The nuclear and mitochondrial genomes of Frieseomelitta varia - a highly eusocial stingless bee (Meliponini) with a permanently sterile worker caste.

BACKGROUND: Most of our understanding on the social behavior and genomics of bees and other social insects is centered on the Western honey bee, Apis mellifera. The genus Apis, however, is a highly derived branch comprising less than a dozen species, four of which genomically characterized. In contrast, for the equally highly eusocial, yet taxonomically and biologically more diverse Meliponini, a full genome sequence was so far available for a single Melipona species only. We present here the genome sequence of Frieseomelitta varia, a stingless bee that has, as a peculiarity, a completely sterile worker caste. RESULTS: The assembly of 243,974,526 high quality Illumina reads resulted in a predicted assembled genome size of 275 Mb composed of 2173 scaffolds. A BUSCO analysis for the 10,526 predicted genes showed that these represent 96.6% of the expected hymenopteran orthologs. We also predicted 169,371 repetitive genomic components, 2083 putative transposable elements, and 1946 genes for non-coding RNAs, largely long non-coding RNAs. The mitochondrial genome comprises 15,144 bp, encoding 13 proteins, 22 tRNAs and 2 rRNAs. We observed considerable rearrangement in the mitochondrial gene order compared to other bees. For an in-depth analysis of genes related to social biology, we manually checked the annotations for 533 automatically predicted gene models, including 127 genes related to reproductive processes, 104 to development, and 174 immunity-related genes. We also performed specific searches for genes containing transcription factor domains and genes related to neurogenesis and chemosensory communication. CONCLUSIONS: The total genome size for F. varia is similar to the sequenced genomes of other bees. Using specific prediction methods, we identified a large number of repetitive genome components and long non-coding RNAs, which could provide the molecular basis for gene regulatory plasticity, including worker reproduction. The remarkable reshuffling in gene order in the mitochondrial genome suggests that stingless bees may be a hotspot for mtDNA evolution. Hence, while being just the second stingless bee genome sequenced, we expect that subsequent targeting of a selected set of species from this diverse clade of highly eusocial bees will reveal relevant evolutionary signals and trends related to eusociality in these important pollinators.

Abdominoplasty and Patient Safety: The Impact of Body Mass Index and Bariatric Surgery on Complications Profile.

BACKGROUND: Abdominoplasty is a frequent aesthetic surgical procedure. Despite its popularity, patient safety requires further attention because of an important risk of complications. Obesity and bariatric surgery are potential risk factors. This study evaluated the impact of body mass index (BMI) and bariatric surgery on complications profile. METHODS: A retrospective review of patients undergoing abdominoplasty over a 5-year period was conducted. Patient demographics, comorbidities, BMI, bariatric surgery, complications (minor and major) and drainage outcomes were evaluated. Univariate and multivariate logistic regression analyses were performed for risk assessment. RESULTS: The 191 study patients showed an overall complication rate of 32.5%, comprising minor (27.7%) and major complications (7.9%). Seroma was the most common complication (20.9%). Mean BMI was 27.1 kg/m2. Obesity was present in 14.1%, and bariatric surgery in 53.4%. Obese patients had significant higher rates of overall complications (74.1%, p < 0.001), minor complications (66.7%, p < 0.001), seroma (51.9%, p < 0.001) and drainage outputs (p < 0.001). Bariatric patients showed an increased time (p = 0.003) and volume of drainage (p < 0.001), without significant differences in complications. Obesity and preoperative BMI were the only independent risk factors for overall complications (OR 8.3; and OR 1.3; p < 0.001), minor complications (OR 7.4; and OR 1.3; p < 0.001) and seroma (OR 4.5; and OR 1.2; p = 0.002). Diabetes was an independent risk for major complications (OR 4.6; p = 0.047). CONCLUSION: Obesity has negatively impacted the profile and risk of complications, whereas bariatric surgery independently did not. Our study provides an accurate understanding of abdominoplasty complications, allowing better risk stratification, candidate selection refinement, morbidity reduction, fair patient counseling and overall improvement in patient safety. EBM LEVEL III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Rats Born to Mothers Treated with Dexamethasone 15 cH Present Changes in Modulation of Inflammatory Process.

