Detection of tumor-associated antigens in sera of canine cancer patients by monoclonal antibodies generated against canine mammary carcinoma cells

Veterinary Immunology and Immunopathology 48 (1995) 193-207

Veterinary immunology ad immunopathology

Detection of tumor-associated antigens in sera of canine cancer patients by monoclonal antibodies generated against canine mammary carcinoma cells Jianyi Wang, Cindy J. Brunner, Aniruddha Gangopadhyay Allison Church Bird, Lauren G. Wolfe * Deparrmenr

of Pathobiology,

College

of Veterinary Medicine,

Auburn

Unir’ersity, AL 36849 - 5519.

I, USA

Accepted 20 January 1995

Abstract

Two murine monoclonal antibodies (MAbs), lAl0 and SB2, generated against a canine mammary carcinoma cell line, were used in a competitive enzyme-linked immunosorbent assay (ELISA) to measure tumor-associated antigens (TAAs) in canine serum samples. Sera were tested from disease-free dogs and from dogs diagnosed with mammary carcinoma, non-mammary carcinoma, sarcoma, benign mammary tumor, benign non-mammary tumor, or non-neoplastic disease. Serum antigen concentrations measured by ELISA were expressed as inhibitory units (IU). The upper limit of normal, defined as the mean plus 2 SD of the TAA concentration in disease-free dogs, was 20 IU with antibody lAl0 and 22 IU with antibody SB2. Compared with disease-free dogs, the frequency of TAA-positive sera was significantly greater (P < 0.05) among dogs with mammary or non-mammary carcinoma when tested with h4Abs lAl0 or SB2, and also with sarcoma when tested with h&41, SB2. Testing a serum sample with both antibodies rather than just one increased the sensitivity of the competitive ELISA for TAA detection. The presence of TAA in serum might serve as a useful marker for certain types of carcinomas or sarcomas in canine cancer patients. Keywords: Canine; Mammary cancer; Carcinoma; Monoclonal antibody; Enzyme-linked immunosorbent assay (ELlSA); Tumor-associated antigen

* Corresponding author. Tel: + l-334-8444539; Fax: + l-334-8442652. I Present address: Deaconess Hospital, Harvard Medical School, Boston, MA 02115, USA. 0165-2427/95/$09.50 SSDI

0165.2427(95)05436-7

0 1995 Elsevier Science B.V. All rights reserved

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1. Introduction Since the development of hybridoma technology in 1975 (Kohler and Milstein, 19751, researchers have produced a variety of monoclonal antibodies (MAbs) that have led to the identification and characterization of novel human tumor-associated antigens (TAAs). Several MAbs that were generated using breast cancer cells or cell lines as immunogens have demonstrated utility in the immunodiagnosis of breast carcinomas and in estimation of tumor burden (Colcher et al., 1981; Hilkens et al., 1986; Linsley et al., 1988; Stacker et al., 1988a; Tjandra et al., 1988). Currently, the most important application of MAbs in the management of human breast cancer is measurement of antigen levels in the serum to assess the response to primary treatment and to detect early evidence of metastasis (Bates, 1991; Schwartz et al., 1993). Interpretation of serum TM concentrations must take into account the lack of absolute specificity of MAbs for cancer detection or for a single type of neoplasm. Also, development of a serum TAA assay requires establishment of a threshold of reactivity by which antigen levels in cancer patients can be distinguished from levels encountered in normal individuals or in patients with non-neoplastic conditions (Jotti and Bombardieri, 1990; Bates, 1991). Compared with human medical oncology, veterinary oncology has not yet profited significantly from the use of MAbs for identification of TAAs or as an adjunct to conventional methods of cancer diagnosis and management. The most common application of MAbs has been in diagnostic pathology, wherein commercially available MAbs are employed to demonstrate tumor differentiation antigens (e.g. intermediate filaments) in tissue sections (Andreasen et al., 1988; Hellman and Lindgren, 1989; Cardona et al., 1989; Moore et al., 1989; Sandusky et al., 1991; Destexhe et al., 1993; Griffey et al., 1993). Other tumor markers that have been identified in canine tumor specimens include carcino-embryonic antigen (Ribas et al., 19891, a carbohydrate antigen (Haines et al., 1989), human breast tumor-associated antigens (Clemo et al., 1993; Mottolese et al., 1994), and TAAs recognized by MAbs generated against a canine mammary carcinoma cell line (Gangopadhyay and Wolfe, 1989), a canine melanoma cell line (Oliver and Wolfe, 19921, or canine mesothelioma cells (Liu et al., 1994). Mammary tumors are the most common type of neoplasm in the female dog, and at least 25% of canine mammary tumors are malignant (Moulton, 1990). Malignant mammary tumors often metastasize and become fatal. Successful clinical management of the canine mammary cancer patient requires early detection of the neoplasm, assessment of treatment success, and monitoring for evidence of remission, recurrence, or metastasis. Physical examination and radiography, the methods used most often for detection and monitoring, are relatively insensitive. Mammary gland tumors smaller than 10 mm in diameter are rarely detected by palpation, and metastatic nodules smaller than 5 mm in diameter cannot be distinguished from surrounding tissue (e.g. lung) by radiography. The rationale for conducting the present study was the need to improve methods of detecting and monitoring tumors in veterinary patients. Our approach was based on reports that progression or remission of human breast cancer could be correlated with levels of TAA in patients’ sera (Salinas et al., 1987; Kim et al., 1988; Tjandra et al.,

