Measurement of Ki-67 antigen in 159 pituitary adenomas using the MIB-1 monoclonal antibody

Pituitary adenomas sometimes show rapid growth and recurrence, and about one third invade the structures surrounding the sella turcica. In an attempt to determine aggressive behavior at an early stage, we used the MIB-1 antibody to identify the Ki-67 antigen. The present study was designed to evaluate pituitary adenomatous tissue in terms of secretion and proliferation and to correlate the Ki-67 index with hormone phenotype and invasive behavior. Material from 159 patients submitted to one or more resections of pituitary adenomas was evaluated. Forty-two non-secretory adenomas and 43 adenomas immunoreactive for growth hormone, 19 for prolactin, 18 for growth hormone and prolactin, 16 for adrenocorticotropic hormone (ACTH), and 21 cases of plurihormonal/gonadotropin adenomas were detected by immunohistochemistry. The MIB-1 antibody was positive in 139 samples and the Ki-67 index ranged from 0.16 to 15.48% (mean = 1.22 ± 2.09%), with no significant difference between genders, age groups, or secretory and non-secretory status. The Ki-67 index was higher in ACTH-secreting adenomas. Invasive pituitary adenomas had a significantly higher Ki-67 index (2.01 ± 3.15%) than macroadenomas with or without supra-sellar extension (1.12 ± 1.87%; P = 0.02). The index was not significantly different in the subgroup of adenomas with invasion of the cavernous sinus compared to groups with other types of invasion. We conclude that tumoral proliferative activity evaluated by the detection of the Ki-67 antigen is significantly higher in invasive than noninvasive adenomas, information which can be useful in therapeutic postoperative management since index cut-off values associated with aggressive behavior can be established.

A DNA enzyme targeting Egr-1 inhibits rat vascular smooth muscle cell proliferation by down-regulation of cyclin D1 and TGF-β1

We have demonstrated that a synthetic DNA enzyme targeting early growth response factor-1 (Egr-1) can inhibit neointimal hyperplasia following vascular injury. However, the detailed mechanism of this inhibition is not known. Thus, the objective of the present study was to further investigate potential inhibitory mechanisms. Catalytic DNA (ED5) and scrambled control DNA enzyme (ED5SCR) were synthesized and transfected into primary cultures of rat vascular smooth muscle cells (VSMCs). VSMC proliferation and DNA synthesis were analyzed by the MTT method and BrdU staining, respectively. Egr-1, TGF-β1, p53, p21, Bax, and cyclin D1 expression was detected by RT-PCR and Western blot. Apoptosis and cell cycle assays were performed by FACS. Green fluorescence could be seen localized in the cytoplasm of 70.6 ± 1.52 and 72 ± 2.73% VSMCs 24 h after transfection of FITC-labeled ED5 and ED5SCR, respectively. We found that transfection with ED5 significantly inhibited cultured VSMC proliferation in vitro after 24, 48, and 72 h of serum stimulation, and also effectively decreased the uptake of BrdU by VSMC. ED5 specifically reduced serum-induced Egr-1 expression in VSMCs, further down-regulated the expression of cyclin D1 and TGF-β1, and arrested the cells at G0/G1, inhibiting entry into the S phase. FACS analysis indicated that there was no significant difference in the rate of apoptosis between ED5- and ED5SCR-transfected cells. Thus, ED5 can specifically inhibit Egr-1 expression, and probably inhibits VSMC proliferation by down-regulating the expressions of cyclin D1 and TGF-β1. However, ED5 has no effect on VSMC apoptosis.

