Publications by authors named "Valeria Martini"

28 Publications

  • Page 1 of 1

Cytology of Feline Nodal Lymphoma: Low Interobserver Agreement and Variable Accuracy in Immunophenotype Prediction.

J Comp Pathol 2021 Apr 22;184:1-6. Epub 2021 Feb 22.

Department of Veterinary Medicine, Veterinary Teaching Hospital, University of Milan, Milan, Italy; Veterinary Teaching Hospital, University of Milan, Milan, Italy.

Nodal lymphomas are less common in cats than in dogs and, consequently, no specific studies have been published. Cytology is the first step in the diagnosis of nodal lymphoma but is highly subjective. Morphological features have been introduced for the cytological classification of canine lymphomas but not for cats. Therefore, the aim of this study was to evaluate interobserver agreement on various cytological features of feline nodal lymphomas and to investigate the accuracy in predicting B or T immunophenotypes. Four veterinary cytologists examined 25 feline nodal and mediastinal lymphoma cytological samples by adapting the criteria used for the evaluation of canine lymphomas and setting histopathology and immunohistochemistry as the gold standard. High interobserver variability was found in the evaluation of most features except for the presence or absence of cytoplasmic vacuoles, which were more common in B-cell lymphomas. Cytology training centre was the major factor influencing the extent of agreement among evaluators. Diagnostic accuracy in predicting lymphoma immunophenotype varied from 35% to 75% and did not appear to be correlated with the experience of the evaluators. We conclude that cytological criteria, commonly used to describe canine lymphomas, are not adaptable to the counterpart feline neoplasms. Cytology-based immunophenotyping of feline lymphomas from different laboratories, and different cytologists within the same laboratory, differ substantially and should not be considered reliable. Specific cytological criteria are needed to describe feline lymphoma.
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http://dx.doi.org/10.1016/j.jcpa.2021.01.007DOI Listing
April 2021

Tumor staging in a Beagle dog with concomitant large B-cell lymphoma and T-cell acute lymphoblastic leukemia.

J Vet Diagn Invest 2021 Apr 22:10406387211011024. Epub 2021 Apr 22.

Department of Veterinary Medicine, University of Milan, Lodi, Italy.

An 8-y-old spayed female Beagle dog was presented with peripheral lymphadenomegaly. Lymph node cytology and flow cytometry led to the diagnosis of large B-cell lymphoma (LBCL). We detected minimal percentages of LBCL cells in peripheral blood and bone marrow samples. However, a monomorphic population of neoplastic cells different from those found in the lymph node was found in the bone marrow. T-cell acute lymphoblastic leukemia was suspected based on flow cytometric immunophenotyping. PCR for antigen receptor rearrangement (PARR) revealed clonal rearrangement of both B-cell and T-cell receptors, and the presence of both neoplastic clones in the lymph node, peripheral blood, and bone marrow. The dog was treated with multi-agent chemotherapy but died 46 d following diagnosis. Tumor staging and patient classification are needed to accurately establish a prognosis and select the most appropriate therapeutic protocol.
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http://dx.doi.org/10.1177/10406387211011024DOI Listing
April 2021

Phenotypical Characterization and Clinical Outcome of Canine Burkitt-Like Lymphoma.

Front Vet Sci 2021 17;8:647009. Epub 2021 Mar 17.

Department of Medical Veterinary Science, University of Bologna, Bologna, Italy.

In dogs, Burkitt-like lymphoma (B-LL) is rare tumor and it is classified as a high-grade B-cell malignancy. The diagnosis is challenging because of the similar histologic appearance with other histotypes, no defined phenotypical criteria and poorly described clinical aspects. The aim of the study was to provide a detailed description of clinical and morphological features, as well as immunophenotypical profile of B-LL in comparison with the human counterpart. Thirteen dogs with histologically proven B-LL, for which a complete staging and follow-up were available, were retrospectively selected. Immunohistochemical expression of CD20, PAX5, CD3, CD10, BCL2, BCL6, MYC, and caspase-3 was evaluated. Histologically, all B-LLs showed a diffuse architecture with medium to large-sized cells, high mitotic rate and diffuse starry sky appearance. B-phenotype of neoplastic cells was confirmed both by flow-cytometry and immunohistochemistry. Conversely, B-LLs were negative for BCL2 and MYC, whereas some cases co-expressed BCL6 and CD10, suggesting a germinal center B-cell origin. Disease stage was advanced in the majority of cases. All dogs received CHOP-based chemotherapy with or without immunotherapy. Despite treatment, prognosis was poor, with a median time to progression and survival of 130 and 228 days, respectively. Nevertheless, ~30% of dogs survived more than 1 year. An increased apoptotic index, a high turnover index and caspase-3 index correlated with shorter survival. In conclusion, canine B-LL shows phenotypical differences with the human counterpart along with features that might help to differentiate this entity from diffuse large B-cell lymphoma.
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http://dx.doi.org/10.3389/fvets.2021.647009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010238PMC
March 2021

Variation of apoptotic and proliferative activity among lymphoma subtypes in dogs: A flow cytometric study.

