ABSTRACT
Adenoid cystic carcinoma (ACC) is a MYB-driven head and neck malignancy with high rates of local recurrence and distant metastasis and poor long-term survival. New effective targeted therapies and clinically useful biomarkers for patient stratification are needed to improve ACC patient survival. Here, we present an integrated copy number and transcriptomic analysis of ACC to identify novel driver genes and prognostic biomarkers. A total of 598 ACCs were studied. Clinical follow-up was available from 366 patients, the largest cohort analyzed to date. Copy number losses of 1p36 (70/492; 14%) and of the tumor suppressor gene PARK2 (6q26) (85/343; 25%) were prognostic biomarkers; patients with concurrent losses (n = 20) had significantly shorter overall survival (OS) than those with one or no deletions (p < 0.0001). Deletion of 1p36 independently predicted short OS in multivariate analysis (p = 0.02). Two pro-apoptotic genes, TP73 and KIF1B, were identified as putative 1p36 tumor suppressor genes whose reduced expression was associated with poor survival and increased resistance to apoptosis. PARK2 expression was markedly reduced in tumors with 6q deletions, and PARK2 knockdown increased spherogenesis and decreased apoptosis, indicating that PARK2 is a tumor suppressor in ACC. Moreover, analysis of the global gene expression pattern in 30 ACCs revealed a transcriptomic signature associated with short OS, multiple copy number alterations including 1p36 deletions, and reduced expression of TP73. Taken together, the results indicate that TP73 and PARK2 are novel putative tumor suppressor genes and potential prognostic biomarkers in ACC. Our studies provide new important insights into the pathogenesis of ACC. The results have important implications for biomarker-driven stratification of patients in clinical trials. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Subject(s)
Carcinoma, Adenoid Cystic , Head and Neck Neoplasms , Humans , Carcinoma, Adenoid Cystic/genetics , Carcinoma, Adenoid Cystic/pathology , Prognosis , Genes, Tumor Suppressor , Head and Neck Neoplasms/genetics , Transcriptome , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolismABSTRACT
ABSTRACT: Extraocular sebaceous carcinoma (ESC) is a rare appendiceal skin tumor. In contrast to ocular sebaceous carcinoma, information about the exact cellular architecture of these lesions is scarce and the histogenesis of ESC is unknown. Here, we extend our previous study and investigate 28 extraocular carcinomas in comparison to 54 benign sebaceous tumors and 8 cases of normal sebaceous glands using a broad spectrum of antibodies against p63, several keratins, adipophilin, EMA, Ki67, androgen receptor, and mismatch repair proteins. This observational study demonstrates that p63- and K5/14-positive basaloid cells are key cells in normal sebaceous gland and in all sebaceous tumors and that these basaloid cells give rise to EMA+, adipophilin+ sebocytes, and K5/14+, K7±, K10± ductal structures. Finally, about half of ESC is associated with superficial in situ neoplasia, which provides evidence that at least part of these carcinomas arises from flat superficial in situ carcinoma. In contrast to the normal sebaceous gland, about half of all sebaceous tumors lack keratin K7. MMR protein IHC-profiles role will be discussed.
Subject(s)
Adenoma/chemistry , Biomarkers, Tumor/analysis , Carcinoma/chemistry , Immunohistochemistry , Sebaceous Gland Neoplasms/chemistry , Adenoma/pathology , Adult , Aged , Aged, 80 and over , Biopsy , Carcinoma/pathology , Female , Humans , Male , Middle Aged , Predictive Value of Tests , Sebaceous Gland Neoplasms/pathologyABSTRACT
Adenosquamous carcinoma of the pancreas (ASCAP) is characterized by conventional pancreatic ductal adenocarcinoma (PDAC) and squamous carcinoma components with at least 30% of the tumour showing squamous differentiation. To get further insight into the histogenesis of these lesions, we analysed the cellular organization of ASCAP compared to PDACs. Using Immunohistochemistry and triple immunofluorescence labelling studies for keratins, p63, p40, MUC1, MUC2, MUC5AC, Ki67, and EGFR we demonstrate that many ASCAPs contain a transitional zone between the K8/18-positive adenocarcinomatous component and the p63+ /p40+ /K5/K14+ squamous component initiated by the expression of p63 in K8/18+ adenocarcinomatous cells and the appearance of basally located p63+ K5/14+ cells. p63+ K5/14+ cells give rise to fully developed squamous differentiation. Notably, 25% of conventional PDACs without histologically recognizable squamous component contain foci of p63+ p40+ and K5/14+ cells similar to the transitional zone. Our data provide evidence that the squamous carcinoma components of ASCAPs originate from pre-existing PDAC via transdifferentiation of keratin K8/18-positive glandular cells to p63-, p40-, and keratin K5/14-positive squamous carcinoma cells supporting the squamous metaplasia hypothesis. Thus our findings provide new evidence about the cellular process behind squamous differentiation in ASCAPs.