As little information about the effect of ultra high dilutions of glucocorticoid in reproduction is available in the literature, pregnant female Wistar rats (N = 12) were blindly subcutaneously treated during all gestational and lactation period with: dexamethasone 4 mg/kg diluted into dexamethasone 15 cH (mixed); or dexamethasone 4 mg/kg diluted in water; or dexamethasone 15 cH, or vehicle. Parental generation had body weight, food and water consumption monitored. The F1 generation was monitored regarding to newborn development. No birth occurred in both groups treated with dexamethasone 4 mg/kg. After 60 days from birth, 12 male F1 rats were randomly selected from each remaining group and inoculated subcutaneously with 1% carrageenan into the footpad, for evaluation of inflammatory performance. Edema and histopathology of the footpad were evaluated, using specific staining methods, immunohistochemistry and digital histomorphometry. Mothers treated with mixed dexamethasone presented reduced water consumption. F1 rats born to dexamethasone 15 cH treated females presented significant increase in mast cell degranulation, decrease in monocyte percentage, increase in CD18+ PMN cells, and early expression of ED2 protein, in relation to control. The results show that the exposure of parental generation to highly diluted dexamethasone interferes in inflammation modulation in the F1 generation.

Bilateral Lower Limb Salvage after Fourth-degree Burns: Subscapular Axis Free and Chimeric Flaps Effectiveness in Complex Reconstruction.

Microsurgery is usually required for reconstruction of complex lower limb defects, preserving otherwise unsalvageable injuries. Fourth-degree burns are severe traumatic injuries. A case of bilateral lower limb salvage through a resourceful use of subscapular axis free and chimeric flaps for acute burn reconstruction of extensive lower leg injuries is reported. A 48-year-old man sustained a 40% surface area flame burn, circumferential and full-thickness at the lower limbs. Debridement of fourth-degree burns of the anterior lower legs resulted in bone exposure of the left and right tibias, right medial malleolus, and Achilles tendon. A latissimus dorsi (LD) flap plus a chimeric subscapular axis free flap with 3 components (LD, serratus anterior (SA), and parascapular) were designed for reconstruction. LD insetting for left tibia coverage with anastomoses to anterior tibial vessels was performed. Right side flap insetting provided tibia coverage with LD; medial malleolus with SA; and Achilles tendon with parascapular flap. An anatomical variation required anastomoses to proximal (chimeric LD + SA) and distal (parascapular) ends of posterior tibial vessels because of an independent origin of the pedicles. At 10-months follow-up after intensive rehabilitation, the patient showed proper functional outcomes at daily-life and work activities with autonomous walking using a single crutch. This case highlights the importance of microsurgery and chimeric flaps for limb salvage in extreme situations. The authors review and discuss the surgical options, emphasizing specific considerations of microsurgical reconstruction in burn patients.

Nail enthesis ultrasound and automated software-guided assessment of bilateral common carotid intima-media thickness in psoriasis and psoriatic arthritis: is there a correlation with clinical and laboratory findings?

BACKGROUND: Ultrasound (US) in psoriasis (PsO) and psoriatic arthritis (PsA) is an important tool in several situations to detect joint ecostructural damage as well as other tissue alterations, such as those that occur in the larger vessels. The objective of this study was to detect and correlate the changes that indicate the inflammatory and atherosclerotic process in two groups of patients, using nail US and carotid artery intima-media thickness radiofrequency (RF) software. METHODS: A total of 30 patients diagnosed with (PsO) and (PsA) were selected. About 15 patients were present in each group, assigned by the Dermatology and Rheumatology Service of the Universidade Pontifícia Católica de Campinas, São Paulo, Brazil, and were assessed using carotid artery US (radiofrequency quality intima-media thickness [RF-QIMT]), joint US, clinical evaluation, and laboratory tests. RESULTS: Spearman and Pearson correlations between US variables per group were Psoriasis Area and Severity Index (PASI) and loss of the nail pattern trilaminar: r=0.658, p=0.015; Framingham Score (FS) and Internal Resistance Index (IR): 0.351 to 0.526, p=0.034 to 0.002; the significant correlations by the Bayesian factor (BF) were those with a BF greater than 2.5, between QIMT expected with FS: r=0.677, BF=10.06, with total cholesterol: r=0.5232, BF=2.60, and QIMT-RF with low density lipoproteins: r=0.633, BF=3.70. CONCLUSION: The use of US in the evaluation of these patients showed significant correlations between clinical and laboratory measures, characterized by QIMT and FS as well as changes in nail insertion. Future studies may demonstrate an even better interaction.

