor cells and immune cells are associated with the efficacy of the chemotherapy for first remission induction.

Use of genome-wide DNA methylation analysis to identify prognostic CpG site markers associated with longer survival time in dogs with multicentric high-grade B-cell lymphoma.

BACKGROUND: DNA methylation analysis might identify prognostic CpG sites in CHOP-treated dogs with multicentric high-grade B-cell lymphoma (MHGL) with heterogenous prognosis. OBJECTIVE: To identify prognostic CpG sites of MHGL through genome-wide DNA methylation analysis with pyrosequencing validation. ANIMALS: Test group: 24 dogs. Validation group: 100 dogs. All client-owned dogs were diagnosed with MHGL and treated with CHOP chemotherapy. METHODS: Cohort study. DNA was extracted from lymph node samples obtained via FNA. Genome-wide DNA methylation analysis using Digital Restriction Enzyme Analysis of Methylation (DREAM) was performed on the test group to identify differentially methylated CpG sites (DMCs). Bisulfite pyrosequencing was used to measure methylation status of candidate DMCs in the validation group. Median survival times (MST) were analyzed using Kaplan-Meier (log-rank) product limit method. RESULTS: DREAM analyzed 101 576 CpG sites. Hierarchical clustering of 16 262 CpG sites in test group identified group with better prognosis (MST = 55-477 days vs 10-301 days, P = .007). Volcano plot identified 1371 differentially methylated CpG sites (DMCs). DMC near the genes of FAM213A (DMC-F) and PHLPP1 (DMC-P) were selected as candidates. Bisulfite-pyrosequencing performed on validation group showed group with methylation level of DMC-F < 40% had favorable prognosis (MST = 11-1072 days vs 8-1792 days, P = .01), whereas group with the methylation level combination of DMC-F < 40% plus DMC-P < 10% had excellent prognosis (MST = 18-1072 days vs 8-1792 days, P = .009). CONCLUSION AND CLINICAL IMPORTANCE: Methylation status of prognostic CpG sites delineate canine MGHL cases with longer MST, providing owners with information on expectations of potential improved treatment outcomes.

Long-term remission and survival in dogs with high-grade, B cell lymphoma treated with chemotherapy with or without sequential low-dose rate half-body irradiation.

BACKGROUND: Standard of care for dogs with high-grade lymphoma, multiagent chemotherapy, achieves good initial responses but long-term remissions are infrequent; previous studies using half-body irradiation suggest improved long-term outcomes. HYPOTHESIS: The addition of low-dose rate half-body irradiation would improve outcomes in dogs with B-cell lymphoma. ANIMALS: Client-owned dogs with stage III or higher, substage a, B-cell lymphoma that achieved complete remission after 4 doses of multiagent chemotherapy. METHODS: A case-controlled design comparing 2-year remission and survival rates between dogs treated with CHOP-based chemotherapy and those treated with chemotherapy and sequential low-dose rate half-body irradiation. RESULTS: Thirty-eight dogs were enrolled with 18 included in final analysis, 9 prospectively-enrolled dogs and 9 case-matched historical controls. The irradiation cohort's 2-year disease-free rate was 56% whereas median duration exceeded the 730-day study period compared with 0% and 261 days in the chemotherapy only group. Remission duration significantly differed between cohorts (P < .01), hazard ratio 0.218 (95% CI: 0.06-0.77). The irradiation cohort's 2-year survival rate was 78% with median overall survival duration exceeding the 730 day study period compared with 11% and 286 days in the chemotherapy only group. Overall survival time significantly differed between cohorts (P < .02), hazard ratio 0.173 (95% CI: 0.03-0.839). CONCLUSIONS AND CLINICAL IMPORTANCE: The improved long-term outcome achieved by dogs administered sequential low-dose rate half-body irradiation in this study is similar to previous observational studies. Where long-term remission is sought in dogs with B-cell lymphoma low-dose rate half-body irradiation could be considered in addition to standard chemotherapy.

