RESUMO
BACKGROUND: Assessment of mucosal healing is important for the management of patients with inflammatory bowel disease (IBD), but endoscopy can miss microscopic disease areas that may relapse. Histological assessment is informative, but no single scoring system is widely adopted. We previously proposed an eight-item histological scheme for the easy, fast reporting of disease activity in the intestine. The aim of the present study was to evaluate the performance of our Simplified Histologic Mucosal Healing Scheme (SHMHS). METHODS: Between April and May 2021 pathologists and gastroenterologists in Italy were invited to contribute to this multicenter study by providing data on single endoscopic-histological examinations for their IBD patients undergoing treatment. Disease activity was expressed using SHMHS (maximum score, 8) and either Simple Endoscopic Score for Crohn's Disease (categorized into grades 0-3) or Mayo Endoscopic Subscore (range 0-3). RESULTS: Thirty hospitals provided data on 597 patients (291 Crohn's disease; 306 ulcerative colitis). The mean SHMHS score was 2.96 (SD = 2.42) and 66.8% of cases had active disease (score ≥ 2). The mean endoscopic score was 1.23 (SD = 1.05), with 67.8% having active disease (score ≥ 1). Histologic and endoscopic scores correlated (Spearman's ρ = 0.76), and scores for individual SHMHS items associated directly with endoscopic scores (chi-square p < 0.001, all comparisons). Between IBD types, scores for SHMHS items reflected differences in presentation, with cryptitis more common and erosions/ulcerations less common in Crohn's disease, and the distal colon more affected in ulcerative colitis. CONCLUSIONS: SHMHS captures the main histological features of IBD. Routine adoption may simplify pathologist workload while ensuring accurate reporting for clinical decision making.
Assuntos
Colite Ulcerativa , Doença de Crohn , Doenças Inflamatórias Intestinais , Doença Crônica , Colite Ulcerativa/tratamento farmacológico , Doença de Crohn/patologia , Endoscopia Gastrointestinal , Humanos , Mucosa Intestinal/patologia , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Crohn's disease (CD) and ulcerative colitis, two forms of inflammatory bowel disease (IBD), are chronic and relapsing conditions of the gastrointestinal tract both characterized by long lasting chronic inflammation and increased risk of dysplasia and colorectal cancer (CRC). The aim of our study was to evaluate the interobserver agreement about IBD-associated dysplasia among pathologists belonging to the Italian Group for Inflammatory Bowel Diseases (IG-IBD P). METHODS: The present multicenter survey was performed using telepathology, supported by an open source E-learning platform. Biopsy specimens from 30 colonoscopies and from 20 patients were included. The glass slides of any case, including clinical and endoscopic data, were digitalized and uploaded on the E-learning platform. All the digital slides were grouped in 54 diagnostic "blocks". Blinded histopathological evaluation on all the digital slides was performed by 20 gastrointestinal pathologists. Closed-ended questions about (1) the occurrence of IBD; (2) the classification of IBD (as UC or CD); (3) the presence of active versus quiescent disease; (4) the presence of dysplasia; (5) the possible association of dysplasia with the sites of disease (dysplasia-associated lesion or mass-DALM vs adenoma-like mass-ALM); (6) the grading of dysplasia according to the ECCO guidelines (negative, indefinite, low grade, high grade categories) and (7) the presence of associated serrated features, were proposed in each case. Inter-observer agreement was evaluated by mean agreement percentage and kappa statistic, when suitable. RESULTS: The diagnosis of IBD was confirmed in 19 of 20 patients, 17 of 19 being classified as UC, 2 as CD. The mean interobserver agreement percentages about (1) the evidence of IBD, (2) the presence of either UC or CD and (3) the activity grading resulted to be 80%, 69% and 86%, respectively. Dysplasia was detected in 8/20 patients, with moderate agreement between pathologists (mean 72%, k 0.48). Particularly, low grade dysplasia was found in 13 biopsies (combined k 0.38), whereas high grade dysplasia in 8 (combined k 0.47). When the endoscopic and histopathological data were combined, features consistent with DALM were found in 6 of 20 patients with low grade dysplasia and those consistent with ALM in 2 patients with low grade dysplasia in a single biopsy (mean agreement: 86%). An associated serrated pattern was discovered in 4 patients (7 biopsies). CONCLUSIONS: Our study showed moderate interobserver agreement about the histopathological detection and classification of IBD-associated dysplasia. Further efforts should be undertaken to integrate the histopathological data with both the ancillary tests and molecular investigations.
