RESUMO Contexto A hepatite C é um relevante problema de saúde pública. A doença pode permanecer clinicamente silenciosa tanto na forma aguda como na crônica e as infecções crônicas podem progredir para doenças avançadas, tais como cirrose e carcinoma hepatocelular (CHC), requerendo tratamentos dispendiosos, comprometendo a qualidade de vida do paciente e até mesmo levando à morte. Por esta razão, é uma das indicações mais frequentes para o transplante hepático. Apesar da introdução do tratamento com antivirais de ação directa (AAD) representar um progresso notável, muitos pacientes não receberam o tratamento e continuam infectados, e mesmo aqueles que eliminaram a infecção viral devem ser seguidos devido às lesões hepáticas anteriores, especialmente no que diz respeito às alterações da arquitetura lobular e dos vasos sanguíneos e linfáticos. Objetivo Avaliar os aspectos imuno-histoquímicos dos brotos linfáticos e dos vasos linfáticos “maduros” com variáveis histológicas de lesão hepática atribuíveis ao vírus da hepatite C (VHC) e à doença gordurosa. Métodos O presente estudo incluiu 72 biópsias hepáticas em pacientes com hepatite C crônica. Foram analisadas alterações estruturais relativas a “estadiamento” e “atividade”. Reações imuno-histoquímicas foram realizadas com anticorpo D2-40 anti-podoplanina. As principais variáveis histológicas também foram semiquantificadas, de modo a permitir a procura de possíveis associações entre os critérios histológicos e imunohistoquímicos, bem como com os genótipos 1 e 3 do VHC. Resultados Os achados histológicos mostraram que os diferentes graus de alterações estrutural estavam bem representados nesta casuística. A atividade necro-inflamatória lobular/parenquimatosa foi predominantemente leve à moderada. A maioria dos casos não apresentava grandes evidências de doença gordurosa, que foi encontrada significativamente mais elevada nos casos infectados com o genótipo 3 do VHC. A quantidade de brotos linfáticos portais aumentou com a progressão de alterações estruturais, sendo máxima na cirrose. Os brotos linfáticos portais, bem como os vasos linfáticos “maduros” portais também mostraram um aumento paralelo ao aumento do grau de infiltrado inflamatório portal/septal. No presente estudo, não foi encontrada qualquer associação significativa entre a proporção de brotos linfáticos portais ou vasos linfáticos maduros portais e o grau de atividade periportal/periseptal. Não foram detectadas relações significativas entre os brotos linfáticos/vasos maduros e a atividade inflamatória periportal ou atividade inflamatória parenquimatosa, nem com infecções devido ao genótipo 1 ou 3 do VHC. Conclusão A reação imunohistoquímica com anticorpo monoclonal D2-40 possibilitou a visualização e a semiquantitação de brotos e vasos linfáticos “maduros” nas amostras obtidas por biópsia hepática. A quantidade de linfáticos aumentou ao longo do processo fibrogênico, significativamente relacionada com a progressão da doença hepática e máxima na cirrose. Não foram detectadas relações significativas com a atividade necro-inflamatória periportal ou parenquimatosa.

