Evaluation of a multiagent chemotherapy protocol combining vincristine, cyclophosphamide, mitoxantrone and prednisolone (CMOP) for treatment of feline intermediate-large cell lymphoma.

OBJECTIVES: The aim of this study was to determine response rates, median progression-free intervals (PFIs) and median survival times (MSTs) for cats with intermediate-large cell lymphoma treated with a vincristine, cyclophosphamide, mitoxantrone and prednisolone (CMOP) protocol. A secondary objective was to determine the tolerability of mitoxantrone used within this multiagent protocol. METHODS: The medical records of 31 cats treated at a single institution between 2009 and 2022 were reviewed to identify suitable cases. Cats were included in the study if they had a confirmed diagnosis of intermediate-large cell lymphoma, had received a CMOP protocol as first-line treatment and had completed at least one 4-week cycle of this protocol. Modifications allowed in the protocol included the use of l-asparaginase, vinblastine substitution for vincristine, chlorambucil substitution for cyclophosphamide and dexamethasone or methylprednisolone substitution for prednisolone. RESULTS: The overall response rate was 74% (n = 23), with 45% (n = 14) achieving complete remission (CR), 29% (n = 9) achieving partial remission (PR) and 26% (n = 8) achieving stable disease (SD). The Kaplan-Meier median PFI and MST were 139 days and 206 days, respectively. Responders (CR or PR) had a significantly longer (P <0.001) median PFI and MST compared with non-responders (SD) (176 days vs 62 days, and 251 days vs 61 days, respectively). Cats that achieved CR had a significantly longer median PFI and MST (P <0.001) at 178 days and 1176 days, respectively. The 6-month and 1- and 2-year survival rates in cats with CR were 64%, 57% and 35%, respectively. Treatment with mitoxantrone was generally well tolerated, with no cats experiencing Veterinary Cooperative Oncology Group adverse effects above grade 2. CONCLUSIONS AND RELEVANCE: The CMOP protocol is an alternative and well-tolerated treatment for cats with intermediate-large cell lymphoma. As demonstrated with previous chemotherapy protocols, cats that respond to treatment, particularly those that achieve CR, are likely to have more durable responses.

Association of serum and fecal microRNA profiles in cats with gastrointestinal cancer and chronic inflammatory enteropathy.

BACKGROUND: Differentiation of gastrointestinal cancer (GIC) from chronic inflammatory enteropathies (CIE) in cats can be challenging and often requires extensive diagnostic testing. MicroRNAs (miRNAs) have promise as non-invasive biomarkers in serum and feces for diagnosis of GIC. HYPOTHESIS/OBJECTIVES: Cats with GIC will have serum and fecal miRNA profiles that differ significantly from healthy cats and cats with CIE. Identify serum and fecal miRNAs with diagnostic potential for differentiation between cats with GIC and CIE as compared to healthy cats. ANIMALS: Ten healthy cats, 9 cats with CIE, and 10 cats with GIC; all client-owned. METHODS: Cats were recruited for an international multicenter observational prospective case-control study. Serum and feces were screened using small RNA sequencing for miRNAs that differed in abundance between cats with GIC and CIE, and healthy cats. Diagnostic biomarker potential of relevant miRNAs from small RNA sequencing and the literature was confirmed using reverse transcription quantitative real-time PCR (RT-qPCR). RESULTS: Serum miR-223-3p was found to distinguish between cats with GIC and CIE with an area under the curve (AUC) of 0.9 (95% confidence interval [CI], 0.760-1.0), sensitivity of 90% (95% CI, 59.6-99.5%), and specificity of 77.8% (95% CI, 45.3-96.1%). Serum miR-223-3p likewise showed promise in differentiating a subgroup of cats with small cell lymphoma (SCL) from those with CIE. No fecal miRNAs could distinguish between cats with GIC and CIE. CONCLUSION AND CLINICAL IMPORTANCE: Serum miR-223-3p potentially may serve as a noninvasive diagnostic biomarker of GIC in cats, in addition to providing a much needed tool for the differentiation of CIE and SCL.