Assuntos
Colite Ulcerativa , Doença de Crohn , Doenças Inflamatórias Intestinais , Colite Ulcerativa/complicações , Doença de Crohn/complicações , Humanos , Itália/epidemiologia , Recidiva Local de Neoplasia , Variações Dependentes do Observador , PatologistasRESUMO
Schistosoma haematobium plays a central role in the development of bladder cancer in Burkina Faso. The objective of this study was to determine the presence of S. haematobium in the bladder cancer and in vector snails. For the first time, formalin-fixed tissues embedded in paraffin were analyzed by immunohistochemistry and PCR. Molecular detection has resulted in 7/7 positive bladder cancer. Finally, as the snail vectors were positive. We suggest the use of molecular methods in the snail vectors for the detection of cysts and in cancerous tissues in larger studies.
Assuntos
Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/parasitologia , Oocistos/patologia , Schistosoma haematobium/isolamento & purificação , Esquistossomose Urinária/complicações , Caramujos/parasitologia , Neoplasias da Bexiga Urinária/etiologia , Neoplasias da Bexiga Urinária/parasitologia , Animais , Burkina Faso , Vetores de Doenças , Ovos , Humanos , Schistosoma haematobium/genética , Schistosoma haematobium/metabolismo , Esquistossomose Urinária/parasitologiaRESUMO
Fixation is a critical step in the preparation of tissues for histopathology. The objective of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved the best fixative for morphology. The morphology obtained with Bouin was comparable to that with formalin. Hollande was the best fixative for histochemistry. Bouin proved equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved possible to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.
Assuntos
Alendronato/farmacologia , Conservadores da Densidade Óssea/farmacologia , Fixadores/farmacologia , Histocitoquímica/métodos , Côndilo Mandibular/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Animais , Matriz Extracelular , Proteínas da Matriz Extracelular/metabolismo , Feminino , Formaldeído/farmacologia , Masculino , Côndilo Mandibular/metabolismo , Proteoglicanas/metabolismo , Ratos , Ratos WistarRESUMO
BACKGROUND: There is recent evidence that mast cells may play important roles in the gut, especially concerning visceral hypersensitivity and motor activity. However, most data are only available for clinical conditions characterised by diarrhoea, where MC have chiefly investigated in the mucosal layer of the colon and there is almost no information concerning constipation. AIM: To investigate mast cells distribution in all colonic layers in controls and severely constipated patients. METHODS: Full-thickness specimens from colons of patients undergoing surgery for slow transit constipation (n=29), compared with controls, were obtained and the number of mast cells (evaluated by specific monoclonal antibodies) counted as a whole and in single colonic segments (caecum, ascending, transverse, descending and sigmoid). RESULTS: Compared with controls, constipated patients revealed significantly higher number of mast cells, both as overall number and in single colonic segments. The distribution of mast cells resulted fairly homogeneous in the various segment of the large bowel, in both controls and patients, and no significant difference in the percentage of degranulated cells was found between groups. CONCLUSIONS: Colonic mast cells display a homogeneous distribution within the viscus. This cell population is shown to increase in severely constipated patients, which might represent a mechanism trying to compensate for the impaired propulsive activity of these patients.