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1988; Jensen et al., 1991; Eskelinen et al., 1992; Geraghty et al., 1992; Kouri et al., 1992). We are aware of only one report describing the measurement of TAA concentrations in canine serum, that being the detection of a heat-labile substance in the serum of dogs with transmissible venereal sarcoma (Yang et al., 1991). The antigen in that study was measured by ELISA using polyclonal rabbit antiserum. We employed two MAbs, lAl0 and SB2, which had been generated in our laboratory against canine mammary carcinoma cells. Our objective was to determine the sensitivity and specificity of MAbs lAl0 and SB2 for detection of TAA by competitive enzyme-linked immunosorbent assay (ELISA) in sera from dogs with mammary cancer and other types of epithelial neoplasms. These MAbs had been shown in preliminary studies utilizing immunohistochemical methods, flow cytometric analysis or competitive ELISA to recognize canine TAA in tissue sections of mammary carcinomas and other selected types of carcinomas, in cytocentrifuge preparations of mammary carcinoma cell lines, and in culture fluid (shed antigens) of mammary carcinoma cell lines.

2. Materials and methods

2.1. Study groups Seven study groups of dogs were established: (1) mammary carcinoma; (2) nonmammary carcinoma; (3) sarcoma; (4) benign mammary tumor; (5) benign non-mammary tumor; (6) non-neoplastic disease; (7) disease-free. The tumor and non-neoplastic disease groups consisted of canine patients admitted to the Auburn University Small Animal Clinic or private veterinary hospitals for diagnosis and treatment. Assignment of a dog to a particular tumor group was based on histopathologic diagnosis. Dogs in the non-neoplastic disease group had a wide variety of medical problems as determined from clinical examinations and laboratory findings. The disease-free dogs were either client-owned or were research animals undergoing a baseline work-up for nutritional or parasitological studies. 2.2. Serum samples The serum samples tested for TAA in this study were obtained either specifically for this project or as surplus serum that was submitted initially to the College of Veterinary Medicine’s clinical pathology laboratory for blood chemistry determinations. The former samples were processed and immediately stored in the Department of Pathobiology’s - 70°C serum bank, whereas the latter samples were transferred to the serum bank after storage for 1 week at - 5°C. 2.3. Monoclonal

antibodies

The MAbs SB2 and 1AlO were generated by the authors (J.W. and A.G., respectively) utilizing viable cells derived from CMT-1 (SB2) or CMT-2 (1AlO) canine mammary carcinoma cell lines (Wolfe et al., 1986) as immunogens, and standard

