Enrichment and isolation of breast cancer stem cells from breast cancer transplantation tumors in nude mice / 肿瘤研究与临床

Objective:Enriching and isolating breast cancer stem cells from breast cancer transplantation tumors in nude mice.Methods:Human breast cancer MDA-MB-231 cells were injected into the right axilla subcutaneous of 20 nude mice, and the tumor growth was observed .After 30 days, tumors were isolated and stained with hematoxylin and eosin, and then tumor cells from tissues were isolated. DMEM medium containing serum was used to cultivate isolated transplantation tumor cells, cell morphology and growth were also observed. Flow cytometry was used to detect the proportion of stem cells (CD44 +/CD24 -/low cells) in transplantation tumor cells. Serum-free DMEM medium containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and B27 cell supplement were used to cultivate transplantation tumor cells and to obtain cell microspheres. The proportion of stem cells on the 10th day in cell microspheres was detected by using flow cell sorter and stem cells were isolated according to the markers of cell surface. Results:After subcutaneously injecting MDA-MB-231 cells into 20 nude mice for 9 days, 17 nude mice had subcutaneous tumors with more parenchymal cells, little interstitial cells, arranged cords tumor cells, large volume of the cell and abundant cytoplasm, the nuclei in different sizes and hyperchromatic state, mitotic more common, the nucleoli clear and obvious pleomorphy. After cultivating transplantation tumor cells with DMEM medium containing serum, the cells began to grow adherent after 24 h, and the adherent proportion rose to 60% after 3 days; after 7 days, the cell proliferation was accelerated; and the cell morphology was more consistent, most of which were spindle shaped and were not significantly different from MDA-MB-231 cells; the proportion of stem cells in transplantation tumor cells was (0.10±0.02)%. After cultivating transplantation tumor cells with serum-free DMEM medium containing cell cultured supplement, the cells grow in spherical patterns, the proportion of stem cells in cell microspheres got up to (70.47±2.03)% on the 10th day.Conclusions:Subcutaneously injecting MDA-MB-231 cells in nude mice can build breast cancer nude mice ectopic transplantation tumor model. Breast cancer stem cells in the transplantation tumors can be enriched from isolated transplantation tumor cells through serum-free medium, and more stem cells can be isolated to provide the research basis for the biological characteristics of breast cancer stem cells.

Oxidative stress in female dogs with mammary neoplasms

ABSTRACT: The result of the reaction of free radicals with biomolecules is the formation of substances with the potential of inducing oxidative damage, a condition known as oxidative stress. There are voluminous literature data reporting the association, both as a cause and as a consequence, between different diseases and oxidative stress. In this study, 144 female dogs with mammary neoplasia were analyzed. The animals were submitted to clinical evaluation for disease staging, hematological evaluation, serum biochemistry (renal and hepatic function tests), and dosage of the oxidative damage biomarker, malondialdehyde (MDA), at the time of its approach and 30 days after treatment. A control group of 100 healthy animals was also submitted to determination of serum MDA levels. The mean age of the animals affected by mammary neoplasms was 9.88±2.95 (4 to 14) years, while in healthy animals it was 2.31±1.90 years (1 to 6). Of the 144 animals, 113 (78.9%) had malignant neoplasms, and 15, 21, 46, 17 and 14 animals were in clinical stage I, II, III, IV and V respectively and the carcinoma in a mixed tumor was the most frequent histological pattern in this group (26%). Thirty-one animals were diagnosed with benign neoplasms and mammary adenoma was the most frequent histological pattern in 15 animals (51.61%). Hematological changes in the preoperative period were observed in 44 (38.9%) and 12 (38.7%) animals with malignant and benign neoplasias, respectively, and there was a positive correlation between anemia and higher levels of MDA (P=0.0008) for animals with malignant tumors. Regarding serum biochemical parameters, the most frequent alterations in animals with malignant neoplasms were elevated ALT levels in 12 animals (10.6%), creatinine in 10 animals (8.84%) and urea in eight animals (7.07%). Females with benign neoplasms presented less occurrence of changes in these parameters. In the group of healthy animals (control), the mean serum MDA values were 12.08±4.18, whereas in the pre-treatment group, mean MDA was 24.80±5.74 for bitches with benign neoplasms and 32.27±10.24 for bitches with malignant tumors. A significant increase (P 0.001) in MDA levels was observed in animals with malignant mammary neoplasms when compared to healthy animals and with benign tumors. In addition, a significant reduction (P 0.001) was observed 30 days after treatment in MDA levels (27.37±7.86) in animals with malignant tumors. In conclusion, our results indicate an association between MDA seric levels and mammary neoplasms in dogs. The results suggest that this factor can be used as a biomarker of oxidative stress with a potential impact in the prognostic of mammary tumors, since significantly higher levels of MDA were detected especially in dogs carrying malignant tumors and presenting anemia.

RESUMO: O resultado da reação de radicais livres com biomoléculas é a formação de substâncias que podem ser utilizadas como marcadores de dano oxidativo, condição mais conhecida como estresse oxidativo. Evidências científicas comprovam a relação, quer como causa, quer como consequência, entre muitas doenças e o estresse oxidativo. Neste estudo, 144 cadelas portadoras de neoplasia de mama, foram submetidas à avaliação clínica para estadiamento da doença, avaliação hematológica, testes de função renal e hepática e dosagem do biomarcador de dano oxidativo, malondialdeído (MDA), no momento de sua abordagem e 30 dias após realização de tratamento. Um grupo controle de 100 cadelas saudáveis foi submetido também à determinação dos níveis séricos de MDA. A idade média dos animais acometidos por neoplasias mamárias foi de 9,88±2,95 (4 a 14) anos, enquanto que nos animais saudáveis foi de 2,31±1,90 anos (1 a 6). Dos 144 animais, 113 (78, 9%) apresentavam neoplasias malignas, sendo que 15, 21, 46, 17 e 14 animais encontravam-se em estadiamento clínico I, II, III, IV e V respectivamente, e o carcinoma em tumor misto foi o padrão histológico mais frequente neste grupo (26%). Trinta e um animais tiveram diagnóstico de neoplasias benignas, sendo que 7 estavam no estádio I, 16 no estádio II e 8 no estádio III e o adenoma mamário foi o padrão histológico mais frequente em 15 animais (51,61%). Alterações hematológicas no período pré-operatório foram observadas em 44 (38,9%) e 12 (38,7%) animais portadores de neoplasias malignas e benignas, respectivamente, sendo que houve correlação positiva entre anemia e níveis mais elevados de MDA (P=0,0008), para os animais com tumores malignos. Em relação aos parâmetros bioquímicos séricos, as alterações mais frequentes nos animais com neoplasias malignas foram a elevação dos níveis de ALT em 12 animais (10,6%), de creatinina em 10 animais (8,84%) e de ureia em oito animais (7,07%) Cadelas portadoras de neoplasias benignas apresentaram menor ocorrência de alterações nesses parâmetros. No grupo controle, a média dos valores séricos de MDA foi 12,08±4,18, enquanto que no grupo pré-tratamento, a média de MDA foi de 24,80±5,74 para as cadelas com neoplasia benigna e 32,27±10,24 para as neoplasias malignas. Verificou-se aumento significativo do valor sérico de MDA em cadelas portadoras de neoplasias malignas em comparação com os animais hígidos ou com neoplasias benignas (P 0,001). Ainda, 30 dias após o tratamento observou-se uma diminuição significativa (P 0,001) no valor médio de MDA (27,37±7,86) nos animais com neoplasias malignas. Em conclusão, os resultados deste estudo evidenciam uma associação entre níveis séricos aumentados de MDA e presença de neoplasias mamárias em cadelas. Os resultados sugerem que este fator pode ser utilizado como biomarcador de estresse oxidativo em cães, com provável impacto no prognóstico dos tumores mamários, uma vez que níveis significativamente mais altos de MDA foram detectados especialmente nas cadelas portadoras de tumores malignos e apresentando anemia.