Res Vet Sci 2021 Mar 20;135:324-328. Epub 2020 Oct 20.

Department of Veterinary Sciences, University of Turin, largo Braccini 2-5, 10095 Grugliasco, Turin, Italy.

Tumor growth depends on both proliferative and apoptotic rate of neoplastic cells. High proliferation index is a well-known negative prognostic factor in canine lymphomas, whereas little is known about apoptotic activity. We describe proliferative and apoptotic rates in different canine lymphoma subtypes at diagnosis. Flow cytometry (FC) was used to assess the percentage of proliferating cells (Ki67%) and of apoptotic cells (AnnV%) in 128 lymph node (LN) aspirates from dogs with lymphoma. Proliferation/apoptosis ratio (PAR) and turnover index (TI; Ki67% + AnnV%) were then calculated for each case. High-grade B-cell lymphomas showed high values for both Ki67% and AnnV%, low-grade B-cell lymphomas showed low Ki67% and high AnnV%, high-grade T-cell lymphomas showed high Ki67% and low AnnV%, and low-grade T-cell lymphomas showed low levels of both parameters. Lymphoblastic lymphomas had the highest PAR values. High-grade B-cell lymphomas had the highest TI values while small clear cells lymphomas the lowest. The panorama of proliferative and apoptotic activity widely varies among lymphoma subtypes. Our results lay the ground for future clinical and pharmacological studies.
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http://dx.doi.org/10.1016/j.rvsc.2020.10.008DOI Listing
March 2021

Flow Cytometric Analysis of Mediastinal Masses in Cats: A Retrospective Study.

Front Vet Sci 2020 7;7:444. Epub 2020 Aug 7.

Department of Veterinary Medicine, University of Milan, Lodi, Italy.

Mediastinal masses occur in dogs and cats and are often investigated with cytology. However, discrimination between the two most common lesions (thymoma and lymphoma) may be challenging, especially when small/medium lymphocytes represent the prevalent population. The aim of the present study is to describe the flow cytometric aspects of mediastinal masses in cats and to assess the ability of flow cytometry (FC) to differentiate lymphoma from non-lymphomatous lesions. We retrospectively describe FC features of fine needle aspiration cytology from cats with mediastinal masses. Cases were grouped in lymphoma and non-lymphoma based on results of cytology, histopathology, PCR for antigen receptor rearrangement (PARR), clinical presentation, and follow-up. Scatter properties, positivities to CD5, CD4, CD8, CD21, CD18, and their co-expressions were recorded using a multicolour approach. Twenty cats were included, 12 lymphomas and eight non-lymphomatous cases. Forward scatter (FSC) of lymphoid cells was higher in the lymphoma group. Double positive CD4+CD8+ T-cells were the dominant population in eight out of 12 lymphomas, whereas non-lymphomatous lesions showed no dominant lymphoid population in five out of eight cases. Unlike dogs, the high prevalence of CD4+CD8+ lymphomas in cats it makes difficult to differentiate lymphoma from non-lymphomatous lesions using FC alone. FC may add interesting information to refine diagnosis in some cases, but PARR and histopathology remain mandatory to solve differential in case of expansion of small-medium sized double positive lymphoid cells.
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http://dx.doi.org/10.3389/fvets.2020.00444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438742PMC
August 2020

Flow cytometry expression pattern of CD44 and CD18 markers on feline leukocytes.

J Vet Diagn Invest 2020 Sep 28;32(5):706-709. Epub 2020 Jul 28.

Department of Veterinary Medicine (Martini, Bernardi, Giordano, Comazzi) and Veterinary Teaching Hospital (Martini, Bernardi, Giordano, Comazzi), University of Milan, Lodi, Italy.

