Elisabetta Antuofermo

Spontaneous Mammary Intraepithelial Lesions in Dogs—A Model of Breast Cancer

Mammary intraepithelial lesions (IEL) are nowadays frequently diagnosed as a result of the success of mammographic screening, education programs, and awareness by women. Establishment of an animal model for these lesions to test treatment or preventive modalities is a prerequisite for human clinical trials. A model for spontaneous IELs, especially for estrogen receptor (ER)-negative lesions, does not exist. This study describes the histologic and immunohistochemical similarity between human and canine mammary IELs. Mammary tumors from 200 dogs were classified and histologic sections of the excisional specimens were evaluated for IELs. IELs, found in specimens from 60 dogs, were categorized as adenosis, sclerosing adenosis, intraductal papilloma, sclerosing papilloma, ductal hyperplasia, atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS; high, intermediate, and low grade). Most proliferative IELs without atypia were associated with benign tumors, whereas IELs with atypia (ADH and DCIS) were generally associated with mammary cancer. ER-α expression was significantly low or absent in most ADH and DCIS lesions as well as in their associated tumors. Ki67 expression was significantly higher in high-grade DCIS than in hyperplasia or low-grade DCIS. Two thirds of high-grade DCIS lesions were positive for HER-2. Canine mammary IELs were strikingly similar to those of the human breast. The frequency of IELs in the dog, their association with spontaneous mammary cancer, their pattern of ER-α and HER-2 expression, and their histologic resemblance to human IELs may make the dog an ideal model to study human ER-negative (both HER-2 positive and negative) breast cancer progression as well as prevention and treatment. (Cancer Epidemiol Biomarkers Prev 2007;16(11):2247–56)

Spontaneous feline mammary intraepithelial lesions as a model for human estrogen receptor- and progesterone receptor-negative breast lesions

BackgroundBreast cancer is the most frequently diagnosed cancer in women. Intraepithelial lesions (IELs), such as usual ductal hyperplasia (UH), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS) are risk factors that predict a womans chance of developing invasive breast cancer. Therefore, a comparative study that establishes an animal model of pre-invasive lesions is needed for the development of preventative measures and effective treatment for both mammary IELs and tumors. The purpose of this study was to characterize the histologic and molecular features of feline mammary IELs and compare them with those in women.MethodsFormalin-fixed, paraffin-embedded specimens (n = 205) from 203 female cats with clinical mammary disease were retrieved from the archives of the Purdue University Animal Disease Diagnostic Laboratory and Veterinary Teaching Hospital (West Lafayette, IN), and the Department of Pathology and Veterinary Clinic, School of Veterinary Medicine (Sassari, Italy). Histologic sections, stained with hematoxylin and eosin (HE), were evaluated for the presence of IELs in tissue adjacent to excised mammary tumors. Lesions were compared to those of humans. Immunohistochemistry for estrogen receptor (ER-alpha), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2/neu) and Ki-67 was performed in IELs and adjacent tumor tissues.ResultsIntraepithelial lesions were found in 57 of 203 (28%) feline mammary specimens and were categorized as UH (27%), ADH (29%), and DCIS (44%). Most IELs with atypia (ADH and DCIS) were associated with mammary cancer (91%), whereas UH was associated with benign lesions in 53% of cases. Feline IELs were remarkably similar to human IELs. No ER or PR immunoreactivity was detected in intermediate-grade or high-grade DCIS or their associated malignant tumors. HER-2 protein overexpression was found in 27% of IELs.ConclusionThe remarkable similarity of feline mammary IELs to those of humans, with the tendency to lose hormone receptor expression in atypical IELs, supports the cat as a possible model to study ER- and PR-negative breast lesions.

Comparability of differential proteomics data generated from paired archival fresh-frozen and formalin-fixed samples by GeLC-MS/MS and spectral counting.

In this study, a Veterinary Department repository composed by paired formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FrFr) sets of the same tissues, routinely archived in the typical conditions of a clinical setting, was exploited to perform a comparative evaluation of the results generated by GeLC-MS/MS (1-DE followed by in-gel digestion and LC-MS/MS) and spectral counting with the two types of archival samples. Therefore, two parallel differential proteomic studies were performed using 3 canine mammary carcinomas and 3 normal controls in a paired fashion (6 FrFr and 6 FFPE in total). As a result, the FrFr and FFPE differential proteomic datasets exhibited fair consistency in differential expression trends, according to protein molecular function, cellular localization, networks, and pathways. However, FFPE samples were globally slightly less informative, especially concerning the high-MW subproteome. As a further investigation, new insights into the molecular aspects of protein fixation and retrieval were obtained. In conclusion, archival FFPE samples can be reliably used for differential proteomics studies employing a spectral counting GeLC-MS/MS approach, although some typical biases need to be taken into account, and FrFr specimens (when available) should still be considered as the gold standard for clinical proteomics.