Low serum folate concentrations in dogs with non-associative immune-mediated haemolytic anaemia.

BACKGROUND: Folate deficiency in people can occur in conditions causing increased demand, including haemolytic anaemia. This has not been investigated in dogs with non-associative immune-mediated haemolytic anaemia (IMHA). METHODS: Cohort study of 15 dogs with non-associative IMHA. Haematocrit (HCT) and serum folate concentrations were measured at presentation and each subsequent venipuncture performed for monitoring. The relationship between serum folate concentrations and HCT was investigated using linear and logistic mixed-effects regression models and in paired samples using a one-tailed paired t-test. RESULTS: Low serum folate concentrations occurred in five of 15 dogs. In 126 samples, a significant positive relationship was found between HCT and corresponding serum folate concentrations. A significant relationship was found between dichotomised folate concentrations (below the reference interval or within/above the reference interval) and HCT and between serum folate concentrations and dichotomised HCT (less than or equal/above 0.30 L/L). For paired samples (available in eight dogs), the mean serum folate concentration of samples with the lowest HCT was significantly lower than that of samples in which the HCT first exceeded 0.30 L/L. CONCLUSIONS: Low serum folate concentrations were observed in some dogs with non-associative IMHA. Further studies are needed to determine the cause and investigate whether folate supplementation would be beneficial.

Primary multifocal muscular T-cell lymphoma with cutaneous involvement in a dog: A case report and review of the literature.

Canine lymphoma represents a heterogeneous group of lymphoid neoplasms, with multicentric nodal lymphoma being the most common presentation. Musculoskeletal involvement is uncommon, and primary muscular lymphoma is a very rare presentation. Only a few cases have been described in dogs, which were of variable classification and immunophenotype. Here, we report the case of a 5-year-old female neutered Beagle that presented with an intramuscular mass on the right shoulder and associated lameness and lethargy. One month after initial presentation, multiple cutaneous nodules appeared on the head, and staging with advanced imaging revealed additional masses affecting other muscles. Cytology, histopathology, immunohistochemistry, and PCR for antigen receptor rearrangements of one of the muscle masses and skin lesions supported a diagnosis of peripheral T-cell lymphoma with large granular lymphocytes at both sites. The dog was euthanized after diagnosis due to the poor prognosis. This is the first report of primary muscular peripheral T-cell lymphoma with large granular lymphocytes and cutaneous involvement in the dog. Despite being a rare presentation, lymphoma must be considered a differential in dogs presenting with a discrete, intramuscular, soft tissue mass.

A matrisome RNA signature from early-pregnancy mouse mammary fibroblasts predicts distant metastasis-free breast cancer survival in humans.

BACKGROUND: During pregnancy, the mouse mammary ductal epithelium branches and grows into the surrounding stroma, requiring extensive extracellular matrix (ECM) and tissue remodelling. It therefore shows parallels to cancer invasion. We hypothesised that similar molecular mechanisms may be utilised in both processes, and that assessment of the stromal changes during pregnancy-associated branching may depict the stromal involvement during human breast cancer progression. METHODS: Immunohistochemistry (IHC) was employed to assess the alterations within the mouse mammary gland extracellular matrix during early pregnancy when lateral branching of the primary ductal epithelium is initiated. Primary mouse mammary fibroblasts from three-day pregnant and age-matched non-pregnant control mice, respectively, were 3D co-cultured with mammary epithelial cells to assess differences in their abilities to induce branching morphogenesis in vitro. Transcriptome analysis was performed to identify the underlying molecular changes. A signature of the human orthologues of the differentially expressed matrisome RNAs was analysed by Kaplan-Meier and multi-variate analysis in two large breast cancer RNA datasets (Gene expression-based Outcome for Breast cancer Online (GOBO) und Kaplan-Meier Plotter), respectively, to test for similarities in expression between early-pregnancy mouse mammary gland development and breast cancer progression. RESULTS: The ECM surrounding the primary ductal network showed significant differences in collagen and basement membrane protein distribution early during pregnancy. Pregnancy-associated fibroblasts (PAFs) significantly enhanced branching initiation compared to age-matched control fibroblast. A combined signature of 64 differentially expressed RNAs, encoding matrisome proteins, was a strong prognostic indicator of distant metastasis-free survival (DMFS) independent of other clinical parameters. The prognostic power could be significantly strengthened by using only a subset of 18 RNAs (LogRank P ≤ 1.00e-13; Hazard ratio (HR) = 2.42 (1.8-3.26); p = 5.61e-09). The prognostic power was confirmed in a second breast cancer dataset, as well as in datasets from ovarian and lung cancer patients. CONCLUSIONS: Our results describe for the first time the early stromal changes that accompany pregnancy-associated branching morphogenesis in mice, specify the early pregnancy-associated molecular alterations in mouse mammary fibroblasts, and identify a matrisome signature as a strong prognostic indicator of human breast cancer progression, with particular strength in oestrogen receptor (ER)-negative breast cancers.