The paucity of specific feline antibodies for flow cytometry (FC) is an ongoing challenge. Flow cytometrists must extrapolate information from relatively few markers. We evaluated the expression pattern of the panleukocyte markers CD18 and CD44 on leukocyte (white blood cell, WBC) subclasses in the peripheral blood (PB) of 14 healthy cats. The degree of expression of CD18 and CD44 was calculated as the ratio between the median fluorescence intensity (MFI) value of antibody-stained cells and autofluorescence. All samples were acquired with the same cytometer with constant photomultiplier setting and compensation matrices. Both molecules were expressed at higher levels on monocytes, intermediate levels on polymorphonuclear cells (PMNs), and lower levels on lymphocytes. CD18-MFI discriminated well among the 3 populations, whereas CD44-MFI mostly overlapped between monocytes and PMNs. However, CD44-MFI had a lower intra-population variability. Evaluation of CD18 and CD44, together with morphologic parameters, was useful for discriminating among WBC subclasses in healthy cats. This information may be helpful for future studies given that an increase in CD18-MFI may indicate reactive changes, whereas fluctuations in CD44-MFI may suggest neoplasia.
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http://dx.doi.org/10.1177/1040638720945670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7488972PMC
September 2020

Blood L-Lactate Concentration as an Indicator of Outcome in Roe Deer () Admitted to a Wildlife Rescue Center.

Animals (Basel) 2020 Jun 20;10(6). Epub 2020 Jun 20.

Department of Veterinary Medicine, University of Milan, 26900 Lodi, Italy.

Roe deer () are among the most frequent patients of rescue centers in Italy. Three outcomes are possible: natural death, euthanasia, or treatment and release. The aim of the present study is to propose blood L-lactate concentration as a possible prognostic biomarker that may assist veterinarians in the decision-making process. Sixty-three roe deer, admitted to one rescue center in the period between July 2018 and July 2019, were sampled and divided into 4 groups according to their outcome: (1) spontaneous death (17 cases), (2) humanely euthanized (13 cases), (3) fully recovered and released (13 cases), and (4) euthanized being unsuitable for release (20 cases). In addition, blood samples from 14 hunted roe deer were analyzed as controls. Whole blood lactate concentrations were measured with a point of care lactate meter. Differences among groups were close to statistical significance ( = 0.51). A cut-off value of 10.2 mmol/L was identified: all the animals with higher values died or were humanely euthanized. The results suggest that roe deer with lactatemia higher than 10.2 mmol/L at admission, have a reduced prognosis for survival during the rehabilitation period, regardless of the reason for hospitalization and the injuries reported. Therefore, humane euthanasia should be considered for these animals.
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http://dx.doi.org/10.3390/ani10061066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341283PMC
June 2020

Immune-complex glomerulonephritis in cats: a retrospective study based on clinico-pathological data, histopathology and ultrastructural features.

BMC Vet Res 2019 Aug 20;15(1):303. Epub 2019 Aug 20.

Istituto Veterinario di Novara, Strada Provinciale 9, 28060 Granozzo con Monticello (NO), Novara, Italy.

Background: Chronic kidney disease (CKD) has typically a non-immune mediated origin in cats and immune-complex glomerulonephritis (ICGN) is scarcely described. Aims of this study were to characterize ICGN by light and electron microscopy and identify associations with clinico-pathological findings. In addition, comparisons between cats with ICGN and non immune-complex glomerulonephritis (non-ICGN) were performed. Renal samples examined between 2010 and 2019 were considered if both light and electron microscopy were performed. Signalment, feline immunodeficiency virus (FIV) and leukemia virus (FeLV) status, serum creatinine concentration, urine protein-to-creatinine (UPC) ratio, systolic blood pressure (SBP) and International Renal Interest Society (IRIS) stage were retrieved and used for comparisons.

Results: Sixty-eight client-owned cats were included. Thirty-seven cats (54.4%) had ICGN and 31 (45.6%) non-ICGN. Eighteen (48.6%) with ICGN had membranous glomerulonephropathy (MGN), 14 (37.8%) membranoproliferative glomerulonephritis (MPGN), and 5 (13.5%) mesangioproliferative glomerulonephritis (MeGN). Clinico-pathological data were not associated with any type of ICGN. Among cats with non-ICGN, 11 (35.5%) had end-stage CKD, 9 (29%) focal segmental glomerulosclerosis, 6 (19.4%) global and multifocal mesangiosclerosis, 2 (6.5%) glomerular atrophy, 2 (6.5%) renal dysplasia and 1 (3.1%) amyloidosis. Eight (25.8%) cats with non-ICGN had chronic interstitial nephritis (CIN) grade 1, 13 (41.9%) grade 2 and 10 (32.3%) grade 3; creatinine and UPC ratio increased with CIN grades (p = 0.001, p < 0.001). Cats with ICGN were more frequently FIV or FeLV-infected (OR:11.4; 95%CI:1.4-94.4; p = 0.024), had higher UPC ratio (OR:6.8; 95%CI:2.5-18.2; p < 0.001) and were younger (OR:0.9; 95%CI:0.7-1.0; p = 0.042) than cats with non-ICGN.