Subject(s)
Carcinoma, Adenosquamous/pathology , Carcinoma, Squamous Cell/pathology , Pancreatic Neoplasms/pathology , Aged , Aged, 80 and over , Carcinoma, Adenosquamous/metabolism , Carcinoma, Squamous Cell/metabolism , Cell Differentiation , Female , Humans , Keratins/metabolism , Male , Middle Aged , Pancreatic Neoplasms/metabolismABSTRACT
Understanding the mechanisms regulating human mammary epithelium requires knowledge of the cellular constituents of this tissue. Different and partially contradictory definitions and concepts describing the cellular hierarchy of mammary epithelium have been proposed, including our studies of keratins K5 and/or K14 as markers of progenitor cells. Furthermore, we and others have suggested that the p53 homolog p63 is a marker of human breast epithelial stem cells. In this investigation, we expand our previous studies by testing whether immunohistochemical staining with monospecific anti-keratin antibodies in combination with an antibody against the stem cell marker p63 might help refine the different morphologic phenotypes in normal breast epithelium. We used in situ multilabel staining for p63, different keratins, the myoepithelial marker smooth muscle actin (SMA), the estrogen receptor (ER), and Ki67 to dissect and quantify the cellular components of 16 normal pre- and postmenopausal human breast epithelial tissue samples at the single-cell level. Importantly, we confirm the existence of K5+ only cells and suggest that they, in contrast to the current view, are key luminal precursor cells from which K8/18+ progeny cells evolve. These cells are further modified by the expression of ER and Ki67. We have also identified a population of p63+K5+ cells that are only found in nipple ducts. Based on our findings, we propose a new concept of the cellular hierarchy of human breast epithelium, including K5 luminal lineage progenitors throughout the ductal-lobular axis and p63+K5+ progenitors confined to the nipple ducts.
Subject(s)
Breast/metabolism , Epithelium/metabolism , Keratin-14/metabolism , Keratin-5/metabolism , Stem Cells/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers/metabolism , Female , Humans , Immunohistochemistry , Middle AgedABSTRACT
Endogenous cell autofluorescence is a common nuisance that complicates the use of fluorescence microscopy. When using fluorescence-labeled antibodies for specific cell labeling in tissue sections of human angioimmunoblastic T-cell lymphoma (AITL), we encountered with a problematic autofluorescence of multiple cells. These cells emitted fluorescence signals in the green, red and deep-red spectral range. Characterization of these autofluorescent cells solely on the basis of their autofluorescence failed. To identify these enigmatic cells residing the lymphoma tissue, we combined two imaging techniques-fluorescence and brightfield microscopy. Combined fluorescence/brightfield imaging of cells immunolabeled with a panel of CD antibodies raised against diverse cellular components allowed us to identify the autofluorescent cells in the AITL as eosinophils. These cells tended to accumulate in the vicinity of capillaries and arterioles apparently mediating the process of angiogenesis resembling other angiogenesis-associated diseases.