Assuntos
Colo/patologia , Constipação Intestinal/patologia , Trânsito Gastrointestinal , Mastócitos/metabolismo , Mastócitos/patologia , Adulto , Idoso , Anticorpos Monoclonais , Estudos de Casos e Controles , Colo/fisiopatologia , Constipação Intestinal/fisiopatologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-IdadeRESUMO
Various studies have described abnormalities of the enteric nervous system (ENS) in tissue samples from patients with chronic idiopathic inflammatory bowel diseases (IBD). The distribution of density of the different cell types of the ENS was however not studied in a systematic way. The aim of this study was to examine the density of neurons, enteroglial cells and interstitial cells of Cajal (ICC) in the different plexuses of the ENS in samples from patients with Crohn's disease (CD), ulcerative colitis (UC) and controls. Tissue samples from 16 patients with CD (ileum) and 16 patients with UC obtained in involved and non-involved areas were studied using immunohistochemistry with antibodies directed against neuron-specific enolase, S100, C-Kit and CD3. Sections were analysed blindly by two pathologists and the number of positive cells was counted for each type. Overall, an increase was noted for neuronal cell bodies, enteroglia and ICC in the deep muscular plexus in CD. In uninvolved areas of CD patients, the number of enteroglial cells was decreased. In UC, an increase of ICC in the muscularis propria and enteroglial cells was observed in diseased tissue. The study confirms the presence of abnormalities of the different cells of the ENS in IBD. The presence of lesions in samples from uninvolved areas, such as a reduction of enteroglia, supports a pathogenetic role of the ENS.
Assuntos
Sistema Nervoso Entérico/anormalidades , Doenças Inflamatórias Intestinais/patologia , Adulto , Animais , Biomarcadores/metabolismo , Colo/citologia , Colo/metabolismo , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/fisiologia , Feminino , Humanos , Íleo/citologia , Íleo/metabolismo , Doenças Inflamatórias Intestinais/fisiopatologia , Masculino , Pessoa de Meia-IdadeRESUMO
AIMS: To study relationships between the number of pseudomelanosis coli cells and that of colonic enteric neurons and interstitial cells of Cajal, which are significantly reduced compared with controls in severely constipated patients. Pseudomelanosis coli is frequent in patients using anthraquinone laxatives. It is not known whether the prolonged use of these compounds damages the enteric nervous system in constipated patients. PATIENTS AND METHODS: The relationship between the number of pseudomelanosis coli cells and that of colonic enteric neurons (as well as that of apoptotic enteric neurons) and of interstitial cells of Cajal was assessed by histological and immunohistochemical methods in 16 patients with chronic use of anthraquinone laxatives undergoing surgery for severe constipation unresponsive to medical treatment. No relationship was found between the number of pseudomelanosis coli cells and that of enteric neurons (and that of the apoptotic ones), nor of interstitial cells of Cajal, in either the submucosal or the myenteric plexus. CONCLUSION: The use of anthraquinone laxatives, leading to the appearance of pseudomelanosis coli, is probably not related to the abnormalities of the enteric nervous system found in severely constipated patients.