Conclusions: MGN and MPGN were the most common morphological diagnoses of ICGN in cats. Unfortunately, none of the investigated findings differentiated ICGN morphological diagnoses. Serum creatinine concentration and UPC ratio were directly associated with grades of CIN (p = 0.001 and p < 0.001, respectively), confirming previous literature. More ICGN than non-ICGN was observed in cats with retroviral infections, younger cats and higher UPC ratio.
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http://dx.doi.org/10.1186/s12917-019-2046-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702729PMC
August 2019

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

Res Vet Sci 2019 Aug 11;125:130-135. Epub 2019 Jun 11.

Department of Veterinary Medicine, University of Milan, via Celoria 10, 20133 Milan, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.rvsc.2019.06.003DOI Listing
August 2019

Opportunities and challenges of active immunotherapy in dogs with B-cell lymphoma: a 5-year experience in two veterinary oncology centers.

J Immunother Cancer 2019 06 7;7(1):146. Epub 2019 Jun 7.

Department of Veterinary Medical Sciences, University of Bologna, Bologna, Italy.

Background: Pet dogs spontaneously develop lymphoma. An anthracycline-based multidrug chemotherapy regimen represents the treatment cornerstone; however, cure is rarely achieved. We have been treating dogs with B-cell lymphoma with an autologous vaccine (APAVAC®) and CHOP-based chemotherapy since 2011.

Methods: To better characterize the safety and efficacy of APAVAC®, and to find the best candidates for immunotherapy, we designed a retrospective study on all dogs treated with chemo-immunotherapy to date and compared them with those dogs treated with chemotherapy only. All dogs were completely staged and re-staged at the end of treatment. The primary endpoint was the effectiveness of chemo-immunotherapy, measured as time to progression (TTP), lymphoma-specific survival (LSS), and 1-, 2-, and 3-year survival rates. The secondary objective was safety.

Results: Three hundred dogs were included: 148 (49.3%) received chemotherapy and 152 (50.7%) chemo-immunotherapy. Overall, the latter survived significantly longer (median LSS, 401 vs 220; P <  0.001). Among dogs with diffuse large B-cell lymphoma, the 1-, 2- and 3-year survival rates were 20, 13 and 8% for chemotherapy, and 51, 19 and 10% for chemo-immunotherapy. The benefit of chemo-immunotherapy was particularly relevant in dogs with concurrent high serum LDH, stage V, substage a disease and not previously treated with steroids (median LSS, 480 vs 85 days; P <  0.001). Among dogs with nodal marginal zone lymphoma, those having at least 3 of the aforementioned characteristics significantly benefited from chemo-immunotherapy (median LSS, 680 vs 160 days, P <  0.001). The 1-, 2- and 3-year survival rates were 30, 16 and 10% for chemotherapy, and 55, 28 and 10% for chemo-immunotherapy. Among dogs with follicular lymphoma, lack of immunotherapy administration was the only variable significantly associated with increased risk of tumor-related death. Chemo-immunotherapy was remarkably well tolerated, with no local or systemic adverse events.

Conclusions: Overall, the addition of immunotherapy to a traditional CHOP protocol is associated with improved outcome in dogs with B-cell lymphoma, regardless of histotype and evaluated prognostic factors. Moreover, the identikit of the best candidate for immune-therapy was delineated for the most common histotypes. The study also confirms the excellent tolerability of the vaccine.
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http://dx.doi.org/10.1186/s40425-019-0624-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554898PMC
June 2019

Blood lymphocyte subpopulations in healthy water buffaloes (Bubalus bubalis, Mediterranean lineage): Reference intervals and influence of age and reproductive history.

Vet Immunol Immunopathol 2019 May 29;211:58-63. Epub 2019 Apr 29.

Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, via Delpino 1, 80137, Naples, Italy. Electronic address:

There is an increasing interest toward infectious diseases and mechanisms of immune response of water buffaloes, mainly because of the growing economic impact of this species and of its high-quality milk. However, little is known about the immune system of these animals in physiological conditions. Recently, a wide number of antibodies cross reacting with buffalo antigens has been validated for use in flow cytometry (FC), allowing detailed characterization of the lymphocytic population in this species. The aim of the present study was to describe the lymphocyte subpopulations in a large number of healthy water buffaloes, providing reference intervals (RIs), and to assess whether the composition of blood lymphocyte population significantly varied with age and reproductive history. Our final aim was to lay the ground for future studies evaluating the role of host immune response in water buffaloes. One-hundred-twelve healthy buffaloes from four different herds in the South of Italy were included in the study. All animals had been vaccinated for Infectious Bovine Rhinotracheitis (IBR), Salmonellosis, Colibacillosis and Clostridiosis, and all herds were certified Brucellosis- and Tuberculosis-free. Venous blood collected into EDTA tubes was processed for FC, and the percentage of cells staining positive for the following antibodies was recorded: CD3, CD4, CD8, CD21, TCR-δ-N24, WC1-N2, WC1-N3 and WC1-N4. Absolute concentration of each lymphoid subclass was then calculated, based on automated White Blood Cell (WBC) Count. Reference Intervals were calculated according to official guidelines and are listed in the manuscript. The composition of the lymphocyte population varied with age and reproductive history, with animals <2-years-old and heifers having higher concentration of most of the subclasses. The present study provides RIs for the main lymphocytic subclasses in healthy water buffaloes, highlighting gross differences between young and old animals. Establishment of age-specific RIs is recommended in water buffaloes. The data we present may be useful as a basis for further studies concerning mechanisms of immune response toward infectious agents in water buffaloes.
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http://dx.doi.org/10.1016/j.vetimm.2019.04.007DOI Listing
May 2019