Subject(s)
Fluorescence , Fluorescent Antibody Technique , Immunoblastic Lymphadenopathy/pathology , Lymphoma, T-Cell/pathology , Aged , Aged, 80 and over , Female , Humans , Immunohistochemistry , Male , Middle AgedABSTRACT
Mast cells are ubiquitous throughout the human tissues and play an essential role in physiology and pathology. For evaluation of patients with pathological conditions, mast cells were primarily detected using metachromatic staining with toluidine blue. In the last decades, the staining arsenal of pathologists was enriched with enzyme histochemical and immunohistochemical methods, and it was established that depending on species and tissue localization mast cells are not similar both in appearance and function. The aim of this study was to characterize different mast cell populations using the up-to-date methods of their identification. We compared standard metachromatic method for mast cells with enzyme histochemical detection of chloroacetyl esterase and with immunohistochemical detection of tryptase and chymase in human and rodent tissues. Combination of these methods allowed us to assay quantitatively mast cell populations in different organs of humans and rodents. Furthermore, we assessed the appropriate implementation of each of these methods for mast cell identification in diagnostic labs.
Subject(s)
Cell Separation/methods , Histocytochemistry/methods , Mast Cells/cytology , Staining and Labeling/methods , Animals , Cell Count , Gerbillinae , Humans , Mast Cells/metabolism , Mice , Tolonium Chloride/chemistryABSTRACT
AIMS: Syringomatous tumour of the nipple and low-grade adenosquamous carcinoma (LGAdSC) of the breast are regarded as distinct entities. To clarify the nature of these two lesions, we compared the expression of different lineage/differentiation markers in 12 syringomatous tumours of the nipple, nine LGAdSCs, and normal breast epithelium. METHODS AND RESULTS: Using triple immunofluorescence labelling and quantitative RT-PCR for keratins, p63, and smooth muscle actin, we demonstrated that syringomatous tumour and LGAdSC contain p63+/K5/14+ tumour cells, K10+ squamous cells, and K8/18+ glandular cells, with intermediary cells being found in both lineages. Identical p63+/K5/14+ cells were also found in the normal breast duct epithelium. CONCLUSIONS: Our data provide evidence that syringomatous tumour of the nipple and LGAdSC are identical or nearly identical lesions. They contain p63+/K5/14+ cells as the key cells from which the K10+ squamous lineage and the K8/18+ glandular lineage arise. On the basis of our findings in normal breast tissue and associated benign lesions, we suggest that p63+/K5/14+ cells of the normal breast duct epithelium or early related cells might play a key role in the neoplastic transformation of both syringomatous tumour and LGAdSC. We propose that the differentiation patterns found in both lesions reflect the early ontogenetic stages of the normal breast epithelium.
Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/pathology , Carcinoma, Adenosquamous/pathology , Nipples/pathology , Syringoma/pathology , Cell Differentiation , Female , Humans , Immunohistochemistry , Neoplasm Grading , Real-Time Polymerase Chain ReactionABSTRACT
Salivary gland-like tumors of the breast show a great variety of architectural patterns and cellular differentiations such as glandular, myoepithelial, squamous, and even mesenchymal phenotypes. However, currently little is known about the evolution and cellular differentiation of these tumors. For that reason, we performed an in situ triple immunofluorescence lineage/differentiation tracing (isTILT) and qRT-PCR study of basal (K5/K14), glandular (K7/K8/18), and epidermal-specific squamous (K10) keratins, p63, and smooth muscle actin (SMA; myoepithelial marker) with the aim to construct and trace different cell lineages and define their cellular hierarchy in tumors with myoepithelial differentiation. isTILT analysis of a series of 28 breast, salivary, and lacrimal gland tumors, including pleomorphic adenomas (n=8), epithelial-myoepithelial tumors (n=9), and adenoid cystic carcinomas (n=11) revealed that all tumor types contained K5/K14-positive progenitor cells in varying frequencies from a few percent up to 15%. These K5/K14-positive tumor cells were found to differentiate to glandular- (K8/18-positive) and myoepithelial-lineage (SMA-positive)-specific cells and were also shown to generate various heterologeous cell differentiations such as squamous and mesenchymal progenies. p63 was co-expressed with K5/K14 in basal-like progenitor cells, myoepithelial, and squamous cells but not in glandular cells. Our results show that the corresponding counterpart tumors of breast and salivary/lacrimal glands have identical cellular compositions. Taken together, our isTILT and RNA-expression data indicate that look-alike tumors of the breast represent a special subgroup of basal-type tumors with benign or usually low malignant potential.