Esôfago de Barrett: aspectos fisiopatológicos e moleculares da seqüência metaplasia-displasia-adenocarcinoma - artigo de revisão

The Barrett's esophagus (BE) is defined as endoscopically visible columnar mucosa at the distal esophagus, of any extension, proved to harbor intestinal metaplasia on biopsy, highlighted by the presence of goblet cells. BE denotes long-standing gastroesophageal reflux disease (GERD) and is an important risk factor for the development of esophageal adenocarcinoma (EAC). Therefore, these patients must be on follow-up, in order to diagnose cancer early. BE patients have frequent alterations in esophageal physiologyc studies. Alkaline duodenogastroesophageal reflux seems to have important role. The development BE occurs in steps, initially with formation of cardiac type mucosa subsequent intestinalization. Futher progression can follow a sequence, from low grade dysplasia, to high grade dysplasia and esophageal adenocarcinoma. Current follow-up is based on the presence of dysplasia. It has limitations, grouping patients heterogeneously. Different steps of carcinogenesis have been studied looking for an ideal prognostic marker. Uncontrolled proliferative activity, apoptosis inhibition, angiogenesis, tissue invasion and metastases formation are all implicated in cancer origin. Some cycle cell molecules have been studied in BE, such as retinoblastoma protein, ciclins, kinase dependent ciclins and cell cycle inhibitors. The P53 protein is one of the most investigated in the metaplasia-adenocarcinoma progression. Growth Factors, apoptotic proteins, telomers and DNA ploidy have also been searched. Increased proliferative activity has been implicated in Barrett's carcinogenesis and the Ki-67 antigen, through imunohistochemical analysis, has become the the method of choice. Present in the nucleus, it is found in proliferative cells only. Some studies suport association between Ki-67 activity and the metaplasia-dysplasia-adenocarcinoma sequence.The results, however, are inconclusive and research should follow this way.

Meningiomas em cães: aspectos clínicos, histopatológicos e imuno-histoquímicos

Resumo: As neoplasias no sistema nervoso central (SNC) de animais de companhia são frequentemente diagnosticadas, no entanto dados sobre prevalência são escassos. O objetivo deste estudo foi avaliar retrospectivamente a ocorrência de neoplasias primárias de SNC em cães atendidos em um Hospital-Escola Veterinário e descrever aspectos clínicos, histopatológicos e imuno-histoquímicos dos tumores mais frequentes. Quatorze casos (prevalência de 0,27%) de neoplasias primárias de SNC foram identificados no período de 1998 a 2013 e destes, 11 tiveram o diagnóstico de meningiomas. A idade média dos animais com meningioma foi 10 anos, sendo machos (7/11) e a raça Boxer (3/11) os mais afetados. Sete meningiomas eram espinhais e quatro intracranianos, sendo os principais sinais clínicos alteração na locomoção e convulsões, respectivamente. Metástase pulmonar ocorreu em dois casos. Em seis animais com meningioma espinhal foi realizada a mielografia, sendo que em um também foi realizada a tomografia. Em todos os casos os exames foram efetivos na visualização de desvio ou interrupção da coluna de contraste, com alterações sugestivas da presença de massa. Em cinco animais realizou-se cirurgia exploratória visando a confirmação da suspeita clínica ou retirada da massa, sendo que a sobrevida variou de 85 a 960 dias. Na avaliação histopatológica, os meningiomas foram classificados em transicional (4/11), meningotelial (2/11), papilar (2/11), angiomatoso (1/11), microcístico (1/11) e anaplásico (1/11). Destes, oito (8/11) apresentaram marcação positiva para tricrômio de Masson e um para vermelho congo nas técnicas histoquímicas. No painel imuno-histoquímico, todos os casos apresentaram imunomarcação positiva para vimentina, mas imunomarcação negativa para fator VIII e p53. A imunomarcação para S100 (6/11), GFAP (5/11) e pancitoqueratina (3/11) foi de intensidade variável. Na graduação histológica, dez meningiomas eram grau I e um grau III. O índice médio de proliferação celular foi de 3,2 figuras de mitose e 3,4% avaliando a expressão de Ki-67. Os resultados confirmaram que os meningiomas são a neoplasia primária de SNC mais prevalente em cães, com variação nos subtipos histológicos. A caracterização histoquímica e imuno-histoquímica contribuiu com a determinação do diagnóstico, no entanto, estudos envolvendo a expressão de outros genes são necessários para auxiliar no prognóstico dessas neoplasias.