A case of spermatic cord B-cell lymphoma relapsing to the brain in a dog.

A 13-year-old, intact male mixed-breed dog was referred to our clinic for lethargy and asthenia following an episode of gastroenteritis. As an incidental finding during abdominal ultrasound, a mass on the right spermatic cord was seen. Cytology of the mass revealed a monomorphic population of large, round cells with a lymphoid appearance. A bilateral orchiectomy was conducted, and histopathology revealed the presence of a B-cell lymphoma in the right spermatic cord. Based on clinical staging, which showed no involvement of other sites, no additional treatment was administered. Recheck evaluations were scheduled for every 3 mo thereafter. Five months after surgery, the dog developed left central vestibular syndrome with a paradoxical right-sided head tilt. An MRI of the brain showed multifocal lesions and, due to a rapidly worsening clinical condition, the dog was humanely euthanized. The histopathology of the brain lesions was consistent with B-cell lymphoma. Key clinical message: This is the first report of a primary spermatic cord lymphoma relapsing to the brain in a dog. Although rare, spermatic cord tumors should be included among the differential diagnoses for masses arising from the spermatic cord. If lymphoma is diagnosed, location to other sites, especially to the central nervous system, should be considered.

Un cas de lymphome à cellules B du cordon spermatique récidivant au cerveau chez un chien. Un chien de race mixte mâle intact de 13 ans a été référé à notre clinique pour léthargie et asthénie à la suite d'un épisode de gastro-entérite. Comme découverte fortuite lors d'une échographie abdominale, une masse sur le cordon spermatique droit a été observée. La cytologie de la masse a révélé une population monomorphe de grosses cellules rondes d'aspect lymphoïde. Une orchidectomie bilatérale a été réalisée et l'histopathologie a révélé la présence d'un lymphome à cellules B dans le cordon spermatique droit. Sur la base du stade clinique, qui n'a montré aucune implication d'autres sites, aucun traitement supplémentaire n'a été administré. Des évaluations de contrôle étaient programmées tous les 3 mois par la suite. Cinq mois après la chirurgie, le chien a développé un syndrome vestibulaire central gauche avec une inclinaison paradoxale de la tête du côté droit. Une IRM du cerveau a montré des lésions multifocales et, en raison d'une détérioration rapide de l'état clinique, le chien a été euthanasié sans cruauté. L'histopathologie des lésions cérébrales correspondait à un lymphome à cellules B.Message clinique clé :Il s'agit du premier rapport d'un lymphome primaire du cordon spermatique récidivant au cerveau chez un chien. Bien que rares, les tumeurs du cordon spermatique doivent être incluses dans les diagnostics différentiels des masses provenant du cordon spermatique. Si un lymphome est diagnostiqué, la localisation vers d'autres sites, en particulier vers le système nerveux central, doit être envisagée.(Traduit par Dr Serge Messier).

Concurrent Mycobacterium genavense infection and intestinal B-cell lymphoma in a pet rabbit (Oryctolaguscuniculus).

A 6-year-old male intact pet rabbit was evaluated for chronic weight loss. A large mass was detected by palpation in the mid-abdomen and ultrasound examination suggested a jejunal location. Explorative laparotomy revealed a nodular mass within the jejunal wall. Histological examination of a biopsy revealed mycobacterial granulomatous enteritis with an atypical lymphoblastic proliferation suggestive of lymphoma. Neoplastic lymphocytes were immunopositive for Pax-5 but negative for CD3, which is diagnostic of a B-cell neoplasm. Numerous acid-fast bacteria were seen within histiocytes and identified by polymerase chain reaction as Mycobacterium genavense, which is a non-tuberculous and opportunistic mycobacterium with zoonotic potential. To the best of our knowledge, this is the first documented case of a concurrent B-cell lymphoma and M. genavense infection in a rabbit. Concomitant mycobacteriosis and lymphoma have been rarely described in animals and the coexistence of neoplasia and mycobacterial infection within the jejunum suggests a potential pathogenetic association. Interestingly, the rabbit owner worked in an anti-tuberculosis clinic, and an anthropic origin of the mycobacterial infection could not be excluded.

