Can TILs be associated with prognostic factors and survival rates in breast cancer? A retrospective analysis

Introduction: The relationship between the tumor inflammatory infiltrate, also known as tumor-infiltrating lymphocytes (TILs), and invasive breast carcinomas has been extensively studied in recent years to verify its association with prognosis and response to treatment. The goal of this study was to associate the presence of TILs with patient's survival time. Methods: We studied prognostic clinicopathological characteristics already established in the literature and their impact on overall five-year survival time of patients with invasive breast cancer treated at Hospital Santa Casa in Belo Horizonte, Minas Gerais, Brazil, in 2011 (n=290). This was an observational and retrospective study. Results: The presence of TILs was associated with tumors of no special type (p=0.018) and with younger age of the patients (p=0.042). Smaller tumor size (HR: 19.24; 95%CI 4.30­86.15; p<0.001), absence of metastasis to the axillary lymph nodes (HR: 2.80; 95%CI 1.02­7.70; p=0.002), positivity for progesterone receptor (HR: 0.39; 95%CI 0.17­0.87; p=0.022), and presence of TILs (HR: 0.23; 95%CI 0.08­0.65; p=0.005) were associated with longer survival times. Conclusions: This study suggests that the presence of TILs, along with other clinicopathological characteristics, is a prognostic factor in breast cancer

COX-2 expression in mammary invasive micropapillary carcinoma is associated with prognostic factors and acts as a potential therapeutic target in comparative oncology.

Pure human and canine mammary invasive micropapillary carcinoma is a rare malignant epithelial tumor accounting for 0.9 to 2% of all invasive mammary carcinomas and present a high rate of lymphatic invasion and metastasis, with unfavorable prognosis. Surgery and chemotherapy are standard treatments for almost all mammary cancer in both species, as well as hormonal and target therapies available for human patients. However, depending on the patient's clinical staging, satisfactory therapeutic results for invasive micropapillary carcinoma are a challenge due to its high capacity of invasion and metastasis. Cyclooxygenase-2 (COX-2) isoform is an important enzyme stimulated by cytokines, growth factors and oncogenes activation to synthetizes prostaglandins in inflammatory process. COX-2 overexpression is associated with angiogenesis and invasion and contributes to cancer development, disease progression, tumor recurrence and regional lymph node metastasis in human and canine mammary carcinomas. This enzyme can be targeted by non-steroidal anti-inflammatory drugs and its inhibition can reduce tumor growth and metastasis in several cancer types. Given the similarity between both species, the present study aims to elucidate the involvement of COX-2 mRNA and protein expression in canine (cIMPC) and human (hIMPC) pure invasive mammary micropapillary carcinoma, with clinicopathological and survival data. Twenty-nine cases of cIMPC and 17 cases of hIMPC were analyzed regarding histologic type, grade, age, tumor size, lymph node condition, extracapsular extension, inflammatory infiltrate and immunophenotype. When available, information on adjuvant treatment, recurrence, metastasis and overall survival were collected. The present study demonstrated COX-2 protein expression in 65.5% of cIMPC and 92.3% of hIMPC, and an association with more advanced histological grades in bitches and higher Ki67 in women. COX-2 mRNA expression was significantly higher in cIMPC than in hIMPC, and its expression was not associated with COX-2 protein expression in both species. COX-2 mRNA expression was associated with negative-ER hIMPC as well as higher Ki67. cIMPC demonstrated proportional early development, more regional metastasis, and a prevalence of negative estrogen receptor, than hIMPC. This is the first time COX-2 expression is associated with negative prognostic factors in both cIMPC and hIMPC, besides the overexpression of COX-2 protein in such unfavorable histological type, which suggests that COX-2 can act as a potential target in IMPC.

Pseudoangiomatous stromal hyperplasia of the breast: a rare condition ­ from diagnosis to treatment

Pseudoangiomatous Stromal Hyperplasia (PASH) of the breast is a rare condition that consists of the proliferation of the breast myofibroblastic stromal cells, lining anastomosing vascular slit-like spaces. This condition is not considered a pre-malignant lesion and affects mainly premenopausal women. Its etiology is still uncertain, but its behavior points to a hormonal cause. It has a varied clinical presentation and can be diagnosed as an incidental finding of biopsies or with the manifestation of clinical signs and symptoms. As for the diagnosis, it can be performed with the correlation between clinical data, imaging and histopathological analysis. Due to its rare nature, there are still no prospective studies regarding treatment, but, in most cases, clinical and radiological follow-up is a safe strategy. The aim of this paper is to synthesize the data available in the literature about this condition, which, although benign in nature, can bring important aesthetic, musculoskeletal and psychological repercussions

High positivity values for bovine leukemia virus in human breast cancer cases from Minas Gerais, Brazil.

Bovine leukemia virus (BLV) is a retrovirus that causes lymphoma in cattle worldwide and has also been associated with breast cancer in humans. The mechanism of BLV infection in humans and its implication as a primary cause of cancer in women are not known yet. BLV infection in humans may be caused by the consumption of milk and milk-products or meat from infected animals. Breast cancer incidence rates in Brazil are high, corresponding to 29.5% a year of cancer cases among women. In 2020, an estimated 66,280 new cases of breast cancer are expected, whereas in 2018 breast cancer has led to 17,572 deaths, the highest incidence and lethality among cancers in women in this country that year. BLV infection occurrence ranges from 60 to 95% in dairy herds. In addition, there are some regions, such as the Minas Gerais State, southeastern Brazil, where the population traditionally consume unpasteurized dairy products. Taken together, this study aimed to verify if there is a higher association between breast cancer and the presence of BLV genome in breast tissue samples within this population that consumes raw milk from animals with high rates of BLV infection. A molecular study of two BLV genes was carried out in 88 breast parenchyma samples, between tumors and controls. The amplified fragment was subjected to BLV proviral sequencing and its identity was confirmed using GenBank. BLV proviral genes were amplified from tumor breast parenchyma samples and healthy tissue control samples from women, revealing a 95.9% (47/49) and 59% (23/39) positivity, respectively. Our results show the highest correlation of BLV and human breast cancer found in the world to date within the population of Minas Gerais, Brazil.