Assuntos
Colo/patologia , Constipação Intestinal/patologia , Sistema Nervoso Entérico/patologia , Melanose/patologia , Adulto , Idoso , Antraquinonas/uso terapêutico , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Apoptose , Catárticos/uso terapêutico , Colo/química , Colo/inervação , Constipação Intestinal/tratamento farmacológico , Constipação Intestinal/fisiopatologia , Sistema Nervoso Entérico/química , Sistema Nervoso Entérico/fisiopatologia , Feminino , Humanos , Imuno-Histoquímica , Mucosa Intestinal/química , Mucosa Intestinal/patologia , Melanose/metabolismo , Pessoa de Meia-Idade , Plexo Mientérico/química , Plexo Mientérico/patologia , Plexo Mientérico/fisiopatologia , Fosfopiruvato Hidratase/análiseRESUMO
BACKGROUND: Idiopathic slow transit constipation is one of the most severe and often intractable forms of constipation. As motor abnormalities are thought to play an important pathogenetic role, studies have been performed on the colonic neuroenteric system, which rules the motor aspects of the viscus. AIMS: We hypothesised that important neuropathological abnormalities of the large bowel are present, that these are not confined to the interstitial cells of Cajal and ganglion cells, and that the previously described reduction of enteric neurones, if confirmed, might be related to an increase in programmed cell death (apoptosis). PATIENTS AND METHODS: Surgical specimens from 26 severely constipated patients were assessed by conventional and immunohistochemical methods. Specific staining for enteric neurones, glial cells, interstitial cells of Cajal, and fibroblast-like cells associated with the latter were used. In addition, gangliar cell apoptosis was evaluated by means of indirect and direct techniques. Data from patients were compared with those obtained in 10 controls. RESULTS: Severely constipated patients displayed a significant decrease in enteric gangliar cells, glial cells, and interstitial cells of Cajal. Fibroblast-like cells associated with the latter did not differ significantly between patients and controls. Patients had significantly more apoptotic enteric neurones than controls. CONCLUSION: Severely constipated patients have important neuroenteric abnormalities, not confined to gangliar cells and interstitial cells of Cajal. The reduction of enteric neurones may in part be due to increased apoptotic phenomena.
Assuntos
Apoptose/fisiologia , Colo/inervação , Constipação Intestinal/patologia , Sistema Nervoso Entérico/patologia , Neuroglia/fisiologia , Adulto , Idoso , Doença Crônica , Colo/metabolismo , Colo/fisiopatologia , Constipação Intestinal/metabolismo , Constipação Intestinal/fisiopatologia , Feminino , Trânsito Gastrointestinal , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Plexo Mientérico/metabolismo , Neuroglia/patologia , Fosfopiruvato Hidratase/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
A novel application of fluorescence in situ hybridization (FISH) to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer with greater sensitivity and specificity than existing FISH methods. In this study, the method is applied to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer. Nuclei were extracted and isolated from formalin fixed, paraffin embedded tissue of 43 different carcinomas (breast, ovary, endometrium, gastrointestinal stromal tumor and malignant mesothelioma). FISH was performed both on sections and extracted nuclei of each tissue using chromosome enumeration probes (CEP) for the centromeric regions of chromosomes 8 and 17, and a locus specific identifier (LSI) for the HER-2/neu oncogene. Differences between ploidy calculated in sections and extracted nuclei were seen in 3 breast carcinomas and 1 gastrointestinal stromal tumor (GIST). Furthermore, 1 breast cancer, previously considered to be borderline for HER-2/neu gene amplification turned out to be clearly amplified. Nuclei extraction and isolation bypass all the problems related to signal interpretation in tissue sections, and the adoption of this new technique, which improves the signal quality in several neoplastic samples, is suggested.
Assuntos
Aneuploidia , Carcinoma/genética , Núcleo Celular/genética , Amplificação de Genes/genética , Hibridização in Situ Fluorescente/métodos , Neoplasias/genética , Inclusão em Parafina , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma/patologia , Núcleo Celular/patologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Genes erbB-2/genética , Humanos , Mesotelioma/genética , Mesotelioma/patologia , Neoplasias/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Estudos Retrospectivos , Sensibilidade e Especificidade , Fixação de Tecidos/métodosRESUMO
Qualitative evaluation of protein content in formalin fixed, paraffin-embedded tissues is usually performed by means of cytofluorimetric analysis. On the other hand, several studies underline the opportunity to measure the concentration of nuclear proteins, which is often accomplished by using complex techniques and instrumentation. In the present work, we suggest a new application for the spectrophotometric evaluation of protein content on extracted and isolated nuclei, based on EDTA treatment of specimens and chemical extraction of proteins, followed by direct spectrophotometric measurement at UV wavelengths. We also demonstrate how this parameter correlates with other diagnostic factors, such as the proliferation index (MIB-1) and the DNA content (ploidy) of cells. This method is simple and effective, yet less expensive than other protein quantitation protocols.
