A Hormone Receptor Positive Mammary Gland Adenocarcinoma In A Llama

 

Bryan M. Waldridge, DVM, MS^†

Leonard H. Billups, DVM^‡

Andra R. Frost, MD^*

David M. McKenzie, DVM, MS^†

Stephen D. Lenz, DVM, PhD§

 

^†Department of Clinical Sciences, School of Veterinary Medicine, Tuskegee, AL

^‡Department of Pathobiology, School of Veterinary Medicine, Tuskegee, AL

^*Department of Pathology, University of Alabama at Birmingham, Birmingham, AL

§Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL

 

Key Words: Camelids, llama, adenocarcinoma, mammary neoplasia, hormone receptors, immunohistochemistry

ABSTRACT

A mammary gland adenocarcinoma was diagnosed in an 8-year-old, ovariectomized, female llama. Biopsy of the mass confirmed a tubular-alveolar mammary gland adenocarcinoma with osseous metaplasia. The llama was euthanized due to extensive local invasion of the mass and poor prognosis for surgical resection. Immunohistochemical staining of the neoplasm was negative for estrogen and progesterone receptors. Normal mammary gland tissue from the llama stained positive for estrogen receptors, but did not stain for progesterone receptors. These results suggest that the carcinoma would be unlikely to respond to hormonal therapy. Mammary gland neoplasia should be included in the differential diagnosis for udder enlargement in female camelids.

Introduction

An 8-year-old female llama was examined for an inguinal mass of unknown duration. The llama had been laparoscopically ovariectomized approximately 5 years earlier as part of a research project to develop the technique in camelids. Physical examination revealed a necrotic mass involving the left inguinal area near the mammary gland and that the llama was underweight (body condition score 4/9).1 A complete blood count revealed anemia (PCV 24.7%, reference range 29.0 to 39.0%; RBC 9.2 X 106/µl, reference range 11.3 to 17.5 X 106/µl) and macrocytosis (MCV 33 fl, reference range 21 to 28 fl). A reticulocyte count was not performed. Serum biochemistries indicated hyperglycemia (203 mg/dL, reference range 90 to140 mg/dL), elevated serum creatinine concentration (3.2 mg/dL, reference range 1.5 to 2.9 mg/dL), hypoalbuminemia (2.5 g/dl, reference range 3.5 to 4.4 g/dL), hypophosphatemia (3.9 mg/dL, reference range 4.6 to 9.8 mg/dL), and hypokalemia (3.5 mEq/L, reference range 4.3 to 5.6 mEq/L).

The llama was anesthetized using a combination of xylazine (0.4 mg/kg, IM), butorphanol (0.04 mg/kg, IM), and ketamine (3.7 mg/kg, IM) to allow a complete examination and biopsy of the inguinal mass. The mass (approximately 15 cm in diameter) extensively involved the left half of the mammary gland. Several draining tracts were present which discharged a fetid, purulent exudate. Palpation revealed that the mass contained several areas of mineralization and extended into the adjacent abdominal wall. Biopsies were obtained using a 6 mm biopsy punch (Miltex Instrument Company, Inc., Lake Success, NY). Histopathological examination indicated a tubular-alveolar mammary gland adenocarcinoma with osseous metaplasia.

The llama was subsequently euthanized due to a poor prognosis for recovery and the lack of likelihood of complete surgical resection of the mass. A complete necropsy was performed and the only significant external finding was the large inguinal mammary mass with multiple surface ulcerations and tracts containing purulent exudate. The mass was lobulated and contained several gritty foci. External inguinal lymph nodes were enlarged and edematous. There were numerous firm, grayish-white, 5 mm-diameter nodules scattered throughout the liver. Pseudomonas aeruginosa was isolated from the udder exudate.

Necropsy specimens were fixed in 10% neutral buffered formalin, processed by routine histological procedures, embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin and eosin. All sections were examined by light microscopy. Microscopic examination of the mammary mass revealed proliferating, solid, acinar, and tubular epithelial structures which were separated into irregular lobules by fibrous connective tissue (Fig. 1). The acinar structures were lined by large epithelial cells several layers thick and formed lobules containing central areas of necrosis (Fig. 2). Tubular structures were occasionally filled with neutrophils and cellular debris. Epithelial cell cytoplasm was abundant and cytoplasmic borders were indistinct. Nuclei varied in size and were often vesicular with prominent nucleoli (Fig. 3). Occasional papillary epithelial projections were observed in tubular structures. The connective tissue stroma frequently contained foci of osseous metaplasia.