Oxidative stress in female dogs with mammary neoplasms / Estresse oxidativo em cadelas com neoplasia mamária

The result of the reaction of free radicals with biomolecules is the formation of substances with the potential of inducing oxidative damage, a condition known as oxidative stress. There are voluminous literature data reporting the association, both as a cause and as a consequence, between different diseases and oxidative stress. In this study, 144 female dogs with mammary neoplasia were analyzed. The animals were submitted to clinical evaluation for disease staging, hematological evaluation, serum biochemistry (renal and hepatic function tests), and dosage of the oxidative damage biomarker, malondialdehyde (MDA), at the time of its approach and 30 days after treatment. A control group of 100 healthy animals was also submitted to determination of serum MDA levels. The mean age of the animals affected by mammary neoplasms was 9.88±2.95 (4 to 14) years, while in healthy animals it was 2.31±1.90 years (1 to 6). Of the 144 animals, 113 (78.9%) had malignant neoplasms, and 15, 21, 46, 17 and 14 animals were in clinical stage I, II, III, IV and V respectively and the carcinoma in a mixed tumor was the most frequent histological pattern in this group (26%). Thirty-one animals were diagnosed with benign neoplasms and mammary adenoma was the most frequent histological pattern in 15 animals (51.61%). Hematological changes in the preoperative period were observed in 44 (38.9%) and 12 (38.7%) animals with malignant and benign neoplasias, respectively, and there was a positive correlation between anemia and higher levels of MDA (P=0.0008) for animals with malignant tumors. Regarding serum biochemical parameters, the most frequent alterations in animals with malignant neoplasms were elevated ALT levels in 12 animals (10.6%), creatinine in 10 animals (8.84%) and urea in eight animals (7.07%). Females with benign neoplasms presented less occurrence of changes in these parameters. In the group of healthy animals (control), the mean serum MDA values were 12.08±4.18, whereas in the pre-treatment group, mean MDA was 24.80±5.74 for bitches with benign neoplasms and 32.27±10.24 for bitches with malignant tumors. A significant increase (P<0.001) in MDA levels was observed in animals with malignant mammary neoplasms when compared to healthy animals and with benign tumors. In addition, a significant reduction (P<0.001) was observed 30 days after treatment in MDA levels (27.37±7.86) in animals with malignant tumors. In conclusion, our results indicate an association between MDA seric levels and mammary neoplasms in dogs. The results suggest that this factor can be used as a biomarker of oxidative stress with a potential impact in the prognostic of mammary tumors, since significantly higher levels of MDA were detected especially in dogs carrying malignant tumors and presenting anemia.(AU)

O resultado da reação de radicais livres com biomoléculas é a formação de substâncias que podem ser utilizadas como marcadores de dano oxidativo, condição mais conhecida como estresse oxidativo. Evidências científicas comprovam a relação, quer como causa, quer como consequência, entre muitas doenças e o estresse oxidativo. Neste estudo, 144 cadelas portadoras de neoplasia de mama, foram submetidas à avaliação clínica para estadiamento da doença, avaliação hematológica, testes de função renal e hepática e dosagem do biomarcador de dano oxidativo, malondialdeído (MDA), no momento de sua abordagem e 30 dias após realização de tratamento. Um grupo controle de 100 cadelas saudáveis foi submetido também à determinação dos níveis séricos de MDA. A idade média dos animais acometidos por neoplasias mamárias foi de 9,88±2,95 (4 a 14) anos, enquanto que nos animais saudáveis foi de 2,31±1,90 anos (1 a 6). Dos 144 animais, 113 (78, 9%) apresentavam neoplasias malignas, sendo que 15, 21, 46, 17 e 14 animais encontravam-se em estadiamento clínico I, II, III, IV e V respectivamente, e o carcinoma em tumor misto foi o padrão histológico mais frequente neste grupo (26%). Trinta e um animais tiveram diagnóstico de neoplasias benignas, sendo que 7 estavam no estádio I, 16 no estádio II e 8 no estádio III e o adenoma mamário foi o padrão histológico mais frequente em 15 animais (51,61%). Alterações hematológicas no período pré-operatório foram observadas em 44 (38,9%) e 12 (38,7%) animais portadores de neoplasias malignas e benignas, respectivamente, sendo que houve correlação positiva entre anemia e níveis mais elevados de MDA (P=0,0008), para os animais com tumores malignos. Em relação aos parâmetros bioquímicos séricos, as alterações mais frequentes nos animais com neoplasias malignas foram a elevação dos níveis de ALT em 12 animais (10,6%), de creatinina em 10 animais (8,84%) e de ureia em oito animais (7,07%) Cadelas portadoras de neoplasias benignas apresentaram menor ocorrência de alterações nesses parâmetros. No grupo controle, a média dos valores séricos de MDA foi 12,08±4,18, enquanto que no grupo pré-tratamento, a média de MDA foi de 24,80±5,74 para as cadelas com neoplasia benigna e 32,27±10,24 para as neoplasias malignas. Verificou-se aumento significativo do valor sérico de MDA em cadelas portadoras de neoplasias malignas em comparação com os animais hígidos ou com neoplasias benignas (P<0,001). Ainda, 30 dias após o tratamento observou-se uma diminuição significativa (P<0,001) no valor médio de MDA (27,37±7,86) nos animais com neoplasias malignas. Em conclusão, os resultados deste estudo evidenciam uma associação entre níveis séricos aumentados de MDA e presença de neoplasias mamárias em cadelas. Os resultados sugerem que este fator pode ser utilizado como biomarcador de estresse oxidativo em cães, com provável impacto no prognóstico dos tumores mamários, uma vez que níveis significativamente mais altos de MDA foram detectados especialmente nas cadelas portadoras de tumores malignos e apresentando anemia.(AU)