[Multicentric B cell lymphoma in an 8-week-old calf]. / Multizentrisches B-Zell-Lymphom bei einem 8 Wochen alten Kalb.

The presented report describes a case of sporadic bovine leukosis and its disease progression in an 8-week old, male cross-breed calf (Red Holstein Fleckvieh). The calf was initially presented due to suspect pulmonary infection. However, generalized enlargement of the subcutaneous lymph nodes was noticed, which is untypical for this disease. Based on the hematologic findings of highly increased numbers of lymphoblasts in peripheral blood as well as the sonographic examination of the lymph nodes, sporadic bovine leukosis was suspected. The calf died suddenly, three weeks after initial presentation. Pathohistological examination revealed a high-degree enlargement of all lymph nodes as well as an infiltration of nearly all organs and tissues with a monomorphic round cell population. These cells were also detected in bone marrow cytology. Immunhistochemical examination was performed and the cells reacted positive for the B-cell markers Pax 5 and CD20. Virologic examination for enzootic bovine leukosis was negative. In conjunction with the diagnosis of multicentric B-cell lymphoma, the test results indicated a juvenile form of sporadic bovine lymphoma.

A retrospective histopathological survey on canine lymphomas subtypes of Porto District.

Background: Lymphomas are dogs' most common hematopoietic neoplasms and represent a heterogeneous group, as occurs in humans. Considering the role of dogs as models of human lymphomas and the geographical correlation of the cases of canine and human lymphoma, it is important to continuously assess the epidemiological distribution of lymphoma subtypes in dogs. Aim: This study aimed to provide a survey of canine lymphoma subtypes diagnosed from 2005 to 2016 in the academic veterinary pathology laboratory of the University of Porto. Methods: A total of 75 canine lymphomas diagnosed by histopathology in the Porto district were included. All cases were immunophenotyped by CD3 and PAX5, classified according to the current classification WHO and coded with Vet-ICD-O-canine-1. Results: Mixed breed dogs were most common (28%), followed by Cocker Spaniels (12%), Boxers (9%), and Labrador Retrievers (6%). The mean age was 9.2 years (SD = 3.3) (10.7 years for small, 8.9 years for medium and large, and 5.7 years for giant breed dogs, p < 0.05). Regarding sex, there was no difference in frequencies or mean age. B-cell lymphomas were more common (57.4%) than T-cell lymphomas (37.3%), and 5.3% were classified as non-B/non-T-cell lymphomas. Of the cases, 49% had a multicentric distribution, followed by splenic (22%), cutaneous (12%), alimentary (12%), and extranodal (3%) forms. The most common B-cell subtypes were diffuse large B-cell lymphoma (DLBCL) (16.3%) and large immunoblastic lymphoma (14%), while T-zone lymphoma (21.4%) and intestinal lymphoma (18%) were the most common T-cell lymphoma subtypes. Conclusion: Our study shows that the Porto district follows the international trend of higher prevalence of B-cell lymphomas in dogs, especially of the DLBCL subtype.

An Unusual Cause of Femoral Nerve Paresis in a Horse: Disseminated B Cell Lymphoma With Plasmacytoid Differentiation and Direct Neuronal Invasion.