A proteinaceous fluid was present in the marginal sinus of the inguinal lymph nodes and there was moderate hyperplasia of the lymphoid follicles. Examination of the liver revealed multiple microabscesses with varying degrees of mineralization, which were walled off by dense, fibrous, connective tissue. There was no evidence of metastasis to regional lymph nodes or other organs.

The mammary gland adenocarcinoma was evaluated for the presence of estrogen receptors and progesterone receptors. Histologic sections (measuring 5.0 µm in thickness) were cut from a paraffin block containing the carcinoma and attached to slides (Superfrost/Plus slides, Fisher Scientific, Pittsburgh, PA) by heating at 60oC for one hour. The sections were deparaffinized in xylene, hydrated in graded alcohols, and placed in Tris buffered saline (0.05 M Tris base, 0.15 M NaCl, Triton X-100 4 drops/L, pH 7.6) before antigen recovery and immunostaining. 

Sections were subjected to low-temperature antigen retrieval with enzymatic pretreatment, which consists of predigestion in 0.1% trypsin (Type II-S from porcine pancreas, Sigma Chemicals, St. Louis, MO) in phosphate-buffered saline for 15 minutes at 37o C followed by incubation in 10 mM citrate buffer, pH 6, for 2 hours at 80oC, as previously described.2,3 After antigen retrieval, sections were incubated with an aqueous solution of 3% hydrogen peroxide followed by incubation with 1% goat serum for 1 hour at room temperature.

Subsequently, sections were incubated with antiestrogen receptor mouse monoclonal antibody (Clone ER88, Biogenex, San Ramon, CA) at 1:30 dilution (0.33 mg/mL total protein) or antiprogesterone receptor mouse monoclonal antibody (Clone PR88, Biogenex, San Ramon, CA) at 1:30 dilution (0.33 mg/mL total protein), diluted in phosphate-buffered saline (pH 7.6) containing 1% bovine serum albumin, 1 mM ethylenediamine tetraacetic acid, and 1.5 mM sodium azide, for 1 hour at room temperature. Primary antibody detection was accomplished using a super-sensitive biotin-streptavidin horseradish peroxidase detection kit (Super Sensitive Biotin-Streptavidin Horseradish Peroxidase Detection Kit, Biogenex, San Ramon, CA). A super-sensitive diaminobenzidine tetrachloride substrate kit (Super Sensitive Substrate Kit, Biogenex, San Ramon, CA) was used to visualize the antibody-antigen complex. Negative controls consisted of histologic sections processed without the addition of primary antibody, but incubated instead with 1% goat serum or mouse IgG (Reagent Grade, 0.33 mg/mL, Sigma Chemical, Saint Louis, MO). Positive-negative human multi-tissue control sections were also included.

There was no staining of the mammary carcinoma with antiestrogen receptor or antiprogesterone receptor. However, a cross section of histologically normal lactiferous duct was present in the immunostained sections. Cells of this duct demonstrated weak to moderate nuclear staining with antiestrogen receptor, but did not stain with antiprogesterone receptor (Fig. 4). Positive staining for antiestrogen receptor was specific only for the nuclei of epithelial cells lining the lactiferous duct. No other cell types in the sections, including epidermis, adnexal structures, or the neoplastic tissue of the mammary gland stained with antiestrogen receptor. This normal duct served as a positive internal control for antiestrogen receptor and indicated the reactivity of this mouse monoclonal antibody with the estrogen receptor in the llama. The negative control sections, which included sections incubated with mouse IgG at the same concentration as the estrogen receptor and progesterone antibodies, had no staining. This pattern of reactivity allows the interpretation of the staining in the mammary carcinoma as truly negative for estrogen receptor. Because there was no staining with antiprogesterone receptor in the normal duct, the ability of this antibody to react with the llama progesterone receptor is uncertain. In a previously reported case of mammary gland adenocarcinoma with extensive multisystem metastasis in a llama, immunohistochemical staining for estrogen receptors was moderately positive in the nucleus and cytoplasm.4

The evaluation of human mammary carcinomas for the presence of estrogen and progesterone receptors is now routine practice and provides information that predicts prognosis and impacts treatment.5 Most investigators6-9 have found that the presence of estrogen receptors in human breast carcinomas (i.e., estrogen receptor-positive breast carcinomas) is a significant, but weak, independent marker of a favorable prognosis. The presence of estrogen and progesterone receptors are considered to be more useful, however, as predictors of responsiveness of a mammary carcinoma to hormonal or antiestrogen therapy. Previous attempts to detect estrogen and progesterone receptors in equine mammary gland adenocarcinomas have been equivocal.10,11 The mammary carcinoma of this llama lacked estrogen receptors and most likely lacked progesterone receptors, although staining results were inconclusive for progesterone receptors. This suggests that the carcinoma would be unlikely to be responsive to hormonal therapy if attempted.