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J. Wanget al./ VeterinaryImmunologyand Immunopathology 48 (1995) 193-207

hybridoma methods as previously described (Harlow and Lane, 1988; Gangopadhyay and Wolfe, 1989). Splenic cells harvested from immunized BALB/c mice were fused with non-secreting P3X63Ag8.653 mouse myeloma cells (Kohler and Milstein, 1975). Hybridoma colonies were screened for antibody production against CMT-2 cells by ELISA and positive clones were expanded and subcloned three times by limiting dilution. Additional screening by competitive ELISA demonstrated that SB2 and 1AlO recognized shed antigens in culture supemates harvested from the CMT-1 and CMT-2 cell lines. Immunohistochemical staining of the cell lines and of formalin-fixed, paraffin embedded tissue sections of canine mammary carcinomas revealed membrane and cytoplasmic staining with MAb SB2 and primarily membrane staining with MAb 1AlO. Isotypic analysis by ELISA using subclass-specific antibodies indicated that MAbs SB2 and 1AlO were IgGl and IgG2b, respectively. Hybridomas producing SB2 and lAl0 were inoculated intraperitoneally (lo7 cells per mouse) into pristane (Sigma Chemical Co., St. Louis, MO) primed BALB/c mice for production of ascitic fluid. The MAbs were purified from ascitic fluid by affinity chromatography using an ImmunoPure Immobilized Protein G Kit (Pierce, Rockford, IL); protein concentrations were determined by the Lowry method (Lowry et al., 19511, and purification was demonstrated by polyacrylamide gel electrophoresis. 2.4. Competitive

ELISA

Canine sera were tested for TAA with a two-step competitive immunoassay. First, the serum specimen was mixed with a predetermined amount of MAb to allow formation of antigen-antibody complexes. Second, the residual (unreacted) MAb in this mixture was detected by its ability to adsorb to solid-phase antigen consisting of tissue culture cells expressing the TAA. CMT-2 mammary carcinoma cells were used to detect residual antibody. They were seeded into 96-well polystyrene tissue culture plates (Corning Glass Works, Corning, NY) at a concentration of 4 x lo4 cells per well, incubated at 37°C overnight, fixed with 0.025% glutaraldehyde for 30 min at 4°C and stored at - 5°C. On the day of an assay, a 96-well plate was retrieved from storage and the cells thawed at room temperature. The wells were washed four times with 0.1% v/v Tween 20 in phosphate-buffered saline (Tween-PBS); 100 ~1 of 10% horse serum in PBS (HS-PBS) were added per well, and the plate was incubated for 30 min to 1 h at room temperature to block non-specific protein-binding sites. Simultaneously with preparation of the plate, 60 ~1 of each test serum were incubated with 60 ~1 of purified MAb lAl0 (10 pg ml-‘) or SB2 (2 pug ml-‘) in a glass tube for 1 h at room temperature. A negative-control sample (pooled sera from disease-free dogs) and a positive-control sample (CMT-2 cell culture supernatant diluted in pooled normal dog serum) were incubated with MAbs lAl0 and SB2 in parallel with test samples. After reaction of the serum with MAb, 50 ~1 of each serum/MAb mixture were transferred in duplicate into wells of the 96-well plate containing the CMT-2 cells. The plate was incubated for 45 min at room temperature, then the wells were washed four times with Tween-PBS and incubated with 50 ~1 of biotinylated horse anti-mouse IgG

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for 15 min. The wells were then washed four times with Tween-PBS and once with distilled water, incubated with 50 ~1 of ABC reagent (Vector Laboratories Inc., Burlingame, CA) for 15 min, and washed again with Tween-PBS and water as in the preceding step. Colored reactant was developed by adding 100 ~1 per well of either 0.1% 2,Zazinobis (3-ethylbenzthiazoline) sulfonic acid (ABTS; Sigma Chemical Co., St. Louis, MO) in 0.05 M citrate-phosphate buffer (pH 5.2) with 0.03% H,O,, or 0.04% o-phenylenediamine (OPD; Sigma Chemical Co.) with 0.02% H,O,. Reactions were stopped by adding 100 ~1 per well of 2% oxalic acid to ABTS solutions or 50 ~1 per well of 2.5 M H,SO, to OPD solutions. Absorbance of the colored product was measured with an ELISA plate reader (Titertek Multiskan, Flow Laboratories, Helsinki, Finland) at 414 nm with ABTS or 492 nm with OPD. 2.5. Standard antibody curve Along with test and control samples, each plate contained dilutions of MAb that encompassed the range of residual antibody concentrations likely to remain after reaction of test serum with MAb. MAb lAl0 was diluted in HS-PBS to protein concentrations of 5.0, 2.5, 1.25, 0.625 and 0 pg ml-‘, and MAb SB2 was diluted to concentrations of 1.0, 0.5, 0.25, 0.125, and 0 pg ml -’ . The serially diluted MAbs were incubated in tubes at room temperature simultaneously with the serum/MAb mixtures, and were similarly dispensed in duplicate and incubated in wells containing CMT-2 cells. Absorbance values generated by the known concentrations of MAb were plotted to construct a standard antibody curve. 2.6. Calculation of inhibitory units The concentration of serum TAA measured by competitive ELISA was expressed as inhibitory units (NJ). The IU values were derived by converting absorbance values in test and control wells to residual antibody concentrations (using the standard antibody curve) and then transforming the residual antibody concentrations to IU using the following formulas: for 1AlO:

IU = (5 - x)/O.1

where 5 is pg of 1AlO ml-’ in the competitive ELISA, x is pg of residual antibody determined from the standard curve and 0.1 is an arbitrary divisor which equates 0.1 pg of inhibited antibody to 1 IU; for SB2:

IU = (1 -x)/O.02

where 1 is pg of SB2 ml-’ in the competitive ELISA, x is pg of residual antibody determined from the standard curve and 0.02 is an arbitrary divisor which equates 0.02 Fg of inhibited antibody to 1 IU. 2.7. Assay rralidation Acceptability of the results from each plate was judged by examining the standard antibody curve as well as the IU values generated from positive- and negative-control

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samples. The standard antibody curve was considered valid if its R linear regression was 0.96 or greater. Limits of acceptability for IU were defined by the mean f 1 SD of positive- and negative-control the first 25 plates having acceptable standard curves. Intra-assay variability was assessed by testing each of two serum on one plate; interassay variability was assessed by testing a panel and negative samples on each of at least 3 different days. 2.8. Establishment

value obtained by values of controls samples tested on samples six times of known positive

of upper limits of normal

Sera from disease-free dogs were tested with MAbs 1AlO (n = 200 dogs) and SB2 (n = 192 dogs) by competitive ELISA to determine the upper limit of TAA concentrations in normal sera. The upper limit of normal (the positive/negative cutoff) was defined as the mean IU & 2 SD of TAA levels. 2.9. Statistical

analysis

Dunnett’s one-tailed t-test was used to determine if the frequency of positive sera within any test group of dogs was significantly greater (P < 0.05) than the frequency of positive sera within the control group of disease-free dogs.

3. Results TAA in sera were detected by their ability to compete with cell-associated antigens for MAb binding. The two serum antigens detected by MAbs 1AlO and SB2 were designated canine tumor-associated serum antigen-l (CTASA-1) and CTASA-2, respectively. 3.1. Antigen levels in disease-pee

dogs

The mean f 1 SD of CTASA-1 concentrations in sera from 200 disease-free dogs was 7.96 f 5.66 IU; the mean f 1 SD of CTASA-2 in sera from 192 disease-free dogs was 8.96 f 6.34 IU. The upper limit of normal, defined as the mean f 2 SD of results obtained with sera from these healthy dogs, was 20 IU for CTASA-1 and 22 IU for CTASA-2. Ten of 200 disease-free dogs (5.0%) had CTASA-1 concentrations above 20 IU, and three of 192 disease-free dogs (1.6%) had CTASA-2 concentrations above 22. 3.2. Antigen levels in dogs with neoplastic disease Serum TAA levels were measured in dogs from which tumors had been excised and examined histopathologically. Based on the histopathologic diagnosis, dogs were assigned to one of five groups: mammary carcinoma, benign mammary tumor, nonmammary carcinoma, sarcoma, or benign non-mammary tumor. Tumors of mammary gland origin were emphasized in this study because canine mammary carcinoma cells

.I. Wang et al. / Veterinary Immunology and Immunopathology 48 (199.5)193-207

:: .:. ... ::. .. : ..::.. ....... .....:.. ....... ....

:: :: . .. .&

.-.-. .J.

.. :: ”

.:. :.

.. Mammary CarClnOma

h=49)

Benw mammarv f”mol,“=23)

2::::::::. .. . . . . . . .. .::::. . . . .. . . . ::::::’ .. . . . .. . ::::.