Recurrence/Regrowth in Grade I Meningioma: How to Predict?

The HLA-G and HLA-E molecules, Ki67, progesterone (PR), estrogen (ER) and androgen receptors (AR), p53, COX-2, and HER2 were studied to assess whether the biological behavior of grade I meningiomas is related to their expression. Tissue samples from 96 patients with grade I intracranial meningiomas were analyzed by immunohistochemistry on tissue microarray blocks (TMA) using antibodies specific for HLA-G, HLA-E, Ki67, PR, ER, AR, p53, COX-2, and HER2. Meningiomas were classified as small (≤2 cm, 1.0%), medium (>2 and ≤4 cm, 32.3%), and large (>4 cm, 66.7%). Tumor size was not related to recurrence/regrowth (p = 0.486), but was significantly correlated with peritumoral edema (p = 0.031) and intratumoral calcifications (p = 0.018). Recurrent meningiomas were observed in 14.6% of cases. Immunostaining for each marker was: HLA-G 100%; HLA-E 95.6%; PR 62%; ER 2.1%; AR 6.5%; p53 92.6%; COX-2 100%; HER2 0%; Ki67, mean 2.61 ± 2.29%, median 2.1%. Primary and recurrent meningiomas showed no significant relation with HLA-E and hormone receptors (p > 0.05), except for Ki67, where a higher median was observed in recurrent tumors than in primary (p = 0.014). The larger the tumor, the more severe the peritumoral edema, and the greater the presence of calcifications. Ki67 appears to be a good biomarker of recurrence/regrowth in grade I meningiomas.

Evaluation of the use of tacrolimus ointment for the prevention of hypertrophic scars in experimental model

Abstract BACKGROUND: Tacrolimus, for its activity on modulation of collagen production and fibroblast activity, may have a role in the prevention of hypertrophic scars. OBJECTIVES: Evaluate macroscopic, microscopic, metabolic, laboratory effects and side effects of the use of topical tacrolimus ointment, in different concentrations, in the prevention of hypertrophic scars. METHODS: Twenty-two rabbits were submitted to the excision of 2 fragments of 1 cm of each ear, 4 cm apart, down to cartilage. The left ear of the animals was standardized as control and Vaseline applied twice a day. The right ear received tacrolimus ointment, at concentrations of 0.1% on the upper wound and 0.03% on the lower wound, also applied twice a day. Macroscopic, microscopic, laboratory criteria and the animals' weight were evaluated after 30 days of the experiment. RESULTS: Wounds treated with tacrolimus, at concentrations of 0.1% and 0.03%, when compared to control, showed a lower average degree of thickening (p = 0.048 and p <0.001, respectively). The average of scar thickness and lymphocyte, neutrophil and eosinophil concentrations are lower in the treated wounds compared to the control (p <0.001, p=0.022, p=0.007, p=0.044, respectively). The mean concentration of lymphocytes is lower in wounds treated with a higher concentration of the drug (p=0.01). STUDY LIMITATIONS: experiment lasted only 30 days. CONCLUSIONS: Tacrolimus at the 2 concentrations evaluated reduced the severity of inflammatory changes and positively altered the macroscopic aspect of the scar in the short term. Its use was shown to be safe, with no evidence of systemic or local adverse effects.

Comparison of the efficacy of losartan, hydrocortisone and acetylsalicylic acid (ASA) in preventing the development of fibrous scar tissue in skeletal muscle / Comparação da eficácia de losartana, hidrocortisona e ácido acetilsalicílico (AAS) na prevenção do desenvolvimento de tecido cicatricial fibroso nos músculos esqueléticos / Comparación de la eficacia de losartán, hidrocortisona y ácido acetilsalicílico (AAS) en la prevención del desarrollo de tejido cicatricial fibroso en los músculos esqueléticos

OBJECTIVE: To analyze fibrous scar tissue inhibition capacity with the use of losartan, hydrocortisone and acetylsalicylic acid. METHOD: The sample consisted of 120 male heterogeneic Wistar rats with a muscle laceration model. The rats were divided into four groups of 30 animals each: control group, losartan group, ASA group and hydrocortisone group. The animals were anesthetized and a 2.5 cm longitudinal incision was made in the left thoracolumbar paravertebral region. The muscles were subjected to a Grade III lesion caused by applying Kelly hemostatic forceps for 60 seconds, followed by sectioning with scissors. The skin was sutured with 3-0 nylon monofilament thread. The animals were placed in individual cages with plenty of food and water. The losartan group received losartan diluted in water at a dose of 0.1 mg/mL (10 mg/kg/day), the ASA Group received a 3 mg/mL ASA solution (300 mg/kg/day), and the hydrocortisone group received a 0.2 mg/mL hydrocortisone solution (20 mg/kg/day). RESULTS: The control, losartan, hydrocortisone and aspirin groups had a fibrotic area of 0.95 ± 0.35 mm, 0.55 ± 0.34 mm, 0.93 ± 0.33 mm, and 0.66 ± 0.36 mm, respectively. We observed a significantly smaller fibrotic area in the losartan group compared to the control (p=0.01) and hydrocortisone (p=0.01) groups. There were no significant differences among the other groups. CONCLUSION: The healing of striated skeletal muscle produced less fibrous scar tissue when exposed to losartan in comparison to the control group or the hydrocortisone group. Level of Evidence I; Randomized double-blind placebo-controlled study.