ABSTRACT Background Viral hepatitis C is a significant public health challenge. The disease may remain clinically silent in both acute and chronic forms, and chronic infections may progress to advanced disease such as cirrhosis and hepatocellular carcinoma, requiring costly treatment, compromising the patient’s quality of life and even leading to death. For this reason, it is one of the most frequent indications for liver transplantation. Although treatment with direct-acting antivirals represents remarkable progress, many patients are still infected and even those who cleared the viral infection must be followed due to their previous hepatic lesions, especially regarding the disturbances of lobular architecture and the sanguineal and lymphatic vessels. Objective To assess immunohistochemical aspects of lymphatic sprouts and mature lymphatic vascularity with histological variables of liver injury attributable to hepatitis C virus (HCV) and fatty disease. Methods The present study included 72 liver biopsies of cases with chronic hepatitis C. Morphologic changes reflecting “staging” and “activity” were analyzed. Immunohistochemical reactions were performed with monoclonal antibody D2-40 anti-podoplanin. Major histological variables were also semiquantified so as to enable the search for possible associations among histological and Immunohistochemical criteria, as well as with genotypes 1 and 3 of HCV. Results Histological findings showed that the different degrees of strutural changes were well represented in this casuistic. Intralobular/parenchymal necro-inflammatory activity was predominantly mild to moderate. Most cases did not show major evidences of fatty disease, which was found significantly higher in cases infected with HCV genotype 3. The amount of portal lymphatic sprouts increased along with the progression of structural changes, maximal at cirrhosis. Portal lymphatic sprouts as well as portal mature lymphatic vessels also showed an increase parallel to the increase in the degree of portal/septal inflammatory infiltrate. In the present study, no significant association was found between the proportion of portal lymphatic sprouts or portal mature lymphatic vessels and the degree of periportal/periseptal activity. No significant relations were detected between lymphatic sprouts/mature vessels and periportal or parenchymal inflammatory activity, nor with infections due to HCV genotype 1 or 3. Conclusion Visualization and semiquantitation of sprouts and mature lymphatic vessels were clearly yielded by Immunohistochemical staining with monoclonal antibody D2-40. The amount of lymphatics was increased along fibrogenic process, significantly related to progression of liver disease and maximal at cirrhosis. No significant relations were detected with necro-inflammatory activity at interface or in the parenchyma.

OBJECTIVES: Whole genome expression profiles allow the stratification of bladder urothelial carcinoma into basal and luminal subtypes which differ in histological patterns and clinical behavior. Morpho-molecular studies have resulted in the discovery of immunohistochemical markers that might enable discrimination between these two major phenotypes of urothelial carcinoma. METHODS: We used two combinations of immunohistochemical markers, i.e., cytokeratin (CK) 5 with CK20 and CK5 with GATA3, to distinguish subtypes, and investigated their association with clinicopathological features, presence of histological variants, and outcomes. Upon searching for tumor heterogeneity, we compared the findings of primary tumors with their matched lymph node metastases. We collected data from 183 patients who underwent cystectomy for high-grade muscle-invasive urothelial carcinoma, and representative areas from the tumors and from 76 lymph node metastasis were organized in tissue microarrays. RESULTS: Basal immunohistochemical subtype (CK5 positive and CK20 negative, or CK5 positive and GATA3 negative) was associated with the squamous variant. The luminal immunohistochemical subtype (CK5 negative and CK20 positive, or CK5 negative and GATA3 positive) was associated with micropapillary and plasmacytoid variants. Remarkably, only moderate agreement was found between the immunohistochemical subtypes identified in bladder tumors and their lymph node metastasis. No significant difference in survival was observed when using either combination of the markers. CONCLUSION: This study demonstrates that these three routinely used immunohistochemical markers could be used to stratify urothelial carcinomas of the bladder into basal and luminal subtypes, which are associated with several differences in clinicopathological features.

The jaw muscles are essential components in the stomatognatic system. Their complex architecture allows them to execute several motor tasks. One of the structural peculiarities is the presence of hybrid and neonatal fibers.We studied the differences of the fiber-type in masseter and temporalis muscles along the first to nineth decades in both genders. Seventy-four (74) samples were analyzed by immunohistochemistry. Slow and fast muscle fibers distribution was similar in both muscles in both genders. Hybrid fiber was observed in all decades, and its frequency decreased significantly (p<0.001) with aging in masseter. Neonatal myosin expression was observed in all decades, its expression was more frequent in masseter (p=0.01), and males in temporalis (p=0.025). Decrease of the cross sectional area of fast and slow fibers, and decrease of capillary density were detected with aging. These morpho-immunohistochemical alterations on masseter and temporalis muscles correlated to the decrease in bite force with aging.