Preliminary assessment of serum clusterin as a potential biomarker for canine lymphoma.

Clusterin (CLU), also known as apolipoprotein J, is a widely expressed, heterodimeric, glycoprotein, important in tumourigenesis, apoptosis and immunoregulation. In humans, CLU expression has been associated with anaplastic large cell and Hodgkin's lymphoma. In this study, serum CLU levels in dogs with multicentric lymphoma (MLSA) were compared with healthy control dogs, using both western blot and enzyme-linked immunosorbent assay (ELISA). Western blot confirmed the presence of CLU in dog sera at the predicted molecular weight and the relative levels detected correlated with the levels detected by ELISA. CLU level analysis by ELISA found treatment naïve dogs with MLSA had a significantly (P < .001) lower serum CLU level compared with healthy controls. However, there was no significant difference between MLSA dogs prior to treatment and in complete remission. The wide variation in serum CLU levels may limit its potential as a single candidate biomarker for MLSA, although any prognostic predictive value of serum CLU concentrations has yet to be assessed.

Owner perceptions of radiotherapy treatment for veterinary patients with cancer.

Veterinary clients may have trepidation about treating their pet with radiotherapy because of concerns about radiation side effects or repeated anaesthetics. The purpose of this study is to assess whether owners' attitudes towards veterinary radiotherapy, including concerns over side effects, change during the course of treatment, and whether radiotherapy was perceived to affect pets' quality of life (QOL). A prospective cohort study of clients from 2012 to 2015 was performed. Pets received palliative or definitive radiotherapy for various tumours. Clients completed questionnaires before, during and after radiotherapy. Questions assessed owner preconceptions before treatment, including side effect expectations, actual side effects experienced and overall satisfaction with the process. In addition, at each time point, the owners assessed their pet's QOL using a simple numerical scale. Forty-nine patients were included. After completing treatment, owners were significantly less concerned about potential side effects of radiotherapy (P < 0.001), side effects associated with repeat anaesthetics (P < 0.001), and about radiotherapy in general (P < 0.001). QOL did not show a significant change at any point during or after treatment. Following treatment, 94% reported that the experience was better than expected and 100% supported the use of radiotherapy in pets. This is the first prospective study evaluating client attitudes and satisfaction before and after radiotherapy treatment in pets. The results indicate that radiotherapy is well tolerated, and the anxiety associated with radiotherapy is significantly alleviated after experiencing the process. These results will help veterinarians allay client concerns, and will hopefully lead to an increase in clients pursuing radiotherapy in pets.

Fibulin-2 is required for basement membrane integrity of mammary epithelium.