Tumorsphere formation and cancer stem cell characterization of REM134 canine mammary carcinoma cells / 대한수의학회지

Canine mammary tumors are among the most frequently observed cutaneous tumors in female dogs. Cancer stem cells (CSCs), referred to as tumor-initiating cells, are thought to have properties similar to normal stem cells such as the ability to self-renewal and to differentiate into various cell types. Biological understanding of CSCs and the critical pathways involved in their maintenance are important in research and therapy for mammary tumors. We conducted the present study on sphere formation from REM134 cells by using methylcellulose to produce tumorspheres on a large scale and compared the specific markers of the spheres-formed and plating-cultured REM134 cells. The results revealed that the tumorspheres cultured in methylcellulose had higher seeding density and improved morphology compared to those produced in normal sphere formation medium. Expression levels of stemness markers and CSC-related markers were higher in tumorsphere-forming cells than in plating-cultured cells. Subsequently, we transplanted the tumorsphere-forming and plating-cultured cells into female nude mice to examine their tumorigenic potential. Tumor volume increased rapidly in mice transplanted with tumorsphere-derived cells compared to plating-cultured cells. We observed a novel sphere-forming condition for REM134 cells and showed that REM134 cell tumorspheres can exhibit improved CSC properties.

Evolución natural del tumor filodes de mama. Reporte de un caso / Natural evolution of breast phyllodes tumor. A case report

Resumen Antecedentes: Los tumores filodes se originan del estroma de la glándula mamaria. Su incidencia es menor de 1% y afecta a mujeres de 35 a 55 años. Suelen aparecer como nódulos móviles, redondeados, indoloros y de crecimiento rápido. El tratamiento de elección para tumores benignos y malignos es la escisión local, con márgenes menores de 1 cm. Caso clínico: Paciente de 42 años, nulípara, sin antecedentes familiares de cáncer de mama, quien percibió un aumento de volumen en la mama izquierda. En la exploración física se palpó una tumoración de gran tamaño, en el cuadrante inferior interno de la mama izquierda, de crecimiento progresivo hasta deformar la glándula. Mediante ultrasonido se clasificó el tumor como BIRADS IV. Se obtuvo una biopsia por trucut, con la que se estableció el diagnóstico histopatológico de tumor filodes de bajo grado de malignidad. Se llevó a cabo el tratamiento radical con mastectomía. El estudio histopatológico final reportó: tumor filodes de bajo grado con metaplasia condroide focal. Conclusión: Es importante profundizar en la estirpe histológica del tumor filodes, con la finalidad de establecer el tratamiento adecuado y detener la evolución natural de la enfermedad, que en casos extremos provoca la destrucción total de la glándula mamaria.

Abstract Background: Phyllodes tumors originate from the stroma of the mammary gland. The frequency is fewer than 1% predominating in women between 35 and 55 years. They typically present as moving, rounded nodules, usually painless, and may have rapid growth. The treatment of choice is the local excision of the lesion, both for benign and malignant forms, with margins of less than 1 cm. Clinical case: Female patient, 42 years old, nulliparous, with no family history of breast cancer. In February 2016 she noticed an increase in volume in the lower internal quadrant of the left breast, which is why she attended with multiple doctors receiving unspecified treatments. He consulted us by presenting a large tumor in the lower internal quadrant of the left breast, whose growth was progressive until the mammary gland was deformed. He underwent ultrasound, which reports BIRADS IV proceeding to perform a Trucut Biopsy; with a histopathological diagnosis of Phyllodes tumor of low grade of malignancy, for which he undergoes radical treatment by means of a mastectomy on February 22, 2017. The final histopathological study reports: Low grade Phyllodes tumor with focal chondroid metaplasia. Conclusion: It is important to deepen the histological strain of the phyllodes tumor, in order to establish the appropriate treatment and stop the natural evolution of the disease, which in extreme cases causes the total destruction of the mammary gland

Differential and correlated expressions of p16/p21/p27/p38 in mammary gland tumors of aged dogs

The inhibitory effect of neutering on mammary gland tumor development in dogs has been well described. However, we observed that the effect of neutering on tumor malignancy may be altered by aging. Therefore, we characterized mammary tumors in aged dogs by analyzing the expression of cellular senescence markers. Expressions of p16, p38, p21, and p27 antibodies, which are senescence-associated markers, were assessed in canine mammary tumors of aged dogs via immunohistochemical analysis. In addition, correlations between those expressions were analyzed. Expression of p16 was negatively associated with strong nuclear p27 expression. Expression of p38 was observed in most of the mammary tumors examined, and negative p38 expression was related to positive p21 expression. Moreover, p21 expression was associated with p27 expression; negative p21 expression was associated with negative p27 expression, while positive p21 expression was associated with positive p27 expression. The results confirm that the p21- and p27-encoding genes have similar expression patterns in the mammary tumors of aged dogs. In the present study, we characterized the expression of cellular senescence markers in these tumors and elucidated the relationships among their expression patterns.