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

Vet J 2019 Apr 10;246:78-84. Epub 2019 Feb 10.

Department of Veterinary Medicine, University of Milan, Via Celoria 10, 20133, Milan, Italy.

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-naïf 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 ≥30% 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 1-20% of 337 days, and those with BM infiltration ≥20% 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.
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http://dx.doi.org/10.1016/j.tvjl.2019.02.002DOI Listing
April 2019

Parachini-Winter et al "A case of canine high-grade T-cell lymphoma immunophenotypically consistent with T-zone lymphoma".

Vet Clin Pathol 2019 03 27;48(1):5-6. Epub 2019 Feb 27.

Centro Oncologico Veterinario, Sasso Marconi, BO, Italy.

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http://dx.doi.org/10.1111/vcp.12718DOI Listing
March 2019

Minimal residual disease in lymph nodes after achievement of complete remission predicts time to relapse in dogs with large B-cell lymphoma.

Vet Comp Oncol 2019 Jun 4;17(2):139-146. Epub 2019 Feb 4.

Centro Oncologico Veterinario, Bologna, Italy.

Most dogs with large B-cell lymphoma (LBCL) that undergo chemotherapy and achieve clinical complete remission (CR) eventually relapse. However, time to relapse (TTR) is unpredictable. The aims of this prospective study were to assess the influence of post-chemotherapy lymph node (LN) infiltration by large CD21+ cells using flow cytometry (FC) on TTR, and to establish a cut-off value of prognostic significance. Dogs with newly-diagnosed, completely staged LBCL in CR after treatment were enrolled. Minimal residual disease (MRD) analysis by FC was performed on LN aspirates. TTR was calculated between MRD and relapse. Thirty-one dogs were enrolled: 4% had stage V disease, and diffuse large B-cell lymphoma was the most common histotype (74%). Based on LN infiltration at MRD evaluation, three groups were created: (a) acellular samples, (b) ≤0.5% infiltration and (c) >0.5% infiltration. Overall median TTR was 154 days (range, 31-1974): 22 (71%) dogs relapsed during the study period, whereas 9 (29%) dogs did not. The difference among the three groups was significant (P = 0.042 log-rank test): median TTR was not reached for dogs with LN infiltration ≤0.5% (range, 195-429 days), 164 days (range 63-1974) for dogs with acellular LN samples, and 118 days (range, 31-232) for dogs with LN infiltration >0.5%. These results demonstrate that MRD assessment by FC on LN aspirates in dogs with LBCL in clinical CR predicts TTR. LN infiltration by >0.5% large CD21+ cells after treatment is an unfavourable prognostic factor.
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http://dx.doi.org/10.1111/vco.12453DOI Listing
June 2019

Development and validation of a method using ultra performance liquid chromatography coupled to tandem mass spectrometry for determination of zoledronic acid concentration in human bone.

J Pharm Biomed Anal 2019 Jan 20;162:286-290. Epub 2018 Sep 20.

Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche, Università degli Studi di Milano, Milan, Italy.

A method for the extraction and quantification of zoledronic acid (ZA) from human bone was set up and validated. This method allowed the quantification of ZA from jawbone sequestrations of patients affected by bisphosphonate-related osteonecrosis of the jaw (BRONJ) associated with ZA treatment. The analyte was extracted from the bone tissues with phosphoric acid and derivatized using trimethylsilyl diazomethane (TMS-DAM). ZA tetramethyl phosphonate was then quantified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), showing high accuracy, repeatability and selectivity. Lower limits of quantification and detection (LLOQ and LLQD) were 3.4 ng/mL and 1 ng/mg, respectively. This study fully described the analytical process for the determination of ZA in human bone sequestrations, representing a pivotal step for further biomedical research on ZA and BRONJ.
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http://dx.doi.org/10.1016/j.jpba.2018.09.042DOI Listing
January 2019

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

J Vet Diagn Invest 2018 Nov 28;30(6):830-836. Epub 2018 Sep 28.