A 21-year-old Quarter Horse mare presented with a chronic, progressively worsening left pelvic limb lameness of 3 weeks duration. The initial examination identified a consistent lameness at a walk. Neurological examination showed sensory and gait abnormalities consistent with left femoral nerve dysfunction. The horse minimally advanced the leg cranially and had a shortened stride length at the walk. During the stance phase, the heels of the left hind foot did not contact the ground and the horse quickly took weight off of the limb. Diagnostic imaging (ultrasound and nuclear scintigraphy) examinations did not reveal a cause. Severe lymphocytosis was identified on complete blood cell count (69,600 cells /uL; reference range: 1,500-4,000 cells/uL), suggestive of lymphoma. Postmortem examination revealed focal swelling of the left femoral nerve. Multiple masses were found in the stomach, large colon, adrenal gland, mesentery, heart, and meninges. The entire left pelvic limb was dissected and did not reveal other causes of the gait deficit. Histologic evaluation of the left femoral nerve revealed disseminated intermediate cell size B cell lymphoma, with an immunophenotype suggestive of plasmacytoid differentiation. These lymphocytes infiltrated the femoral nerve at the location of the focal nerve swelling, in addition to other peripheral nerves. This case highlights a horse with an atypical diagnosis of femoral nerve paresis caused by direct neoplastic lymphocyte infiltration, deriving from disseminated B cell lymphoma with plasmacytoid differentiation (neurolymphomatosis). Though rare, disseminated lymphoma with direct nerve infiltration should be considered in horses with peripheral neuropathies.

A series of heterogeneous lymphoproliferative diseases with CD3 and MUM1 co-expressed in cats and dogs.

Lymphoma diagnosis in dogs and cats is continually evolving as new subtypes and human correlates are being recognized. In humans, T-cell lymphomas with MUM1 expressed and plasma cell neoplasia or B-cell lymphomas with CD3 expressed aberrantly are reported only rarely. We report here a case series of tumors in dogs and cats with CD3 and MUM1 co-expressed as determined by immunocytochemistry or immunohistochemistry. Lineage was assigned for these tumors by 3 board-certified pathologists and a veterinary immunologist based on review of clinical and cellular features and the results of ancillary testing including PCR for antigen receptor rearrangements, flow cytometry, and serum protein electrophoresis with immunofixation. In cats, 7 of 7 tumors, and in dogs, 3 of 6 tumors with CD3 and MUM1 co-expressed had clonal rearrangement of the immunoglobulin gene or serum monoclonal immunoglobulin, consistent with a diagnosis of a plasma cell neoplasia or myeloma-related disorder with CD3 expressed aberrantly. Disease was often disseminated; notably, 3 of 7 feline cases had cutaneous and/or subcutaneous involvement in the tarsal area. In dogs, 3 of 6 cases had a clonal T-cell receptor gamma result and no clonal immunoglobulin gene rearrangement and were diagnosed as a T-cell tumor with MUM1 expressed. The use of multiple testing modalities in our series of tumors with plasma-cell and T-cell antigens in dogs and cats aided in the comprehensive identification of the lymphoproliferative disease subtype.

Clinical outcome and Ki67 evaluation in dogs with nodal small cell B-cell lymphoma diagnosed by flow cytometry.

BACKGROUND: Nodal small cell B-cell lymphoma subtypes in dogs cannot be distinguished by flow cytometry and information regarding treatment, prognosis, and outcome are limited. HYPOTHESIS/OBJECTIVES: Objectives were to describe outcome in dogs with nodal small cell B-cell lymphoma diagnosed by flow cytometry and correlate clinical and laboratory data with survival. We hypothesized that B-cell Ki67 expression measured by flow cytometry is associated with shorter progression free survival (PFS) and overall survival (OS). ANIMALS: Forty-nine dogs with nodal small cell B-cell lymphoma, defined by >80% CD21+ B-cells by flow cytometry and small-sized B-cells by forward scatter. METHODS: Retrospective study reviewing treatment and outcome data extracted from medical records. Percentage of Ki67-expressing B-cells was measured by flow cytometry. Clinical, laboratory, and flow cytometry data were assessed for association with outcome. RESULTS: Median percentage of B-cell Ki67 was 41% (range, 3%-97%). Median PFS was 119 days and median OS was 222 days (n = 49). Among cases treated with CHOP-based chemotherapy (n = 32), median PFS was 70 days, median OS was 267 days, and 50% of cases achieved complete response. Low percentage of B-cell Ki67 (≤11%) was associated with prolonged OS by univariable analysis. Greater age, substage B, high B-cell CD25 expression and low B-cell CD21 and class II major histocompatibility complex expression by flow cytometry were independently associated with shorter OS. CONCLUSIONS AND CLINICAL IMPORTANCE: Most nodal small cell B-cell lymphoma cases had aggressive disease. Low Ki67 expression can help identify cases with better prognosis. Age, substage, and flow cytometry variables are useful prognostic factors.