Subject(s)
Adenoma, Pleomorphic/pathology , Breast Neoplasms/pathology , Carcinoma, Adenoid Cystic/pathology , Myoepithelioma/pathology , Salivary Gland Neoplasms/pathology , Adenoma, Pleomorphic/metabolism , Biomarkers, Tumor/analysis , Breast Neoplasms/metabolism , Carcinoma, Adenoid Cystic/metabolism , Cell Differentiation , Cell Lineage , Diagnosis, Differential , Female , Fluorescent Antibody Technique , Humans , Myoepithelioma/metabolism , Real-Time Polymerase Chain Reaction , Salivary Gland Neoplasms/metabolism , Stem Cells/metabolism , Stem Cells/pathologyABSTRACT
Adenoid cystic carcinoma (ACC) is an aggressive head and neck malignancy characterized by a t (6;9) translocation resulting in an MYB-NFIB gene fusion or, more rarely, an MYBL1 fusion. The true frequency and clinical significance of these alterations are still unclear. Here, we have used tissue microarrays and analyzed 391 ACCs and 647 non-ACC salivary neoplasms to study the prevalence, expression, and clinical significance of MYB/MYBL1 alterations by FISH and immunohistochemistry. Alterations of MYB or MYBL1 were found in 78% of the cases, of which 62% had MYB alterations and 16% had MYBL1 rearrangements. Overexpression of MYB/MYBL1 oncoproteins was detected in 93% of the cases. MYB split signal, seen in 39% of the cases, was specific for ACC and not encountered in non-ACC salivary tumors. Loss of the 3'-part of MYB was enriched in grade 3 tumors and was a significant independent prognostic biomarker for overall survival in multivariate analyses. We hypothesize that loss of the 3'-part of MYB results from an unbalanced t(6;9) leading to an MYB-NFIB fusion with concomitant loss of the segment distal to the MYB breakpoint in 6q23.3. Our study provides new knowledge about the prevalence and clinical significance of MYB/MYBL1 alterations and indicates the presence of genes with tumor suppressive functions in 6q23.3-qter that contribute to poor prognosis and short overall survival in ACC.
ABSTRACT
BACKGROUND/AIM: Polymorphous adenocarcinoma (PAC) is a low-grade salivary gland malignancy in contrast to variants with papillary (PAP) or cribriform (CASG) architecture and confers the second most common malignancy of minor salivary glands. Our study aimed to identify prognostic factors and to evaluate histomorphological and molecular diagnostic criteria of PACs. PATIENTS AND METHODS: A series of 155 PACs, including 10 PAPs and 12 CASGs from the population-based Cancer Registry of North Rhine-Westphalia (LKR-NRW) and the Hamburg Salivary Gland Reference Centre (HRC) were analyzed. RESULTS: One fifth of the tumors were located in the major salivary glands and PACS/CASGS invariably lacked p40 expression. Fifty-two percent of PACs showed a PRKD1 E710D mutation. Ordinary PACs had a disease-specific 10-year survival probability of 97% compared to 90% when combining PAPs and CASGs. T-stage at diagnosis was a prognostic factor with 98% for stages T1/T2 versus 75% for T3/T4. CONCLUSION: Diagnostic algorithms for the PAC/CASG spectrum of tumors need to be improved and should include molecular markers.
