General and species-specific recommendations for minimal requirements for the use of cephalopods in scientific research.

Here we list species-specific recommendations for housing, care and management of cephalopod molluscs employed for research purposes with the aim of contributing to the standardization of minimum requirements for establishments, care and accommodation of these animals in compliance with the principles stated in Directive 2010/63/EU. Maximizing their psychophysical welfare was our priority. General recommendations on water surface area, water depth and tank shape here reported represent the outcome of the combined action of the analysis of the available literature and an expertise-based consensus reached - under the aegis of the COST Action FA1301 - among researchers working with the most commonly used cephalopod species in Europe. Information on water supply and quality, environmental conditions, stocking density, feeding and handling are also provided. Through this work we wish to set the stage for a more fertile ground of evidence-based approaches on cephalopod laboratory maintenance, thus facilitating standardization and replicability of research outcomes across laboratories, at the same time maximizing the welfare of these animals.

DNA methylation profiling reveals common signatures of tumorigenesis and defines epigenetic prognostic subtypes of canine Diffuse Large B-cell Lymphoma.

Epigenetic deregulation is a hallmark of cancer characterized by frequent acquisition of new DNA methylation in CpG islands. To gain insight into the methylation changes of canine DLBCL, we investigated the DNA methylome in primary DLBCLs in comparison with control lymph nodes by genome-wide CpG microarray. We identified 1,194 target loci showing different methylation levels in tumors compared with controls. The hypermethylated CpG loci included promoter, 5'-UTRs, upstream and exonic regions. Interestingly, targets of polycomb repressive complex in stem cells were mostly affected suggesting that DLBCL shares a stem cell-like epigenetic pattern. Functional analysis highlighted biological processes strongly related to embryonic development, tissue morphogenesis and cellular differentiation, including HOX, BMP and WNT. In addition, the analysis of epigenetic patterns and genome-wide methylation variability identified cDLBCL subgroups. Some of these epigenetic subtypes showed a concordance with the clinical outcome supporting the hypothesis that the accumulation of aberrant epigenetic changes results in a more aggressive behavior of the tumor. Collectively, our results suggest an important role of DNA methylation in DLBCL where aberrancies in transcription factors were frequently observed, suggesting an involvement during tumorigenesis. These findings warrant further investigation to improve cDLBCL prognostic classification and provide new insights on tumor aggressiveness.

Analytical and diagnostic validation of a flow cytometric strategy to quantify blood and marrow infiltration in dogs with large B-cell lymphoma.

BACKGROUND: Lymph node (LN), peripheral blood (PB), and bone marrow (BM) samples are commonly analyzed by flow cytometry (FC) for the immunophenotyping and staging of canine lymphomas. A prognostic value for FC BM infiltration in dogs with large B-cell lymphoma (LBCL) was demonstrated. Aim of this study was to define the analytical performances of this technique, and to establish a cutoff suitable to safely discriminate between infiltrated and noninfiltrated PB and BM samples. METHODS: Large B-cells were added to control PB and BM samples, to achieve twelve different large B-cells concentrations, ranging from 0 to 50%. The percentage of large B-cells was recorded for each dilution, using a BD Accuri C6 FC. Accuracy was evaluated by Passing-Bablok regression analysis. Intra-assay precision was assessed at 0%, 1, 3, and 10% dilutions evaluating the CVs of 10 repeated acquisitions. ROC curves were drawn to identify the cutoffs most suitable to discriminate between 25 infiltrated (PARR-positive) and 25 noninfiltrated (PARR-negative) PB and BM samples, respectively. RESULTS: Optimal analytical accuracy and precision were achieved. Almost all CVs were <10%. Negative controls had up to 0.5% large B-cells, with 50 and 22% CV in PB and BM samples, respectively, 0.56 and 2.45% cutoffs were selected based on the ROC curves for PB and BM samples, respectively. CONCLUSIONS: Quantification of large B-cells in PB and BM samples by FC is reliable and analytical performances met the acceptance criteria. Assessment of performances of different instruments and protocols is warranted. © 2016 International Clinical Cytometry Society.

Array-based comparative genomic hybridization analysis reveals chromosomal copy number aberrations associated with clinical outcome in canine diffuse large B-cell lymphoma.