Ovis aries Papillomavirus 3: A prototype of a novel genus in the family Papillomaviridae associated with ovine squamous cell carcinoma

Papillomaviruses play an important role in human cancer development, and have been isolated from a number of animal malignancies. However, the association of papillomaviruses with tumors has been poorly investigated in sheep. In this study, a novel ovine Papillomavirus, OaPV3, was cloned from sheep squamous cell carcinoma. Unlike the already known ovine papillomaviruses, belonging to the Delta genus, OaPV3 lacks the E5 open reading frame and maintains the conserved retinoblastoma motif in the E7 gene. OaPV3 infects exclusively epithelial cells, and was found in skin of healthy sheep of geographically separated flocks located in Sardinia (Italy). This new virus is transcriptionally active in tumors and shares low homology with all the other papillomaviruses, establishing a new genus. Taken together, the co-occurrence of OaPV3 and tumors, its cell and tissue tropism, and its gene repertoire, suggests a role for this virus in development of sheep squamous cell carcinoma.

Prevalence and classification of spontaneous mammary intraepithelial lesions in dogs without clinical mammary disease.

Mammary intraepithelial lesions (IELs) are noninvasive epithelial proliferations that include ductal hyperplasia (DH), atypical DH (ADH), and ductal carcinoma in situ (DCIS). In women, IELs are associated with increased risk of invasive breast cancer and form a basis for therapeutic decisions. Similarly, in female dogs, IELs are common in tumor-bearing glands and in non-tumor-bearing glands. To determine the prevalence and types of spontaneous IELs, mammary glands from 108 female dogs without clinical mammary disease were evaluated histologically and immunohistochemically. Within this population, 56 dogs (52%) had at least one type of spontaneous IEL, including DH (49 dogs), ADH (14 dogs), low-grade DCIS (19 dogs), intermediate-grade DCIS (12 dogs), and high-grade DCIS (1 dog). Twenty-one dogs had two or more different IEL types. In 23 of 24 dogs with atypical IELs (ADH or DCIS), immunohistochemical expression was determined for estrogen receptor α (ER-α), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2/neu), and Ki-67. For all markers examined, low-grade DCIS had significantly lower scores than did adjacent nonlesional gland; PR expression was significantly decreased in low-grade DCIS compared to other atypical lesions. Sixty-one lesions were ER-α negative (12 ADH, 36 low-grade DCIS, 13 intermediate-grade DCIS), and no lesions overexpressed HER-2/neu. Based on the dog’s prevalence of spontaneous mammary IELs that precede clinical mammary disease, the remarkable histologic similarity between canine and human IELs, and the loss of ER expression in certain IELs in both species, the dog shows promise as a model for human breast preneoplasia.

Application of 2D-DIGE to formalin-fixed diseased tissue samples from hospital repositories: results from four case studies

In the recent past, the potential suitability of fixed samples to 2D‐DIGE studies has been demonstrated on model tissues, but not on “real‐world” archival tissues. Therefore, this study was aimed to assess the quality of the results delivered by 2D‐DIGE on samples retrieved from hospital tissue repositories.

Mammography and Ultrasound Imaging of Preinvasive and Invasive Canine Spontaneous Mammary Cancer and Their Similarities to Human Breast Cancer

Understanding the evolution of proliferative breast disease such as atypical hyperplasia and carcinoma in situ is essential for clinical management of women diagnosed with these lesions. Therefore, an animal model that faithfully represents human breast disease in every aspect from spontaneity of dysplasia onset, histopathologic features, and genetics to clinical outcome is needed. Previously, we studied canine spontaneous atypical hyperplasia and ductal carcinoma in situ (low, intermediate, and high grade) and reported their similarities to human lesions in histopathologic and molecular features as well as prevalence. To further validate the resemblance of these lesions to humans, we examined their mammographic and sonographic characteristics in comparison with those of humans as well as the potential of the human Breast Imaging Reporting and Data System (BI-RADS) to predict canine disease. Nonlesional, benign, and malignant mammary glands of dogs presented to Sassari Veterinary Hospital were imaged using mammography and ultrasonography. The images where then analyzed and statistically correlated with histopathologic findings and to their similarities to humans. Our results showed that canine mammary preinvasive lesions, benign, and malignant tumors have mammographic abnormalities, including the presence, pattern, and distribution of macrocalcification and microcalcification, similar to their human counterparts. BI-RADS categorization is an accurate predictor of mammary malignancy in canine, with 90% sensitivity and 82.8% specificity. The similarities of mammographic images and the ability of BI-RADS to predict canine mammary malignances with high specificity and sensitivity further confirm and strengthen the value of dog as a model to study human breast premalignancies for the development of prognostic biomarkers. Cancer Prev Res; 4(11); 1790–8. ©2011 AACR.