OBJETIVO: Analisar a capacidade de inibição de formação de tecido cicatricial fibroso com losartana, hidrocortisona e AAS. MÉTODOS: A amostra consistiu em 120 ratos Wistar heterogênicos machos com modelo de laceração muscular. Os ratos foram distribuídos em quatro grupos de 30 animais: grupo controle, grupo losartana, grupo AAS e grupo hidrocortisona. Os animais foram anestesiados e submetidos a uma incisão em sentido longitudinal de 2,5 cm de extensão na região paravertebral toracolombar esquerda, e os músculos sofreram uma lesão grau III com pinça hemostática de Kelly durante 60 segundos e posterior secção com tesoura. A pele foi suturada com nylon monofilamentar 3-0. Os animais foram colocados em gaiolas individuais, com água e alimento à vontade. O grupo losartana recebeu losartana diluída em água na dose de 0,1 mg/ml (10 mg/kg/dia), o grupo AAS recebeu solução de AAS 3 mg/ml (300 mg/kg/dia), o grupo hidrocortisona recebeu solução de hidrocortisona 0,2 mg/ml (20 mg/kg/ dia). RESULTADOS: Os grupos controle, losartana, hidrocortisona e AAS apresentaram área fibrótica de0,95 ± 0,35 mm, 0,55 ± 0,34 mm, 0,93 ± 0,33 mm, 0,66 ± 0,36 mm, respectivamente. Observou-se área fibrótica significativamente menor do grupo losartana em comparação com o grupo controle (p = 0,01) e hidrocortisona (p = 0,01). Nos demais grupos não houve diferença significativa. CONCLUSÃO: A cicatrização do músculo estriado esquelético produziu menos tecido cicatricial fibroso quando exposto à losartana do que quando comparado com o grupo controle ou o grupo hidrocortisona. Nível de Evidência I; Estudo duplo-cego randomizado controlado por placebo.

OBJETIVO: Analizar la capacidad de inhibición de formación de tejido cicatricial fibroso con losartán, hidrocortisona y AAS (ácido acetilsalicílico). MÉTODOS: La muestra consistió en 120 ratas Wistar heterogéneas machos con modelo de laceración muscular. Las ratas fueron distribuidas en cuatro grupos de 30 animales: grupo control; grupo losartán; grupo AAS y grupo hidrocortisona. Los animales fueron anestesiados y sometidos a una incisión longitudinal de 2,5 cm de extensión en la región paravertebral toracolumbar izquierda y los músculos sufrieron una lesión de grado III con pinza hemostática de Kelly durante 60 segundos y posterior sección con tijera. La piel se suturó con monofilamento de nylon 3-0. Los animales fueron dispuestos en jaulas individuales con abundante comida y agua. El grupo losartán recibió losartán diluido en agua a una dosis de 0,1 mg/ml (10 mg/kg/día), el grupo AAS recibió solución de AAS de 3 mg/ml (dosis 300 mg/kg/día), el grupo hidrocortisona recibió solución hidrocortisona de 0,2 mg/ml (20 mg/kg/día). RESULTADOS: Los grupos control, losartán, hidrocortisona y AAS mostraron área fibrótica de 0,95 ± 0,35 mm, 0,55 ± 0,34 mm, 0,93 ± 0,33 mm, 0,66 ± 0,36 mm, respectivamente. Se observó área fibrótica significativamente menor del grupo losartán en comparación con el grupo control (p = 0,01) e hidrocortisona (p = 0,01). En los demás grupos no hubo diferencias significativas. CONCLUSIÓN: La cicatrización del músculo estriado esquelético produjo menos tejido cicatricial fibroso cuando fue expuesto a losartán que cuando fue comparado con el grupo control o el grupo hidrocortisona. Nivel de Evidencia I; Estudio doble ciego aleatorio controlado por placebo.

Comparative study of intraperitoneal adhesions related to light-weight polypropylene mesh and type I polymerized and purified bovine collagen coated light-weight polypropylene mesh in rabbits.

PURPOSE: To compare the effectiveness of light-weight polypropylene mesh coated with polymerized and purified bovine type I collagen (Surgidry HNB) in the treatment of abdominal wall defect and the degree of adhesion formation. METHODS: Two types of polypropylene mesh were implanted after creation of defect measuring 6.0cm X 5.5cm in the anterior abdominal wall of 32 male New Zealand breed rabbits, divided in two groups (n = 32): (1) light-weigh macroporous polypropylene, (2) type I polymerized and purified bovine collagen coated light-weigh macroporous polypropylene. These animals were further accessed for adhesions, histological evaluation of inflammation and wall's thickness. RESULTS: The percentage of the area adhered in group 1 (62.31 ± 16.6) was higher compared to group 2 (22.19 ± 14.57) (p <0.05). There was an association between the percentage of the covered area by adhesions and the type of adhesion, toughness and the scores obtained by the adhesion score by correlation analysis (p <0.05). There was no difference between the groups in any variables in relation to the degree of inflammation. CONCLUSION: The purified type I bovine collagen coated light-weigh polypropylene mesh showed to be effective in the repair of abdominal wall defects and reducing adhesion formation.

Comparative study of intraperitoneal adhesions related to light-weight polypropylene mesh and type I polymerized and purified bovine collagen coated light-weight polypropylene mesh in rabbits

Abstract Purpose: To compare the effectiveness of light-weight polypropylene mesh coated with polymerized and purified bovine type I collagen (Surgidry HNB) in the treatment of abdominal wall defect and the degree of adhesion formation. Methods: Two types of polypropylene mesh were implanted after creation of defect measuring 6.0cm X 5.5cm in the anterior abdominal wall of 32 male New Zealand breed rabbits, divided in two groups (n = 32): (1) light-weigh macroporous polypropylene, (2) type I polymerized and purified bovine collagen coated light-weigh macroporous polypropylene. These animals were further accessed for adhesions, histological evaluation of inflammation and wall's thickness. Results: The percentage of the area adhered in group 1 (62.31 ± 16.6) was higher compared to group 2 (22.19 ± 14.57) (p <0.05). There was an association between the percentage of the covered area by adhesions and the type of adhesion, toughness and the scores obtained by the adhesion score by correlation analysis (p <0.05). There was no difference between the groups in any variables in relation to the degree of inflammation. Conclusion: The purified type I bovine collagen coated light-weigh polypropylene mesh showed to be effective in the repair of abdominal wall defects and reducing adhesion formation.

Association between HER2 status in gastric cancer and clinicopathological features: a retrospective study using whole-tissue sections.