Estudo comparativo de aspectos histológicos e imunohistoquímicos entre o colesteatoma espontâneo do meato acústico externo e o colesteatoma adquirido da orelha média

OBJETIVO: Neste trabalho foram avaliadas as características histológicas e imunohistoquímicas (Ki-67 e CK-16) do colesteatoma espontâneo do meato acústico externo comparando-as com as do colesteatoma adquirido da orelha média. FORMA DE ESTUDO: Caso controle. MATERIAL E MÉTODO: Fragmentos de colesteatoma do meato acústico externo foram submetidos a estudo histológico e imunohistoquímico com o intuito de verificar a expressão da CK16 e do antígeno nuclear Ki-67 nas células de sua matriz, e os resultados foram comparados com os encontrados no colesteatoma adquirido da orelha média. RESULTADOS: Os aspectos histológico e imunohistoquímico quanto à presença da CK16 e do antígeno nuclear Ki-67 na matriz do colesteatoma do meato acústico externo foram idênticos àqueles encontrados no colesteatoma adquirido da orelha média. DISCUSSÃO: Acreditamos que a ocorrência do colesteatoma do meato acústico externo seja devido ao comportamento anormal de células com potencial hiperproliferativo no epitélio do meato. Este potencial estaria relacionado com a presença da CK16 em uma região onde estes marcadores não são usualmente encontrados. Esta característica hiperproliferativa do colesteatoma do meato fica patente pela presença do antígeno nuclear Ki-67 nas células suprabasais de sua epiderme. Possivelmente esta doença deve ser desencadeada pela interação desta citoqueratina (CK16) com citocinas presentes no tecido conjuntivo subepitelial inflamado, como a TGF-µ. CONCLUSÃO: As características histológicas (presença de cones epiteliais) e histoquímicas (expressão da CK16 e do antígeno nuclear KI-67) são idênticas no colesteatoma adquirido da orelha média e no colesteatoma espontâneo do meato acústico externo.

Müllerian adenosarcoma of the uterus with sarcomatous overgrowth following tamoxifen treatment for breast cancer

Müllerian adenosarcoma with sarcomatous overgrowth presented by a 52-year-old female patient after adjuvant tamoxifen treatment for breast carcinoma is described. The diagnosis was made on histological basis after curettage and complementary total hysterectomy with bilateral salpingo-oophorectomy. The immunohistochemical study showed high expression of estrogen receptors in the epithelial component of the lesion and irregularly positive findings in the stroma. The proliferative activity evaluated by Ki-67 immunoexpression was higher in the stroma than the epithelium. Some of the stromal cells showed rhabdomyoblastic differentiation. The association of tamoxifen use and development of mesenchymal neoplasms is discussed.

Four whole-istic aspects of schistosome granuloma biology: fractal arrangement, internal regulation, autopoietic component and closure

This paper centers on some whole-istic organizational and functional aspects of hepatic Schistosoma mansoni granuloma, which is an extremely complex system. First, it structurally develops a collagenic topology, originated bidirectionally from an inward and outward assembly of growth units. Inward growth appears to be originated from myofibroblasts derived from small portal vessel around intravascular entrapped eggs, while outward growth arises from hepatic stellate cells. The auto-assembly of the growth units defines the three-dimensional scaffold of the schistosome granulomas. The granuloma surface irregularity and its border presented fractal dimension equal to 1.58. Second, it is internally regulated by intricate networks of immuneneuroendocrine stimuli orchestrated by leptin and leptin receptors, substance P and Vasoactive intestinal peptide. Third, it can reach the population of ± 40,000 cells and presents an autopoietic component evidenced by internal proliferation (Ki-67+ Cells), and by expression of c-Kit+ Cells, leptin and leptin receptor (Ob-R), granulocyte-colony stimulating factor (G-CSF-R), and erythropoietin (Epo-R) receptors. Fourth, the granulomas cells are intimately connected by pan-cadherins, occludin and connexin-43, building a state of closing (granuloma closure). In conclusion, the granuloma is characterized by transitory stages in such a way that its organized structure emerges as a global property which is greater than the sum of actions of its individual cells and extracellular matrix components.