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.

Lymph vascular invasion in invasive mammary carcinomas identified by the endothelial lymphatic marker D2-40 is associated with other indicators of poor prognosis.

BACKGROUND: Immunohistochemical studies of lymphatic vessels have been limited by a lack of specific markers. Recently, the novel D2-40 antibody, which selectively marks endothelium of lymphatic vessels, was released. The aim of our study is to compare lymphatic and blood vessel invasion detected by hematoxylin and eosin (H&E) versus that detected by immunohistochemistry, relating them with morphologic and molecular prognostic factors. METHODS: We selected 123 cases of invasive mammary carcinomas stratified into three subgroups according to axillary lymph node status: macrometastases, micrometastases, and lymph node negative. Lymphatic (LVI) and blood (BVI) vessel invasion were evaluated by H&E and immunohistochemistry using the D2-40 and CD31 antibodies, and related to histologic tumor type and grade, estrogen and progesterone receptors, E-cadherin, Ki67, p53, and Her2/neu expression. RESULTS: LVI was detected in H&E-stained sections in 17/123 cases (13.8%), and in D2-40 sections in 35/123 cases (28.5%) (Kappa = 0.433). BVI was detected in H&E-stained sections in 5/123 cases (4.1%), and in CD31 stained sections in 19/123 cases (15.4%) (Kappa = 0.198). LVI is positively related to higher histologic grade (p = 0.013), higher Ki67 expression (p = 0.00013), and to the presence of macrometastases (p = 0.002), and inversely related to estrogen (p = 0.0016) and progesterone (p = 0.00017) receptors expression. CONCLUSION: D2-40 is a reliable marker of lymphatic vessels and is a useful tool for lymphatic emboli identification in immunostained sections of breast carcinomas with higher identification rates than H&E. Lymphatic vessel invasion was related to other features (high combined histologic grade, high Ki67 score, negative hormone receptors expression) associated with worse prognosis, probable reflecting a potential for lymphatic metastatic spread and aggressive behavior.

Rabbit monoclonal antibodies show higher sensitivity than mouse monoclonals for estrogen and progesterone receptor evaluation in breast cancer by immunohistochemistry.

A novel generation of rabbit monoclonal antibodies for estrogen (ER) and progesterone (PR) receptor evaluation in breast cancer by immunohistochemistry has been released recently. We compared the novel RabMab anti-ER and anti-PR antibodies with the mouse monoclonal antibodies using a tissue microarray of breast carcinomas. Two cylinders (2mm diameter) of formalin-fixed, paraffin-embedded tissue were obtained from 24 invasive breast cancers and were immunostained using anti-ER mouse (1D5 and 6F11) and rabbit antibodies (SP1 and B644), and anti-PR mouse (PgR312 and PgR636) and rabbit antibodies (SP2 and B645). The immunohistochemistry was evaluated by considering positive those tumors in which more than 10% of the tumor cell nuclei stained independently on the staining intensity. Our results demonstrated that rabbit antibodies against ER have a similar staining pattern compared to the 6F11, but better than 1D5 from three different suppliers. The rabbit antibodies against PR (SP2 and B645) provide a stronger and sharper immunohistochemical signal compared to mouse antibodies (PgR636 and PgR312). Both ER and PR rabbit antibodies allow a lower cost per test because of higher working dilutions compared to mouse antibodies using the same procedure. The novel rabbit antibodies against ER and PR are highly sensitive for immunohistochemical testing of breast carcinomas.

[Molecular features of breast cancer predictive of lymph node metastases]. / Marcadores moleculares em câncer de mama preditivos de metástases axilares.