Canine Diffuse Large B-cell Lymphoma (cDLBCL) is an aggressive cancer with variable clinical response. Despite recent attempts by gene expression profiling to identify the dog as a potential animal model for human DLBCL, this tumor remains biologically heterogeneous with no prognostic biomarkers to predict prognosis. The aim of this work was to identify copy number aberrations (CNAs) by high-resolution array comparative genomic hybridization (aCGH) in 12 dogs with newly diagnosed DLBCL. In a subset of these dogs, the genetic profiles at the end of therapy and at relapse were also assessed. In primary DLBCLs, 90 different genomic imbalances were counted, consisting of 46 gains and 44 losses. Two gains in chr13 were significantly correlated with clinical stage. In addition, specific regions of gains and losses were significantly associated to duration of remission. In primary DLBCLs, individual variability was found, however 14 recurrent CNAs (>30%) were identified. Losses involving IGK, IGL and IGH were always found, and gains along the length of chr13 and chr31 were often observed (>41%). In these segments, MYC, LDHB, HSF1, KIT and PDGFRα are annotated. At the end of therapy, dogs in remission showed four new CNAs, whereas three new CNAs were observed in dogs at relapse compared with the previous profiles. One ex novo CNA, involving TCR, was present in dogs in remission after therapy, possibly induced by the autologous vaccine. Overall, aCGH identified small CNAs associated with outcome, which, along with future expression studies, may reveal target genes relevant to cDLBCL.

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<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<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<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.

Minimal residual disease detection by flow cytometry and PARR in lymph node, peripheral blood and bone marrow, following treatment of dogs with diffuse large B-cell lymphoma.

The most promising techniques for detecting minimal residual disease (MRD) in canine lymphoma are flow cytometry (FC) and polymerase chain reaction amplification of antigen receptor genes (PARR). However, the agreement between these methods has not been established. MRD was monitored by FC and PARR following treatment of dogs affected with diffuse large B-cell lymphoma (DLBCL), comparing results in lymph node (LN), peripheral blood (PB) and bone marrow (BM) samples. The prognostic impact of MRD on time to relapse (TTR) and lymphoma-specific survival (LSS) was also assessed. Fourteen dogs with previously untreated DLBCL were enrolled into the study; 10 dogs eventually relapsed, while four dogs with undetectable MRD were still in remission at the end of the study. At diagnosis, the concordance rate between FC and PARR was 100%, 78.6%, and 64.3% for LN, PB and BM, respectively. At the end of treatment, the agreement rates were 35.7%, 50%, and 57.1% for LN, PB and BM, respectively. At least one of the follow-up samples from dogs experiencing relapse was PARR(+); conversely, FC was not able to detect MRD in seven of the dogs that relapsed. PARR was more sensitive than FC in predicting TTR, whereas the combination of PARR and FC was more sensitive than either technique alone in predicting LSS using PB samples. The results suggest that immunological and molecular techniques should be used in combination when monitoring for MRD in canine DLBCL.

The role of vascular endothelial growth factor and matrix metalloproteinases in canine lymphoma: in vivo and in vitro study.

BACKGROUND: Canine lymphoma represents the most frequent haematopoietic cancer and it shares some similarities with human non-Hodgkin lymphoma. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play a coordinated role during invasion and proliferation of malignant cells; however, little is known about their role in canine haematologic malignancies. The aim of this study was to investigate the mRNA and protein expression of VEGF and the most relevant MMPs in canine lymphoma. Lymph node aspirates from 26 B-cell and 21 T-cell lymphomas were collected. The protein expression levels of MMP-9, MMP-2 and VEGF-A were evaluated by immunocytochemistry, and the mRNA levels of MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2, RECK, VEGF-A and VEGF-164 were measured using quantitative RT-PCR. RESULTS: MT1-MMP, TIMP-1 and RECK mRNA levels were significantly higher in T-cell lymphomas than in B-cell lymphomas. Higher mRNA and protein levels of MMP-9 and VEGF-A were observed in T-cell lymphomas than in B-cell lymphomas and healthy control lymph nodes. A positive correlation was found between MMP-9 and VEGF-A in T-cell lymphomas. Moreover, MMP-9, MT1-MMP, TIMP-1 and VEGF-A were expressed at the highest levels in high-grade T-cell lymphomas. CONCLUSIONS: This study provides new information on the expression of different MMPs and VEGF in canine lymphoma, suggesting a possible correlation between different MMPs and VEGF, immunophenotype and prognosis.

