4 ERGEBNISSE
4.2 Manuskript II
4.2.3 Material and methods
4.2.3.1 Study Population and Sample Preparation
Prior to the analysis of VEGF in dogs with mammary gland tumours, serum and plasma concentrations of 10 healthy and 38 dogs with different diseases were compared in order to establish the best source for measuring VEGF. VEGF was not detectable in serum and plasma of the healthy group, whereas 17 dogs with different diseases showed positive serum VEGF concentrations. Only 2 of them had detectable plasma VEGF level, therefore serum samples were used in our study.
Currently there are no available studies comparing the human and canine ELISA in dogs.
Therefore we measured serum VEGF in 18 dogs with different diseases using both kits, to test if there is a difference in the detection and concentration of VEGF.
Thirty-two dogs with mammary gland tumours presented for surgery at the Small Animal Clinic of the University of Göttingen were included in this prospective study. Preoperative serum VEGF concentrations were measured from blood samples collected from the cephalic
50
vein. Before surgical intervention thoracic radiographs including right and left lateral views were performed to evaluate potential metastasis. Serum samples were also collected from 23 healthy dogs as a control group. The dogs were confirmed to be healthy on the basis of a physical examination, clinical pathology (complete blood count (CBC), serum chemistry) and abdominal ultrasonography. Serum samples were processed following the R&D Systems protocol. All samples were kept at room temperature for 2 hours to allow them to clot and were then centrifuged (30 min, 1000 g). Samples were stored at -20°C until required for analysis. SALVEN et al. (1997) reported that serum VEGF concentrations were unaffected by a freeze-thaw cycle.
Histopathology from mammary gland tumours was performed on surgical biopsies that were fixed in 10% buffered formalin and routinely embedded in paraffin. Sections were stained with haematoxylin and eosin (H.& E.). The tumours were classified by a board-certified pathologist (European College of Veterinary Pathologists, ECVP) according to the World Health Organization (WHO) histological classification.
4.2.3.2 VEGF assay
The Serum VEGF levels were measured using a commercially available canine VEGF quantitative enzyme-linked immunosorbent assay kit (Quantikine, R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.
To determine the serum concentration of VEGF 100 μl of assay diluent (RD1W) was added to each well of a microtitre plate, which had been coated with monoclonal antibody specific for VEGF. Then 100 μl of VEGF standard or serum sample were pipetted into each well and incubated for 2 hours at room temperature so that any VEGF present in the sample was bound by the immobilized antibody. The plate was washed three times with wash buffer solution to remove unbound substances. Two hundred microliters of an enzyme-linked polyclonal antibody specific for VEGF (VEGF conjugate) were then added to the wells and incubated for 2 hours at room temperature to bind to the immobilized VEGF.
The plate was washed three times again, and then 200 μl of a substrate solution (Colour Reagent A and Colour Reagent B) were added to the wells and colour developed in proportion to the amount of VEGF bound in the initial step. The preparation was incubated for 25 minutes at room temperature and was protected from light. Finally, 50 μl of a stop solution was added and the intensity of the colour was measured.
51
Samples were analyzed with the use of a TECAN microplate reader (Fa. TECAN Austria GmbH, Grödig, Österreich) at an optical density of 450 nm and a wavelength correction of 570 nm.
Calibration on the microtitre plate included standard series dilutions of recombinant canine VEGF. All assays were conducted in triplicate and the mean values were calculated. The average zero standard optical density was subtracted. The optical density of the standard solutions was plotted against their corresponding concentrations to generate a standard curve and allow the determination of VEGF concentrations from all samples. Concentrations are reported as pg/ml.
According to the manufacturer’s instructions, the assay measures the predominant isoform VEGF 164, and the minimum detectable dose (MDD) of VEGF was defined as typically less than 19.5 pg/ml. The assay shows no cross-reactivity with a series of cytokines and growth factors. The manufacturer reported mean intra- and inter-assay coefficients of variation of 5.4 and 7.3%, respectively, for this kit.
Values read as ≤ 0 pg/ml were all considered negative for circulating VEGF.
4.2.3.3 Statistical analysis
Statistical calculations were performed using a commercial software package (SAS-system, version 9.3, Cary, NC, USA.). For the purposes of analysis, dogs with VEGF values ≤ 0 pg/ml were set at 0 pg/ml. VEGF concentrations were not normally distributed.