.. . . . . . ........... .. . Non-mammary CarClnOma

,n=1681

.... :: .. ..+. . . .. . . ...... .. ... ... ..... .... .... ....... .. ... ..... .. .. ..... . .... .. . .. .... . .. .... . ..“..

.: .. ..:..

.A. . .... . ... . ..:: y:::.. ..::.. . ... .... ... . . . . ..

. ..... . . . . . . . . .-.Y.:. . . . . . . Sarcoma

,” = ,471

199

.;;I.. .. . . . . . . .. . . . . . . , Benlg”

non-mammary tUrnOrI” = 100)

Fig. 1. Concentration of CTASA-1 in serum of dogs with neoplastic disease. Each point represents a serum sample from a different patient. Assignment to groups was based on histopathologic diagnosis. The horizontal line indicates the upper limit of normal (mean + 2 S.D. of CTASA-I in 200 disease-free dogs equals 20).

had been used as the immunogen to generate MAbs lAl0 and SB2. Most serum samples were collected within 2 days prior to surgical excision of the tumor. CTASA-1 concentrations detected with MAb lAl0 were above the normal limit in 15 of 49 dogs with mammary carcinoma (30.6%), zero of 23 with benign mammary tumor, 40 of 168 with non-mammary carcinoma (23.8%), 16 of 147 with sarcoma (10.9%), and eight of 100 with benign non-mammary tumor (8.0%). The distribution of IU values is illustrated in Fig. 1. Compared with the control group of disease-free dogs, the frequency of sera positive for CTASA-1 was significantly greater (P < 0.05) among dogs with mammary or non-mammary carcinoma but was not significantly different in the other study groups. Table 1 displays the frequency of positive samples, as well as the mean and range of CTASA-1 concentrations in positive versus negative sera, from dogs with selected types of carcinoma. The percentage of samples positive for CTASA-1 ranged from 0% (prostatic carcinoma, adrenocortical carcinoma) to 100% (bile duct carcinoma). CTASA-2 concentrations measured with MAb SB2 were above the normal limit in 18 of 44 dogs with mammary carcinoma (40.9%), two of 19 with benign mammary tumor (10.5%), 39 of 136 with non-mammary carcinoma (28.7%), 12 of 117 with sarcoma (10.3%), and seven of 70 with benign non-mammary tumor (10.0%) (Fig. 2). Compared with the control group, the frequency of sera positive for CTASA-2 was significantly greater (P < 0.05) among dogs with mammary carcinoma, non-mammary carcinoma, and sarcoma, but was not significantly different in the other groups. Among dogs with mammary carcinoma, the distribution of CTASA-2 concentrations was bimodal (Fig. 2). The percentage of positive samples from dogs with selected types of carcinoma, and the mean and range of CTASA-2 concentrations in positive versus negative sera from those dogs, are displayed in Table 1. The percentage of samples positive for CTASA-2 ranged from 0% (hepatocellular carcinoma, adrenocortical carcinoma) to 100% (bile duct carcinoma).

of CTASA-1

Mammary gland Integumentary system Squamous cell Sweat gland Ceruminous gland Anal sac Perianal gland Respiratory system Nasal Lung Alimentary system Gastric Intestinal Bile duct Hepatocellular Urinary bladder Prostate Endocrine gland Adrenal cortex Pancreatic islet

Tumor

Table 1 Concentrations

29.0 (21-39) 36.7 (36-38) 29.5 29.6 (21-39) 36.3 (30-43) 36.2 29.2 (21-35)

33% 33%

20% 25% 100% 33% 33% 0%

0% 40%

24 6

5 12 3 3 9 5

5 5 26.3 (25-28)

35.8 (21-39) 28.0 25.6 20.6 25.9

17% 14% 25% 33% 13%

35 7 4 3 8

3 10 3 3 7 5

7.8 (O-17) 9.0 (O-19)

9.6 (O-16) 7.1 (O-13)

5 5

19 5

8.9 (O-19) 9.5 (3-14)

7.5 (4-11) 12.8 (l-17) 8.0 (O-14)

28 5 3 3 7

44

(O-19) (O- 9) (7-14) (4-10) (O-16)

10.1 3.9 11.4 7.0 9.0

10.0 (O-201

(Range)

29.4 (20-39)

(Range)

31%

49

0% 40%

67% 20% 100% 0% 29% 20%

47% 40%

32% 0% 67% 67% 14%

41%

% Pos

37.9 (36-40)

26.3 (25-28) 32.5

32.4 (30-35) 31.3 (28-34) 42.9 (38-46)

28.3 (23-40) 36.3 (32-39)

28.7 (26-32) 30.8 (27-35) 25.5 (n/a>

33.9 (23-48)

32.9 (24-42)

(Range)

Positive IU Mean

No.