Matrix metalloproteinases and vascular endothelial growth factor expression in canine leukaemias.

Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play a coordinated role during neoplastic invasion and proliferation. VEGF and MMPs expression was investigated in canine leukaemias by immunocytochemistry (MMP-9, MMP-2, VEGF-A) and quantitative RT-PCR (MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2, RECK, VEGF-A, VEGF-164). Blood samples were obtained from dogs with acute leukaemia (AL; n = 11) and chronic lymphocytic leukaemia (CLL; n = 12). Levels of MMP-9, TIMP-1 and VEGF-A were higher in CLL than AL and controls. Expression of TIMP-2 and MT1-MMP mRNA was significantly higher in AL than CLL. Significant positive correlations were found between MMP-9 and TIMP-1 and between MMP-9 and VEGF-A in CLL. These results suggest a potential role of MMP-9, MT1-MMP, TIMP-1, TIMP-2 and VEGF in tissue migration and angiogenesis in canine leukaemia.

Matrix metalloproteinases and their inhibitors in canine mammary tumors.

BACKGROUND: Malignant canine mammary tumors represent 50% of all neoplasms in female dogs. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in tumor progression, and they are also associated with the reactive stroma, which provides structural and vascular support for tumor growth. RESULTS: MMP-2, MMP-9 and MT1-MMP were expressed at both the mRNA and protein levels in tumor samples. MMP-2 and MMP-9 immunohistochemical reactions were evident both in the epithelial tumor cells and in the stromal compartment to varying degrees; in particular, the intensity of the MMP-2 staining was stronger in the stromal fibroblasts close to epithelial tumor cells in simple carcinomas than in adenomas. These data were supported by gelatin-zymography; bands for the active form of MMP-2 were found in 94% of carcinoma samples, compared with 17% of benign tumor samples. The gene expression and immunohistochemical results for MT1-MMP were comparable to those for MMP-2. The immunoreactivity for MMP-13 and TIMP-2 was lower in carcinomas than in adenomas, confirming the mRNA data for MMP-13 and the other MMP inhibitors that were evaluated. The active form of MMP-9, but not the active form of MMP-2, was identified in the plasma of all of the tested dogs. CONCLUSIONS: Our findings suggest that MMP-9, MMP-2 and MT1-MMP, which are synthesized by epithelial cancer cells and cancer-associated fibroblasts, play an important role in malignant canine mammary tumors. The reduction of MMP-13 and TIMP-2 could also be a significant step in malignant transformation. MMP-2 and MT1-MMP could be further evaluated as future biomarkers for predicting the progression and prognosis of canine mammary tumors.

Chondroitin sulfate proteoglycan-4: a biomarker and a potential immunotherapeutic target for canine malignant melanoma.

Chondroitin sulfate proteoglycan-4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA), is a membrane-bound chondroitin sulfate proteoglycan highly expressed by human melanoma cells. This phylogenetically conserved tumour antigen plays an important biological role in human melanoma, where it is used as a marker to diagnose forms with unusual characteristics, such as desmoplastic melanoma, and to detect melanoma cells in lymph nodes and peripheral blood, and as a target for immunotherapy because of its restricted distribution in normal tissues. To identify suitable targets to develop novel approaches of treating canine melanoma, CSPG4 was studies to see whether it is expressed in canine malignant melanomas. Immunohistochemical staining of 65 canine malignant melanomas with an anti-human CSPG4-specific antibody detected CSPG4 in 37 cases (56.9%). Positive staining was more frequent, albeit not significantly, in amelanotic compared to melanotic tumours and was statistically associated with tumours having both melanin and the epithelioid histotype. The frequency of CSPG4 expression was similar to that of other melanoma antigens used as diagnostic markers for canine malignant melanoma, such as Melan A and the protein recognized by the PNL2 monoclonal antibody. The results suggest that CSPG4 constitutes a new potential immunohistochemical marker of canine malignant melanoma and may represent an immunotherapeutic target as in humans.