Correlation between PET/CT results and histological and immunohistochemical findings in breast carcinomas

Objective To correlate the results of 18F-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) performed with a specific protocol for assessment of breasts with histological/immunohistochemical findings in breast carcinoma patients. Materials and Methods Cross-sectional study with prospective data collection, where patients with biopsy-confirmed breast carcinomas were studied. The patients underwent PET/CT examination in prone position, with a specific protocol for assessment of breasts. PET/CT findings were compared with histological and immunohistochemical data. Results The authors identified 59 malignant breast lesions in 50 patients. The maximum diameter of the lesions ranged from 6 to 80 mm (mean: 32.2 mm). Invasive ductal carcinoma was the most common histological type (n = 47; 79.7%). At PET/CT, 53 (89.8%) of the lesions demonstrated anomalous concentrations of 18F-FDG, with maximum SUV ranging from 0.8 to 23.1 (mean: 5.5). A statistically significant association was observed between higher values of maximum SUV and histological type, histological grade, molecular subtype, tumor diameter, mitotic index and Ki-67 expression. Conclusion PET/CT performed with specific protocol for assessment of breasts has demonstrated good sensitivity and was associated with relevant histological/immunohistochemical factors related to aggressiveness and prognosis of breast carcinomas.

Imunossupressão com tacrolimus favorece a regeneração hepática induzida por hepatectomia ampla em ratos

OBJETIVO: Avaliar em dois momentos distintos da regeneração hepática a influência do Tacrolimus sobre o fenômeno da regeneração hepática desencadeada pela ressecção de 70% do parênquima hepático em ratos plenamente desenvolvidos. MÉTODOS: Utilizaram-se 40 ratos Wistar com peso médio de 510,08 g ± 11.66 g distribuidos aleatoriamente em dois grupos de 20, cada grupo subdividido em dois subgrupos conforme o dia da morte após a hepatectomia. De acordo com o grupo os animais receberam por gavagem solução aquosa de Tacrolimus 0,1 mg/kg/dia ou solução salina no mesmo volume. Após três dias de pré-terapia todos foram submetidos à hepatectomia de 70% pela ressecção dos lobos hepáticos mediano e lateral esquerdo que foram pesados para posterior cálculo da regeneração hepática pela fórmula de Kwon. Vinte e quatro horas ou sete dias após a hepatectomia, 10 animais de cada grupo foram mortos, os fígados remanescentes (regenerados) foram pesados e amostrados para realização de índice mitótico por hematoxilina-eosina e estudo imunoistoquímico com os marcadores PCNA e Ki-67. RESULTADOS: Os animais que receberam tacrolimus mostraram índice maior de regeneração hepática, atingindo significância estatística quando comparado ao subgrupo de animais mantidos com placebo quando analisados pelos parâmetros: fórmula de Kwon, índice mitótico e marcador PCNA. A tendência para o marcador Ki-67 foi idêntica aos outros parâmetros mas não alcançou significância estatística. CONCLUSÃO: A imunossupressão com tacrolimus possui efeito estimulatório no processo de regeneração hepática desencadeado pela hepatectomia 70% em ratos Wistar adultos, plenamente desenvolvidos.

Sirolimus influence on hepatectomy-induced liver regeneration in rats

OBJECTIVE: To evaluate the influence of sirolimus on liver regeneration triggered by resection of 70% of the liver of adult rats.METHODS: we used 40 Wistar rats randomly divided into two groups (study and control), each group was divided into two equal subgroups according to the day of death (24 hours and seven days). Sirolimus was administered at a dose of 1mg/kg in the study group and the control group was given 1 ml of saline. The solutions were administered daily since three days before hepatectomy till the rats death to removal of the regenerated liver, conducted in 24 hours or 7 days after hepatectomy. Liver regeneration was measured by the KWON formula, by thenumber of mitotic figures (hematoxylin-eosin staining) and by the immunohistochemical markers PCNA and Ki-67.RESULTS: there was a statistically significant difference between the 24h and the 7d groups. When comparing the study and control groups in the same period, there was a statistically significant variation only for Ki-67, in which there were increased numbers of hepatocytes in cell multiplication in the 7d study group compared with the 7d control group (p = 0.04).CONCLUSION: there was no negative influence of sirolimus in liver regeneration and there was a positive partial effect at immunohistochemistry with Ki-67.