Los músculos cráneomandibulares son componentes esenciales en el sistema estomatognático. Su arquitectura compleja les permite ejecutar variadas tareas motoras. Una de sus características estructurales es la presencia de fibras musculares híbridas y neonatales. Se estudiaron las diferencias del tipo de fibra en los músculos masetero y temporal en la primera a novena décadas dela vida en ambos sexos. Setenta y cuatro (74) muestras se analizaron por inmunohistoquímica. La distribución de lãs fibras musculares lentas y rápidas fue similar en ambos músculos en ambos sexos. Fibras musculares híbridas fueron observadas en todas las décadas, pero su frecuencia disminuyó significativamente (p <0,001) con el envejecimiento en el masétero. La expresión de miosina neonatal se observo em todas los grupos analizados, aunque su expresión era más frecuente en los músculos maseteros (p = 0,01) y en varones en el temporal (p = 0,025). Se observo una disminución del área de la sección transversal de las fibras rápidas y lentas, y disminución de la densidad capilar al aumentar La edad. Estas alteraciones morfológicas en los músculos masetero y temporal se correlacionan con la disminución de la fuerza asociada al envejecimiento.

OBJECTIVES: We investigated four components of the Wnt signaling pathway in medulloblastomas. Medulloblastoma is the most common type of malignant pediatric brain tumor, and the Wnt signaling pathway has been shown to be activated in this type of tumor. METHODS: Sixty-one medulloblastoma cases were analyzed for β-catenin gene (CTNNB1) mutations, β-catenin protein expression via immunostaining and Wnt signaling pathway-related gene expression. All data were correlated with histological subtypes and patient clinical information. RESULTS: CTNNB1 sequencing analysis revealed that 11 out of 61 medulloblastomas harbored missense mutations in residues 32, 33, 34 and 37, which are located in exon 3. These mutations alter the glycogen synthase kinase-3β phosphorylation sites, which participate in β-catenin degradation. No significant differences were observed between mutation status and histological medulloblastoma type, patient age and overall or progression-free survival times. Nuclear β-catenin accumulation, which was observed in 27.9% of the cases, was not associated with the histological type, CTNNB1 mutation status or tumor cell dissemination. The relative expression levels of genes that code for proteins involved in the Wnt signaling pathway (CTNNB1, APC, AXIN1 and WNT1) were also analyzed, but no significant correlations were found. In addition, large-cell variant medulloblastomas presented lower relative CTNNB1 expression as compared to the other tumor variants. CONCLUSIONS: A small subset of medulloblastomas carry CTNNB1 mutations with consequent nuclear accumulation of β-catenin. The Wnt signaling pathway plays a role in classic, desmoplastic and extensive nodularity medulloblastoma variants but not in large-cell medulloblastomas.

INTRODUCTION: Limb-girdle muscular dystrophy presents with heterogeneous clinical and molecular features. The primary characteristic of this disorder is proximal muscular weakness with variable age of onset, speed of progression, and intensity of symptoms. Sarcoglycanopathies, which are a subgroup of the limb-girdle muscular dystrophies, are caused by mutations in sarcoglycan genes. Mutations in these genes cause secondary deficiencies in other proteins, due to the instability of the dystrophin-glycoprotein complex. Therefore, determining the etiology of a given sarcoglycanopathy requires costly and occasionally inaccessible molecular methods. OBJECTIVE: The aim of this study was to identify phenotypic differences among limb-girdle muscular dystrophy patients who were grouped according to the immunohistochemical phenotypes for the four sarcoglycans. METHODS: To identify phenotypic differences among patients with different types of sarcoglycanopathies, a questionnaire was used and the muscle strength and range of motion of nine joints in 45 patients recruited from the Department of Neurology - HC-FMUSP (Clinics Hospital of the Faculty of Medicine of the University of São Paulo) were evaluated. The findings obtained from these analyses were compared with the results of the immunohistochemical findings. RESULTS: The patients were divided into the following groups based on the immunohistochemical findings: a-sarcoglycanopathies (16 patients), b-sarcoglycanopathies (1 patient), y-sarcoglycanopathies (5 patients), and nonsarcoglycanopathies (23 patients). The muscle strength analysis revealed significant differences for both upper and lower limb muscles, particularly the shoulder and hip muscles, as expected. No pattern of joint contractures was found among the four groups analyzed, even within the same family. However, a high frequency of tiptoe gait was observed in patients with a-sarcoglycanopathies, while calf pseudo-hypertrophy was most common in patients with non-sarcoglycanopathies. The a-sarcoglycanopathy patients presented with more severe muscle weakness than did y-sarcoglycanopathy patients. CONCLUSION: The clinical differences observed in this study, which were associated with the immunohistochemical findings, may help to prioritize the mutational investigation of sarcoglycan genes.