Fibulin-2 (FBLN2) is a secreted extracellular matrix glycoprotein which has been associated with tissue development and remodelling. In the mouse mammary gland, FBLN2 can be detected during ductal morphogenesis in cap cells and myoepithelial cells at puberty and early pregnancy, respectively. In an attempt to assign its function, we knocked down Fbln2 in the mouse mammary epithelial cell line EpH4. FBLN2 reduction led to an increase in the size of spheroidal structures when compared to scrambled control shRNA-transduced cells plated on Matrigel matrix. This phenotype was associated with a disruption of the collagen IV sheath around the epithelial spheroids and downregulation of integrin ß1, suggesting a role for FBLN2 in stabilizing the basement membrane (BM). In contrast to mice, in normal adult human breast tissue, FBLN2 was detected in ductal stroma, and in the interlobular stroma, but was not detectable within the lobular regions. In tissue sections of 65 breast cancers FBLN2 staining was lost around malignant cells with retained staining in the neighbouring histologically normal tissue margins. These results are consistent with a role of FBLN2 in mammary epithelial BM stability, and that its down-regulation in breast cancer is associated with loss of the BM and early invasion.

The receptor protein tyrosine phosphatase PTPRB negatively regulates FGF2-dependent branching morphogenesis.

PTPRB is a transmembrane protein tyrosine phosphatase known to regulate blood vessel remodelling and angiogenesis. Here, we demonstrate that PTPRB negatively regulates branching morphogenesis in the mouse mammary epithelium. We show that Ptprb is highly expressed in adult mammary stem cells and also, although at lower levels, in oestrogen receptor-positive luminal cells. During mammary development, Ptprb expression is downregulated during puberty, a period of extensive ductal outgrowth and branching. In vivo shRNA knockdown of Ptprb in the cleared mammary fat pad transplant assay resulted in smaller epithelial outgrowths with an increased branching density and also increased branching in an in vitro organoid assay. Organoid branching was dependent on stimulation by FGF2, and Ptprb knockdown in mammary epithelial cells resulted in a higher level of fibroblast growth factor receptor (FGFR) activation and ERK1/2 phosphorylation, both at baseline and following FGF2 stimulation. Therefore, PTPRB regulates branching morphogenesis in the mammary epithelium by modulating the response of the FGFR signalling pathway to FGF stimulation. Considering the importance of branching morphogenesis in multiple taxa, our findings have general importance outside mammary developmental biology.

A metastatic secretory gastric plasmacytoma with aberrant CD3 expression in a dog.

A 10-year-old crossbred dog was presented with a 6-week history of hematemesis, melena, anorexia, and lethargy. Clinical evaluation revealed a gastric mass with a regional lymphadenomegaly as well as a monoclonal gammopathy manifesting as hyperglobulinemia. Cytologic and histopathologic analyses were consistent with a round cell neoplasm; neoplastic cells showed nuclear immunoreactivity for MUM1 and diffuse cytoplasmic reactivity for CD3. Polymerase chain reactions performed on fixed and fresh tissue identified a clonal rearrangement with an IgH primer set. An extramedullary plasmacytoma (EMP) was confirmed by cellular morphology and molecular diagnostics. Following an objective response to chemotherapy, the dog was euthanized 8 months after diagnosis, and a postmortem examination confirmed the clinical findings. This is the first reported case of a monoclonal gammopathy secondary to a gastric EMP coupled with aberrant expression of CD3 in an aggressive plasmacytic tumor, and highlights the utility of molecular diagnostics for classifying atypical hemolymphoid neoplasms.

RNA Profiling of Non-cultured Fibroblasts Isolated from Pubertal Mouse Mammary Gland Sections.

The epithelium of the pubertal mouse mammary gland grows and invades the mammary fat pad to form a primary ductal network. This outgrowth is tightly controlled by epithelial and stromal factors that are present in the environment around the terminal end buds (TEB) at the growth front and the newly formed ducts. Identifying the contribution that each cell type makes to this regulation is a major challenge. To identify the role that fibroblasts play during this process we have optimised a fibroblast isolation procedure, followed by cell cleanup, RNA extraction, and amplification from non-cultured, freshly isolated fibroblasts from around the TEB as well as the subtending ducts. This was facilitated by the use of mice that constitutively expressed EGFP, which allowed the visualization of the growth front of the pubertal mammary tree under UV light. The isolated RNA is of sufficiently high quality, giving reproducible qRT-PCR results, for transcriptome analysis after RNA amplification.