BACKGROUND: Gastric cancer is usually diagnosed in an advanced stage of disease and treatment options are sparse. Trastuzumab was recently approved for metastatic or locally advanced carcinomas arising in the stomach or in the gastroesophageal junction in patients with HER2-positive tumors. However, data on the frequency of HER2-positive cases among Brazilian patients are limited. Our aim was to characterize HER2 protein and gene status in a series of Brazilian patients with gastric cancer and to evaluate its association with clinicopathological data. METHODS: Histological slides from 124 primary gastrectomies were reviewed and their pathological reports were retrieved from the files at a Brazilian university hospital. Automated immunohistochemistry for HER2 was performed on whole-tissue sections from each tumor. HER2-equivocal cases by immunohistochemistry were submitted to automated dual in situ hybridization for gene amplification evaluation. HER2 status was confronted with clinicopathological parameters in order to assess statistically significant associations. RESULTS: Immunohistochemistry analysis revealed that 13/124 cases (10.5 %) were HER2 positive (3+), 10/124 cases (8.1 %) were equivocal (2+) and 101/124 cases (81.4 %) were negative, being 7 cases 1+. None of the equivocal cases showed gene amplification. The overall HER2 positivity rate was 10.5 %. There was an association between HER2 expression and Laurén's intestinal histological subtype (P = 0.048), well to moderately differentiated tumors (P = 0.004) and presence of lymphovascular invasion (P = 0.031). No association was found between HER2 status and tumor topography. CONCLUSIONS: Confronted with data published by other authors, the lower percentage of HER2-positive cases found in our series might be partially explained by the lower frequency of tumors arising at the gastroesophageal junction in comparison with distal gastric carcinomas in Brazilian patients. This could also account for the lack of statistically significant association between HER2 status and tumor topography in our study.

False positivity in HER2 testing of breast cancer: novel paths for approaching an old dilemma.

AIMS: Variability in determining HER2 status has been reported, especially, differences in sensitivity and specificity among commercially available antibodies, with false positive and false negative results. We compared the sensitivity and specificity of five anti-HER2 antibodies by immunohistochemistry (IHC), using the new dual colour brightfield in situ hybridisation (DDISH) as the gold standard, on invasive breast carcinomas (IBC) arrays. MATERIAL AND METHODS: Serial sections from tissue microarrays (TMA) containing 200 preselected primary IBC were submitted to DDISH (VENTANA INFORM HER2 Dual ISH assay), and immunohistochemistry, using Dako A0485 and HercepTest (polyclonal), Novocastra CB11 (mouse monoclonal), NeoMarkers SP3 and Ventana 4B5 (rabbit monoclonal). RESULTS: From the initial 200 cases, 184 were assessed by DDISH and IHC. The concordance among the antibodies was considered very good (kappa statistics varied from 0.82 to 0.9). The overall concordance between IHC and DDISH ranged from 94.1% for CB11 to 96.6% for A0485. The antibodies A0485, HercepTest, SP3 and 4B5 were over 95% sensitive and specific. CB11 was the most specific antibody (97.1%). 60% (CB11) to 83.3% (SP3) of the 2+ cases showed no gene amplification by DDISH. False negative cases varied from 0.5% (A0485) to 3.8% (CB11) of the cases, and false positive from 1.6% (CB11) to 2.7% (HercepTest, SP3 and 4B5) of the 184 cases. CONCLUSIONS: There was very good agreement among the five anti-HER2 antibodies. CB11 was the most specific antibody, but showed more false negative cases. A0485, SP3, 4B5 and HercepTest were highly sensitive and specific, but showed more false positive cases.

Rabbit antibodies for hormone receptors and HER2 evaluation in breast cancer.

BACKGROUND: Novel rabbit monoclonal antibodies (RabMab) for estrogen (ER), progesterone (PR) receptors and HER2 evaluation by immunohistochemistry have recently been commercially released. We compared the RabMab anti-ER, anti-PR and anti-HER2 to mouse monoclonal antibodies (Mab) using tissue microarrays (TMA) of breast carcinomas. METHODS: Two TMA containing breast carcinomas were built. Sections were immunostained using anti-ER and anti-PR, Mab and RabMab. The sections stained for ER and PR were evaluated considering positive those tumors in which more than 1% of the tumor cell nuclei stained moderate or strong. For HER2, the immunostained sections were evaluated using the ASCO/CAP guidelines for HER2. Chromogenic in situ hybridization (CISH) was used as the gold standard for HER2 evaluation. CISH was evaluated using the Zymed HER2 CISH interpretation guidelines. RESULTS: RabMab against ER have similar staining patterns compared to the 6F11 (Mab), but stronger than 1D5 (Mab) from three different suppliers. The RabMab against PR provide stronger and sharper immunohistochemical signals compared to Mab. The detection of HER2 protein overexpression was more prevalent with the polyclonal antibodies and RabMab than with the Mab. These were more specific than the RabMab, which were more sensitive when compared to CISH. CONCLUSION: The novel RabMab against ER and PR showed higher intensity of staining than the Mab. The RabMab against HER2 is more sensitive than Mab, however, Mab presented more specificity than RabMab when compared to CISH for HER2 evaluation of breast carcinomas.

Efeito da suplementação nutricional com glicina e glutamina, por via oral, na cicatrização colônica em coelhos / Effect of the nutritional supplementation with glicine and glutamine, oral feeding, in the colonic cicatrization in rabbits

OBJETIVO: Avaliar o efeito de dois aminoácidos, glicina e glutamina, na epitelização da mucosa colônica, tamanho da área de cicatriz, fibroplasia e resistência tênsil da anastomose intestinal, após colectomia parcial e anastomose término-terminal. MÉTODOS: Quarenta e cinco coelhos adultos, com idade entre 24 e 28 semanas, do sexo masculino, com peso inicial médio de 2.362 g, foram distribuídos aleatoriamente, de forma dupla desconhecida, em cinco grupos, de acordo com o tipo de suplemento. Dois animais morreram em cada grupo por causas diversas. Os grupos de coelhos foram assim distribuídos: Grupo 1 - (n = 9) receberam suplementação alimentar com glutamina durante sete dias antes e cinco dias após a operação; Grupo 2 - (n = 9) receberam suplementação alimentar com glicina durante sete dias antes e cinco dias após a operação; Grupo 3 - (n = 9) receberam suplementação alimentar com glutamina durante cinco dias após a operação; Grupo 4 - (n = 9) receberam suplementação alimentar com glicina durante cinco dias após a operação; Grupo 5 - (n = 9) não receberam suplementação alimentar. A resistência da anastomose foi medida por meio de pressão de ruptura por insuflação de ar intraluminar. RESULTADOS: O Grupo 2 foi o único grupo que apresentou valores de ruptura superiores aos do Grupo 5 (p < 0,05). Não houve diferença entre os grupos quanto aos aspectos histológicos estudados. CONCLUSÃO: A suplementação oral com glicina, no período pré e pós-operatório, aumenta a resistência tênsil anastomótica colônica após colectomia parcial em coelhos.