OBJECTIVE: The aim of our study was to analyze morphologic and molecular markers of breast cancer relating them to the presence of metastases in axillary lymph nodes. METHODS: We selected 123 cases of invasive mammary carcinomas stratified into three subgroups: with macrometastases, with micrometastases, and lymph node negative. Presence of metastases was evaluated relating them with morphologic factors (size of primary tumor, type and grade, presence of lymphatic and blood vessel invasion in hematoxylin and eosin-stained slides) and molecular factors of primary tumor (estrogen and progesterone receptors, E-cadherin, Ki67, p53, Her2 expression, and the presence of lymphatic and blood vessel invasion in immunostained sections for D2-40 and CD31). RESULTS: Axillary lymph node metastases were positively related to the presence of lymphatic vessel invasion in hematoxylin and eosin (H&E)-stained slides, when analyzed with or without metastases (p=0.04) and when analyzed in the three subgroups (p=0.002). Lymph node metastases were also positively related to presence of blood vessel invasion identified by immunohistochemistry (IHC) for CD31 (p=0.02). However other morphologic and molecular factors were not related to the presence of axillary node metastases. CONCLUSION: Lymphatic and blood vessel invasion identified in H&E and IHC-stained slides are positively related to the rmetastatic status of axillary lymph nodes and are predictive of axillary lymph node metastases in breast cancer.

Qualidade de Vida de Estudantes de Medicina em um Curso que Adota Metodologias Ativas de Ensino-Aprendizagem / Quality of Life of Medical Students on a Course that Adopts Active Teaching-Learning Methods

RESUMO A qualidade de vida (QV) de estudantes de Medicina tem sido alvo de estudos, mas não encontramos na literatura trabalhos que avaliem a QV de estudantes de escolas médicas que adotam exclusivamente metodologias ativas de ensino-aprendizagem. O presente estudo avaliou a qualidade de vida dos estudantes do primeiro ao quarto ano do curso de Medicina da Universidade Federal de São Carlos. Como instrumento de coleta de dados, foi utilizado o questionário WHOQoL-100, em sua versão completa, traduzida e validada em português, que avalia seis domínios, além da QV global. Foram participantes todos os 182 estudantes matriculados do primeiro ao quarto ano do curso. Quatro estudantes foram excluídos da amostra por preenchimento incompleto do questionário, resultando em cinco grupos: grupo controle com 32 alunos recém-ingressos no curso de Medicina; grupo primeiro ano com 38 alunos concluintes; grupo segundo ano com 35 concluintes; grupo terceiro ano com 35 concluintes; e grupo quarto ano com 38 concluintes. A análise descritiva dos resultados foi apresentada como média ± erro padrão da média. A significância das diferenças foi determinada por análise de variância, seguida pelo teste de comparações múltiplas de Newman-Keuls. Para a análise de correlação, foi utilizado o método de correlação de Spearman. O nível de significância adotado foi de 5%. A QV global apresentou melhor pontuação no grupo controle (77,54 ± 2,153); seguida, em ordem decrescente, pelos grupos segundo ano (65,71 ± 3,923), quarto ano (65,63 ± 3,306), primeiro ano (65,46 ± 3,289) e terceiro ano (52,86 ± 2,776). Houve diferença significativa na QV global do grupo terceiro ano em relação aos demais (p < 0,05). Entre todos os domínios avaliados, o psicológico influenciou significativamente a QV, apresentando forte correlação com a QV nos grupos do primeiro ao quarto ano.

ABSTRACT The quality of life (QoL) of medical students has been the subject of studies, but we did not find any works in the literature that assess the QoL of students of medical schools that exclusively adopt active teaching methods. This study therefore evaluates quality of life in students from the 1st to 4th year of medical school at the Universidade Federal de São Carlos. The complete version WHOQoL-100 questionnaire was used as the data collection instrument, translated and validated for Portuguese. This questionnaire evaluates six domains, in addition to global QoL. All 182 students enrolled from the 1st to 4th year were interviewed. Four students were excluded from the sample due to incomplete filling out of the questionnaire, resulting in five groups: a control group with 32 students recently admitted to medical college; 1st year group with 38 students who had just completed the 1st year; a 2nd year group with 35 students who had just completed the 2nd year; a 3rd year group with 35 students who had just completed the 3rd year; and a 4th year group with 38 students who had just completed the 4th year. Descriptive analysis of the results was presented as means ± SEM. The significance of the differences was determined by analysis of variance followed by the Newman-Keuls multiple comparison test. For the correlation analysis, the Spearman correlation method was used. The level of significance was set at 5%. Global QoL showed a better score in the control group (77.54 ± 2.153) followed, in descending order, by the 2nd year (65.71 ± 3.923), 4th year (65.63 ± 3.306), 1st year (65.46 ± 3.289) and 3rd year (52.86 ± 2.766). There was a significant difference in the Global QoL for the 3rd year group in relation to the others (p <0.05). Among all the evaluated domains, the psychological domain had a significant influence on QoL, presenting strong correlation in the 1st to the 4th year groups.