Efeitos de diferentes doses de tamoxifeno sobre a proliferação celular do epitélio mamário

OBJETIVO: analisar quantitativamente a imunorreação do anticorpo monoclonal Ki-67 no epitélio de tecido mamário adjacente ao fibroadenoma de mulheres no menacme tratadas com tamoxifeno, durante 50 dias, nas doses de 5, 10 e 20 mg/dia. MÉTODOS: foram incluídas prospectivamente mulheres no menacme. O tamoxifeno foi administrado durante 50 dias, nas doses de 5, 10 e 20 mg/dia. Empregou-se a imunorreação do anticorpo monoclonal Ki-67 (clone Ki-S5) no epitélio mamário adjacente ao fibroadenoma. Avaliou-se, de forma aleatória, em duplo-cego, 58 pacientes eumenorréicas, que foram divididas em quatro grupos: Grupo A (n=13; placebo), Grupo B (n=16; 5 mg tamoxifeno/dia), Grupo C (n=14; 10 mg/dia) e Grupo D (n=15; 20 mg/dia). Todas receberam a medicação a partir do primeiro dia do ciclo menstrual e as cirurgias foram realizadas no último dia da medicação. Contaram-se os núcleos corados e não corados pelo imunorreagente por microscopia óptica (400) acoplada a sistema digital de captura e análise de imagem. RESULTADOS: a porcentagem média de núcleos corados foi calculada para todos os grupos: no Grupo A foi de 2,0 com erro padrão (EP) de 0,3; no Grupo B, de 0,7 (EP=0,2); no Grupo C, de 0,4 (EP=0,2) e, no Grupo D, de 0,1 (EP=0). A análise de variância revelou haver diferença estatística entre os grupos (p<0,001), e o teste de comparações múltiplas de Tukey confirmou haver redução significativa da imunorreação do anticorpo monoclonal Ki-67 nos Grupos B, C e D. CONCLUSÕES: o tamoxifeno, administrado nas doses de 5, 10 e 20 mg/dia durante 50 dias, reduziu de forma significante a imunorreação do anticorpo monoclonal Ki-67 no epitélio mamário de mulheres no menacme. Não houve diferença significante entre os grupos que receberam 5, 10 e 20 mg ao dia de tamoxifeno.

Comparison of nuclear grade and immunohistochemical features in situ and invasive components of ductal carcinoma of breast

PURPOSE:To compare the prognostic and predictive features between in situ and invasive components of ductal breast carcinomas. METHODS:We selected 146 consecutive breast samples with ductal carcinoma in situ (DCIS) associated with adjacent invasive breast carcinoma (IBC). We evaluated nuclear grade and immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6), and epidermal growth factor receptor (EGFR) in both components, in situ and invasive, and the Ki-67 percentage of cells in the invasive part. The DCIS and IBC were classified in molecular surrogate types determined by the immunohistochemical profile as luminal (RE/PR-positive/ HER2-negative), triple-positive (RE/RP/HER2-positive), HER2-enriched (ER/PR-negative/HER2-positive), and triple-negative (RE/RP/HER2-negative). Discrimination between luminal A and luminal B was not performed due to statistical purposes. Correlations between the categories in the two groups were made using the Spearman correlation method. RESULTS:There was a significant correlation between nuclear grade (p<0.0001), expression of RE/RP (p<0.0001), overexpression of HER2 (p<0.0001), expression of EGFR (p<0.0001), and molecular profile (p<0.0001) between components in situ and IBC. CK 5/6 showed different distribution in DCIS and IBC, presenting a significant association with the triple-negative phenotype in IBC, but a negative association among DCIS. CONCLUSIONS: Our results suggest that classical prognostic and predictive features of IBC are already determined in the preinvasive stage of the disease. However the role of CK5/6 in invasive carcinoma may be different from the precursor lesions.