Negative Me&

Positive Mean

No.

% Pos

Samples tested for CTASA-2

in serum samples from dogs with carcinoma

Samples tested for CTASA-1

and (JTASA-2

11.7 (2-21) 8.3 (7-10)

9.8 (5-14) 8.5 (6-13) 9.5 (O-7)

4.1 8.5 (2-18)

10.9 (O-19) 8.6 (O-20)

9.5 (O-19) 9.9 (7-15) 9.5 12.1 7.6 (O-16)

8.9 (l-18)

(Range)

Negative IU Mean

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Fig. 2. Levels of CXASA-2 in serum of dogs with neoplastic disease. Each point represents a serum sample fry” a .different patient. Assignment to groups was based on histopathologic diagnosis. The horizontal line imhcates the upper limit of normal (mean k 2 SD. of CTASA-2 in 192 disease-free dogs equals 22).

Measurement of both CTASA-1 and CTASA-2, rather than one or the other, increased the sensitivity for identification of antigen-positive sera from dogs with carcinomas (Table 2). Moreover, test specificity declined only slightly. Among sera from 177 disease-free dogs that were tested with both MAbs, 94.9% were negative with lA10, 98.3% were negative with SB2, and 93.2% were negative with both MAbs. The sarcoma group encompassed 15 different types of malignant mesenchymal tumors. Sixteen dogs with sarcoma were positive for CTASA-1 (10.9%, mean IU value: 27.0), and 12 dogs were positive for CTASA-2 (10.3%, mean IU value: 26.2). The mean IU values of samples negative for CTASA-1 or CTASA-2 were 8.4 and 10.4, respec-

Table 2 Increased assay sensitivity with carcinoma Tumor

Mammary gland (43) a Skin Squamous cell (27) Respiratory system Nasal (18) Pulmonary (5) Alimentary system Rectal (7) Other Transitional cell (7) Islet cell (5)

conferred

by measuring

two antigens

rather than a single antigen in sera of dogs -

Number (and %) with positive TAA levels CTASA- 1

CTASA-2

CTASA-1 and/or CTASA-2

14 (32.6)

18 (41.9)

22 (51.2)

4 (14.8)

9 (33.3)

11 (40.7)

7 (38.9) 2 (40.0)

8 (44.4) 2 (40.0)

9 (50.0) 3 (60.0)

3 (42.9)

l(14.3)

3 (42.9)

4 (57.1) 2 (40.0)

2 (28.6) 2 (40.0)

5 (71.4) 3 (60.0)

a Number of dogs tested with both monoclonal

antibodies.

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tively. Of the six most common types of sarcoma in this study (osteosarcoma, mast cell tumor, hemangiosarcoma, lymphosarcoma, fibrosarcoma, chondrosarcoma), hemangiosarcoma and fibrosarcoma were over-represented among the positive samples. Dogs positive for CTASA-1 and/or CTASA-2 numbered seven of 25 (28%) with hemangiosarcoma and three of ten (30%) with fibrosarcoma. Histologic diagnosis in the benign non-mammary tumor group included the spectrum of benign epithelial and mesenchymal tumors. Eight of 100 dogs with benign nonmammary tumor were positive for CTASA-1 (8.0%). The mean of the positive samples was 28.1, while the mean of the negative samples was 9.0. Of 70 dogs with benign non-mammary tumor that were tested with MAb SB2, seven were positive (10.0%); the mean of the positive sera was 25.0 and the mean of the negative sera was 11.0. 3.3. Antigen levels in dogs with non-neoplastic