Assuntos
Neoplasias da Mama/química , Proteínas Nucleares/análise , Espectrofotometria/métodos , Neoplasias da Mama/patologia , Núcleo Celular/química , Núcleo Celular/patologia , DNA de Neoplasias/análise , DNA de Neoplasias/isolamento & purificação , Feminino , Fixadores/química , Citometria de Fluxo , Formaldeído/química , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Inclusão em Parafina , Ploidias , Fixação de TecidosRESUMO
Mammalian cells respond to DNA insults by activating cell-cycle checkpoints. This may result in a temporary cell growth arrest which allows DNA repair before proliferation or induces apoptosis. p53 is one of the main contributors in regulating these activities. To get a better insight on the molecular mechanism underlying these activities we studied the role of p53 in apoptosis and neurogenesis of brain cells from adult p53(+/+) or p53(-/-) mice exposed to gamma-irradiation. Apoptosis and neurogenesis were assessed up to 14 days following the injury. Five-ten hours following gamma-irradiation, cells with TUNEL positive nuclei were identified within the subgranular zone of dentate gyrus (DG) of both p53(+/+) and p53(-/-) mice. At the same time-points, pyknotic and shrinking nuclei were visualized by Hoechst 33258 staining. Furthermore, gamma-irradiation increased the number of proliferating cell nuclear antigen (PCNA) positive cells with a peak at 5-10 h in both animal groups. PCNA immunoreactivity was detected in cells exhibiting condensed nuclei as visualized by Hoechst 33258 staining. Neurogenesis, assessed by mitotic marker p34(cdc2) immunoreactivity, showed a biphasic response to gamma-irradiation both in p53(+/+) and p53(-/-) mice which was characterized by an early inhibition and a delayed stimulation. In p53(-/-) mice, the time required by DG granule cells to recover from the lesion and to stimulate proliferation was significantly shortened in comparison with wild-type mice thus resulting in an accelerated neurogenesis. Our data indicate that following gamma-radiation p53 plays a role in regulating cell-cycle progression rate but it is dispensable for promoting apoptosis of DG granule cells.
Assuntos
Apoptose/fisiologia , Giro Denteado/citologia , Neurônios/fisiologia , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/efeitos da radiação , Divisão Celular/fisiologia , Divisão Celular/efeitos da radiação , Reparo do DNA/fisiologia , Raios gama , Expressão Gênica/fisiologia , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Antígeno Nuclear de Célula em Proliferação/genética , Células-Tronco/citologia , Células-Tronco/fisiologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Cytokine activation and endothelial dysfunction are typical phenomena of congestive heart failure (CHF). We tested the hypothesis that incubating human umbilical vein endothelial cells with serum from patients with CHF will downregulate endothelial constitutive nitric oxide synthase (eNOS) and induce apoptosis. METHODS AND RESULTS: We studied 21 patients with severe CHF. Levels of tumor necrosis factor-alpha (TNF-alpha) and several neuroendocrine parameters were assessed. eNOS was measured by Western Blot analysis and apoptosis by optical microscopy and flow cytometry. We observed (1) eNOS downregulation (difference versus healthy subjects at 24 hours [P<0.05] and 48 hours [P<0.001]), (2) nuclear morphological changes typical of apoptosis; and (3) a high apoptotic rate with propidium iodide (increasing from 2.1+/-0.4% to 11.3+/-1.2% at 48 hours; P<0.001 versus healthy subjects) and annexin V. An anti-human TNF-alpha antibody did not completely counteract these effects. A strong correlation existed between eNOS downregulation and apoptosis (r = -0.89; P<0.001). CONCLUSIONS: Serum from patients with severe CHF downregulates eNOS expression and increases apoptosis. High levels of TNF-alpha likely play a role, but they cannot be the only factor responsible.