VEGF results of dogs with mammary gland tumours and healthy dogs were compared using Fisher’s exact test.
The statistical significance was defined as P <0.05.
4.2.4 Results
4.2.4.1 Dogs with mammary gland tumours
Thirty-two sera obtained from dogs with mammary gland tumours were assayed for VEGF concentrations. The median age of the dogs with mammary gland tumours was 10 years (range, 3-16 years).The dogs sampled belonged to a variety of breeds, including mixed-breed (=13), Golden Retriever (=4), German Shepherd (=2), Bavarian Mountain Scenthound (=2), Old German Cattledog (=2), Labrador Retriever (=1), Boxer (=1), Border Collie (=1), Beagle (=1), Poodle (=1), German Spaniel Dog (=1), Catalan Shepherd Dog (=1),German
52
Shorthaired Pointer (=1) and Shapendoes (=1). Five dogs of the 32 female dogs with mammary gland tumours in our study were neutered and twenty-seven dogs were intact.
In 27 of the studied dogs (n=32), the mammary gland tumours were malignant, while only 5 benign tumours were observed.
The malignant mammary gland tumours included the following histopathologies: complex carcinoma (=12), simple carcinoma (=8), spindle cell carcinoma (=3), carcinosarcoma (=2) and adenocarcinoma (=2).
The benign mammary gland tumours included the following histopathologies: complex adenoma (=2), fibroadenoma (=2) and fibroma (=1).
In addition 4 dogs had mammary mixed tumours.
Only one case had a lymph node metastasis among the 27 malignant mammary tumours and thoracic radiographs showed no evidence of lung metastasis.
4.2.4.2 Healthy control group
Twenty-three serum samples of healthy dogs were assayed for VEGF concentrations. The control group (n=23) consisted of 12 female (5 neutered females, 7 intact females) and 11 male (3 neutered males, 8 intact males) dogs with a median age of 5 years (range, 7 month-13 years). The breeds included mixed breed (=9), German Shepherd (=3), Jack Russell (=2), Rhodesian Ridgeback (=1), Weimaraner (=1), Bavarian Mountain Scenthound (=1), Australian Shepherd (=1), Bearded Collie (=1), Briard (=1), Dalmatian (=1), Hovawart (=1) and Entlebuch Mountain Dog (=1).
4.2.4.3 VEGF results
Measureable concentrations of VEGF were found in 2 of 32 serum samples from dogs with mammary gland tumours in the study (6.9 and 10.3 pg/ml). In the control group, only 2 of 23 healthy dogs had detectable concentrations (3 and 17 pg/ml). There was no significant difference between dogs with mammary gland tumours and healthy dogs related to VEGF detection (Fig. 1).
To compare the sensitivity between the human and canine ELISA kit from R&D Systems, we measured serum VEGF in 18 dogs with different diseases (Table 1). With the use of the human ELISA 6 dogs had detectable VEGF concentrations (range, 4.9 ‒ 92.2 pg/ml), while only one of these dogs were positive for VEGF (71 pg/ml), when using the canine ELISA.
53 4.2.5 Discussion
In the present study, we measured serum VEGF levels in dogs with mammary gland tumours and healthy control subjects using a canine ELISA kit, which mainly detects the predominant canine isoform VEGF 164. The aim of the study was to find out if this ELISA is useable for dogs with mammary gland tumours.
To our knowledge, there is only 1 study available in veterinary oncology using the same canine ELISA kit. The reason herefore is its recent commercial availability. ARESU et al.
(2012) showed that plasma VEGF concentrations were significant higher in lymphoma-affected dogs than in controls.
To detect VEGF-levels in serum and/or plasma from dogs most studies used a commercial ELISA test kit for human VEGF (Quantikine, R&D Systems, Minneapolis, MN, USA) (CLIFFORD et al. 2001; WERGIN u. KASER-HOTZ 2004; GENTILINI et al. 2005; KATO et al. 2007; ROSSMEISL et al. 2007; THAMM et al. 2008; SILVERSTEIN et al. 2009). This human VEGF commercial kit is designed to measure VEGF 165 levels and has been already validated for use in the dog (SCHEIDEGGER et al. 1999; TROY et al. 2006).