disease

To test the specificity of the competitive ELISA for neoplastic disease, sera were evaluated from dogs that were diagnosed clinically as having a variety of medical problems exclusive of neoplasia. The mean + 1 SD of CTASA-1 concentrations within this group was 9.23 IU &-7.59; the mean + 1 SD of CTASA-2 concentrations was 10.38 IU f 7.15, Positive IU concentrations were detected with MAb lAl0 in 15 of 216 dogs (6.9%) and with MAb SB2 in 14 of 212 dogs (6.6%). Tumors were not apparent in any dogs at the time of admission, and review of hospital records of the antigen positive dogs did not reveal evidence of neoplasia during the 2 years since the serum samples were collected. The most common diagnoses among the 26 dogs in this group that were positive with at least one of the MAbs were hepatic disorders: seven antigen-positive dogs were diagnosed histologically as having hepatitis or hepatopathy at the time of serum collection. 3.4. Reproducibility

of serum antigen assay

Variability within an assay was determined by testing two serum samples in replicates of six on a plate. The coefficients of variation for the two samples were 21.7% and 5.3% with MAb lA10, and 13.5% and 6.5% with MAb SB2. Interassay variability was evaluated by testing three positive and eight negative samples with MAb 1AlO on each of at least 3 different days. The coefficients of variation ranged from 5.4% to 11.0% among the positive samples and from 17.1% to 62.1% among the negative samples. Accuracy of the competitive ELISA for CTASA-1 was also assessed by testing at least five serum samples that had been collected on different days from each of 12 TAA-negative dogs with neoplastic disease (total of 83 samples). None of the 83 samples had a CTASA-1 concentration above 20.

4. Discussion The results of this study indicate that dogs with certain types of epithelial cancer have TAA in their serum that can be detected with a MAb-based ELISA. The finding of

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serum TAA in canine cancer patients is consistent with reports that some human cancer patients have circulating TAA detectable by immunoassay (Bates, 1991; Sell, 1992). The competitive ELISA for serum TAA in this study was less sensitive than immunoassays described in similar studies of human TAA (Stacker et al., 1988a; Tjandra et al., 1988; Devine et al., 1993). However, the sensitivity was enhanced by use of two MAbs as a panel. Application of an antibody panel has proven valuable in detection of serum TM in human patients when no single monoclonal or polyclonal antibody possesses sufficient sensitivity (Hirota et al., 1985; Jensen et al., 1991; Nicolini et al., 1991; Guadagni et al., 1993). In addition to their use as a panel, MAbs lAl0 and SB2 might be more effective if combined as a cocktail prepared by mixing optimal dilutions of each antibody (Shitara et al., 1989). For this approach to be reliable, however, each MAb must recognize a distinct epitope so that antigenic competition does not occur (Linsley et al., 1988). Regardless of the sensitivity that a panel of MAbs might provide, serum TAA testing is not likely to be used for cancer screening in dogs. Serum TM assays generally have unacceptably low predictive values in clinically healthy individuals (Bates, 1991). With the exception of tests for prostate-specific antigen (Catalona et al., 1991), serum TAA assays are not used for routine cancer screening in human medicine (Van der Schouw et al., 1992; Wobbes et al., 1992). Some information useful to prognosis might be gained from determining the serum TAA concentration at the time of diagnosis. Generally, a high serum TAA concentration correlates with large tumor mass, extensive metastasis, or advanced disease (Hilkens et al., 1986; Salinas et al., 1987; Colomer et al., 1989). Preoperative levels of TAA can be used to predict the likelihood of recurrence and the duration of remission (Eskelinen et al., 1992; Geraghty et al., 1992; Kouri et al., 1992). Seeking such a relationship between TAA concentration and survival, we reviewed the hospital records of 95 canine carcinoma patients whose serum we had tested for TAA. We were unable to find an association between the IU values of serum TAA and the clinical stage of the tumor or the likelihood of survival. Serial measurement of serum TAA in canine cancer patients might be of value during follow-up after initial diagnosis and treatment. A decreasing concentration of serum TAA in a human cancer patient is thought to reflect successful treatment; conversely, a sustained high concentration is interpreted as incomplete response to treatment (Tjandra et al., 1988; Nicolini et al., 1991). A serum TAA level that falls after treatment but then rises significantly some time later can portend recurrence even before clinical signs appear (Hilkens et al., 1986; Stacker et al., 1988b; Tjandra et al., 1988; Todini et al., 1988; Nicolini et al., 1991). We were not able to determine whether the competitive ELISA described in this report could be used prognostically. Nearly all the patients entered into this study were discharged within a week after diagnosis and lost to follow-up, or were euthanized. One exception was a dog that was positive with both h4Ab lAl0 and MAb SB2 at the time of initial diagnosis of mammary cancer and was monitored for 15 months after surgery. Clinically, the animal showed no signs of recurrence. Competitive ELISA testing of serum samples collected at bimonthly intervals over the 15 month period revealed no reversion to positive serum TAA status. This patient succumbed to accidental injury, and at necropsy there was no gross or histologic evidence of tumor recurrence or metastasis.