Avaliação higienicossanitária de açougues de rede atacadista do município de Guarulhos / Hygienic sanitary evaluation of butcher in the city of Guarulhos

O modelo atual de sociedade com sua dinâmica e características, impacta diretamente a vida das pessoas e, consequentemente, nos seus hábitos alimentares; esse contexto tem favorecido o consumo de produtos industrializados ou preparados fora do lar. As carnes comercializadas nos açougues dos hipermercados e atacadistas devem apresentar condições adequadas para a comercialização e consumo, já que representam o principal veículo de patógenos na alimentação humana. O consumo de carne bovina é influenciado principalmente pela renda per capita da população, pelos preços do produto e das demais carnes substitutas. Nos abatedouros e frigoríficos, o uso de Boas Práticas de Manufatura (BPM) e o uso de sistemas de gestão da qualidade, são fatores importantes para obter a confiança do consumidor. Desta maneira, este trabalho objetivou avaliar as condições higienicossanitárias de açougues de rede atacadista do município de Guarulhos. Foram inspecionados 05 (cinco) açougues localizados em uma rede atacadista, onde foram avaliadas as condições higienicossanitárias, por meio de um Roteiro de Inspeção ­ Açougues e Peixaria estabelecido na Portaria nº 2619/11. Neste estudo, os estabelecimentos foram classificados em: insatisfatórios, quando apresentaram um total de pontos abaixo de 50%; e satisfatório quando igual ou acima de 76% de pontos. Todos os 05 (cinco) açougues estudados foram classificados como satisfatórios, apresentando um total de pontos acima de 65 (A1 = 75; A2 = 76; A3 = 76; A4 = 80 e A5 = 87). Foram avaliadas as etapas do processo que envolve o alimento (carne bovina) desde o recebimento até a distribuição relatando questões sobre edificações e instalações, manipuladores, equipamentos e utensílios, entre outras, revelando os seguintes dados: foi verificado não conformidade no recebimento em 03 açougues; foi verificado não conformidade no armazenamento em 02; em 03, péssimas condições de conservação; em 03, os manipuladores não atendiam às recomendações legais; em 03, verificaram-se vestígios da presença de pragas e vetores; em 04, os manipuladores não tinham o hábito de lavar as mãos quando trocava de função; em todos, as instalações de pessoal mostrou precariedade. Os resultados revelam a necessidade de adequação à legislação pertinente, além da importância da adoção das boas práticas de fabricação nos açougues da rede atacadista estudada.

[Importance of a second opinion in breast surgical pathology and therapeutic implications]. / Importância da segunda opinião em patologia cirúrgica mamária e suas implicações terapêuticas.

PURPOSE: to evaluate the agreement between histopathologic diagnoses of breast lesions made by general pathologists and by a specialist in breast pathology. METHODS: a cohort retrospective study comparing histopathologic diagnoses of 329 cases of breast lesions received in consultation for a second opinion was carried out. The material received for consultation included slides (152 cases), paraffin blocks (59 cases) or slides and blocks (118 cases). Cases were reviewed and the original diagnoses and diagnoses from a specialist in breast pathology were compared. The main diagnoses, nuclear grade of ductal carcinoma in situ, and the histopathologic grade of invasive mammary carcinomas were evaluated. The kappa index and percentual concordance were used in the statistical analyses. RESULTS: a moderate agreement was observed between the original histopathologic diagnoses and the second opinion (kappa index=0.48; percentual concordance=59.9%). The diagnosis of malignancy was confirmed in 185/225 cases (82.2%) and diagnosis of benign lesions was confirmed in 89/104 cases (85.6%). The highest agreement was observed in the diagnosis of invasive mammary carcinomas (81%) and the highest disagreement was observed among diagnoses of ductal carcinoma in situ with microinvasion (74%), lobular carcinoma in situ (70%), and atypical epithelial hyperplasias (61%). There was a moderate agreement in the nuclear grade of ductal carcinoma in situ (kappa index=0.52; percentual concordance=68.8%), and good concordance in the histologic grade of invasive carcinomas (kappa index=0.61; percentual concordance=74.3). CONCLUSIONS: the results show higher concordance rate in the diagnosis of invasive carcinomas and lower concordance in the diagnosis of ductal carcinoma in situ with microinvasion and premalignant breast lesions, especially lobular neoplasia in situ, and atypical epithelial hyperplasias.