OBJECTIVE: To evaluate the effect of two amino-acids, glycine and glutamine, in the epithelialization of the colonic mucosa, size of the scar area, fibrous as well as the tensile strength of the intestinal anastomoses, after the removal of 17 cm of the colon and an end-to-end with interrupted suture. METHODS: Forty five male adult rabbits varying between 24 and 28 weeks of age with the starting average weight of 2,362 grams were randomly distributed into five groups, according to their respective type of feeding. Two animals died in each group due to different complications. The rabbits were divided into the following groups, according to the procedures performed on them: Group 1 - (n = 9) received supplemental feeding with glutamine for seven days before and as five days after the operation; Group 2 - (n = 9) received supplemental feeding with glycine for seven days before and five days after the operation; Group 3 - (n = 9) received supplemental feeding with glutamine for five days after the operation; Group 4 - (n = 9) received supplemental feeding with glycine for five days after the operation; Group 5 - (n = 9) did not receive any supplemental feeding. The resistance of the anastomoses was evaluated through the means of assay for pressure of rupture during intraluminal air insufflation. RESULTS: Group 2 was the only group that presented rupture values above those of Group 5 (p < 0.05). There was no difference among the groups concerning the histological aspects. CONCLUSION: Oral supplementation with glycine, in the pre and post-operative periods increases the tensile strength of colonic anastomoses after partial colectomy in rabbits. The oral supplementation with glutamine did not present detectable effect on the colonic anastomoses in rabbits.

Rabbit antibodies for hormone receptors and HER2 evaluation in breast cancer / Anticorpos de coelho para avaliação de receptores hormonais e HER2 em câncer de mama

BACKGROUND: Novel rabbit monoclonal antibodies (RabMab) for estrogen (ER), progesterone (PR) receptors and HER2 evaluation by immunohistochemistry have recently been commercially released. We compared the RabMab anti-ER, anti-PR and anti-HER2 to mouse monoclonal antibodies (Mab) using tissue microarrays (TMA) of breast carcinomas. METHODS: Two TMA containing breast carcinomas were built. Sections were immunostained using anti-ER and anti-PR, Mab and RabMab. The sections stained for ER and PR were evaluated considering positive those tumors in which more than 1 percent of the tumor cell nuclei stained moderate or strong. For HER2, the immunostained sections were evaluated using the ASCO/CAP guidelines for HER2. Chromogenic in situ hybridization (CISH) was used as the gold standard for HER2 evaluation. CISH was evaluated using the Zymed HER2 CISH interpretation guidelines. RESULTS: RabMab against ER have similar staining patterns compared to the 6F11 (Mab), but stronger than 1D5 (Mab) from three different suppliers. The RabMab against PR provide stronger and sharper immunohistochemical signals compared to Mab. The detection of HER2 protein overexpression was more prevalent with the polyclonal antibodies and RabMab than with the Mab. These were more specific than the RabMab, which were more sensitive when compared to CISH. CONCLUSION: The novel RabMab against ER and PR showed higher intensity of staining than the Mab. The RabMab against HER2 is more sensitive than Mab, however, Mab presented more specificity than RabMab when compared to CISH for HER2 evaluation of breast carcinomas.

OBJETIVOS: Novos anticorpos monoclonais de coelho (RabMab) para a avaliação imuno-histoquímica de receptores de estrógeno (RE), progesterona (RP) e HER2 foram lançados comercialmente. Comparamos os RabMab anti-RE, anti-RP e anti-HER2 com os anticorpos monoclonais de camundongo (Mab) utilizando tissue microarrays (TMA) de carcinomas de mama. MÉTODOS: Foram construídos dois TMAs de carcinomas de mama. As secções foram marcadas usando anti-RE, anti-RP e anti-HER2, Mab e RabMab através de imuno-histoquímica. As secções marcadas para RE e RP foram avaliadas considerando positivos aqueles tumores nos quais mais de 1 por cento dos núcleos coraram moderadamente ou forte. Para HER2, as secções foram avaliadas utilizando as recomendações da ASCO/CAP para HER2. Hibridização in situ cromogênica (CISH) foi usada como padrão-ouro para avaliação de HER2. CISH foi avaliado utilizando as recomendações da Zymed. RESULTADOS: Os RabMab anti-RE apresentam intensidade de coloração semelhante ao 6F11 (Mab), porém maior que o 1D5 (Mab) proveniente de três diferentes fabricantes. Os RabMab anti-RP apresentaram sinal imunoistoquímico mais forte e delimitado comparado aos Mab. A detecção da superexpressão da proteína HER2 foi mais prevalente entre os anticorpos policlonais e RabMab, que se mostraram mais sensíveis quando comparados com o CISH. CONCLUSÃO: Os novos RabMab anti-RE e RP proporcionaram maior intensidade de coloração que os Mab. O RabMab anti-HER2 apresentou maior sensibilidade que os Mab, porém os Mab apresentaram maior especificidade quando comparados com o CISH para a avaliação de HER2 em carcinomas de mama.

Predictive factors of breast cancer evaluated by immunohistochemistry: [review] / Fatores preditivos do câncer de mama avaliados pela imuno-histoquímica: [revisão]

Hormone receptor and Her2 protein overexpression evaluated by immunohistochemistry (IHC) is widely validated as a predictive factor in breast cancer. The quality of the IHC reaction is influenced by tissue fixation and processing. Over- and underfixation deeply affect IHC results. Antigen retrieval may improve IHC but it does not recover tissue from autolysis or overfixation. The choice of primary antibody for IHC as to its sensitivity and specificity in relation to therapeutic response represents an important stage. Apart from mouse monoclonal antibodies, new rabbit monoclonal antibodies are commercially available, such as clones anti-ER SP1 and B644, anti-PR SP2 and B645 and anti-Her2 SP3 and 4B5. They represent an alternative to hormone receptor and Her2 evaluation by IHC. New polymeric non-biotinylated detection systems are also available and allow accurate and strong marking with no stromal and no non-specific cytoplasmic staining due to endogenous biotin. The most recommended cut off for estrogen and progesterone receptors (ER and PR) is more than 1 percent of positive cells with moderate or strong staining intensity (Allred's scoring system). New guidelines for Her2 evaluation by IHC show a cut off of more than 30 percent of positive cells with strong intensity (3+) that correlates better with gene amplification. The 2+ cases are now considered indeterminate and should be confirmed by fluorescence in situ hybridisation (FISH) or chromogenic in situ hybridisation CISH. A quality control of pre-analytical, analytical and post-analytical phases of IHC is recommended in order to optimize results.