Hofbauer cells morphology and density in placentas from normal and pathological gestations

PURPOSE: In placentas from uncomplicated pregnancies, Hofbauer cells either disappear or become scanty after the fourth to fifth month of gestation. Immunohistochemistry though, reveals that a high percentage of stromal cells belong to Hofbauer cells. The aim of this study was to investigate the changes in morphology and density of Hofbauer cells in placentas from normal and pathological pregnancies. METHODS: Seventy placentas were examined: 16 specimens from normal term pregnancies, 10 from first trimester's miscarriages, 26 from cases diagnosed with chromosomal abnormality of the fetus, and placental tissue specimens complicated with intrauterine growth restriction (eight) or gestational diabetes mellitus (10). A histological study of hematoxylin-eosin (HE) sections was performed and immunohistochemical study was performed using the markers: CD 68, Lysozyme, A1 Antichymotrypsine, CK-7, vimentin, and Ki-67. RESULTS: In normal term pregnancies, HE study revealed Hofbauer cells in 37.5% of cases while immunohistochemistry revealed in 87.5% of cases. In first trimester's miscarriages and in cases with prenatal diagnosis of fetal chromosomal abnormalities, both basic and immunohistochemical study were positive for Hofbauer cells. In pregnancies complicated with intrauterine growth restriction or gestational diabetes mellitus, a positive immunoreaction was observed in 100 and 70% of cases, respectively. CONCLUSIONS: Hofbauer cells are present in placental villi during pregnancy, but with progressively reducing density. The most specific marker for their detection seems to be A1 Antichymotrypsine. It is remarkable that no mitotic activity of Hofbauer cells was noticed in our study, as the marker of cellular multiplication Ki-67 was negative in all examined specimens.

Loss of PTEN expression and AKT activation in HER2-positive breast carcinomas

PURPOSE: To examine the expression of AKT and PTEN in a series of HER2-positive primary invasive breast tumors using immunohistochemistry, and to associate these expression profiles with classic pathologic features such as tumor grade, hormone receptor expression, lymphatic vascular invasion, and proliferation.METHODS: A total of 104 HER2-positive breast carcinoma specimens were prepared in tissue microarrays blocks for immunohistochemical detection of PTEN and phosphorylated AKT (pAKT). Original histologic sections were reviewed to assess pathological features, including HER2 status and Ki-67 index values. The associations between categorical and numeric variables were identified using Pearson's chi-square test and the Mann-Whitney, respectively.RESULTS: Co-expression of pAKT and PTEN was presented in 59 (56.7%) cases. Reduced levels of PTEN expression were detected in 20 (19.2%) cases, and these 20 tumors had a lower Ki-67 index value. In contrast, tumors positive for pAKT expression [71 (68.3%)] were associated with a higher Ki-67 index value.CONCLUSION: A role for AKT in the proliferation of HER2-positive breast cancers was confirmed. However, immunohistochemical detection of PTEN expression did not correlate with an inhibition of cellular proliferation or control of AKT phosphorylation, suggesting other pathways in these mechanisms of control.

Nova classificação dos carcinomas da mama: procurando o luminal A

OBJETIVO:Comparar a distribuição das pacientes segundo os subtipos clínico-patológicos de carcinomas de mama luminais like segundo o consenso de St. Gallen 2011 e 2013.MÉTODOS:Foram selecionadas 142 pacientes com carcinoma invasivo da mama que eram positivas para receptor de estrógeno, diagnosticadas e tratadas no estado de São Paulo, região Sudeste do Brasil. A expressão dos receptores de estrógeno, progesterona (RP) e Ki-67 foi avaliada por imunoistoquímica em microarranjo de tecidos. A expressão de HER-2 foi avaliada por hibridização fluorescente in situ.RESULTADOS:Observamos que 29 casos classificados como luminais A na classificação de St. Gallen 2011 eram luminais B na classificação de 2013. Dentre os 65 casos luminais B like da classificação de 2013, além dos 29 (45%) casos que migraram, observamos 15 casos (20%) com Ki-67 >14% e pelo menos 20% das células coradas; e 21 casos (35%) com Ki-67 >14% e RP positivo em mais de 20% das células coradas.CONCLUSÕES:Comparando a distribuição das pacientes com carcinomas luminais da mama segundo a classificação de St. Gallen 2011 e 2013 observamos que houve um aumento no número de carcinomas da mama luminais B like. Consequentemente, estima-se um aumento nas indicações de quimioterapia adjuvante e no custo do tratamento.