Pubertal Ductal Morphogenesis: Isolation and Transcriptome Analysis of the Terminal End Bud.

The terminal end bud (TEB) is the growing part of the ductal mammary epithelium during puberty, enabling the formation of a primary epithelial network. These highly proliferative bulbous end structures that drive the ductal expansion into the mammary fat pad comprise an outer cap cell layer, containing the progenitor cells of the ductal myoepithelium, and the body cells, which form the luminal epithelium. As TEB make up only a very small part of the whole mammary tissue, TEB-associated factors can be easily missed when whole-tissue sections are being analyzed. Here we describe a method to enzymatically separate TEB and ducts, respectively, from the surrounding stroma of pubertal mice in order to perform transcriptomic or proteomic analysis on the isolated structures and identify potential novel regulators of epithelial outgrowth, or to allow further cell culturing. This approach has previously allowed us to identify novel TEB-associated proteins, including several axonal guidance proteins. We further include protocols for the culturing of isolated TEB, processing of mammary tissue into paraffin and immunohistochemical/fluorescent staining for verification, and localization of protein expression in the mammary tissue at different developmental time points.

Optimisation and validation of a PCR for antigen receptor rearrangement (PARR) assay to detect clonality in canine lymphoid malignancies.

PCR for antigen receptor gene rearrangements (PARR) analysis is being increasingly used to assist diagnosis of canine lymphoma. In this study, PARR was carried out on consecutive samples received as part of routine diagnostic practice from 271 patients: 195 with lymphoid malignancies, 53 with reactive conditions and 23 with other neoplasms. Initially, published primer sets were used but later minor primer modifications were introduced and primers were rationalised to give a PARR panel that provides a good compromise between sensitivity and cost. Results were compared to diagnoses made by histology or cytology, coupled with immunophenotyping by flow cytometry or immunohistochemistry where possible. After exclusion of 11 poor quality samples, 230/260 (88%) gave a clear result with 162/163 (99%) of samples classified as clonal and 56/67 (84%) classified as polyclonal giving results concordant with the cytological/histological diagnosis. Among 30 samples with equivocal results, 21 had clonal peaks in a polyclonal background and nine showed little amplification. These were from patients with a range of neoplastic and non-neoplastic conditions emphasising the need to interpret such results carefully in concert with other diagnostic tests. The combination of primer sets used in this study resulted in a robust, highly specific and sensitive assay for detecting clonality.

Genomic and proteomic profiling for cancer diagnosis in dogs.

Global gene expression, whereby tumours are classified according to similar gene expression patterns or 'signatures' regardless of cell morphology or tissue characteristics, is being increasingly used in both the human and veterinary fields to assist in cancer diagnosis and prognosis. Many studies on canine tumours have focussed on RNA expression using techniques such as microarrays or next generation sequencing. However, proteomic studies combining two-dimensional polyacrylamide gel electrophoresis or two-dimensional differential gel electrophoresis with mass spectrometry have also provided a wealth of data on gene expression in tumour tissues. In addition, proteomics has been instrumental in the search for tumour biomarkers in blood and other body fluids.

Cancer immunology and canine malignant melanoma: A comparative review.