A superexpressão de receptores hormonais e Her2 avaliada pela imuno-histoquímica (IHQ) é amplamente validada como fator preditivo em câncer de mama. A qualidade da reação imuno-histoquímica é influenciada pela fixação do tecido e seu processamento. A fixação insuficiente ou demasiada afeta profundamente os resultados da IHQ. A reativação antigênica pode melhorar os resultados da IHQ, porém não recupera tecidos com autólise ou com excessiva fixação. A escolha do anticorpo primário para a IHQ, considerando sua sensibilidade e sua especificidade de acordo com a resposta terapêutica, representa uma importante etapa. Além de anticorpos monoclonais de camundongo, novos anticorpos monoclonais de coelho são comercialmente disponíveis, tais como clones SP1 e B644 anti-RE, SP2 e B645 anti-RP, e SP3 e 4B5 anti-Her2. Eles representam uma alternativa para avaliação de receptores hormonais e Her2 através da IHQ. Novos sistemas de detecção poliméricos não-biotinilados também são disponíveis e permitem marcação exata e forte sem marcação estromal ou citoplasmática inespecífica devido à biotina endógena. O cut off mais recomendado para receptor de estrogênio (RE) e receptor de progesterona (RP) é acima de 1 por cento de células positivas com marcação moderada ou forte (sistema de escore de Allred). Novas recomendações para avaliação de Her2 através da IHQ apontam um cut off de mais de 30 por cento de células positivas com marcação forte (3+), que melhor se relaciona com amplificação gênica. Os casos 2+ são agora considerados indeterminados e devem ser confirmados por hibridação in situ por fluorescência (FISH) ou hibridização in situ colorimétrica (CISH). Um controle de qualidade de fases pré-analítica, analítica e pós-analítica da IHQ é recomendado para a otimização dos resultados.

A comparison between the novel rabbit monoclonal antibodies (SP1 and B644) and mouse antibodies for evaluating estrogen receptor in breast tumors / Uma comparação entre os novos anticorpos monoclonais de coelho (SP1 e B644) e anticorpos de camundongo para detecção de receptores de estrógeno em carcinomas mamários

BACKGROUND: A novel generation of rabbit monoclonal antibodies has been released recently for estrogen (ER) and progesterone (PR) receptor evaluation in breast cancer by immunohistochemistry. Aims: We compared novel rabbit monoclonal antibodies anti-ER SP1 (LabVision®) and B644 (Cell Marque®) to mouse monoclonal antibodies 1D5 (Dako®) and 6F11 (Novocastra®) using a tissue microarray of breast carcinomas. METHODS: Two cylinders (2 mm diameter) of formalin-fixed paraffin embedded tissue were obtained from 24 invasive breast carcinomas and immunostained by using the anti-ER rabbit and mouse antibodies and the streptavidin-biotin detection system (Biogenex®). Immunostaining was evaluated considering positive those tumors in which more than 10 percent of the tumor cell nuclei stained. The stain intensity was also evaluated as weak (1), moderate (2), and strong (3). Results: Both rabbit antibodies against ER have similar staining pattern to each other and also to 6F11, but significantly stronger scores compared to mouse 1D5. The rabbit antibodies allow better cost/benefit because of higher working dilutions compared to mouse antibodies using the same procedure. CONCLUSION: The new rabbit antibodies against ER are highly sensitive and reliable in clinical and research immunohistochemical testing of breast carcinomas.

INTRODUÇÃO: Uma nova geração de anticorpos monoclonais de coelho tem sido produzida para detecção de receptores de estrógeno (RE) e progesterona (RP) pela imuno-histoquímica em câncer de mama. OBJETIVO: Comparamos os novos anticorpos monoclonais de coelho anti-RE SP1 (LabVision®) e B644 (Cell Marque®) com anticorpos monoclonais de camundongo 1D5 (DAKO®) e 6F11 (Novocastra®) utilizando um tissue microarray de carcinomas mamários. METODOLOGIA: Dois cilindros (2 mm de diâmetro) de tecido fixado em formol e embebido em parafina foram retirados de 24 carcinomas mamários invasivos e corados pela imuno-histoquímica utilizando-se os anticorpos de coelho e de camundongo anti-RE e o sistema de detecção estreptavidina-biotina peroxidase (Biogenex®). A coloração imuno-histoquímica foi avaliada considerando positivos os tumores nos quais mais de 10 por cento dos núcleos das células tumorais estivessem corados. A coloração também foi classificada em fraca (1), moderada (2) e forte (3). RESULTADOS: Ambos os anticorpos monoclonais de coelho contra RE apresentaram intensidade de coloração semelhante àquela pelo anticorpo de camundongo 6F11, porém os anticorpos de coelho apresentaram intensidades de coloração significativamente mais fortes que as do clone de camundongo 1D5. As altas diluições possíveis utilizando anticorpos de coelho permitem melhor custo/benefício quando comparadas com as diluições possíveis utilizando anticorpos de camundongo. CONCLUSÃO: Os novos anticorpos monoclonais de coelho anti-RE são altamente sensíveis e fidedignos para testes imuno-histoquímicos tanto para a clínica quanto para pesquisa de tumores mamários.