Subject(s)
Adenocarcinoma, Papillary , Adenocarcinoma , Biomarkers, Tumor , Salivary Gland Neoplasms , Adenocarcinoma/chemistry , Adenocarcinoma/genetics , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Adenocarcinoma, Papillary/chemistry , Adenocarcinoma, Papillary/genetics , Adenocarcinoma, Papillary/mortality , Adenocarcinoma, Papillary/pathology , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Child , Female , Germany , Humans , Male , Middle Aged , Mutation , Neoplasm Staging , Registries , Salivary Gland Neoplasms/chemistry , Salivary Gland Neoplasms/genetics , Salivary Gland Neoplasms/mortality , Salivary Gland Neoplasms/pathology , Sex Factors , Time Factors , Tumor Burden , Young AdultABSTRACT
OBJECTIVES: Information of the clinicopathological characteristics and outcome data of patients with adenosquamous carcinoma of the pancreas (ASCAP) remains limited. This study's aim is to describe the clinical, pathological, and molecular characteristics of 25 resected ASCAPs. METHODS: Of all 25 cases, patient characteristics, follow-up data, and pathological/immunohistological features were reviewed and analyzed. RESULTS: In this 3-institutional retrospective analysis of 562 pancreatic cancer patients, we identified 25 cases with histologically confirmed ASCAP (4.4%). Follow-up was available in 21 ASCAP and 50 pancreatic ductal adenocarcinoma control patients with a median overall survival of 8.2 and 21 months, respectively. Age, tumor size, localization in the tail, lymph node status, and resection margin seem to be the most significant factors of survival in our ASCAP cohort. In contrast to pancreatic ductal adenocarcinoma, positive expression of p63, keratins K5/14, and the epidermal growth factor receptor are a robust marker profile of these tumors. CONCLUSIONS: Adenosquamous carcinoma of the pancreas comprises a group of neoplasms in which stage and adverse morphological features contribute to its bad prognosis. Further work must be pursued to improve detection and treatment options to reduce mortality. Specifically, differences in biology might become a target for the development of possible therapies.
Subject(s)
Carcinoma, Adenosquamous/surgery , Carcinoma, Pancreatic Ductal/surgery , Pancreatic Neoplasms/surgery , Aged , Aged, 80 and over , Carcinoma, Adenosquamous/drug therapy , Carcinoma, Pancreatic Ductal/drug therapy , Combined Modality Therapy , Drug Therapy/methods , Female , Humans , Male , Middle Aged , Pancreatic Neoplasms/drug therapy , Prognosis , Retrospective Studies , Survival Analysis , Treatment OutcomeABSTRACT
Recent studies have shown that intraductal prostate carcinoma (IDC-P) should be considered as a separate lesion distinct from prostatic intraepithelial neoplasia (PIN). The purpose of the present study was to analyze the genetic relationship between benign prostatic tissue, PIN, invasive cancer, IDC-P, and extracapsular tumor tissue to get further information about the role of IDC-P in the development of prostate cancer. One hundred five radical prostatectomy specimens were investigated immunohistochemically, 77 cases were analyzed by PCR for LOH of the tumor suppressor genes TP53 and RB1, and 11 cases of IDC-P and 10 cases of PIN were investigated using comparative genomic hybridization (CGH). At CGH analysis, IDC-P showed several chromosomal imbalances in contrast to PIN, where no changes were found. We could demonstrate a significant increase of LOH for TP53 or RB1 from benign tissue to PIN. LOH of both TP53 and RB1 were frequently found in IDC-P (52%), followed by extracapsular tumor tissue (44%), invasive cancer (24%), PIN (19%), and benign prostatic tissue (17%). Increased immunohistochemical expression was found in invasive cancer for TP53, RB1, and for PTEN. Decreased expression could be demonstrated in extracapsular tumor tissue and in IDC-P. Our results indicate that IDC-P in general follows the genetic pathway from normal epithelium over PIN lesion. IDC-P represents a separate prostatic lesion and should be graded as a poorly differentiated carcinoma.