Oral canine malignant melanoma (CMM) is a spontaneously occurring aggressive tumour with relatively few medical treatment options, which provides a suitable model for the disease in humans. Historically, multiple immunotherapeutic strategies aimed at provoking both innate and adaptive anti-tumour immune responses have been published with varying levels of activity against CMM. Recently, a plasmid DNA vaccine expressing human tyrosinase has been licensed for the adjunct treatment of oral CMM. This article reviews the immunological similarities between CMM and the human counterpart; mechanisms by which tumours evade the immune system; reasons why melanoma is an attractive target for immunotherapy; the premise of whole cell, dendritic cell (DC), viral and DNA vaccination strategies alongside preliminary clinical results in dogs. Current "gold standard" treatments for advanced human malignant melanoma are evolving quickly with remarkable results being achieved following the introduction of immune checkpoint blockade and adoptively transferred cell therapies. The rapidly expanding field of cancer immunology and immunotherapeutics means that rational targeting of this disease in both species should enhance treatment outcomes in veterinary and human clinics.

Gammaherpesviruses and canine lymphoma: no evidence for direct involvement in commonly occurring lymphomas.

Lymphoma is the most common haematopoietic malignancy in dogs, but little is known about the aetiology of this heterogeneous group of cancers. In humans, the Epstein-Barr virus (EBV) is associated with several lymphoma subtypes. Recently, it was suggested that EBV or an EBV-like virus is circulating in dogs. We therefore investigated whether EBV, or a novel herpesvirus, is associated with canine lymphoma using both serological and molecular techniques. In an assay designed to detect antibodies to EBV viral capsid antigens, 41 % of dogs were positive. Dogs with cancers, including lymphoma, were more frequently positive than controls, but no particular association with B-cell lymphoma was noted. EBV-specific RNA and DNA sequences were not detected in lymphoma tissue by in situ hybridization or PCR, and herpesvirus genomes were not detected using multiple degenerate PCR assays with the ability to detect novel herpesviruses. We therefore found no evidence that herpesviruses are directly involved in common types of canine lymphoma although cannot exclude the presence of an EBV-like virus in the canine population.

Expression of RUNX1 correlates with poor patient prognosis in triple negative breast cancer.

The RUNX1 transcription factor is widely recognised for its tumour suppressor effects in leukaemia. Recently a putative link to breast cancer has started to emerge, however the function of RUNX1 in breast cancer is still unknown. To investigate if RUNX1 expression was important to clinical outcome in primary breast tumours a tissue microarray (TMA) containing biopsies from 483 patients with primary operable invasive ductal breast cancer was stained by immunohistochemistry. RUNX1 was associated with progesterone receptor (PR)-positive tumours (P<0.05), more tumour CD4+(P<0.05) and CD8+(P<0.01) T-lymphocytic infiltrate, increased tumour CD138+plasma cell (P<0.01) and more CD68+macrophage infiltrate (P<0.001). RUNX1 expression did not influence outcome of oestrogen receptor (ER)-positive or HER2-positive disease, however on univariate analysis a high RUNX1 protein was significantly associated with poorer cancer-specific survival in patients with ER-negative (P<0.05) and with triple negative (TN) invasive breast cancer (P<0.05). Furthermore, multivariate Cox regression analysis of cancer-specific survival showed a trend towards significance in ER-negative patients (P<0.1) and was significant in triple negative patients (P<0.05). Of relevance, triple negative breast cancer currently lacks good biomarkers and patients with this subtype do not benefit from the option of targeted therapy unlike patients with ER-positive or HER2-positive disease. Using multivariate analysis RUNX1 was identified as an independent prognostic marker in the triple negative subgroup. Overall, our study identifies RUNX1 as a new prognostic indicator correlating with poor prognosis specifically in the triple negative subtype of human breast cancer.

RUNX2 correlates with subtype-specific breast cancer in a human tissue microarray, and ectopic expression of Runx2 perturbs differentiation in the mouse mammary gland.