KATO et al. (2007) reported that serum VEGF where significantly higher in dogs with mammary gland tumours compared to a healthy control group with the use of the described human ELISA kit. Furthermore, SOBCZYNSKA-RAK (2009) detected plasma VEGF in dogs suffering from skin and subcutaneous tissue tumours with the use of a mouse VEGF ELISA kit (Quantikine, R&D Systems, Minneapolis, MN, USA). This can be explained because the predominant canine isoform VEGF 164 shares, according to the manufacturer, 95% and 90%
amino acid sequence identity with human VEGF and mouse VEGF, respectively (SCHEIDEGGER et al. 1999).
Nevertheless, these three commercial kits from R&D Systems differ in their sensitivity. The limit of detection in the mouse ELISA was defined as 3 pg/ml, whereas the minimum detectable dose (MDD) of VEGF in the human and canine ELISA was defined as typically less than 9.0 pg/ml and 19.5 pg/ml, respectively. In comparison with the human and mouse ELISA, the canine ELISA showed a worse sensitivity and this may be one reason why we couldn’t confirm the observations of KATO et al. (2007). In his study serum VEGF concentrations in dogs with mammary gland tumours ranged from ND-992.9 pg/ml with a median of 14.85 pg/ml.
54
Our results when comparing VEGF concentrations from 18 dogs with different diseases measured with a human and canine ELISA from R&D Systems demonstrated a better sensitivity of the human ELISA related to VEGF detection (Table 1). This examination supports our hypothesis of an inadequate sensitivity of the canine ELISA for mammary gland tumours and may explain why serum VEGF was undetectable in 30 of 32 dogs with mammary gland tumours, contrary to our expectations.
Nonetheless, because of the small sample size and the inhomogeneity of the group further investigations are required.
A technique fault in the implementation of the canine ELISA, that could explain why the 30 tumour-bearing dogs have no detectable VEGF concentrations, is excluded, since a total of 175 dogs with various internal diseases were analyzed for their individual VEGF concentration. Thus, serum samples from dogs with mammary gland tumours were distributed on several assays and significant increased VEGF-levels were found for several other diseases in comparison to healthy controls (unpublished data).
Alternatively to a deficient sensitivity, it is possible that VEGF 164 may not be the primary angiogenic isoform involved in mammary gland tumour growth or that other pro-angiogenic factors have a predominant role in angiogenesis.
Altogether, 5 isoforms of VEGF have been isolated in dogs. The canine splice variants include VEGF 120, VEGF 144, VEGF 164, VEGF 188 and VEGF 205, while human VEGF consist of nine isoforms (121, 145, 148, 162, 165, 165b, 183, 189 and 206). VEGF 164 and VEGF 120 are soluble proteins, while VEGF 144 and VEGF 188 are associated with the extracellular matrix or bound to the cell surface by heparan sulphate. Therefore VEGF 144 and VEGF 188 do not exist in a soluble form (SCHEIDEGGER et al. 1999; ROBINSON u.
STRINGER 2001).
The manufacturer reported that VEGF 164 is measured by the canine ELISA as it is the dominant isoform in dogs. Other isoforms are potentially also detected but this was not tested.
In our study serum VEGF was detectable in 2 of the 32 dogs with mammary gland tumors and the detectable concentrations were 6.9 and 10.3 pg/ml, which is below the MDD of 19.5 pg/ml.
55
Both dogs had malignant mammary gland tumours and one of them also had a chronic fistula at the neck, diagnosed histopathologically as a chronic inflammation.
VEGF is known also as VPF (Vascular Permeability Factor), based on its ability to increase vascular permeability (SENGER et al. 1983). Therefore VEGF expression is also up-regulated in inflammatory disorders and this may be a cause for the detectable VEGF concentration in the dog with an inflammatory process (BENAV et al. 1995).
It is not known why the second dog with mammary gland tumour had a measureable VEGF concentration. Another plausible hypothesis for the two positive values is that the isoform VEGF 164 may have been the primary angiogenic factor for tumour growth in these two dogs.
Two healthy dogs (n=23) also showed a positive VEGF concentration of 3 and 17 pg/ml.
Both (one female mixed breed and one male Bavarian Mountain Scenthound) were intact, under 1 year old, and had a low-grade leukocytosis. The serum sample of the female dog with a VEGF level of 17 pg/ml was collected after a longer period of heat because of ovarian cysts. VEGF as an inducer of angiogenesis is involved in a variety of physiological processes, like wound healing and also the female reproductive cycle. This is a plausible explanation for this relative high value in our study (FERRARA 1999).