Subject(s)
Chromosomal Instability , Loss of Heterozygosity , PTEN Phosphohydrolase/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Retinoblastoma Protein/metabolism , Tumor Suppressor Protein p53/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Aged , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Intraductal, Noninfiltrating/genetics , Carcinoma, Intraductal, Noninfiltrating/metabolism , Carcinoma, Intraductal, Noninfiltrating/pathology , Humans , Immunoenzyme Techniques , Male , Middle Aged , Neoplasm Invasiveness , Prognosis , Prostatic Intraepithelial Neoplasia/genetics , Prostatic Intraepithelial Neoplasia/metabolism , Prostatic Intraepithelial Neoplasia/pathology , Prostatic Neoplasms/pathologyABSTRACT
The histogenesis of extraocular sebaceous carcinomas is - in contrast to ocular sebaceous carcinomas - unclear, and information about the exact cellular architecture of these lesions and even of the normal sebaceous gland is still scarce. This study attempts to elucidate the histogenesis of sebaceous tumors, using multicolor immunofluorescence stainings to analyze 21 cases of sebaceous tumors (six each of extraocular sebaceous carcinoma, sebaceous adenoma and sebaceoma, and three cases of steatocystomas) and eight cases of normal sebaceous glands for p63, several keratins, androgen receptor, adipophilin, epithelial membrane antigen (EMA) and Ki-67. The data of this observational study provide evidence for the existence of two subpopulations of progenitors in normal sebaceous glands: (i) p63+ K5+ progenitors which generate the K10+ luminal cells of sebaceous ducts; and (ii) p63+ K5+ K7+ progenitors which finally generate K7+ adipophilin+ EMA+ sebocytes. Without exception, all types of sebaceous tumors contained p63+ K5+ cells. Furthermore, these tumors showed a cellular hierarchy and differentiation to adipophilin+ and/or EMA+ mature sebocytes and to K10+ ductal cells through intermediary cells. Notably, a considerable number of sebaceous tumors lack the K7 pathway of cell maintenance in the normal sebaceous lobule. Based on our data, we propose a cellular algorithmic model of the hierarchy of normal sebaceous glands and of sebocytic tumors in which p63+ K5+ cells play a major role.
Subject(s)
Adenocarcinoma, Sebaceous/pathology , Biomarkers, Tumor/metabolism , Sebaceous Gland Neoplasms/pathology , Stem Cells/pathology , Adult , Aged , Aged, 80 and over , Cell Differentiation , Cohort Studies , Female , Humans , Keratin-5/metabolism , Keratin-7/metabolism , Male , Membrane Proteins/metabolism , Middle Aged , Sebaceous Glands/cytology , Sebaceous Glands/pathologyABSTRACT
The concept of endothelium-derived relaxing factor (EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. However, it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts sufficient to account for the vasorelaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle.
Subject(s)
Arteries/physiology , Endothelium, Vascular/physiology , Muscle, Smooth, Vascular/physiology , Vasodilation , Acetylcholine/pharmacology , Animals , Arteries/drug effects , Arteries/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Free Radical Scavengers/pharmacology , Male , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type III/metabolism , Oxidative Stress , Superoxides/metabolismABSTRACT
A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method with primary antibodies originating from the same host species. When primary antibodies are raised in the same host species, the secondary species-specific antibodies can cross-react with each of the primary antibodies. This obstacle can however be avoided with the use of striping buffers eluting the primary/secondary antibody complex. After elution of the previous primary/secondary antibody complex, the next primary antibody from the same host species can be applied. Recently, a group from VENTANA (Tucson, AZ, USA) presented a fully automated multiplex protocol for fluorescent immunohistochemistry on the platform of VENTANA's BenchMark ULTRA slide stainer using the same species antibodies in combination with tyramide signal amplification. We adapted the automated protocol of VENTANA for the use in a routine histochemical laboratory and present here a standard procedure with a manual mode of operation for simultaneously detecting two or more antigens from the same host species.
Subject(s)
Antibody Specificity , Signal Detection, Psychological , Antibodies , Humans , Immunohistochemistry/methods , Intestine, Small/chemistry , Mammary Glands, Human/chemistry , Reference Standards , Skin/chemistry , Species Specificity , Staining and Labeling/methodsABSTRACT
Nitric oxide (NO) is generated by a family of enzymes termed NO synthases (NOS) that convert L-arginine to NO and citrulline. The role of NO as an important biological mediator and recognition of the pathophysiological significance of superoxides/NO interaction has led to an intensive research and development of therapies based on the interception of the NO signaling cascade in the pancreatitis course. However, the presence and localization of the NO-generating enzymes in various organs including pancreas are subject to controversy. We assumed that this controversy might reflect rather the diversity of experimental approaches and an insufficient sensitivity of the methods used. Applying tyramide signal amplification (TSA) immunohistochemical technology, we were able detect all three NOS isoforms both in exocrine and endocrine compartments and in the vasculature in the normal pancreas and in pancreatitis. This also allowed us to demonstrate that oxidative stress runs ahead of NOS up-regulation, which implies that the NO enhancement in the course of pancreatitis is likely to be an adaptive mechanism aimed at maintaining the homeostatic cellular level of the bioactive NO. The aims of this minireview are to describe normal intrapancreatic NO pathways and the role of NO in the pancreatitis course.