RUNX2, a master regulator of osteogenesis, is oncogenic in the lymphoid lineage; however, little is known about its role in epithelial cancers. Upregulation of RUNX2 in cell lines correlates with increased invasiveness and the capacity to form osteolytic disease in models of breast and prostate cancer. However, most studies have analysed the effects of this gene in a limited number of cell lines and its role in primary breast cancer has not been resolved. Using a human tumour tissue microarray, we show that high RUNX2 expression is significantly associated with oestrogen receptor (ER)/progesterone receptor (PR)/HER2-negative breast cancers and that patients with high RUNX2 expression have a poorer survival rate than those with negative or low expression. We confirm RUNX2 as a gene that has a potentially important functional role in triple-negative breast cancer. To investigate the role of this gene in breast cancer, we made a transgenic model in which Runx2 is specifically expressed in murine mammary epithelium under the control of the mouse mammary tumour virus (MMTV) promoter. We show that ectopic Runx2 perturbs normal development in pubertal and lactating animals, delaying ductal elongation and inhibiting lobular alveolar differentiation. We also show that the Runx2 transgene elicits age-related, pre-neoplastic changes in the mammary epithelium of older transgenic animals, suggesting that elevated RUNX2 expression renders such tissue more susceptible to oncogenic changes and providing further evidence that this gene might have an important, context-dependent role in breast cancer.

RUNX2 in mammary gland development and breast cancer.

Runx2 is best known as an essential factor in osteoblast differentiation and bone development but, like many other transcription factors involved in development, is known to operate over a much wider tissue range. Our understanding of these other aspects of Runx2 function is still at a relatively early stage and the importance of its role in cell fate decisions and lineage maintenance in non-osseous tissues is only beginning to emerge. One such tissue is the mammary gland, where Runx2 is known to be expressed and participate in the regulation of mammary specific genes. Furthermore, differential and temporal expression of this gene is observed during mammary epithelial differentiation in vivo, strongly indicative of an important functional role. Although the precise nature of that role remains elusive, preliminary evidence hints at possible involvement in the regulation of mammary stem and/or progenitor cells. As with many genes important in regulating cell fate, RUNX2 has also been linked to metastatic cancer where in some established breast cell lines, retention of expression is associated with a more invasive phenotype. More recently, expression analysis has been extended to primary breast cancers where high levels of RUNX2 align with a specific subtype of the disease. That RUNX2 expression correlates with the so called "Triple Negative" subtype is particularly interesting given the known cross talk between Runx2 and estrogen receptor signaling pathways. This review summaries our current understanding of Runx2 in mammary gland development and cancer, and postulates a role that may link both these processes.

Agarose gel serum protein electrophoresis in cats with and without lymphoma and preliminary results of tandem mass fingerprinting analysis.

BACKGROUND: Serum electrophoretic profiles in cats are poorly characterized with respect to the proteins that comprise the globulin fractions, and interpretation of the electrophoretograms is routinely done in the absence of information about identity of the proteins found within each fraction. OBJECTIVES: The aims of this study were to compare protein fractions separated by serum protein electrophoresis (SPE) in healthy cats and in cats with lymphoma and to confirm some component proteins in the major fractions following SPE using tandem mass fingerprinting analysis (TMFA). METHODS: Total protein concentration was measured and agarose gel SPE performed on serum from 14 healthy cats and 14 cats with lymphoma. The absolute protein concentration within each fraction was compared between the 2 groups. Bands corresponding to the SPE fractions were excised from the gels of 2 control cats and 1 cat with lymphoma and analyzed by liquid chromatography coupled to mass spectrometry. Results were compared with sequences in the National Center for Biotechnology Information protein database. RESULTS: Median albumin concentrations were significantly decreased and median ß-globulin concentrations were significantly increased in cats with lymphoma. Narrow electrophoretic spikes were present in the ß/γ-globulin fraction in 3 cats with lymphoma. Following TMFA, multiple proteins were identified in each fraction, and their mobility agreed with results from previous studies generated using alternative techniques. Inter-α (globulin) inhibitor 4 was identified in feline serum for the first time. CONCLUSIONS: Cats with lymphoma had lower albumin and higher ß-globulin concentrations than did healthy cats. Despite limitations of one-dimensional agarose gel SPE, TMFA provided preliminary data to confirm the protein components of the various fractions.