Prevalência de obesidade, hipertensão arterial e níveis glicêmicos alterados em mulheres com câncer de mama de um núcleo de reabilitação do interior de São Paulo / Prevalence of obesity, high blood pressure and altered glucose levels in women with breast cancer from a rehabilitation center in the heartland of São Paulo State

As modalidades terapêuticas para o câncer de mama têm promovido avanços no seu tratamento, levando ao aumento da sobrevida das mulheres. Contudo, com o aumento do tempo de vida, há também o risco do aumento de comorbidades relacionadas à idade, ao tratamento e à doença, sendo as principais: a obesidade, hipertensão arterial sistêmica (HAS) e diabetes mellitus (DM). Assim, realizou-se o presente estudo com o objetivo de identificar a prevalência de obesidade, HAS e níveis glicêmicos alterados entre mulheres com câncer de mama que frequentam um núcleo de reabilitação e avaliar a distribuição destas comorbidades em relação à idade, tempo de diagnóstico do câncer de mama, circunferência da cintura, hormonioterapia, tratamento quimioterápico e hábitos de vida. Trata-se de um estudo observacional, descritivo e transversal, no qual foram incluídas 67 mulheres com câncer de mama. Para a coleta de dados utilizou-se um instrumento com variáveis sociodemográficas e clínicas, além das avaliações antropométricas, pregas cutâneas, bioimpedância, pressão arterial sistêmica e coletado sangue para glicemia de jejum, no período de setembro a novembro de 2015. Foi realizada análise descritiva, resultando em tabelas de frequência para variáveis qualitativas; para identificar a relação entre as variáveis qualitativas ordinais ou nominais foi empregado o teste exato de Fischer e para estudar a relação entre as medidas contínuas e categóricas da pesquisa foram empregadas técnicas de estatística não paramétrica com o teste de Mann-Whitney (MW) e Kruskal-Wallis (KW). Em relação à obesidade, encontrou-se que 34,3% das participantes eram pré-obesas e 29,9% apresentavam índice de massa corpórea (IMC) com parâmetros de obesidade leve a grave; 53,7% eram hipertensas e 20,9% diabéticas. Encontrou-se associação entre idade e HAS; circunferência da cintura e HAS; níveis glicêmicos e circunferência da cintura; IMC e circunferência da cintura; idade e DM; e idade e circunferência da cintura. Os resultados encontrados mostram alta prevalência de obesidade, hipertensão arterial e níveis glicêmicos alterados nas mulheres participantes, assim como possibilitaram identificar os fatores associados às comorbidades, sendo estes a idade, hábitos de vida e tratamentos realizados para o câncer de mama

The therapeutic modalities for breast cancer have promoted advances in its treatment leading to increased survival of women. However, with the increase in longevity, there is also the risk of comorbidities related to age, to treatment and to the disease, the main ones being: obesity, systemic arterial hypertension (SAH) and diabetes mellitus (DM). Thus, the objective of the present study was to indentify the prevalence of obesity, SAH and altered glucose levels among women with breast câncer who attend a rehabilitation center and to evaluate the distribution of such comorbidities in relation to age, time of breast cancer diagnosis, waist circumference, hormone therapy, chemotherapy treatment and life habits. This is an observational, descriptive and transversal study, which included 67 women with breast cancer. An instrument with sociodemographic and clinical variables was used for the collection of data, in addition to anthropometric evaluations, skin folds, bioimpedance, systemic arterial pressure and blood collected for fasting blood glucose test, from September to November, 2015. A descriptive analysis was perfomed resulting in frequency tables for qualitative variables; the Fischer exact test was employed to identify the relationship between the ordinal or nominal qualitative variables, whereas non-parametric statistical techniques with the Mann-Whitney (MW) and Kruskal-Wallis (KW) test were employed to study the relationship between continuous and categorical measures of the survey. In relation to obesity, 34.3% of participants were found to be pre-obese, 29.9% had a body mass índex (BMI) with mild to severe obesity parameters, 53.7% were hypertensive and 20.9% had diabetes. Association was found between age and SAH; waist circumference and SAH; blood glucose levels and waist circunference; BMI and waist circunference; age and DM; and age and waist circunference. The findings show high prevalence of obesity, high blood pressure and altered glucose levels in the participating women, and allow for identification of the factors associated with comorbidities, namely age, life habits and breast cancer treatments

Expression of mitochondrial apoptotic pathway-related factors in Sprague-Dawley rats with spontaneous mammary neoplasms / 中国实验动物学报

Objective To investigate the expression of AIF, CYT C, PAF-1, caspase-3, and XIAP in Sprague-Dawley rats with spontaneous mammary neoplasms.Methods One-hundred and thirty 3-4-week old SPF Spargue-Dawley rats (♀∶♂=1∶1) were fed in a specific pathogen free (SPF) breeding barrier for 60 weeks.The occurrence of spontane-ous breast tumors was recorded and histopathology was performed to identify the types of tumors.The rats were divided into 3 groups:rats with normal breast tissue ( group I) , with benign tumors ( group II) and with malignant tumors ( group III) . The expression of AIF, CYT C, APAF-1, caspase-3 and XIAP proteins and mRNAs were detected by immunhistochemistry ( IHC) and RT-PCR assay.Results Among these 130 SD rats, 14 rats were observed having spontaneous mammary neo-plasms with the incidence rate of 10.77%(14/130).In these neoplasm cases, 7 cases were mammary fibroadenomas, 7 cases of breast carcinoma, both with an incidence rate of 5.38%.Immunohistochemistry showed that, compared with the group I, the positive expressions of AIF, APAF-1, caspase-3 were decreased significantly (P<0.01), and the CYT C and XIAP expressions were significantly increased in the group II.The positive expression of all genes except XIAP was de-creased in the group III(P<0.01).Compared with the group II, APAF-1 and XIAP were significantly higher in the group III (P<0.01), and the positive expression of AIF, Cyt C, and caspases-3 were significantly decreased (P<0.01).In the results of RT-PCR assay, except APAF-1 which showed significant correlation with the results of immunohistochemistry ( P<0.05 ) , all the others showed an extremely significant correlation with immunohistochemical results ( P <0.01 ) . Conclusions Mammary tumors are most common spontaneous neoplasms in SD rats.Abnormal expression of mitochondrial apoptotic pathway-related factors AIF, CytC, APAF-1, caspase-3, and XIAP are correlated with the carcinogenesis and de-velopment of breast tumors.