Mycobacterium chelonae associated with tumor-like skin and oral masses in farmed Russian sturgeons ( Acipenser gueldenstaedtii )

BackgroundNon-tuberculous mycobacteria responsible for piscine mycobacteriosis usually produce visceral granulomas in both freshwater and marine species. In this study, the first occurrence of Mycobacterium chelonae associated with tumor-like lesions in the Russian sturgeon (Acipenser gueldenstaedtii) is reported. Fifteen sturgeons from an Italian fish farm showing skin and oral cauliflower-like masses were investigated by histopathology, bacterial culture and molecular analyses.ResultsA total of 20 masses different in size located in the mouth and in pectoral and caudal fins (characterized by abundant calcium deposits and by mild to moderate granulomatous inflammation) were observed with a significant different degree of histological severity. All internal organs of the fish were negative for mycobacteria, Ziehl-Neelsen was positive in only one of the oral masses, whereas bacterial and PCR analyses detected the presence of M. chelonae for almost all the skin and oral masses. Based on these results, a calcinosis of dystrophic origin associated with a chronic granulomatous inflammation was considered as a primary diagnosis consequent to tissue injury in areas susceptible to trauma.ConclusionsWe hypothesized that the occurrence of M. chelonae in farmed sturgeons was only a secondary event related to its presence in a stressful rearing environment and subsequent to a dystrophic calcinosis occurred in previously damaged tissues.

Brain renin-angiotensin system modifies the blood pressure response to intracerebroventricular cadmium in rats.

In order to elucidate the involvement of the brain renin-angiotensin system (RAS) in cadmium intracerebroventricular (ICV) hypertension, we evaluated the effects of a pretreatment with different drugs: clonidine, an α2 adrenergic agonist, enalapril and captopril, both ACE inhibitors, and saralasin, a competitive nonselective AT1 and AT2 receptor antagonist. We used a rat strain with low levels of kallikrein (LKR) that was more sensitive to ICV cadmium hypertension, compared with normal kallikrein rats (NKRs), the control strain. The interplay between the kallikrein-kinin system and the RAS in the LKR strain caused various hemodynamic alterations, which we believe were the result of elevated RAS activity in these animals. Moreover, we suggest that the defective kallikrein-kinin system in LKR may also cause an alteration in the activation of brain RAS in these animals. The LKR displayed elevated concentrations of plasma AII, hypertrophy of the myocardium, and initial alterations in the renal glomerulotubular system. With the exception of clonidine, all of the other drugs showed greater antihypertensive effects of differing statistical significance in LKR, compared with NKR. Both ACE inhibitors were able to significantly reduce pressor response to cadmium ICV in LKR throughout the experiment, whereas in NKR, they were only able to reduce the hypertensive peak of cadmium. A significant protective effect was also observed in LKR pretreated with saralasin, while no effect was observed in NKR. These findings confirm the presence of brain RAS activation in LKR and its contribution to the central control of pressor response to cadmium ICV.

Bilateral Ovarian Malignant Mixed Müllerian Tumor in a Dog

Malignant mixed Müllerian tumor (MMMT) is a rare neoplasm of the female genital tract. We report a case of bilateral ovarian MMMT in a 10-year-old female dog. Ovaries were only moderately enlarged with a papillary surface and firm nodules. Multiple metastases were observed in the abdominal cavity and pulmonary parenchyma. Histologically, both ovaries had intermingled carcinomatous and sarcomatous components with cartilage and bone. Metastatic lesions were not mixed. The peritoneal metastases were carcinomatous; pulmonary metastases were sarcomatous. Carcinomatous elements of the MMMT were immunohistochemically positive for cytokeratin (CK) AE1/AE3, CK7, and vimentin and estrogen receptors. Conversely, the sarcomatous cells were positive for vimentin, but negative for CKs. Chondrocytes also expressed S-100 protein. On the basis of similarities to human ovarian MMMT, the diagnosis was heterologous malignant mixed Müllerian tumor of the ovary.