Concordância interobservador na interpretação imuno-histoquímica da superexpressão do Her2 detectada por cinco diferentes anticorpos em array de carcinomas mamários / Interobserver agreement in scoring immunohistochemical overexpression of Her2 detected by five different antibodies on tissue microarrays of breast carcinomas

OBJETIVO: Estudar a concordância interobservador na interpretação da superexpressão imuno-histoquímica para a proteína Her2 empregando diferentes anticorpos em array de carcinomas mamários. MATERIAL E MÉTODO: Foi construído um array contendo dois cilindros (2 mm de diâmetro cada) de 25 carcinomas mamários. Cortes histológicos seriados do array foram submetidos à imuno-histoquímica utilizando-se cinco anticorpos anti-Her2: SP3 (NeoMarkers), HercepTest e A0485 (Dako), CB11 (Novocastra) e 4D5 (Genentech). Uma lâmina corada por cada anticorpo (total = cinco lâminas) foi submetida à avaliação individual por cinco observadores seguindo-se o sistema de escore proposto no HercepTestTM. Para a avaliação interobservador os resultados foram interpretados em três diferentes análises: I (0; 1+; 2+; 3+); II (0 e 1+; 2+ e 3+) e III (0 e 1+; 2+; 3+) e aplicado o teste estatístico de kappa. RESULTADOS: A concordância interobservador foi boa quando os casos foram avaliados em quatro categorias (0; 1+; 2+; 3+). Quando avaliados em duas categorias (0 e 1+; 2+ e 3+), a concordância interobservador foi boa para os casos corados por SP3 e CB11 e muito boa para os corados por A0485, HercepTest e 4D5. Na análise III (0 e 1+; 2+; 3+), a concordância interobservador foi considerada moderada para os casos corados por CB11 e boa para os corados pelos outros anticorpos. CONCLUSÃO: A concordância interobservador foi considerada entre moderada e muito boa na avaliação dos cinco anticorpos. A menor concordância interobservador ocorreu nos casos com marcações fraca (1+) e moderada (2+). A experiência dos observadores influenciou as taxas de concordância.

AIM: To examine interobserver agreement in immunohistochemical evaluation of Her2 overexpression using five different antibodies on breast cancer array. MATERIAL AND METHOD: Material and method: One array was built with two cores (2 mm diameter each) from 25 breast carcinomas. Serial sections from the array were submitted to immunohistochemistry using five anti-Her2 antibodies: SP3 (NeoMarkers), HercepTest and A0485 (Dako), CB11 (Novocastra), and 4D5 (Genentech). One slide immunostained for each antibody (total = five slides) were independently scored by five observers following HercepTestTM scoring system. Interobserver agreement was evaluated in three different analysis: I (0; 1+; 2+; 3+); II (0 and 1+; 2+ and 3+) and III (0 and 1+; 2+; 3+), and the kappa statistics was applied. RESULTS: There was a good rate of interobserver agreement when the four scores were considered (0; 1+; 2+; 3+). When the scores were considered in two categories (0 and 1+; 2+ and 3+) the interobserver agreement rate was considered substantial for cases stained for SP3 and CB11, and almost perfect for cases stained for A0485, HercepTest and 4D5. For analysis III (0 and 1+; 2+; 3+), a moderate rate of interobserver agreement was considered for cases stained for CB11, and a substantial rate for other antibodies. CONCLUSION: The overall interobserver agreement was considered moderate to substantial in the evaluation of cases stained for the five antibodies. The lowest rate of agreement was obtained in the evaluation of the cases scored as weak (1+) and moderate (2+). The observers experience altered the concordance rates.

Morfologia e resistência cicatriciais da parede abdominal após laparotomias longitudinal e transversal em coelhos / Abdominal wall morphology and healing resistance after longitudinal and transversal laparatomy in rabbits

OBJETIVO: Foram estudadas comparativamente as laparotomias longitudinais paramediana e transversal avaliando-se a resistência cicatricial da parede abdominal e seu aspecto histológico, com peritônio suturado ou não. MÉTODO: 30 coelhos foram distribuídos em dois grupos: Grupo 1 (n = 10) - laparotomia longitudinal, Subgrupo 1A (n = 5) sutura das bainhas anterior e posterior do músculo reto abdominal, bem como do peritônio, Subgrupo 1B (n = 5) sutura da bainha anterior do músculo reto abdominal. Grupo 2 (n = 20) - laparotomia transversa, Subgrupo 2A (n = 5) sutura das bainhas anterior e posterior do músculo reto abdominal, bem como do peritônio, Subgrupo 2B (n = 5) sutura apenas da bainha anterior do músculo reto abdominal, Subgrupo 2C (n = 5) sutura, em plano único, do músculo reto abdominal e de sua bainha anterior, Subgrupo 2D (n = 5) síntese, da bainha posterior do músculo reto abdominal junto com o peritônio e, em seguida, sutura do músculo reto abdominal, complementada por sutura da bainha anterior desse músculo. Após 17 dias, foram retirados dois segmentos peritonio-aponeurotico-musculares da cicatriz para avaliação da resistência e de seus aspectos histológicos. RESULTADOS: O valor da resistência para cada um dos grupos avaliados mostrou 1A > 1B, 1A > 2A e 1B > 2B, e 2B > 2C > 2D > 2A (p = 0,014). Deiscência, infecção e aderências foram mais freqüentes no Grupo 2. A histologia mostrou degeneração e necrose muscular, com sua substituição por tecido conjuntivo fibroso maduro cicatricial. CONCLUSÃO: Esses dados indicam que o corte muscular transversal provoca maior enfraquecimento muscular e que o peritônio deixado aberto não altera a resistência cicatricial.

BACKGROUND: The abdominal wall is a main topic of investigation in terms of its healing. The purpose of this investigation was to assess the healing of transverse and longitudinal laparotomies. METHODS: Thirty rabbits were distributed into two groups: Group 1 (n = 10) longitudinal laparotomy, Subgroup 1A (n = 5) suture of the anterior and posterior sheaths of the abdominal rectal muscle and peritoneum, Subgroup 1B (n = 5) suture of the anterior abdominal rectal muscle sheath. Group 2 (n = 20) transverse laparotomy, Subgroup 2A (n = 5) suture of the anterior and posterior sheaths of the rectal muscle and peritoneum, Subgroup 2B (n = 5) suture of only the anterior rectal muscle sheath, Subgroup 2C (n = 5) suture of the anterior rectal muscle sheath of the, Subgroup 2D (n = 5) suture of the anterior and posterior rectal muscle sheaths, including the peritoneum. After 17 days, two peritoneal-aponeurotic-muscle segments with the scar were removed in order to verify their resistance and histological aspects. RESULTS: The resistance values detected on each group showed 1A > 1B, 1A > 2A and 1B > 2B, and 2B > 2C > 2D > 2A (p = 0.014). Dehiscence, infections and adhesions were more frequent in Group 2. Histology revealed muscle degeneration and necrosis, with mature fibrous connective scar tissue replacing the muscle. CONCLUSION: Transverse muscle section provokes a greater muscle weakening; the peritoneum closure does not modify the scar resistance.