Department of Veterinary Medicine, University of Milan, Milan, Italy (Martini, Cozzi, Bernardi, Comazzi).

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.
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http://dx.doi.org/10.1177/1040638718804301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505842PMC
November 2018

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

Sci Rep 2017 09 14;7(1):11591. Epub 2017 Sep 14.

Department of Comparative Biomedicine and Food Science, University of Padova, Legnaro (PD), Italy.

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.
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http://dx.doi.org/10.1038/s41598-017-11724-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599585PMC
September 2017

Flow cytometry for feline lymphoma: a retrospective study regarding pre-analytical factors possibly affecting the quality of samples.

J Feline Med Surg 2018 06 4;20(6):494-501. Epub 2017 Jul 4.

Department of Veterinary Medicine, University of Milan, Milan, Italy.

Objectives Flow cytometry (FC) is becoming increasingly popular among veterinary oncologists for the diagnosis of lymphoma or leukaemia. It is accurate, fast and minimally invasive. Several studies of FC have been carried out in canine oncology and applied with great results, whereas there is limited knowledge and use of this technique in feline patients. This is mainly owing to the high prevalence of intra-abdominal lymphomas in this species and the difficulty associated with the diagnostic procedures needed to collect the sample. The purpose of the present study is to investigate whether any pre-analytical factor might affect the quality of suspected feline lymphoma samples for FC analysis. Methods Ninety-seven consecutive samples of suspected feline lymphoma were retrospectively selected from the authors' institution's FC database. The referring veterinarians were contacted and interviewed about several different variables, including signalment, appearance of the lesion, features of the sampling procedure and the experience of veterinarians performing the sampling. Statistical analyses were performed to assess the possible influence of these variables on the cellularity of the samples and the likelihood of it being finally processed for FC. Results Sample cellularity is a major factor in the likelihood of the sample being processed. Moreover, sample cellularity was significantly influenced by the needle size, with 21 G needles providing the highest cellularity. Notably, the sample cellularity and the likelihood of being processed did not vary between peripheral and intra-abdominal lesions. Approximately half of the cats required pharmacological restraint. Side effects were reported in one case only (transient swelling after peripheral lymph node sampling). Conclusions and relevance FC can be safely applied to cases of suspected feline lymphomas, including intra-abdominal lesions. A 21 G needle should be preferred for sampling. This study provides the basis for the increased use of this minimally invasive, fast and cost-effective technique in feline medicine.
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http://dx.doi.org/10.1177/1098612X17717175DOI Listing
June 2018

Identification of peripheral blood involvement in dogs with large B-cell lymphoma: Comparison of different methods.

Res Vet Sci 2017 Dec 31;115:288-293. Epub 2017 May 31.

Department of Veterinary Sciences, University of Turin, Largo Braccini 2, Grugliasco, Turin, Italy.

Stage V lymphoma is defined as the presence of neoplastic cells in peripheral blood (PB), bone marrow, or any other non-lymphoid tissue. Still, official guidelines do not specify which technique should be used to assess infiltration. We assessed the agreement among flow cytometry (FC), blood smear evaluation, and ADVIA120 (LUC and BASO) to quantify PB infiltration in 100 dogs with large B-cell lymphoma (LBCL). Significant errors were found for all methods compared to FC. A moderate agreement was present between FC and blood smear evaluation, whereas LUC and BASO had excellent specificity but unsatisfactory sensitivity in detecting FC infiltrated PB samples. The different techniques should not be used alternatively. We support the use of LUC/BASO as a speedy preliminary test to detect infiltrated samples, and the joined use of blood smear evaluation and FC to quantify definitively the infiltration. Our results are valid only within canine LBCL staging workup, once the diagnosis has been confirmed.
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http://dx.doi.org/10.1016/j.rvsc.2017.05.027DOI Listing
December 2017

Strategies to exclude subjects who conceal and fabricate information when enrolling in clinical trials.

Contemp Clin Trials Commun 2017 Mar 18;5:67-71. Epub 2016 Dec 18.

Department of Psychiatry, Boston University School of Medicine, USA.

