Prognostic role of non-neoplastic lymphocytes in lymph node aspirates from dogs with diffuse large B-cell lymphoma treated with chemo-immunotherapy

Dogs with Diffuse Large B-Cell Lymphoma (DLBCL) benefit from the addition of active immunotherapy to traditional chemotherapy. We hypothesized that immune cells within neoplastic lymph nodes (LNs) may play a role in the tumor pathobiology and treatment response. The present study describes the composition and prognostic role of non-neoplastic lymphocytes in LNs of 59 dogs with treatment-naive DLBCL receiving chemo-immunotherapy. The percentage of small non-neoplastic cells and of CD5+, CD21+, CD4+ and CD8+ small cells was recorded via flow cytometry. CD4+/CD8+ and CD5+/large CD21+ cell ratios were calculated. The likelihood of progression significantly diminished with increasing percentage of small cells, CD5+ and CD8+ small cells, and CD5+/large CD21+ cell ratio, with decreasing CD4+/CD8+ ratio and in non-anemic dogs. Active immunotherapy is more effective in dogs with higher percentage of non-neoplastic lymphocytes at diagnosis. We lay the ground for future studies assessing the role of the immune system in the pathobiology of canine DLBCL

Prognostic significance of peripheral blood and bone marrow infiltration in newly-diagnosed canine nodal marginal zone lymphoma

Canine nodal marginal zone lymphoma (nMZL) is infrequent and is typically diagnosed at an advanced disease stage. However, it is currently unknown whether different levels of peripheral blood (PB) and bone marrow (BM) infiltration may provide prognostic stratification in dogs with nMZL. The aims of the present prospective study were to assess the influence of PB and BM infiltration detected by flow cytometry (FC) on time to progression (TTP) and lymphoma-specific survival (LSS) in dogs with newly-diagnosed multicentric nMZL, and to establish a cut-off value of prognostic significance. Forty-five completely staged and treatment-na\ueff dogs with histologically-confirmed nMZL were enrolled. After staging, dogs received chemo-immunotherapy or chemotherapy. PB infiltration was significantly associated with TTP (p = 0.001): dogs with PB infiltration <30% had a median TTP of 186 days, whereas dogs with PB infiltration 6530% had a median TTP of 43 days. Additionally, vaccinated dogs had a significantly (p = 0.012) longer TTP (399 days) compared with dogs receiving chemotherapy only (211 days). BM infiltration was significantly associated with LSS (p < 0.001): dogs with BM infiltration <1% had a median LSS of 1403 days, those with BM infiltration 1\u201320% of 337 days, and those with BM infiltration 6520% of 188 days. Normal LDH levels and the administration of chemo-immunotherapy also significantly improved LSS (560 vs 211 days, and 399 vs 211 days, respectively; p < 0.001). PB and BM flow cytometric evaluation is an integral part of staging work-up in dogs with nMZL and has prognostic relevance

A retrospective study of flow cytometric characterization of suspected extranodal lymphomas in dogs

Flow cytometry (FC) is widely applied to characterize and stage nodal lymphomas in dogs because it has a short turnaround time, requires minimally invasive sampling, and allows contemporary evaluation of neoplastic cells in the primary lesion and of blood and marrow involvement. We investigated advantages and limitations of FC in suspected extranodal lymphomas in dogs. The likelihood of obtaining a suitable FC sample was significantly lower for aspirates of extranodal lesions than for lymph node aspirates. However, we noted no differences among different extranodal lesion sites. We also describe FC results for 39 samples compatible with extranodal lymphoma. A dominant population of large cells was easily identified on morphologic FC scattergrams in many cases. Phenotypic aberrancies were frequently present, mainly in T-cell lymphomas. Lymphoma cells were distinguishable from normal residual lymphocytes in >85% of cases, facilitating the quantification of putative blood and marrow involvement by FC. Despite the high percentage of non-diagnostic samples (32 of 73, >40%), we support the inclusion of FC in the diagnostic workup of suspected extranodal lymphomas in dogs, in conjunction with histopathology. Histopathology is the gold standard for diagnosing lymphoma, provides relevant information, including tissue invasion and epitheliotropism, but has a longer turnaround time

The Italian-Canine Cancer (ICC) Biobank: our 10-year challenge

none10nononeAresu L.; Buracco P.; De Maria R.; Iussich S.; Martano M.; Morello E.; Bettini G.; Comazzi S.; Riondato F.; Marconato L.Aresu L.; Buracco P.; De Maria R.; Iussich S.; Martano M.; Morello E.; Bettini G.; Comazzi S.; Riondato F.; Marconato L

Epigenetic silencing of TFPI-2 in canine diffuse large B-cell lymphoma

Epigenetic modifications are important early events during carcinogenesis. In particular, hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a well-known mechanism of gene silencing that contributes to cancer development and progression. Tissue factor pathway inhibitor 2 (TFPI-2) is a tumor suppressor involved in invasiveness inhibition. Although TFPI-2 transcriptional silencing, through promoter hypermethylation, has been widely reported in several human malignancies, it has never been explored in lymphoma. In the present study TFPI-2 methylation and gene expression have been investigated in canine Diffuse Large B-cell lymphomas (cDLBCL). The methylation level of 23 CpGs located within the TFPI-2 promoter was investigated by bisulfite-specific PCR and next generation amplicon deep sequencing (GS Junior 454, Roche) in 22 cDLBCLs and 9 controls. For the same specimens, TFPI-2 gene expression was assessed by means of Real-time RT-PCR. Sequence analysis clearly demonstrated that TFPI2 is frequently hypermethylated in cDLBCL. Hypermethylation of the TFPI-2 promoter was found in 77% of DLBCLs ( 17 out of 22) and in one normal lymph node. Globally, dogs with DLBCL showed a mean methylation level significantly increased compared to controls (p&lt;0.01) and analysis of hypermethylation by site identified 19 loci out of 23 ( 82%) with mean methylation levels from 2- to 120-fold higher in cDLBCL. Gene expression analysis confirmed a significant down-regulation of TFPI-2 ( p&lt;0.05) in DLBCLs compared with normal lymph nodes, suggesting that TFPI-2 hypermethylation negatively regulates its transcription. In addition, a significant positive correlation ( p&lt;0.01) was found between TFPI-2 methylation levels and age providing the first indication of age-associated epigenetic modifications in canine DLBCL. To conclude, our findings demonstrated that epigenetic dysregulation of TFPI-2, leading to its reduced expression, is frequently detected in canine DLBCL. In the next future, the aberrant TFPI-2 promoter hypermethylation may be considered in association with prognosis and therapy