Construção de arrays de tecido com equipamento alternativo e de baixo custo para estudo imuno-histoquímico de tumores mamários / Construction of low cost tissue microarrays using alternative equipment for immunohistochemical study of breast tumors

INTRODUÇÂO: Tissue microarrays (TMA) são blocos contendo numerosos cilindros de tecido parafinado organizados em linhas e colunas que permitem analisar muitas amostras numa única lâmina. Equipamentos disponíveis comercialmente são importados e têm alto custo (entre 11 e 24 mil dólares). OBJETIVOS: Descrever uma forma alternativa de construção de arrays de tumores mamários de baixo custo e relatar nossa experiência na sua utilização em estudo imuno-histoquímico (IIQ). METODOLOGIA: Utilizou-se modelo que consiste numa minirretífica com agulha de biópsia hepática de 2mm de diâmetro acoplada à bancada com suporte (Dremel). Inicialmente preparou-se o bloco receptor fazendo o número de furos desejável (55). Cilindros de tecido foram obtidos com o mesmo dispositivo e colocados nos orifícios do bloco receptor. De cada bloco doador, obteve-se dois cilindros de diferentes áreas representativas do tumor. Em cada array foram incluídos cilindros de tumor (controle positivo e negativo) para cada anticorpo testado na IIQ, e cilindro marcador de iniciação da leitura da lâmina (fígado). Cortes sequenciais de 4µm obtidos do array foram submetidos à IIQ. A primeira e a última lâmina foram coradas pela hematoxilina e eosina (HE) para avaliar: número de discos de tecido, preservação tecidual e adequabilidade da amostra. Foi realizada IIQ empregando anticorpos anti-receptores de estrógeno, progesterona, Ki67, p53 e Her2. RESULTADOS: O custo total do equipamento foi apenas US$180,00. Cortes histológicos do array apresentaram boa preservação tecidual, sendo adequados para avaliação morfológica e suficientes para confirmação diagnóstica. A qualidade das realizações IIQ foi semelhante à obtida nos blocos doadores. CONCLUSÃO: Esse equipamento e a técnica representam uma alternativa econômica aos equipamentos comerciais.

BACKGROUND: Tissue microarrays (TMA) are blocks containing numerous cylinders of paraffinized tissue organized in lines and columns allowing analysis of numerous samples in one slide. Commercially available equipment is imported and have high cost (US$ 11,000.00 to 24,000.00). AIM: we describe a low cost breast-tumor TMA and our experience in its use for immunohistochemistry (IHC). MATERIAL AND METHODS: A model that consists of a work station (Dremel) to which a liver biopsy needle of 2mm of diameter was connected. A receptor block was prepared perforating it until the desired number of rows (55) was reached. Then, the cylinders of tissue were obtained using the same equipment and included in the holes of the receptor block. Two samples were obtained from different tumor areas of each donor block. Cylinders of previously tested positive control tumors for each antibody and one marker (liver sample) that indicated the beginning of slide reading were also included. IHC was performed in sequential 4µm sections from the same array using antibodies against estrogen and progesterone receptors, Ki67, p53 and Her2. The first and the last slides were stained by hematoxylin and eosin to evaluate: number of tissue discs, tissue preservation, and adequacy of the tissue sample. RESULTS: The equipment total cost was US$ 180,00. The slides showed fine tissue preservation, adequate for morphologic evaluation, and sufficient to confirm diagnosis. The IHC quality was similar to the donor blocks. CONCLUSION: This equipment and technique represent an economical alternative when compared to commercial equipments.

Efeito da orquiectomia na resistência cicatricial cutânea em ratos / The effect of orchiectomy in healing of skin wounds in rats

O objetivo do presente trabalho foi verificar o efeito da depleção de testoterona na cicatrização de lesão cirúrgica cutânea em diferentes faixas etárias e distintos períodos pós-operatórios. Foram utilizados 42 ratos Wistar machos, distribuídos em 2 grupos: Grupo 1 (n=21) - ratos jovens, com 30 dias de vida e peso entre 55 e 80 gramas e Grupo 2 (n=21) - ratos adultos, entre 3 e 4 meses de idade e peso entre 250 e 350 gramas. Os animais de ambos os grupos foram distribuídos em dois subgrupos: Bubgrupo 1A (n= 11) - controle jovem, não operado; Subgrupo 1B (n= 10) - ratos jovens submetidos a orquiectommia bilateral; Subgrupo 2A (n=10) - controle adulto, não operado e e Subgrupo 2B(n=11) - ratos adultos submetidos a orquiectomia bilateral. Após seis meses, realisou-se incisão linear no dorso dos animais, atingindo a pele e o subcutâneo, em direção longitudinal mediana. A resistência das cicatrizes foi medida com um tensiômetro, utilizando um fragmento de pele transversal à cicatriz, tendo a cicatriz em sua parte média. Esse procedimento foi feito no 7º e no 21º dias após a realização das incisões. A resistência da cicatriz foi maior no Subgrupo 1A do que no Subgrupo 1B após 7 dias (p<0,05); a resistência cicatricial após 21 dias foi maior do que a encontrada após 7 dias em ambos os grupos (p<0,05). Não se verificou diferença entre as resistências cicatriciais dos ratos jovens e adultos. A orquiectomia bilateral diminuiu a resistência cicatricial nos animais jovens no 7º dia pós-operatório. Nos demais grupos, não se evidenciaram alterações na resistência cicatricial da pele decorrentes da deficiência de testosterona