Clinical trials within the US face an increasing challenge with the recruitment of quality candidates. One readily available group of subjects that have high rates of participation in clinical research are subjects who enroll in multiple trials for the purpose of generating income through study payments. Aside from issues of safety and generalizability, evidence suggests that these subjects employ methods of deception to qualify for the strict entrance criteria of some studies, including concealing information and fabricating information. Including these subjects in research poses a significant risk to the integrity of data quality and study designs. Strategies to limit enrollment of subjects whose motivation is generating income have not been systematically addressed in the literature. The present paper is intended to provide investigators with a range of strategies for developing and implementing a study protocol with protections to minimize the enrollment of subjects whose primary motivation for enrolling is to generate income. This multifaceted approach includes recommendations for advertising strategies, payment strategies, telephone screening strategies, and baseline screening strategies. The approach also includes recommendations for attending to inconsistent study data and subject motivation. Implementing these strategies may be more or less important depending upon the vulnerability of the study design to subject deception. Although these strategies may help researchers exclude subjects with a higher rate of deceptive practices, widespread adoption of subject registries would go a long way to decrease the chances of subjects enrolling in multiple studies or more than once in the same study.
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http://dx.doi.org/10.1016/j.conctc.2016.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936691PMC
March 2017

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

Cytometry B Clin Cytom 2016 11 3;90(6):525-530. Epub 2016 Feb 3.

Department of Veterinary Sciences and Public Health, University of Milan, Italy.

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.
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http://dx.doi.org/10.1002/cyto.b.21353DOI Listing
November 2016

Lymphocyte subpopulations and Treg cells in dogs with atopic dermatitis receiving ciclosporin therapy: a prospective study.

Vet Dermatol 2016 Feb 11;27(1):17-e5. Epub 2015 Dec 11.

Department of Veterinary Sciences and Public Health, Università degli Studi di Milano, Via Celoria 10, 20133, Milano, Italy.

Background: Canine atopic dermatitis (CAD) is a chronic dermatological disease partly due to dysregulation of the immune system. Inappropriate activation of CD4(+) lymphocytes could favour and promote the allergic response. An inadequate activation system of regulatory T cells (Tregs) is suspected to be a key immunological feature of the allergic response in atopic dogs.

Hypothesis/objectives: To evaluate the difference in the CD4/CD8 lymphocyte ratio and the percentage of Tregs in healthy dogs, in a breed predisposed to CAD, and in dogs affected by CAD before and during therapy with ciclosporin (CsA). Additionally to assess the improvement in pruritus and skin lesions during therapy with CsA, and to compare this with CD4/CD8/Treg values.

Animals: Ten atopic dogs of different breed, sex and age, ten healthy dogs and ten English bulldogs were included.

Methods: Peripheral blood from all dogs was tested using flow cytometry to assess the CD4/CD8 ratio and percentage of Tregs. For atopic dogs, sampling was repeated after 30 and 90 days of therapy with CsA.

Results: The CD4/CD8 ratio was not significantly different between the three groups. The Treg percentage was higher, but not statistically significant, in atopic dogs compared with controls. Therapy with CsA led to clinical improvement; it was not associated with statistically significant differences in haematological variables.

Conclusion And Clinical Importance: This study suggests that Tregs may be involved in the pathogenesis of CAD and that ciclosporin therapy does not affect the circulating lymphocyte subpopulations.
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http://dx.doi.org/10.1111/vde.12277DOI Listing
February 2016

Peripheral blood lymphocyte/monocyte ratio as a useful prognostic factor in dogs with diffuse large B-cell lymphoma receiving chemoimmunotherapy.

Vet J 2015 Nov 14;206(2):226-30. Epub 2015 Jul 14.

Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell'Università 16, 35020 Agripolis Legnaro (PD), Italy.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent canine lymphoid neoplasm. Despite treatment, the majority of dogs with DLBCL experience tumour relapse and consequently die, so practical models to characterise dogs with a poor prognosis are needed. This study examined whether the lymphocyte/monocyte ratio (LMR) can predict outcome in dogs with newly diagnosed DLBCL with regard to time-to-progression (TTP) and lymphoma specific survival (LSS). A retrospective study analysed the prognostic significance of LMR obtained at diagnosis by flow cytometry (based on morphological properties and CD45 expression) in 51 dogs that underwent complete staging and received the same treatment, comprising multi-agent chemotherapy and administration of an autologous vaccine. Dogs with an LMR ≤ 1.2 (30% of all cases) were found to have significantly shorter TTP and LSS, and it was concluded that LMR was a useful independent prognostic indicator with biological relevance in dogs with DLBCL treated with chemoimmunotherapy.
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http://dx.doi.org/10.1016/j.tvjl.2015.07.009DOI Listing
November 2015

Analytic errors in Sysmex-generated hematology results in blood from a dog with chronic lymphocytic leukemia.

Vet Clin Pathol 2015 Sep 2;44(3):337-41. Epub 2015 Jul 2.

Department of Veterinary Sciences and Public Health, University of Milan, Milan, Italy.