Avaliação da imunomarcação de células-tronco tumorais em carcinossarcomas mamários e carcinomas em tumores mistos em cadelas / Evaluation of immunostaining of tumor stem cells in mammary carcinosarcomas and carcinomas in mixed tumors in bitches

As células-tronco tumorais (CTTs) pertencem a uma pequena população de células dentro do tumor com propriedades de autorrenovação e diferenciação em outros tipos celulares. Neste estudo avaliou-se o comportamento tanto das porções mesenquimais quanto das epiteliais de seis carcinossarcomas (CSs), 11 carcinomas em tumores mistos (CTMs) grau I, 11 grau II e 10 grau III. Nas porções epiteliais dos CS e CTM foram observadas imunomarcações para os anticorpos CD44, CD24, Oct-4 e ALDH-1. Nas porções mesenquimais dos CS, nas porções epiteliais dos CTMs graus II e III não houve imunomarcação para o ALDH-1. Concluiu-se que as CTTs são expressas em proporções iguais tanto nas porções mesenquimais quanto nas epiteliais dos CSs e ausentes nas porções mesenquimais bem diferenciadas de CTMs.

Cancer stem cells belong to a small population of cells within the tumor with properties of self-renewal and differentiation into other cell types. In this study, the behavior of both portions, mesenchymal and epithelial, was evaluated. Six carcinosarcomas (CSs), 11 carcinomas within mixed tumors (CWMTs) grade I, 11 grade II, and 10 grade III were evaluated. In the epithelial portions of the CS and CWMTs was observed immunostaining for antibodies CD44, CD24, Oct-4 and ALDH-1. In the mesenchymal portions of the CS, in the epithelial portions of CMTs grades II and III no immunostaining for ALDH-1 was found. It was concluded that the tumor stem cells are expressed in equal proportions in the epithelial and mesenchymal portions of the CS. No immunostaining in the mesenchymal portions of well-differentiated CWMTs was seen.

Estudo retrospectivo de 207 casos de tumores mamários em gatas / A retrospective study of 207 cases of mammary tumors in queens

Este estudo teve como objetivos determinar os tumores mais prevalentes em gatos e relacionar os tumores mamários a alguns de seus fatores prognósticos. Os arquivos do Laboratório de Patologia Veterinária (LPV) da Universidade Federal de Santa Maria (UFSM) foram revisados e um total de 1.427 protocolos de biopsias e necropsias de felinos, entre 2000 e 2011, foi encontrado. Com base nas informações dos arquivos, foi estabelecida a relação entre os tumores e alguns fatores como sexo, idade, raça, estado reprodutivo, uso de contraceptivos, número e localização das glândulas afetadas, ulcerações, tamanho do neoplasma, metástases distantes e para os linfonodos. Assim, observou-se que os tumores de mama foram o segundo diagnóstico mais prevalente, após os tumores de pele. Todos os gatos com tumores mamários eram fêmeas, sendo os sem raça definida e os idosos os mais afetados. Os neoplasmas malignos foram diagnosticados com maior frequência, seguidos pelos tumores não neoplásicos e pelos neoplasmas benignos. Os tumores menores eram, na sua maioria, carcinomas. Ulcerações estavam presentes não só em neoplasmas malignos, mas também em alterações não neoplásicas. Metástases distantes foram encontradas principalmente nos pulmões e na pele.

This study aimed to determine the most prevalent mammary tumors in cats and associate them to some prognostic factors. The files from the Laboratório de Patologia Veterinária (LPV) of the Universidade Federal de Santa Maria (UFSM) were reviewed, and 1.427 feline biopsies and autopsy protocols between the years 2000 and 2011 were found. Based on the information retrieved from the files, a relationship was established among the tumors and some prognostic factors such as sex, age, breed, reproductive status, use of contraceptives, number and location of affected glands, ulcers, size of the neoplasm, distant metastases, and affected lymph nodes. Thus, it was observed that mammary cancer is the second most prevalent diagnosis, following skin tumors. All cats with mammary tumors were female, being the elderly and mixed breed the most affected. Malignant neoplasms were the most frequently diagnosed, followed by non-neoplastic tumors, and benign neoplasms. Smaller tumors were mostly carcinomas. Ulcerations were present not only in malignant neoplasms but also in non-neoplastic changes. Distant metastases were found mainly to the lungs and skin.

Estudo clínico, histopatológico e imunoistoquímico de neoplasias mamárias em cadelas / Clinical, histopathological and immunohistochemical study of mammary neoplasm in bitches

O objetivo foi realizar um estudo clínico e epidemiológico de neoplasias mamárias em cadelas, considerando-se histórico reprodutivo, exame físico, diagnóstico histopatológico e imunoistoquímico. Utilizaram-se 60 neoplasias mamárias, divididas em grupos (grupo 1 - benigno, e grupo 2 - maligno). Avaliaram-se dados do histórico reprodutivo, o exame físico e achados histopatológicos e imunoistoquímicos para fator de crescimento endotelial vascular. Ao estudo do histórico reprodutivo, encontraram-se 90% dos animais com irregularidade de cio, 86,63% das cadelas não foram medicadas com contraceptivos e 83,33% não eram castradas. Ao exame físico, não foi verificada diferença (p>0,05) entre grupos ao se avaliar consistência das massas, regularidade da superfície tumoral e localização anatômica dos tumores. Quanto ao tamanho das massas, verificou-se diferença entre os grupos (p=0,0077), com 0,78±1,13cm para o grupo 1 e 1,81±2,29cm para o grupo 2. Diagnosticaram-se 40% de massas benignas e 60% de malignas, de acordo com os tipos de neoplasias. Para VEGF, verificaram-se valores médios de 2,22±0,89 para tumores malignos e 1,66±0,91 para benignos, com diferença entre grupos (p=0,0315). As neoplasias mamárias em cadelas não apresentam características de histórico reprodutivo e de exame clínico que auxiliem o diagnóstico diferencial, sendo a histopatologia o único método para conclusão do diagnóstico e a imunoistoquímica podendo ser utilizada para prognóstico da lesão.

The objective was to conduct a clinical and epidemiological study of mammary cancer in bitches, considering their reproductive history, physical examination, histopathological and immunohistochemical diagnosis. We used 60 breast tumors which were divided into groups (group 1 - group 2 and benign - malignant). We evaluated data from the reproductive history, physical examination and histopathology and immunohistochemistry for VEGF. The study of reproductive history had 90% of irregular estrus, 86.63% of the dogs were not tested with contraceptives and 83.33% were not castrated. On physical examination, there was no difference (p>0.05) between groups regarding the consistency of the masses, surface regularity of the tumor and anatomic location of tumors. As for the masses, there was a difference between groups (p=0.0077), with 0.78±1.13cm for group 1 and 1.81±2.29 cm for group 2. 40% of benign masses and 60% of malignant masses were diagnosed, according to the types of malignancies. For VEGF, the average values were 2.22±0.89 for malignant tumors and 1.66±0.91 for benign, with differences between groups (p=0.0315). The mammary tumors do not exhibit characteristics of reproductive history and clinical examination to help the differential diagnosis, and histopathology is the only method for completion of diagnosis and immunohistochemistry which can be used for injury prognosis.