Genome wide exploration of the methylome in aggressive B-cell lymphoma in Golden Retrievers reveals a conserved hypermethylome.

Few recurrent DNA mutations are seen in aggressive canine B cell lymphomas (cBCL), suggesting other frequent drivers. The methylated island recovery assay (MIRA-seq) or methylated CpG-binding domain sequencing (MBD-seq) was used to define the genome-wide methylation profiles in aggressive cBCL in Golden Retrievers to determine if cBCL can be better defined by epigenetic changes than by DNA mutations. DNA hypermethylation patterns were relatively homogenous within cBCL samples in Golden Retrievers, in different breeds and in geographical regions. Aberrant hypermethylation is thus suspected to be a central and early event in cBCL lymphomagenesis. Distinct subgroups within cBCL in Golden Retrievers were not identified with DNA methylation profiles. In comparison, the methylome profile of human DLBCL (hDLBCL) is relatively heterogeneous. Only moderate similarity between hDLBCL and cBCL was seen and cBCL likely cannot be accurately classified into the subtypes seen in hDLBCL. Genes with hypermethylated regions in the promoter-TSS-first exon of cBCL compared to normal B cells often also had additional hyper- and hypomethylated regions distributed throughout the gene suggesting non-randomized repeat targeting of key genes by epigenetic mechanisms. The prevalence of hypermethylation in transcription factor families in aggressive cBCL may represent a fundamental step in lymphomagenesis.

Bovine leukemia virus-associated B cell lymphoma with severe pleomorphism in a steer.

We examined a 26-month-old steer with neoplastic lesions in the spleen, lymph nodes, heart and kidneys, characterized by pleomorphic lymphoid cells that were immunohistochemically positive for CD20. The presence of bovine leukemia virus (BLV) at >200,000 copies per 100,000 cells by quantitative RT-PCR was considered to be due to random integration of the provirus into the neoplastic cells´ genomes. Inverse PCR identified the presence of one, two, two and three different malignant clones in the heart, spleen, mesenteric node and blood, respectively. Because BLV can rapidly induce lymphoma and a high proviral load facilitates B-cell carcinogenesis, multiclonal tumor development was suspected in the present case.

Allogeneic peripheral blood haematopoietic stem cell transplantation for the treatment of dogs with high-grade B-cell lymphoma.

Autologous peripheral blood haematopoietic stem cell transplantation (HCT) cures 33%-40% of dogs with high-grade B-cell lymphoma. We hypothesized, based on human allogeneic bone marrow transplantation literature, that transplanting dogs using canine donor leukocyte-matched CD34+ cells would lead to fewer relapses and increased cure rates. We retrospectively reviewed medical records of dogs diagnosed with high-grade B-cell lymphoma who received an identical allogeneic HCT. A total of 15 dogs transplanted at four facilities were identified. Five of fifteen dogs relapsed before transplant. The mean number of donor CD34+ cells/kg harvested and infused into recipient dogs was 8.0 × 106 /kg (range: 2.08 × 106 /kg-2.9 × 107 /kg). The median disease-free interval and overall survival of all dogs was 1095 days (range: 9-2920 days) and 1115 days (range: 9-2920 days), respectively. Two of five dogs, not in remission at transplant, died in the hospital. The median disease-free interval and overall survival of the remaining three dogs was 25 days (range: 15-250 days) and 1100 days (range: 66-1902 days), respectively. The median disease-free interval and overall survival of the 10 dogs who had not relapsed was 1235 days (range: 19-2920 days) and 1235 days (range: 19-2920 days), respectively. One dog died soon after discharge of presumed gastric-dilatation-volvulus. Eight of nine remaining dogs lived >4 yrs post-alloHCT, leading to a cure rate of 89%. Acute graft versus host disease was seen in three dogs. These results suggest that allogeneic HCT can cure ~50% more dogs than those treated with autologous HCT.