ABSTRACT
BACKGROUND: The planned nationwide implementation of mammography screening 2007 in Germany will increase the occurrence of mammographically detected breast abnormalities. These abnormalities are normally evaluated by minimal invasive core biopsy. To minimize false positive and false negative histological findings, quality assurance of the pathological evaluation of the biopsies is essential. Various guidelines for quality assurance in breast cancer diagnosis recommend applying the B-classification for histopathological categorization. However, to date there are only few studies that reported results about reliability and validity of B-classification. Therefore, objectives of our study are to determine the inter- and intraobserver variability (reliability study) and construct and predictive validity (validity study) of core biopsy evaluation of breast abnormalities. This paper describes the design and objectives of the DIOS Study. METHODS/DESIGN: All consecutive asymptomatic and symptomatic women with breast imaging abnormalities who are referred to the University Hospital of Halle for core breast biopsy over a period of 24 months are eligible. According to the sample size calculation we need 800 women for the study. All patients in the study population underwent clinical and radiological examination. Core biopsy is performed by stereotactic-, ultrasound- or magnetic resonance (MR) guided automated gun method or vacuum assisted method. The histopathologic agreement (intra- and interobserver) of pathologists and the histopathologic validity will be evaluated. Two reference standards are implemented, a reference pathologist and in case of suspicious or malignant findings the histopathologic result of excision biopsy. Furthermore, a self administrated questionnaire which contains questions about potential risk factors of breast cancer, is sent to the participants approximately two weeks after core biopsy. This enables us to run a case-control-analysis (woman with breast cancer histological verified after excision are defined as cases, woman without malignant breast lesions are defined as controls) to investigate the predictive values of various risk factors on breast cancer risk. CONCLUSION: The analysis of reliability and validity of the histopathological evaluation of core biopsy specimens of breast abnormalities is intended to provide important information needed for a high quality in breast cancer diagnostic and for planning of treatment strategies.
Subject(s)
Breast Neoplasms/pathology , Carcinoma in Situ/pathology , Carcinoma, Ductal, Breast/pathology , Carcinoma, Intraductal, Noninfiltrating/pathology , Carcinoma, Lobular/pathology , Mass Screening/methods , Biopsy, Needle/standards , Breast Diseases/diagnosis , Breast Diseases/pathology , Breast Neoplasms/diagnosis , Calcinosis/diagnosis , Calcinosis/pathology , Carcinoma in Situ/diagnosis , Carcinoma, Ductal, Breast/diagnosis , Carcinoma, Intraductal, Noninfiltrating/diagnosis , Carcinoma, Lobular/diagnosis , Diagnosis, Differential , False Negative Reactions , False Positive Reactions , Female , Germany , Humans , Immunohistochemistry , Mammography , Observer Variation , Reference Standards , Reproducibility of Results , Retrospective Studies , Sensitivity and Specificity , Stereotaxic TechniquesABSTRACT
BACKGROUND: Recently, we were able to show that amplifications of the epidermal growth factor receptor (egfr) gene and the overexpression of EGFR were associated with the initiation and progression of phyllodes tumours. METHODS: In order to gain further insights into regulation mechanisms associated with egfr amplifications and EGFR expression in phyllodes tumours, we performed global gene expression analysis (Affymetrix A133.2) on a series of 10 phyllodes tumours, of these three with and seven without amplifications of an important regulatory repeat in intron 1 of egfr (CA-SSR I). The results were verified and extended by means of immunohistochemistry using the tissue microarray method on an extensively characterized series of 58 phyllodes tumours with antibodies against caveolin-1, eps15, EGF, TGF-alpha, pErk, pAkt and mdm2. RESULTS: We were able to show that the presence of egfr CA-SSR I amplifications in phyllodes tumours was associated with 230 differentially expressed genes. Caveolin-1 and eps15, involved in EGFR turnover and signalling, were regulated differentially on the RNA and protein level proportionally to egfr gene dosage. Further immunohistochemical analysis revealed that the expression of caveolin-1 and eps15 were also significantly correlated with the expression of pAkt (p<0.05), pERK (p<0.05), mdm2 (p<0.01) and EGF (p<0.001 for caveolin-1). Eps15 and pERK were further associated with tumour grade (p<0.01 and p<0.001, respectively). CONCLUSION: Our results show that amplifications within regulatory sequences of egfr are associated with the expression of eps15 and caveolin-1, indicating an increased turnover of EGFR. The interplay between EGFR and caveolin-1, eps15, pAkt, mdm2 and pERK therefore seems to present a major molecular pathway in carcinogenesis and progression of breast phyllodes tumours.