Tratamento quimioterápico das neoplasias mamárias em cadelas e gatas / Chemotherapeutic treatment of mammary neoplasms in female dogs and cats

A neoplasia mamária é um tumor freqüentemente observado na rotina da clínica veterinária. A ocorrência desta neoplasia está relacionada principalmente a fatores hormonais. O diagnóstico desta enfermidade é confirmado por biópsia excisional e exame histopatológico. Exame físico e radiografias torácicas devem ser realizados para pesquisa de metástase. Os tumores mamários devem ser avaliados quanto ao seu diâmetro, envolvimento de linfonodos regionais e presença de metástase. O tratamento de eleição é a excisão cirúrgica do tumor. A quimioterapia é um tratamento adjuvante, cuja finalidade é evitar recidivas locais e metástases, prolongando desta forma, a sobrevida do paciente. Para a escolha da droga quimioterápica, deve-se considerar: classificação histopatológica do tumor, classificação de acordo com o sistema TNM (tumor-nódulo-metástase), estado geral do animal e os efeitos colaterais que podem decorrer da aplicação dos fármacos, como mielossupressão e toxicidade gastrointestinal. Os principais quimioterápicos antineoplásicos empregados no tratamento das neoplasias mamárias são a doxorrubicina, a ciclofosfamida e o 5-Fluorouracil.

Mammary gland tumors are often observed in the practice of veterinary clinics. The occurrence of this tumor is specialy related to hormonal factors. The diagnosis is confirmed by excisional biopsy and histopathologic evaluation. Physical examination and chest radiographs should be taken to evaluate possible metastasis. Mammary gland tumors must be evaluated for tumor size, regional lymphnodes involvement and presence of metastasis. The treatment of choice is surgical excision of the tumor. Chemotherapy is an adjuvant treatment, whose goal is to avoid local recurrence and metastasis, extending this way, the patient's survival. To choice the chemotherapy drug, it must consider: tumor histopathologic classification, classification according to the TNM system (tumor-node-metastasis), animal's general health and the side effects that can occur from the use of the drugs, like myelossupression and gastrointestinal toxicity. The prime chemotherapy drugs used in the treatment of mammary gland tumors are doxorubicin, cyclophosphamide and 5-fluorouracil.

Mammary adenocarcinoma in a mare

A non-lactating 17-year-old grey barren Mangalarga Marchador mare was referred to the Large Animal Veterinary Hospital of the Federal Rural University of Rio de Janeiro (UFRRJ), presenting enlargement of the mammary gland. The owner reported that the animal had a lesion in the mammary gland for at least two years, diagnosed and treated as chronic mastitis. Initially only the right gland was involved, presenting ulceration and exudation on the cutaneous surface. After 20 months, the left gland also became affected. The diagnosis of tubulo-papillary adenocarcinoma was based on the typical microscopic lesions. This work shows the importance of the histopathological examination in the differential diagnosis between the neoplasms and the chronic inflammation in the mammary gland of mares, as well as, to show that the cytological examination cannot detect the tumor, in case the puncture is made in areas of secondary infection.

Uma égua tordilha, Mangalarga Marchador, de 17 anos, não-lactante e não-prenhe, foi encaminhada ao Hospital de Grandes Animais da Universidade Federal Rural do Rio de Janeiro, com histórico de mastite crônica há dois anos. No início da lesão, apenas a glândula mamária direita estava muito aumentada, com exsudação sero-hemorrágica e ulcerada. Após 20 meses, a mama esquerda também estava comprometida. A égua foi tratada para mastite crônica, porém o exame histopatológico revelou tratar-se de um adenocarcinoma túbulo-papilar. Este trabalho evidencia a importância do exame histopatológico no diagnóstico diferencial entre neoplasias e inflamação crônica da mama de éguas, uma vez que o exame citopatológico pode não detectar o tumor, quando a punção é feita em áreas de infecção secundária.

Estudo quantitativo e morfológico das regiões organizadoras de nucléolo coradas pela prata (AgNORs) em neoplasias benignas e malignas da glândula mamária da espécie canina / Quantitative and morphological study of silver staining nucleolar organizer regions (AgNORs) in benign and malignant neoplasms of the canine mammary gland

O alvo deste estudo foi apresentar um novo método de avaliação das regiões organizadoras de nucléolos (NORs), objetivando verificar a correlação entre malignidade das células tumorais com proliferação celular. Vinte e nove tumores foram analisados quanto aos aspectos morfológicos e quantitativos de regiões organizadoras de nucléolos coradas pela prata (AgNORs) e comparados à quatro glândulas mamárias normais utilizadas como controle. Os dados obtidos foram tratados pelos testes não-paramétrico de Kruskal-Wallis e ôUõ de Mann-Whitney. Os números médios de NORs dos grupos neoplásicos malignos incluindo carcinomas tubulares (2,14 ± 0,54), carcinomas papilíferos (1,79 ± 0,24) e carcinomas sólidos (1,95 ± 0,30) mostraram diferença significativa quando comparados ao grupo controle (1,16 ± 0,08). A análise morfológica das AgNORs mostrou que o padrão de apresentação da NORs do tipo I prevaleceu nos grupos neoplásicos benignos (75% dos casos de adenoma tubular e 50% dos casos de carcinoma tubular), enquanto o tipo II prevaleceu nos grupos neoplásicos de comportamento maligno (54,5 % dos carcinomas papilíferos e 100% dos carcinomas sólidos). O grupo controle mostrou na sua totalidade (100% dos casos) predominância de NORs do tipo IV. O padrão do Tipo III não predominou em nenhum dos grupos envolvidos neste estudo. A análise da morfologia das AgNORs é uma ferramenta útil na determinação do prognóstica de alterações neoplásicas da glândula mamária canina.