Assuntos
Apoptose , Insuficiência Cardíaca/sangue , Óxido Nítrico Sintase/antagonistas & inibidores , Fator de Necrose Tumoral alfa/fisiologia , Idoso , Células Cultivadas , Regulação para Baixo , Endotélio Vascular/fisiologia , Citometria de Fluxo , Humanos , Pessoa de Meia-Idade , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo IIIRESUMO
Two new cases of chromophobe renal cell carcinoma were diagnosed on the basis of their morphology and their karyotype complemented by flow cytometry. In one of these cases, however, all these investigations were not sufficient and additional histochemistry investigation had to be used to completely rule out other renal tumors such as oncocytoma, the prognosis of which is totally different.
Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Citometria de Fluxo , Humanos , Cariotipagem , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
UNLABELLED: The quantitation of DNA and growth fraction in the different step from dysplastic to neoplastic process in large bowel is the aim of this study. 70 colonic polyps were studied. The fresh specimens were processed and DNA analysis was carried out using a Partec CA II flow cytometer and the growth fraction was tested with KI-67 monoclonal antibody. The percentage of S-phase cells has been calculated with the Multicycle program. Our results demonstrated that 7 adenomas were tubulo-villous with mild dysplasia, 39 with mild-moderate dysplasia, 1 with severe dysplasia, 5 were polypoid carcinomas, 2 juvenile polyps, 1 polypoid leiomyoma, 1 inflammatory fibroid polyps. DNA analysis showed a diploid DNA content in non adenomatous polyps, in all adenomas with mild dysplasia, in 37 with mild-moderate dysplasia, in 8 cases with moderate-severe dysplasia and 1 cancer. Aneuploidy was discovered in 2 cases with mild-moderate dysplasia, in 6 cases with moderate-severe dysplasia, in the case of severe dysplasia and in 4 cases of carcinomas. Best indexes of linear correlation (Pearson's r) has been found between S-phase and DNA index (r = .75) and between S-phase and KI-67 (r = .82). IN CONCLUSION: 1) No relationship was found between DNA content and age, sex, size and location of polyps. 2) Aneuploidy is strictly related to moderate-severe grade of dysplasia therefore it is an important element in the development of adenomacarcinoma sequence. 3) DNA-index, S-phase and KI-67 are strictly related.
Assuntos
Adenocarcinoma/patologia , Adenoma/patologia , Neoplasias do Colo/patologia , Pólipos do Colo/patologia , DNA de Neoplasias/genética , Citometria de Fluxo , Adenocarcinoma/genética , Adenoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Aneuploidia , Biomarcadores Tumorais/análise , Ciclo Celular , Núcleo Celular/química , Neoplasias do Colo/genética , Pólipos do Colo/genética , Progressão da Doença , Feminino , Humanos , Antígeno Ki-67/análise , Leiomioma/genética , Leiomioma/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas de Neoplasias/análiseRESUMO
Five specimens of normal mammary tissue and 53 primary breast carcinoma lesions were tested for expression of HLA antigens and components of the antigen-processing machinery by immunohistochemical staining. The expression of transporter associated with antigen processing (TAP) 1, TAP2, and HLA class I antigens in breast carcinoma lesions was significantly associated with tumor grading. Like normal mammary tissue, the 16 low-grade (G1) breast carcinoma lesions showed strong staining for TAP1, TAP2, and HLA class I antigens. In contrast, only 12 (32%) of 37 high-grade (G2 and G3) breast carcinoma lesions displayed the normal staining pattern. In 14 (38%) of 37 high-grade lesions, HLA class I antigen down-regulation was observed without loss of low molecular mass polypeptide and/or TAP staining. Congruent down-regulation of HLA class I antigen and TAP1 or TAP2 was found in 8 (22%) of 37 high-grade lesions. Complete loss of HLA class I antigens, TAP1, and TAP2 was observed in 3 (8%) of 37 high-grade lesions. No lesion was negative for TAP1 and/or TAP2 staining while positive for HLA class I antigen staining. These data demonstrate an association of HLA class I antigen and TAP down-regulation