OBJETIVO: Avaliar a resolutividade da imuno-histoquímica (IHQ) no diagnóstico de neoplasias em um laboratório de referência da rede de saúde pública e determinar potenciais influências da comunicação entre a fonte requisitante e o laboratório executante sobre tal resolutividade. MATERIAL E MÉTODOS: Mil casos oncológicos oriundos de oito hospitais públicos foram submetidos a exame IHQ, sendo 500 imediatamente anteriores e 500 posteriores à intervenção para aprimoramento da comunicação entre os requisitantes e o laboratório de IHQ. A recuperação de antígenos foi realizada pelo calor úmido sob pressão, e o sistema de detecção foi o LSAB plus System. Os resultados foram expressos como positivo, negativo ou inconclusivo. Foram coletadas informações sobre recebimento do bloco de parafina e fixador utilizado, guia de requisição de serviços de diagnóstico e terapia (SADT), laudo anatomopatológico original, topografia, idade e sexo do paciente. RESULTADOS E CONCLUSÃO: Foram coradas 5.719 lâminas pela IHQ de 989 casos considerados. A IHQ mostrou-se resolutiva em 919/989 (92,9%) casos. O número de antígenos por caso variou de um a 22, com média de 5,8 marcadores. A pesquisa de até cinco antígenos resolveu 534 casos e foram necessários até 10 marcadores para esclarecer 919 casos. Ainda hoje, informações básicas, como laudo anatomopatológico, idade e sexo do paciente, topografia, hipótese diagnóstica precisamente exposta, fixador e blocos de parafina em boas condições nem sempre são disponibilizadas aos laboratórios de referência. O aprimoramento dessa comunicação é essencial para a racionalização do fluxo dos exames IHQ e o incremento na qualidade da conclusão diagnóstica.

OBJECTIVES: The aim of this study was to evaluate the resolutivity of immunohistochemical (IHC) assays on neoplastic samples in a public health laboratory and to assess the influence of communication between the requiring institution and reference one on the quality of the IHC diagnosis. MATERIAL AND METHODS: One thousand oncological samples from eight public hospitals were submitted to IHC assay: 500 immediately before and 500 after an intervention demanding further information from the requiring pathologist. Antigen retrieval was performed in a pressure cooker and the detection system was LSAB plus System. The results were expressed as positive, negative or inconclusive. Information about paraffin blocks, fixatives, official requiring form, the attachment of original anatomopathological report, topography of the lesion, age and gender of the patients was collected. RESULTS AND CONCLUSION: From 989 samples, 5,719 slides were stained by IHC, leading to a resolutivity for 919/989 (92.9%). The number of antigen tested ranged from one to 22 - average of 5.8 for case. Panels with up to five antigens per case resolved 534 samples, whereas up to 10 markers were necessary to resolve 919 samples. Even nowadays, basic information, such as anatomopathological reports, age, gender, representative and technically superior paraffin block are not always available to the reference laboratory. The improvement of such communication is essential to rationalize the flux of immunohistochemical tests and to enhance the quality of diagnostic conclusions.