Comprehensive Comparison of Novel Bovine Leukemia Virus (BLV) Integration Sites between B-Cell Lymphoma Lines BLSC-KU1 and BLSC-KU17 Using the Viral DNA Capture High-Throughput Sequencing Method.

Bovine leukemia virus (BLV) infects cattle and integrates into host DNA, causing enzootic bovine leukosis (EBL), an aggressive B-cell lymphoma. Here, we developed a novel proviral DNA-capture sequencing (proviral DNA-capture-seq) method investigating BLV proviral integration in two B-cell lymphoma lines, BLSC-KU1 and BLSC-KU17, derived from BLV-infected cattle with EBL. We designed BLV-specific biotinylated probes to capture the provirus genome and enrich libraries for next-generation sequencing. Validation showed high specificity and efficient enrichment of target sequence reads as well as identification of three BLV proviral integration sites on BLV persistently infected FLK-BLV cells as a positive control. We successfully detected a single BLV proviral integration site on chromosome 19 of BLSC-KU1 and chromosome 9 of BLSC-KU17, which were confirmed by standard PCR and Sanger sequencing. Further, a defective provirus in BLSC-KU1 and complete BLV proviral sequence in BLSC-KU17 were confirmed using long PCR and sequencing. This is the first study to provide comprehensive information on BLV proviral structure and viral integration in BLSC-KU1 and BLSC-KU17. Moreover, the proposed method can facilitate understanding of the detailed mechanisms underlying BLV-induced leukemogenesis and may be used as an innovative tool to screen BLV-infected cattle at risk at an earlier stage than those that have already developed lymphoma.

Lymphoma in Miniature Dachshunds: A retrospective multicenter study of 108 cases (2006-2018) in Japan.

BACKGROUND: Miniature Dachshunds (MD) are predisposed to lymphoma with disease onset of young age and long-term survival. OBJECTIVES: To compare clinical features and survival time of lymphoma in MD and non-MD. ANIMALS: One hundred and eight MDs with lymphoma and 149 non-MD breed dogs with lymphoma were included in the study. METHODS: This was a retrospective multicenter observational study. Lymphoma was classified based on signalment, histopathology/cytology, and anatomical site of the disease. For each type of lymphoma, median survival time was analyzed by Kaplan-Meier estimates and life table analysis. Prognostic factors for large-cell gastrointestinal lymphoma (LGIL) were analyzed using Cox regression. RESULTS: Gastrointestinal lymphomas were more common in MDs (53/108) compared to non-MDs (41/149). The multicentric lymphoma was most common in non-MD breed dogs (74/149) compared to MDs (33/108). The median age that dog developed lymphoma in MD and non-MD were both 10 years old; however, lymphomas were more frequently observed in younger dogs (<4 years) in MDs (20/108) compared to non-MDs (9/149; P = .002). Seventy percent were diagnosed with B-cell with median age of diagnosis was 3 (1-14) years. Mott cell differentiation was observed in 6 dogs. Age <4 years and B-cell phenotype were significant factors for longer survival time in MD with LGIL. CONCLUSIONS AND CLINICAL IMPORTANCE: Lymphomas in MDs involved gastrointestinal lesions at higher frequency compared to other dog breeds examined. B-cell lymphoma was more common in early-onset LGIL in MD and cases that involved Mott cell differentiation were observed. Awareness of this specific presentation of lymphoma in dogs will possibly affect the treatment decision process for the owners of MD with LGIL.

Urinary bladder wall mass with neoplastic lymphoid cells in the urine: Diagnosis of an IgG secretory B-cell lymphoma with Bence-Jones proteinuria in a dog.