with tumor progression in breast carcinoma. This association suggests that loss of HLA and/or TAP may represent an escape from the host's immune pressure or may reflect the accumulation of abnormalities associated with neoplastic progression. This accumulation of defects in antigen processing and presentation may in turn be responsible for reduced recognition of malignant cells by putative clinically relevant tumor-specific T cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apresentação de Antígeno , Neoplasias da Mama/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias da Mama/metabolismo , Regulação para Baixo , HumanosRESUMO
Cellular DNA content of solid tumors can be determined either from fresh, frozen, or formalin fixed, paraffin-embedded tissues. However, discordant results have been obtained using the paraffin-embedded technique, and lack of abnormal DNA stemlines in the paraffin-embedded as compared to either fresh or frozen tissues has been reported. In this study we evaluated the validity of nuclear extraction method from paraffin-embedded tissues, using 75 breast carcinomas whose DNA content was previously analyzed from frozen tissue and resulted either normal (12 cases) or abnormal (63 cases). From representative paraffin blocks, nuclei were extracted following Hedley's technique. The results revealed excellent cell counting and good histogram resolution from all paraffin samples; the loss of G2M abnormal peak in eight histograms with abnormal stemline did not compromise the correct interpretation of DNA content. In addition, the comparison between DNA indices obtained from corresponding paraffin and frozen samples showed a good correlation in 69 cases (r = 94); discordance in six cases was demonstrated to be related to tumor heterogeneity. In conclusion the paraffin extraction method is a sensible, and reliable technique, which can be applied for DNA flow cytometric studies on archival cases, as well as whenever fresh sample from the tumor is not obtainable.
Assuntos
DNA/isolamento & purificação , Citometria de Fluxo , Secções Congeladas , Inclusão em Parafina , Aneuploidia , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Contagem de Células , Núcleo Celular/química , DNA de Neoplasias/isolamento & purificação , Reações Falso-Negativas , Feminino , Fase G2 , Humanos , Metáfase , Pepsina A , Solventes , Fixação de Tecidos , XilenosAssuntos
Região Organizadora do Nucléolo , Ploidias , Mama/química , Mama/ultraestrutura , Neoplasias da Mama/genética , Neoplasias da Mama/ultraestrutura , DNA/análise , DNA de Neoplasias/análise , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/ultraestrutura , Endométrio/química , Endométrio/ultraestrutura , Feminino , Citometria de Fluxo , Humanos , Coloração pela PrataRESUMO
Flow cytometric DNA analysis is an important prognostic tool in breast cancer. We evaluated the possibility of performing DNA analysis on cell suspensions obtained by scraping the cut surface of breast tumors; 31 breast tumor nodules, including six benign and 25 malignant lesions, were studied. From each case, cell suspensions acquired by mechanical mincing of a fresh frozen tissue fragment and by two different scrapings (central and peripheral) from the cut surface of the tumor were analyzed via flow cytometry. In all cases, comparison of the DNA histograms for three samples showed no significant differences in the appearance of debris or in the value of coefficient of variation of the G0-G1 peak. All benign nodules showed a normal DNA stemline in all specimens. In 23 of 25 cases of breast carcinoma, the ploidy of the three preparations was similar, with a concordance in 12/14 (85, 71%) cases in DNA nondiploid tumors. Linear regression analysis showed a good correlation in DNA index between either scraping sample and the tissue fragment (r = .955 and r = .905). The results indicate that the scraping technique provides excellent cell suspensions and DNA histograms comparable to those obtained from mechanical mincing of tissue fragments. The technique minimizes preparation time and avoids consuming much tissue and, thus, is the method of choice when very small cancers have to be analyzed.