INTRODUÇÃO: O grande incremento do uso da imuno-histoquímica (IHQ) em numerosos laboratórios de anatomia patológica amplia o poder de resolução diagnóstica, mas traz certo grau de heterogeneidade de procedimentos e resultados. Seguindo recentes propostas da literatura internacional, participantes do Clube de IHQ da Sociedade Brasileira de Patologia desenvolvem ações de controle de qualidade, aplicando protocolos de sua rotina à pesquisa de antígenos que sirvam como indicadores de qualidade da reação. MATERIAL E MÉTODO: Um total de dez laboratórios participou das duas etapas deste estudo, cujos marcadores foram pancitoceratinas e receptores de estrógeno. Com lâminas controle recebidas dos laboratórios, cada participante efetuou a técnica de IHQ conforme sua prática diária, retornando as lâminas juntamente com o formulário de procedimento. A avaliação semiquantificada de 0 a 4 da intensidade da reação específica e de 0 a 3 da coloração de fundo e da qualidade da técnica histológica foram atribuídas individual e sigilosamente durante a projeção em data show em reunião do Clube de IHQ, gerando um escore final. RESULTADOS E DISCUSSÃO: As variações na imunocoloração de citoceratinas e receptor de estrógeno não comprometeram sua detecção nas lâminas preparadas nos diversos laboratórios. Tais variações associaram-se à diversidade de sistemas de recuperação antigênica e de amplificação, resultando ora em imunopositividade menos intensa, ora em maior fundo. Outros estudos devem abordar questões de interpretação, incluindo-se critérios para semiquantificação.

BACKGROUND: The expansion of the use of immunohistochemistry (IHC) in numerous laboratories extends the power of diagnostic resolution, but may impart heterogeneity in procedures and results. Following recent international proposals, participants of the Club of IHC of the Brazilian Society of Pathology developed actions of quality control applying routine protocols for detection of some of the most relevant antigens as quality probes. OBJECTIVES: Ten laboratories participated on the present study on keratins and estrogen receptors. MATERIAL AND METHOD: On control-slides sent from each laboratory, each participant performed IHC reaction according to his daily practice, returning the slides with the filled procedure form. The evaluation of the intensity of the specific reaction and back-ground was performed individually during data show projection in a meeting of the Club of IHC, yielding a final score of performance. DISCUSSION AND CONCLUSION: The methodology adopted was found useful to ascribe the performance to the heterogeneous systems of antigenic retrieval and amplification, leading to the identification of moderate variation in intensity of immunoreaction and in back-ground, but not compromising the antigen detection in any of these laboratories. Other studies must approach interpretative questions, including semi-quantitation and cut-offs.

RACIONAL: O pólipo inflamatório fibróide é uma lesão localizada que se origina na submucosa do trato gastrointestinal, mais freqüentemente do estômago. Embora se acredite que represente condição reativa e não neoplásica, sua histogênese permanece controversa. OBJETIVO: Estudar o pólipo inflamatório fibróide por imunoistoquímica na tentativa de contribuir para o entendimento de sua histogênese. PACIENTES AND MÉTODOS: Foram estudados nove casos por imunoistoquímica usando-se um painel de anticorpos contra actina de músculo liso, vimentina, proteína S-100, fator VIII e macrófago (HAM-56). RESULTADOS: Houve um padrão difuso de coloração nas células fusiformes, fortemente positivo para vimentina. Um padrão focal de positividade para actina de músculo liso foi observado nessas células. O estudo revelou reação heterogênea com HAM-56. Nas áreas edematosas, as células positivas para HAM-56 mostraram citoplasma volumoso e núcleos reniformes. Nas áreas ricamente celulares, as células positivas para HAM-56 apresentaram citoplasma fusiforme. As reações para S-100 e fator VIII foram negativas nos elementos proliferantes. CONCLUSÕES: O presente estudo imunoistoquímico afasta a hipótese de natureza neural ou vascular da lesão. A forte positividade para vimentina em todos os casos sugere um componente principal de células fusiformes, melhor reconhecidas como fibroblastos. Estes resultados favoreceriam a existência de um espectro de padrões morfológicos e imunoistoquímicos, indicando, possivelmente, fases evolutivas de uma reação inflamatória.