The aim of this study was try to present a new method of evaluation of the nucleolar organizer regions (NORs) being related its morphology and distribution, objectifying to verify the correlation between malignancy of tumoral cells with cellular proliferation. Twenty-nine tumors were analyzed with regard to quantitative and morphological aspects of the silver staining nucleolar organizer regions (AgNORs) and compared to four normal mammary glands used as control. The data obtained were submitted to non-parametric Kruskal- Wallis and Mann-Whitney ôUõ tests. The mean NORs counts of the malignant neoplastic groups tubular carcinomas (2.14 ± 0.54), papillary carcinomas (1.79 ± 0.24) and solid carcinomas (1.95 ± 0.30) showed significant difference when compared with the control group (1.16 ± 0.08). Morphological analysis of AgNORs revealed that the expression pattern of type I NORs predominate in the benign histological groups (75% of the tubular adenoma cases and 50% of the tubular carcinoma cases), whereas type II NORs pattern prevailed in the malignant behavior neoplastic groups (54,5% of the papillary carcinoma and 100% of the solid carcinoma cases). Control group showed total predominance (100% of the cases) of type IV NORs. Expression pattern of the type III NORs did not prevail in neither histological group encompassed by this study. Evaluation of AgNORs morphology reveals an useful tool for prognosis assessment of neoplastic alterations of the canine mammary gland.

Estudo quantitativo e morfológico das regiões organizadoras de nucléolo coradas pela prata (AgNORs) em neoplasias benignas e malignas da glândula mamária da espécie canina / Quantitative and morphological study of silver staining nucleolar organizer regions (AgNORs) in benign and malignant neoplasms of the canine mammary gland

O alvo deste estudo foi apresentar um novo método de avaliação das regiões organizadoras de nucléolos (NORs), objetivando verificar a correlação entre malignidade das células tumorais com proliferação celular. Vinte e nove tumores foram analisados quanto aos aspectos morfológicos e quantitativos de regiões organizadoras de nucléolos coradas pela prata (AgNORs) e comparados à quatro glândulas mamárias normais utilizadas como controle. Os dados obtidos foram tratados pelos testes não-paramétrico de Kruskal-Wallis e ôUõ de Mann-Whitney. Os números médios de NORs dos grupos neoplásicos malignos incluindo carcinomas tubulares (2,14 ± 0,54), carcinomas papilíferos (1,79 ± 0,24) e carcinomas sólidos (1,95 ± 0,30) mostraram diferença significativa quando comparados ao grupo controle (1,16 ± 0,08). A análise morfológica das AgNORs mostrou que o padrão de apresentação da NORs do tipo I prevaleceu nos grupos neoplásicos benignos (75% dos casos de adenoma tubular e 50% dos casos de carcinoma tubular), enquanto o tipo II prevaleceu nos grupos neoplásicos de comportamento maligno (54,5 % dos carcinomas papilíferos e 100% dos carcinomas sólidos). O grupo controle mostrou na sua totalidade (100% dos casos) predominância de NORs do tipo IV. O padrão do Tipo III não predominou em nenhum dos grupos envolvidos neste estudo. A análise da morfologia das AgNORs é uma ferramenta útil na determinação do prognóstica de alterações neoplásicas da glândula mamária canina.

The aim of this study was try to present a new method of evaluation of the nucleolar organizer regions (NORs) being related its morphology and distribution, objectifying to verify the correlation between malignancy of tumoral cells with cellular proliferation. Twenty-nine tumors were analyzed with regard to quantitative and morphological aspects of the silver staining nucleolar organizer regions (AgNORs) and compared to four normal mammary glands used as control. The data obtained were submitted to non-parametric Kruskal- Wallis and Mann-Whitney ôUõ tests. The mean NORs counts of the malignant neoplastic groups tubular carcinomas (2.14 ± 0.54), papillary carcinomas (1.79 ± 0.24) and solid carcinomas (1.95 ± 0.30) showed significant difference when compared with the control group (1.16 ± 0.08). Morphological analysis of AgNORs revealed that the expression pattern of type I NORs predominate in the benign histological groups (75% of the tubular adenoma cases and 50% of the tubular carcinoma cases), whereas type II NORs pattern prevailed in the malignant behavior neoplastic groups (54,5% of the papillary carcinoma and 100% of the solid carcinoma cases). Control group showed total predominance (100% of the cases) of type IV NORs. Expression pattern of the type III NORs did not prevail in neither histological group encompassed by this study. Evaluation of AgNORs morphology reveals an useful tool for prognosis assessment of neoplastic alterations of the canine mammary gland.

Spectrum of Axillary Disorders (Excluding Metastasis from Breast Cancer): Radiological and Pathological Correlation: A Pictorial Essay

Axillary disorders originate from an axillary lymph node, subcutaneous fat layer, accessory breast, nerve, vessel and muscle. The most common causes of a palpable axillary mass are a lymph node pathology containing a benign axillary lymphadenopathy, and malignant lymph nodes such as a metastatic lymphadenopathy from breast cancer and a malignant lymphoma. For the detection of masses in the axilla, mammography and sonography are the imaging modalities of choice. We present a spectrum of various axillary masses with correlative radiological imaging and pathological findings in this pictorial essay. Knowledge of the radiological findings of various axillary disorders is useful for a differential diagnosis and for preventing unnecessary invasive procedures.

Study on the Expression of MCP-1mRNA,MIP-1?mRNA and its Clinicopathological Significances in the Benign and Malignant Lesions of Mammary Gland / 医学研究杂志

Objective To study on the expressions of MCP-1mRNA,MIP-1?mRNA and detect their clinicopathological significances in the benigr and malignant lesions of marnmary gland. Methods In situ hybridization was used for the detecting expressions of MCP-1mRNA and MIP-1?mRNA on the routinely paraffin-embedded sections of the specimen of the benign and malignant lesions of mammary gland. Results The positive rate of MCP-1mRNA,MIP-1?mRNA was significantly higher in the breast cancer than that in the benign lesion of mammary gland (62.5% vs 13.3%,?2=17.05,P2cm and with-metastasis of lymphnode in the breast cancer (P0.05). The closely positive correlation was found between the expression of MCP-1mRNA and MIP-1?mRNA in breast cancer (P

Expressão de PRSS11, MTSS1, CLPTM1, SMYD2, NOTCH1 e RPL37A em fatias de carcinoma de mama de cadelas e correlação com resposta à doxorrubicina in vitro / Expression of PRSS11, MTSS1, CLPTM1, SMYD2, NOTCH1 and RPL37A in slices of bitch mammary cancer and correlation with response to exposure doxorubicin in vitro