A blood sample from a 14-year-old dog was submitted to the veterinary diagnostic laboratory of the University of Milan for marked leukocytosis with atypical cells. A diagnosis of chronic T-cell lymphocytic leukemia (CLL) was made based on blood smear evaluation and flow cytometric phenotyping. A CBC by Sysmex XT-2000iV revealed a moderate normocytic normochromic anemia. Red blood cells counted by optic flow cytometry (RBC-O) resulted in a higher value than using electrical impedance (RBC-I). The relative reticulocyte count based on RNA content and size was 35.3%, while the manual reticulocyte count was < 1%. The WBC count of 1,562,680 cells/μL was accompanied by a flag. Manual counts for RBC and WBC using the Bürker chamber confirmed the Sysmex impedance results. Finally the manual PCV was lower than HCT by Sysmex. While Sysmex XT can differentiate between RBC and WBC by impedance, even in the face of extreme lymphocytosis due to CLL, RBC-O can be affected by bias, resulting in falsely increased RBC and reticulocyte numbers. Overestimation of RBC-O may be due to incorrect Sysmex classification of leukemic cells or their fragments as reticulocytes. This phenomenon is known as pseudoreticulocytosis and can lead to misinterpretation of regenerative anemia. On the other side PCV can be affected by bias in CLL due to the trapping of RBC in the buffy coat, resulting in a pink hue in the separation area. As HGB concentration is not affected by flow cytometric or other cell-related artifacts it may represent the most reliable variable to assess the degree of anemia in cases of CLL.
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http://dx.doi.org/10.1111/vcp.12270DOI Listing
September 2015

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.

Vet J 2014 May 31;200(2):318-24. Epub 2014 Mar 31.

Centro Oncologico Veterinario, via San Lorenzo 1-4, 40037 Sasso Marconi (BO), Italy.

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.
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http://dx.doi.org/10.1016/j.tvjl.2014.03.006DOI Listing
May 2014

Assessment of bone marrow infiltration diagnosed by flow cytometry in canine large B cell lymphoma: prognostic significance and proposal of a cut-off value.

Vet J 2013 Sep 2;197(3):776-81. Epub 2013 Jun 2.

Centro Oncologico Veterinario, via San Lorenzo 1-4, 40037 Sasso Marconi, BO, Italy. Electronic address:

The aims of this study were to assess the prognostic significance of bone marrow (BM) infiltration in canine large B cell lymphoma (LBCL) and to establish cut-off values for designating the BM as infiltrated by lymphoid blasts. The degree of BM infiltration by large CD21 positive cells in dogs with LBCL was assessed by flow cytometry (FC) and related to time to progression (TTP) and lymphoma-specific survival (LSS). Forty-six dogs were prospectively enrolled, staged and treated with a dose-intense chemotherapeutic protocol. BM infiltration was directly correlated with peripheral blood infiltration (P=0.001), high lactate dehydrogenase activity (P=0.0024) and substage b disease (P<0.001). In the univariate analysis, there was a significant association between BM infiltration diagnosed by FC and both TTP (P=0.001) and LSS (P<0.001). Substage was the only factor associated with TTP in the multivariate analysis (P=0.002), whereas substage (P<0.001) and anaemia (P=0.008) were associated with LSS. A cut-off of 3% BM infiltration had the strongest prognostic value, since it discriminated between dogs with a poorer prognosis (median TTP 69 days; median LSS 155 days) and dogs with a better prognosis (median TTP 149 days; median LSS 322 days). BM analysis is an essential step in the staging of LBCL. The presence of BM infiltration by FC at diagnosis is a negative prognostic indicator in canine LBCL.
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http://dx.doi.org/10.1016/j.tvjl.2013.05.003DOI Listing
September 2013

Grey matter lesions in MS: from histology to clinical implications.

Prion 2013 Jan-Feb;7(1):20-7. Epub 2012 Oct 23.

The Multiple Sclerosis Center, Deptartment of Neurosciences, University Hospital of Padova, Padua, Italy.

Although multiple sclerosis (MS) is a chronic inflammatory-demyelinating disease of the white matter (WM) of the central nervous system, several pathological and magnetic resonance imaging (MRI) studies have shown that a large amount of lesions are located in the cortical and deep gray matter. The histopathological and immunological characteristics of cortical lesions differ significantly from those located in the WM, which suggests a location-dependent expression of the MS immunopathological process. More recently, the availability of not-conventional MRI sequences having higher sensitivity for the gray matter has allowed to depict in vivo a portion of such lesions. The available MRI data obtained on large cohorts of patients, having different clinical forms of the disease, indicate that cortical lesions can be detected early in the disease course, sometimes even before the appearance of WM lesions, and correlate with the severity of physical disability and cognitive impairment, and with the evolution of the disease toward the secondary progressive phase. This review provides a summary of the main histopathological and MRI findings of cortical lesions in MS and discusses their possible clinical implications.
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http://dx.doi.org/10.4161/pri.22580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609046PMC
August 2013