Ocorrência de acidentes de trabalho com trabalhadores no corte manual da cana-de-açúcar / Accidentes de trabajo ocurridos con los trabajadores del corte manual de la cana de azúcar / Work accidents occurrences with workers on the manual sugar cane cutting

Introdução: A economia da região Noroeste do Estado de São Paulo é baseada no cultivo da cana-de-açúcar e o corte de cana é um processo que envolve diversas situações de risco à saúde do trabalhador. Acidentes de trabalho com trabalhadores rurais no corte manual da cana são problemas frequentes e pouco discutidos...

Insuficiência respiratória aguda secundária a bócio mergulhante da tireoide / Acute respiratory insufficiencysecondary to substernal thyroid goiter

Relato de caso de bócio mergulhante (BM) em idosa com longa história de bócio nodular tóxico (BNT) e que evoluiu com insuficiência respiratória aguda enquanto aguardava cirurgia eletiva para extirpação do bócio. A evolução das complicações respiratórias dessa paciente sugere que o tratamento cirúrgico do BM em idosos deveria ser feito o mais breve possível, após o surgimento de sintomatologia respiratória, para evitar complicações.

Case report of substernal thyroid goiter in an elderly woman with long record of toxic nodular goiter that progressed to acute respiratory insufficiency while waiting for elective surgery for extirpation of goiter. The development of respiratory complications suggests that the surgical treatment of substernal thyroid goiter affecting elderly people should be pursuit as soon as possible after the appearance of respiratory symptomatology as a means of avoiding complications.

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.

Importância da segunda opinião em patologia cirúrgica mamária e suas implicações terapêuticas / Importance of a second opinion in breast surgical pathology and therapeutic implications

OBJETIVO: avaliar a concordância dos diagnósticos histopatológicos de lesões mamárias entre patologistas da comunidade e especialista em patologia mamária. MÉTODOS: foi realizado um estudo observacional, tipo coorte retrospectivo de 329 casos de lesões mamárias recebidas em consultoria para segunda opinião. Foram recebidas lâminas originais (152 casos), blocos de parafina (59 casos) ou lâminas e blocos (118 casos). Os casos foram revistos e os diagnósticos originais e os de revisão comparados, se avaliando a concordância no diagnóstico principal e na graduação nuclear dos casos de carcinoma ductal in situ e graduação histológica dos casos de carcinoma invasivo. Foram incluídos apenas casos com requisição de exame contendo dados clínicos e laudo anatomopatológico original final ou provisório. Para análise estatística foram usados o índice de kappa e a concordância percentual. RESULTADOS: a comparação geral entre os diagnósticos histopatológicos originais e pós-revisão evidenciou moderada concordância (índice de kappa=0,48) e concordância percentual de 59,9 por cento. Os diagnósticos de malignidade foram confirmados em 185/225 casos (82,2 por cento) e de lesões benignas em 89/104 casos (85,6 por cento). A maior concordância diagnóstica foi observada nos casos de carcinoma invasor (81 por cento) e as maiores discordâncias nos casos de carcinoma ductal in situ com microinvasão (74 por cento), carcinoma/neoplasia lobular in situ (70 por cento) e hiperplasias epiteliais atípicas (61 por cento). A análise do grau nuclear de 61 casos de carcinoma ductal in situ evidenciou moderada concordância (índice de kappa=0,52, concordância percentual=68,8 por cento). Foi observada boa concordância em relação ao grau histológico no carcinoma invasivo (índice de kappa=0,61, concordância percentual=74,3 por cento). CONCLUSÕES: os resultados mostram maior concordância no diagnóstico do carcinoma invasivo e menor concordância no diagnóstico de carcinoma ductal ...