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We had hoped that information from hospital records might help to explain an apparent bimodal frequency distribution of CTASA-2 concentrations among patients with mammary carcinoma, but it did not. One cause of such a pattern might be the production of antibodies against mammary carcinoma antigens by some dogs (Rutten et al., 1990). Our assay system may not distinguish between a sample in which no TAA was present, and a sample in which TAA was complexed with endogenous canine antibodies that masked the epitopes recognized by the MAbs. A bimodal distribution of CTASA-2 concentrations could also result from genetic factors. In humans, production of some serum TAA is associated with inheritance of Lewis blood group antigens (Magnani et al., 1982; Van der Schouw et al., 1992). Individuals who lack a particular genetic allele are unable to synthesize the TAA. Such a relationship can only be discovered after complete biochemical characterization of the TAA. Although the sensitivity of the MAb-based competitive ELISA was lower than we had hoped, the specificity was quite high. Among approximately 200 healthy dogs, 5.0% were positive for CTASA-1 and 1.6% were positive for CTASA-2. Positive/negative cutoffs were established with values obtained from those healthy dogs, but specificity of the assay was confirmed by testing more than 200 patients diagnosed with disorders other than neoplasia. Twenty-six dogs with non-neoplastic disease were positive with at least one of the MAbs; the frequency of sera positive with lAl0 was 6.9% and with SB2 was 6.6%. Although tumors were not apparent in these dogs at the time of admission or during limited follow-up the possibility of occult neoplastic disease could not be excluded. There did appear to be an association between CIASA production and benign hepatic disorders, especially hepatitis and corticosteroid-induced hepatopathy. Investigators studying serum TAA in human patients have also observed high TAA concentrations in individuals diagnosed with a variety of benign liver diseases (Nicolini et al., 1991; Collazos et al., 1993). Paradoxically, of the three dogs in our study that were diagnosed with hepatocellular carcinoma, only one was positive for CTASA-1 and none was positive for CTASA-2. Properties of antigens recognized by MAbs lAl0 and SB2 have been partially characterized. CMT-2 cell lysates or CMT-2 culture fluid applied in immunoblot analyses (Liu et al., 1994) indicated that MAb lAl0 binds to a 91-kDa peptide and SB2 binds to a 66-kDa peptide (A. Church Bird, unpublished data, 1994). Our attempts to detect immunoreactive peptides of similar molecular masses in serum by electrophoresis and immunoblotting have been thwarted by their low concentration and by small sample volumes. Flow cytometric analysis and immunohistochemical assays applied to canine mammary carcinoma cell lines or tissue sections of canine carcinomas demonstrated localization of immunoreactivity to plasma membranes. Through production of highly specific MAbs and their application in a competitive ELISA, we have demonstrated that dogs with certain types of malignant epithelial tumors have TAA in their serum. Further investigation, especially monitoring of patients after tumor excision or other treatment, will be necessary to establish whether this assay can be used to improve the clinical outcome in dogs with mammary carcinoma or other malignant neoplasms.

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Acknowledgments This study was supported by the Morris Animal Foundation, Englewood, CO, and by the Scott-Ritchey Research Center at the College of Veterinary Medicine, Auburn University, AL. The authors are grateful to Drs. Ralph A. Henderson, William G. Brewer, Gerald H. Hankes, Eva A. Sartin, Elizabeth M. Whitley, and Martha Thomas for their assistance with clinical diagnoses, treatment, and sample accessions. We greatly appreciate the technical assistance of Lisa Parsons, Monica Stevenson and Angela Kuykendall. This report is published as Publication No. 2497, College of Veterinary Medicine, Auburn University.

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