BACKGROUND: Inflammatory fibroid polyp is a localized lesion, which arises in the submucosa of the gastrointestinal tract, most often in the stomach.Although it is generally believed to represent a reactive, nonneoplastic condition, its histogenesis remains controversial. AIM: To study inflammatory fibroid polyp by immunohistochemistry in an attempt to further clarify their histogenesis. MATERIAL AND METHODS: Nine cases were studied by immunohistochemistry using a panel of antibodies against smooth-muscle actin, vimentin, S-100 protein, factor VIII- R.Ag and macrophage (HAM-56). RESULTS: There was a strong diffuse positive staining pattern in the spindle cells with vimentin antibody. A patchy staining for smooth-muscle actin was observed in these cells. Immunophenotyping revealed a heterogeneous reaction with HAM-56. In edematous areas, HAM-56-positive cells show voluminous cytoplasm and reniform nuclei. In cell-rich areas, the HAM-56-positive cells had fusiform cytoplasm. Stains for S-100 and factor VIII RAg were negative in the proliferating elements. CONCLUSIONS: The present immunohistochemical study refutes the suggested neural or vascular nature of the lesion. The strong positivity for vimentin in all cases suggests a major component of spindle cells best recognizable as fibroblasts. These results would favor the existence of a span of morphological and immunohistochemical patterns possibly indicating evolutive phases of an inflammatory reaction.

O carcinoma hepatocelular (CHC) é um importante tipo de câncer relacionado etiologicamente a alguns vírus, carcinógenos químicos e outros fatores ambientais que causam danos crônicos ao fígado em humanos. A freqüência de mutação do gene p53 em CHC é altamente heterogênea (0-52%) nos diversos países. OBJETIVO: O objetivo deste estudo foi determinar, imuno-histologicamente, a freqüência da expressão anômala de p53 em CHCs em pacientes cirróticos versus não-cirróticos, bem como em displasia hepática e hiperplasia adenomatosa. Para isso, foram estudados 84 pacientes com carcinoma hepatocelular ou cirrose. RESULTADOS: Foram detectadas expressões do p53 alterado em 58,3% dos pacientes com CHC graus III-IV, contrastando com os 22,2% dos pacientes com CHC graus I-II (p = 0,02). Áreas não tumorais, tanto nas proximidades do CHC como nos 30 casos de cirrose não mostraram expressão nuclear alterada do p53, mesmo nas displasias ou hiperplasias adenomatosas. Quando se considerou HBV, HCV ou alcoolismo nos casos estudados, não se encontrou diferença significativa. CONCLUSÃO: A elevada freqüência de imuno-expressão de p53 nesta população é próxima à relatada na China e África, tornando necessárias outras pesquisas para explicar as diferenças com os CHC estudados na Europa e na América do Norte.

Hepatocellular carcinoma (HCC) is an important type of cancer etiologically related to some viruses, chemical carcinogens and other host or environmental factors associated to chronic liver injury in humans. The tumor suppressor gene p53 is mutated in highly variable levels (0-52%) of HCC in different countries. OBJECTIVE: The objective of the present study was to compare the frequency of aberrant immunohistochemical expression of p53 in HCC occurring in cirrhotic or in non-cirrhotic patients as well as in liver cell dysplasia and in adenomatous hyperplasia. We studied 84 patients with HCC or cirrhosis. RESULTS: We detected p53 altered immuno-expression in 58.3% of patients in Grade III-IV contrasting to 22.2% of patients in Grade I-II (p = 0.02). Nontumorous areas either in the vicinity of HCC or in the 30 purely cirrhotic cases showed no nuclear p53 altered expression, even in foci of dysplasia or adenomatous hyperplasia. No significant difference was found among cases related to HBV, HCV or alcohol. CONCLUSION: The high frequency of p53 immunoexpression in this population is closer to those reported in China and Africa, demanding further studies to explain the differences with European and North American reports.