PURPOSE: to evaluate the agreement between histopathologic diagnoses of breast lesions made by general pathologists and by a specialist in breast pathology. METHODS: a cohort retrospective study comparing histopathologic diagnoses of 329 cases of breast lesions received in consultation for a second opinion was carried out. The material received for consultation included slides (152 cases), paraffin blocks (59 cases) or slides and blocks (118 cases). Cases were reviewed and the original diagnoses and diagnoses from a specialist in breast pathology were compared. The main diagnoses, nuclear grade of ductal carcinoma in situ, and the histopathologic grade of invasive mammary carcinomas were evaluated. The kappa index and percentual concordance were used in the statistical analyses. RESULTS: a moderate agreement was observed between the original histopathologic diagnoses and the second opinion (kappa index=0.48; percentual concordance=59.9 percent). The diagnosis of malignancy was confirmed in 185/225 cases (82.2 percent) and diagnosis of benign lesions was confirmed in 89/104 cases (85.6 percent). The highest agreement was observed in the diagnosis of invasive mammary carcinomas (81 percent) and the highest disagreement was observed among diagnoses of ductal carcinoma in situ with microinvasion (74 percent), lobular carcinoma in situ (70 percent), and atypical epithelial hyperplasias (61 percent). There was a moderate agreement in the nuclear grade of ductal carcinoma in situ (kappa index=0.52; percentual concordance=68.8 percent), and good concordance in the histologic grade of invasive carcinomas (kappa index=0.61; percentual concordance=74.3). CONCLUSIONS: the results show higher concordance rate in the diagnosis of invasive carcinomas and lower concordance in the diagnosis of ductal carcinoma in situ with microinvasion and premalignant breast lesions, especially lobular neoplasia in situ, and atypical epithelial hyperplasias.

D2-40, a novel lymphatic endothelial marker: identification of lymphovascular invasion and relationship with axillary metastases in breast cancer

BACKGROUND: Studies of lymphatic vessels were limited by the lack of specific markers. Recently, they have become possible due to the release of new D2-40 antibody, a selective marker for lymphatic endothelium. The aim of our study was to compare neoplastic invasion in lymphatic and blood vessels detected in hematoxylin and eosin (H&E) and immunohistochemistry-stained sections. METHODOLOGY: A total of 123 cases of invasive mammary carcinomas were studied and sorted out into three subgroups according to axillary staging (macrometastasis, micrometastasis and lymph node negative). Lymphatic vessel invasion (LVI) and blood vessel invasion (BVI) were initially evaluated in histological H&E and immunohistochemistry-stained sequential sections. Lymphatic and blood vessel invasions were assessed by immunohistochemistry, employing D2-40 and CD31 antibodies, respectively. LVI and BVI were related to size, type, histologic grade of primary tumors, and the presence of metastasis. RESULTS: LVI was detected through H&E staining procedure in 17/123 cases (13.8 percent), and through immunohistochemistry procedure in 35/123 cases (28.5 percent) (kappa = 0.433). BVI was detected through H&E in 5/123 cases (4.1 percent), and through immunohistochemistry in 19/123 cases (15.4 percent) (kappa = 0.198). LVI and BVI were positively related to higher histologic grade of primary tumors (p < 0.05). LVI was also positively related to the presence of macrometastasis. CONCLUSION: The detection of lymphatic and blood vessel invasions through immunohistochemistry employing D2-40 and CD31 was higher than the detection through H&E, and it was related to higher tumor grade and metastasis in axillary lymph nodes.