In this study, we describe a multimodal approach to diagnose a unique case of myeloma-related disease, extranodal secretory B-cell lymphoma with urinary bladder involvement, an IgG4 monoclonal gammopathy, and Bence-Jones proteinuria in a dog with a 6-year history of hyperglobulinemia that had not been further evaluated. A 12-year-old dog was presented for evaluation of a 1-week history of tenesmus. Urine sediment cytologic evaluation revealed low to moderate numbers of intermediate to large-sized lymphocytes. We describe a technique that yielded adequate numbers of viable neoplastic cells in shipped urine sediment for PARR and flow cytometry. Those studies demonstrated a clonal immunoglobulin gene rearrangement and an expansion of CD21-positive and MHC Class II-negative B cells, respectively. Protein electrophoresis with immunofixation and proteomic evaluation revealed a serum and urine IgG4 monoclonal gammopathy with Bence-Jones proteinuria. MUM1 immunocytochemistry performed on the urine sediment slides failed to label the neoplastic cells; thus, a plasma cell tumor was considered unlikely. Lack of response to a cyclophosphamide, vincristine, and prednisone chemotherapy regimen led to euthanasia without necropsy 21 days after diagnosis. Lymphoma is the most common hematopoietic malignancy and accounts for up to a quarter of all neoplasms in dogs, but lymphoid neoplasms arising primarily from extranodal sites are infrequently reported. Urinary tract neoplasia can be diagnosed by urine evaluation in about one-third of canine cases, but the diagnosis of lymphoid neoplasia via urine evaluation is rarely reported. This case highlights the utility of ancillary diagnostics on urine for detection of lymphoid malignancies.

Primary nervous system lymphoma in cats.

Here we characterize the neuroanatomic distribution, neuropathology, and immunophenotype of 10 cases of primary nervous system lymphoma in cats. Cases were retrospectively searched from 2 academic institutions. Selected cases were reviewed and subjected to immunohistochemistry (IHC) for CD3, CD20, and Pax5. The mean age of affected cats was 9.1 y, and no sex or breed predilection was observed. The most common clinical sign was ataxia (8 cases). Gross changes reported in 8 cases consisted of white-to-tan masses (7 cases) or swelling (1 case) within the neuroparenchyma (5 cases) or epidural spaces (3 cases). Histologically, intraparenchymal lymphomas occurred in the gray and white matter or perivascular spaces (7 cases); extraparenchymal lymphomas (6 cases) consisted of neoplastic cell infiltration of the perivascular spaces in the leptomeninges, choroid plexus, or epidural spaces. Nerve lymphomas were diffusely infiltrative. Tumors occurred in the brain (4 cases), spinal cord and nerves (3 cases), spinal cord (2 cases), and brain, spinal cord, and nerves (1 case). IHC was consistent with a B-cell lymphoma in 5 cases and with a T-cell lymphoma in 5 cases.

Optimization of Culture Conditions for the Generation of Canine CD20-CAR-T Cells for Adoptive Immunotherapy.

BACKGROUND/AIM: Chimeric antigen receptor (CAR) T cell therapy targeting CD20 has the potential to become a promising novel treatment for canine B cell lymphoid malignancy. However, the optimal approach for producing potent CAR-T cells with favorable phenotype for dogs remains unknown. In this study, we assessed several culture conditions and their effects on the phenotypic characteristics of CD20-CAR-T cells. MATERIALS AND METHODS: Canine CAR-T cells were generated by incubating with several mitogens in the presence or absence of Akt inhibitor. Gene transduction efficiency and phenotypic characteristics were determined by flow cytometry. RESULTS: Comparison of several kinds of mitogens revealed that stimulation with phytohemagglutinin has high transduction efficacy, whereas stimulation with concanavalin A was superior in memory T cell formation. Akt inhibition at the initial stage of CAR-T production tended to enhance transduction efficiency and memory T cell formation. CONCLUSION: This study provides a significant insight into the understanding of the ex vivo expansion of canine T cells in adoptive immunotherapy.

Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy.

Anti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identified six amino acid differences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived single-domain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.