Os tumores da glândula mamária constituem as neoplasias mais frequentes em cadelas e a doxorrubicina é uma das principais drogas quimioterápicas utilizadas no tratamento deste tipo de câncer. A quimioterapia neoadjuvante com doxorrubicina possibilita a redução do tumor e permite a avaliação da resposta ao tratamento quimioterápico in vivo. A procura por marcadores preditivos de resposta tem o objetivo de identificar as pacientes responsivas e não responsivas ao tratamento com o quimioterápico de modo a permitir a indicação de uma abordagem terapêutica específica para cada paciente. A expressão de alguns trios de genes, incluindo PRSS11, MTSS1 e CLPTM1 e, PRSS11, MTSS1 e SMYD2 em mulheres com câncer de mama que receberam tratamento neoadjuvante com doxorrubicina permitiu a classificação das pacientes em sensíveis e resistentes ao tratamento. Outros genes diferencialmente expressos entre amostras consideradas sensíveis e resistentes à doxorrubicina foram NOTCH1 e RPL37A. Avaliamos no presente trabalho se a expressão de trios de genes permitia a classificação de resposta ao tratamento quimioterápico em outro modelo, constituído por carcinoma de mama de cadela, mantido em cultura de tecido na forma de fatias e exposto à doxorrubicina in vitro. A cultura de fatias de tecidos permite a manutenção da interação epitélio-mesênquima e é considerado um modelo mais próximo ao observado in vivo. Foram obtidas amostras de tumor mamário de 38 pacientes caninas atendidas no Hospital Veterinário da Faculdade de Medicina Veterinária da Universidade Metodista de São Paulo (UMESP), em São Bernardo do Campo, durante a cirurgia de mastectomia. Os proprietários deram seu consentimento livre e esclarecido para a realização do estudo. A idade mediana das pacientes foi de 10,4 anos e, 55% e 18,4% destas pacientes eram animais sem raça definida (SRD) e da raça poodle, respectivamente. Oito pacientes haviam sido submetidas previamente à cirurgia de esterilização...

Mammary gland tumors are the most common neoplasias of bitches and doxorubicin is one the mainstay drugs used in their treatment. Neoadjuvant chemotherapy with doxorubicin not only reduces the primary tumor as well as allows an in vivo evaluation of tumor response. Predictive markers of response, if identified, might allow a tailored treatment of each patient. In women with breast cancer submitted to neoadjuvant chemotherapy with doxorubicin, expression of groups of three genes, including PRSS11, MTSS1 and CLPTM1 and PRSS11, MTSS1 and SMYD2, classified the patients in responsive and resistant. Other genes differentially expressed between responsive and non-responsive tumors were NOTCH1 and RPL37A. We have then evaluated whether the expression of trios of these genes could allow the classification of tumors according to their response to chemotherapy, in another animal model constituted by mammary carcinoma of bitches, cultured as tissue slices and exposed to doxorrubicin in vitro. Culture of tissue slices preserves epithelial-mesenchymal interactions, in a similar way to that observed in vivo. Tumor samples were obtained from 38 canine patients treated at Hospital Veterinaria da Faculdade de Medicina Veterinária da Universidade Metodista de São Paulo (UMESP), São Bernardo do Campo, during mastectomy. Animal owners gave their informed consent to this study. Median age of patients was 10,4 years and 55% and 18,4% of them were mixed and poodle breed, respectively. Eight patients were previously spayed. Patients were classified in clinical stage III (39,47%), II (28,9%), I (18,4%) and IV (pulmonary metastasis only) (13,1%). Carcinomas were classified as cystic-papillary (34,2%), tubular (34,2%), tubular-papillary (18,42%) and solid (10,52%). Tumors were sliced and cultured in the absence or in the presence of doxorubicin (1 uM) for 24 hours and response evaluation was performed by cell counting of treated and untreated samples...

Simple Method for Preparing Tissue Chip and Its Application in Pathological Study / 广州中医药大学学报

【Objective】To establish a simple method for preparing tissue chip.【Methods】One hundred and thirty mammary cancer and intestinal cancer specimens were fixed by formaldehyde(40?g/L) and embedded by wax,and then were made into routine tissue slices and stained by haematoxylin and eosin(HE).Wax slices with tissue chip were prepared by fixing a tissue slice(0.1 cm in diameter)into the hole(0.2 cm in diameter and 2～4mm in depth) of a blank wax slice and were sliced into 4?m-thick slices.The tissue chips were mounted on the slide and stained by immunohistochemistry after treatment.【Results】A slide held 45 tissue chips and 130 specimens could be all detected only on 3 slides,which saved time and the amount of reagent.The immunohistochemical results showed that the positive location was accurate and the background was clear.【Conclusion】This method for preparing tissue chip is simple and effective,and can save the amount of reagent.It is easy for the comparative and separated detection of specimens.

HER-2/neu Oncogene Amplification by Chromogenic in situ Hybridization and Immunohistochemical Expression of Topoisomerase II-alpha in the Breast Cancer

BACKGROUND: Amplifications of the HER-2/neu oncogene and the Topoisomerase II-alpha gene are important determiners of the response to chemotherapy in the breast cancer. For detecting HER-2/neu amplification, fluorescent in situ hybridization and immunohistochemistry are currently regarded as standard methods. Chromogenic in situ hybridization (CISH) is investigated as a new modification of in situ hybridization. The purpose of this study is to compare the efficacy of CISH and immunohistochemistry (IHC) in detecting HER-2/neu oncogene amplification and to investigate the prognostic significance of the HER-2/neu oncogene and the Topoisomerase II-alpha gene in breast cancer. METHODS: Using CISH and IHC the amplifications and protein expressions of the HER-2/neu oncogene were studied on paraffin sections of 43 infiltrating duct carcinomas. The expression of the Topoisomerase II-alpha gene was studied immunohistochemically. RESULTS: Of the 43 infiltrating duct carcinomas, amplifications of the HER-2/neu oncogene by CISH were observed in 8 cases (18.6%), and the HER-2/neu protein was deemed overexpressed by IHC in 9 cases (20.9%). The amplifications of the HER-2/neu oncogene showed a statistically significant correlation with tumor size, histological grade, and the Topoisomerase II-alpha index. The Topoisomerase II-alpha index showed a statistically significant correlation with tumor size, lymph node status, stage, histologic grade, and estrogen receptor status. CONCLUSIONS: CISH is a useful alternative for determining HER-2/neu amplification, especially for confirming the immunohistochemical staining results. HER-2/neu amplification and the Topoisomerase II-alpha gene index may be prognostic factors of breast cancer.