INTRODUÇÃO: Estudos de vasos linfáticos eram limitados pela ausência de marcadores endoteliais linfáticos específicos. Recentemente, eles se tornaram possíveis após liberação comercial do novo anticorpo D2-40, marcador seletivo para endotélio linfático. O objetivo do nosso estudo foi comparar invasão neoplásica em vasos linfáticos e sanguíneos detectada em secções coradas pela hematoxilina e eosina (HE) e imuno-histoquímica (IIQ). MATERIAIS E MÉTODOS: Foram estudados 123 casos de carcinomas mamários invasores subdivididos em três subgrupos de acordo com o estadiamento axilar: macrometástases (Mac-Met), micrometástases (Mic-Met) e linfonodo negativo (LNN). Invasão de vasos linfáticos (IVL) e de vasos sangüíneos (IVS) foi inicialmente avaliada em secções histológicas coradas pela HE e através da IIQ realizada em cortes seqüenciais. A invasão de vasos linfáticos e sanguíneos foi avaliada pela imuno-histoquímica, empregando-se respectivamente os anticorpos D2-40, e CD31. IVL e IVS foram relacionadas com tamanho tumoral, tipo e grau histológico dos tumores primários e com a presença de metástases. RESULTADOS: IVL foi observada pela HE em 17/123 casos (13,8 por cento) e pela IIQ em 35/123 casos (28,5 por cento) (kappa = 0,433). IVS foi observada pela HE em 5/123 casos (4,1 por cento) e pela IIQ em 19/123 casos (15,4 por cento) (kappa = 0,198). IVL e IVS estavam positivamente relacionadas com maior grau histológico dos tumores primários (p < 0,05). IVL também estava positivamente relacionada com a presença de macrometástases. CONCLUSÃO: A detecção IIQ, respectivamente por D2-40 e CD31, de invasão de vasos linfáticos e sanguíneos foi maior que a detecção feita em cortes corados pela HE e relacionou-se com maior grau tumoral e metástases em linfonodos axilares.

Marcadores moleculares em câncer de mama preditivos de metástases axilares / Molecular features of breast cancer predictive of lymph node metastases

OBJETIVO: Avaliar e relacionar fatores morfológicos e moleculares de câncer de mama preditivos de metástases em linfonodos axilares. MÉTODOS: Selecionamos 123 casos de carcinomas mamários invasores subdivididos em três grupos de acordo com o status axilar (pacientes com macrometástases, com micrometástases e linfonodo-negativas). Avaliamos e correlacionamos a presença de metástases axilares com fatores morfológicos (tamanho do tumor, tipo e grau histológicos, invasão linfática e sangüínea em lâminas coradas pela hematoxilina e eosina) e moleculares do tumor primário (receptores de estrógeno e progesterona, Ki67, p53, E-caderina, Her2, e invasão linfática e sangüínea em lâminas coradas pela imunoistoquímica, para D2-40 e CD31). RESULTADOS: A ocorrência de metástases axilares esteve positivamente relacionada à embolização neoplásica em vasos linfáticos em lâminas coradas pela hematoxilina e eosina (HE), quando analisamos os casos com metástases e sem metástases (p=0,04), e, quando eles eram analisados em três subgrupos (p=0,002). Também identificamos relação positiva e estatisticamente significativa entre a presença de metástases axilares e invasão de vasos sangüíneos em lâminas coradas pelo CD31 (p=0,02). As demais variáveis moleculares e morfológicas não mostraram relação estatisticamente significativa com a presença de metástases. CONCLUSÃO: A invasão neoplásica em vasos linfáticos e sangüíneos identificadas em cortes histológicos corados pela HE e por marcadores imunoistoquímicos relaciona-se positivamente com a ocorrência de metástases, e é preditivo de metástases em linfonodos axilares em câncer de mama.

OBJECTIVE: The aim of our study was to analyze morphologic and molecular markers of breast cancer relating them to the presence of metastases in axillary lymph nodes. METHODS: We selected 123 cases of invasive mammary carcinomas stratified into three subgroups: with macrometastases, with micrometastases, and lymph node negative. Presence of metastases was evaluated relating them with morphologic factors (size of primary tumor, type and grade, presence of lymphatic and blood vessel invasion in hematoxylin and eosin-stained slides) and molecular factors of primary tumor (estrogen and progesterone receptors, E-cadherin, Ki67, p53, Her2 expression, and the presence of lymphatic and blood vessel invasion in immunostained sections for D2-40 and CD31). RESULTS: Axillary lymph node metastases were positively related to the presence of lymphatic vessel invasion in hematoxylin and eosin (H&E)-stained slides, when analyzed with or without metastases (p=0.04) and when analyzed in the three subgroups (p=0.002). Lymph node metastases were also positively related to presence of blood vessel invasion identified by immunohistochemistry (IHC) for CD31 (p=0.02). However other morphologic and molecular factors were not related to the presence of axillary node metastases. CONCLUSION: Lymphatic and blood vessel invasion identified in H&E and IHC-stained slides are positively related to the rmetastatic status of axillary lymph nodes and are predictive of axillary lymph node metastases in breast cancer.

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.

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.