This case is the third reported case of a mammary gland carcinoma in a llama. The neoplasm of this report differs from that reported by Leichner et al4 in that the carcinoma that we report had more differentiated glandular features with frequent acinar and tubular epithelial structures and osseous metaplasia. Additionally, estrogen receptor expression was not detectable in our case. The carcinoma reported by Leichner et al4 was poorly differentiated with rare acinar structure. Estrogen receptor expression was detectable in approximately 30% of carcinoma cells. Hormone receptor immunohistochemistry was not performed on a metastatic mammary gland adenocarcinoma of ductal origin briefly reported by Smith.12 These findings indicate some degree of heterogeneity in mammary neoplasms in llamas, which may be reflected in differences in the aggressiveness of the neoplasia and potential responsiveness to treatment.

References

1. Hilton CD, Pugh DG, Wright JC, et al: How to determine and when to use body weight estimates and body condition scores in llamas. Vet Med 93:1015-1018, 1998.

2. Elias JM, Margiotta M: Low temperature antigen restoration of steroid hormone receptor proteins in routine paraffin sections. J Histotech 22:103-106, 1999.

3. Frost AR, Sparks D, Grizzle WE: Methods of antigen recovery vary in their usefulness in unmasking specific antigens in immunohistochemistry. Appl Immunohistochemistry 8:236-243, 2000.

4. Leichner TL, Turner O, Mason GL, et al: Cutaneous metastases of a mammary carcinoma in a llama. Can Vet J 42:204-206, 2001.

5. Pertschuk LP, Axiotis CA: Steroid hormone receptor immunohistochemistry in breast cancer: past, present, and future. Breast J 5:3-12, 1999.

6. Fisher B, Redmond CK, Wicherham DL, et al: Relation of estrogen and/or progesterone receptor content in breast cancer to patient outcome following adjuvant therapy. Breast Cancer Res Treat  3:335-364, 1983.

7. Menendez-Botet CJ, Schwartz MK: Estrogen and progesterone receptor proteins in patients with breast cancer. Adv Clin Chem 30:185-225, 1993.

8. Nicholson RI, Campbell FC, Blamey RW, et al: Steroid receptors in early breast cancer: value in prognosis. J Steroid Biochem 15:193-199, 1981.

9. Vollenweider-Zerargui L, Barrelet L, Wong Y, et al: The predicative value of estrogen and progesterone receptor concentrations on the clinical behavior of breast cancer in women. Cancer 57:1171-1180, 1986 .

10. Reppas GP, McClintock SA, Canfield PJ, et al: Papillary ductal adenocarcinoma in the mammary glands of two horses. Vet Rec 138:518-519, 1996.

11.  Seahorn TL, Hall G, Brumbaugh GW, et al: Mammary adenocarcinoma in four mares. J Am Vet Med Assoc 200:1675-1677, 1992.

12.  12.   Smith JA. Noninfectious diseases, metabolic diseases, toxicities, and neoplastic diseases of South American camelids. Vet Clin North Am Food Anim Pract 5:101-143, 1989.

 

Figure 1. Mammary mass, llama. Branching, solid cords and duct-like structures lined by proliferating epithelium and separated by abundant fibroblastic stroma. Bar = 70 µm.

 

Figure 2. Mammary mass, llama. Irregular acinar and tubular structures of neoplastic epithelium with central necrosis. Bar = 70 µm.

 

Figure 3. Mammary mass, llama. Neoplastic epithelial cells with prominent cell and nuclear atypia. Bar= 20 µm.

 

Figure 4. Immunohistochemical staining for estrogen receptors. (A) Normal lactiferous duct contains epithelial cells with detectable estrogen receptor expression in the nucleus, as indicated by scattered brown-staining nuclei (arrows). (B) The adenocarcinoma demonstrates negative staining and therefore does not express detectable estrogen receptors. 600X magnification.