In addition VEGF is stored in platelets and leukocytes and is released during blood clotting and it has been considerable debate if serum VEGF or plasma VEGF should be used for analysis (SALVEN et al. 1999; JELKMANN 2001). Therefore an increase in serum VEGF could perhaps be explained by the presence of leukocytosis or thrombocytosis. Nevertheless six tumor-bearing dogs in our study with leukocytosis were negative for serum VEGF.
Serum VEGF concentrations in cancer patients and healthy controls are higher when compared with plasma VEGF levels in the same patient, but platelets and leukocytes of cancer patients contain more VEGF than those of healthy controls (SALVEN et al. 1999;
SALGADO et al. 2001). For this reason some authors support the use of serum for the measurement of circulating VEGF, because it has a higher biological significance. The majority of published studies measured preoperative serum VEGF concentrations and showed a positive correlation between high serum VEGF levels and tumour progression and/or survival (LEE et al. 2000; POON et al. 2003).
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Due to the fact that in both groups only two dogs had detectable VEGF concentrations, a correlation between VEGF concentration, white blood cell (WBC) count and platelet count were not tested.
In our research, we measured serum VEGF concentrations in dogs with mammary gland tumours and a healthy control group using a commercial canine ELISA. Serum VEGF was neither detectable in most dogs with mammary gland tumours nor in healthy dogs.
The results of this study suggest that the measurement of serum VEGF using a canine ELISA cannot be used as a diagnostic and/or prognostic marker for mammary gland tumours.
4.2.6 References
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BENAV, P., L. J. CROFFORD, R. L. WILDER u. T. HLA (1995):
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57
CLIFFORD, C. A., D. HUGHES, M. W. BEAL, A. J. MACKIN, C. J. HENRY, F. S. SHOFER u. K. U. SORENMO (2001):
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NIELSEN, T. TAKSIR, R. K. JAIN u. B. SEED (1998):
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58 JELKMANN, W. (2001):
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KATO, Y., K. ASANO, T. MOGI, K. KUTARA, K. TESHIMA, K. EDAMURA, S. TSUMAGARI, A. HASEGAWA u. S. TANAKA (2007):
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ROBINSON, C. J. u. S. E. STRINGER (2001):
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ROSSMEISL, J. H., R. B. DUNCAN, W. R. HUCKLE u. G. C. TROY (2007):
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59
SALGADO, R., I. BENOY, J. BOGERS, R. WEYTJENS, P. VERMEULEN, L. DIRIX u. E.
VAN MARCK (2001):
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BALLMER-HOFER u. R. JAUSSI (1999):
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60 SOBCZYNSKA-RAK, A. (2009):
Correlation between Plasma Vegf and Angiogenesis of Skin and Subcutaneous Tissue Cancer in Dogs.
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ROBERTSON u. D. L. WARD (2006):
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In Vivo 18, 15-19
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Fig. 1 Box plot of serum VEGF concentrations in dogs with mammary gland tumours (n=32;
group 1) and healthy dogs (n=23; group 2)
Median 25%-75%
Non-Outlier Range Outliers
Extremes
1 2
-2 0 2 4 6 8 10 12 14 16 18
Serum VEGF (pg/ml)
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Table 1 Serum VEGF concentrations in dogs with different diseases, measured with a canine and human ELISA
2 Jack Russell Terrier Diabetes mellitus and Pancreatitis
9 Australian Shepherd Polycystic liver 0 0
10 Golden Retriever Mammary gland tumour
(Carcinosarcoma)
0 4,9
11 Shapendoes Mammary gland
tumour (Complex
13 Labrador Retriever Mammary gland tumour
(Adenocarcinoma)
0 0
63
Continuation of table 1 Serum VEGF concentrations in dogs with different diseases, measured with a canine and human ELISA
No. Dogs Diagnosis
(Histopathology)
Canine ELISA
Serum VEGF (pg/ml)
Human ELISA
Serum VEGF (pg/ml)
14 Mixed breed Mammary gland
tumour (Simple carcinoma)
0 0
15 Old German
Cattledog
Mammary gland tumour (Complex carcinoma)
0 0
16 Mixed breed Splenic lesion
(Splenitis)
0 5,1
17 Golden Retriever Mast cell tumour 0 0
18 German Shorthaired Pointer
Pyometra 0 0
64