Subject(s)
Breast Neoplasms/genetics , ErbB Receptors/genetics , Gene Amplification , Gene Expression Regulation, Neoplastic , Phyllodes Tumor/genetics , Breast Neoplasms/pathology , Female , Humans , Immunohistochemistry , Mitogen-Activated Protein Kinase 3/genetics , Oligonucleotide Array Sequence Analysis , Phyllodes Tumor/pathology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-mdm2/genetics , RNA, Neoplasm/genetics , Tissue Array AnalysisABSTRACT
The interaction between nitric oxide (NO) and superoxides is critical in the development of an acute pancreatitis. Previously, we reported that the expression of superoxides and of the NO-generating enzyme (NO synthase, NOS) was up-regulated in the human pancreatitis, especially within the exocrine compartment indicating an exceptional susceptibility of the exocrine parenchyma to oxidative stress. The aim of the present study was to compare the regulation of NO signalling pathways in the human pancreatitis and in an animal model of an acute pancreatitis induced by pancreatic duct ligation (PDL) in rats. In the PDL-induced rat pancreatitis, we revealed a similar pattern of oxidative stress and NOS up-regulation in acinar and in ductal compartments, like in the human pancreatitis. This demonstrates that the PDL-induced rat pancreatitis is a proper model for further studies of acute pancreatitis development in humans.
Subject(s)
Nitric Oxide/metabolism , Oxidative Stress , Pancreatic Ducts/surgery , Pancreatitis/physiopathology , Animals , Humans , Immunohistochemistry , Ligation , Male , RatsABSTRACT
We contend that knowledge about the cellular composition of normal breast epithelium is a prerequisite for understanding proliferative breast disease. Against this background, we used multicolor immunofluorescence to study normal breast epithelium and two types of intraepithelial proliferative breast lesion for expression of the p63, basal keratin K5, glandular keratin K8/18, SMA, ER-alpha, and Ki67. We studied eight normal breast epithelium samples, 12 cases of usual ductal hyperplasia, and 33 cases of low-grade intraepithelial neoplasia (9 flat epithelial atypia, 14 low-grade ductal carcinoma in situ and 10 cases of lobular neoplasia). Usual ductal hyperplasia showed striking similarity to normal luminal breast epithelium including p63+ and/or K5+ luminal progenitor cells and the full spectrum of luminal progeny cells. In normal breast epithelium and usual ductal hyperplasia, expression of ER-alpha was associated with lack of expression of the proliferation antigen Ki67. In contrast, we found in both types of low-grade intraepithelial neoplasia robust expression of keratin K8/18 and a positive association between ER-alpha and Ki67 expression. However, these lesions were consistently negative for p63 and/or K5. Our observational study supports the view that usual ductal hyperplasia and low-grade intraepithelial neoplasia are different entities rather than part of a spectrum of the same disease. We propose a new operational model of cell differentiation that may serve to better understand correlations between normal breast epithelium and proliferative breast diseases. From our data we conclude that p63+ and/or K5+ progenitor cells contribute to maintenance of normal epithelium and usual ductal hyperplasia, but not to low-grade intraepithelial neoplasia of the breast.