Evaluation of formalin-free tissue fixation for RNA and microRNA studies

FineFix, RCL-2 and HOPE, three formalin-free fixatives, were compared to the common used formalin fixed tissue samples of lung cancer and were evaluated for their effects on quality, quantity and integrity of RNA and microRNA.Two commercially available RNA extraction Kits (RNeasy FFPE by Qiagen and RecoverAll™ Nucleic Acid Isolation by Ambion) were tested and optimized in order to determine an extraction protocol for RNA as well as miRNA independent of the fixative. Two selected miRNAs were quantified via TaqMan MicroRNA assays.The optimized RNA extraction protocol for Qiagen's Kit leads to similar results for RNA quality and integrity for all fixatives. Highest RNA yield was obtained for formalin and the highest average miRNA ratio was found for FineFix. RNA fragments smaller than 500 bases were detected in FineFix, formalin and RCL2 fixed tissues; HOPE was the only fixative showing long fragments in one third of the samples.Our findings demonstrate that formalin-free fixatives are in general not superior for RNA studies. With our optimized RNA extraction protocol, there is no difficulty in extracting great amounts of RNA with high quality. According to the quality obtained, quantitative real-time PCR analysis can be performed without any negative impact. Similar results can be achieved for the tested fixatives and therefore no fixative seems to represent a new “gold-standard” for tissue fixation.     

The effects of chemotherapy on breast cancer tissue in locally advanced breast cancer

It is well known that chemotherapy induces cytomorphological changes in neoplastic and non-neoplastic tissue. Thirty-one stage III breast-carcinoma patients, treated with both pre-operative chemotherapy and mastectomy, were evaluated to define the effects of systemic chemotherapeutic agents in tumours, non-neoplastic breast tissue, and lymph nodes. Histological changes were compared with those observed in patients who had been treated by surgery alone. Cytoplasmic vacuolization was the most striking change in the tumour cells (59%). Chemotherapy was especially effective in the terminal duct lobular unit in non-neoplastic breast tissue. Lobular atrophy was observed in 20 (65%) cases, and lobular cellular atypia was seen in 16 (52%). The rate of ductal cellular atypia (42%) was not different from the control group. The most important changes seen in the non-neoplastic stromal component were fibrosis and hyalinization. These were found in 31 out of 727 evaluated lymph nodes. In serial sections, metastatic deposits were seen in or around these fibrotic or hyalinized areas. Chemotherapy is widely used in the treatment of early and locally advanced breast carcinomas. Familaritiy with chemotherapy induced changes in breast tissue and lymph nodes have considerable importance in the accurate interpretation of these specimens.     

Investigation of targeted biomolecules in a micro-fluxgate-based bio-sensing system

An investigation of targeted biomolecules was accomplished by combining a micro-fluxgate-based bio-sensing system and Dynabeads. The fluxgate sensor for biomolecule detection was fabricated by Micro Electro-Mechanical system technology, including thick photoresist lithography, electroplating and chemical wet etching. The magnetic core of the sensor was made of Fe-based amorphous ribbon core and three dimension solenoid coils were used as magnetic sensitive elements. The micro-fluxgate-based bio-sensing system was characterized firstly in different concentrations of Dynabeads, and a concentration as low as 100 ng/ml was detected with an external dc magnetic field in the range of 525 μT to 875 μT. Sandwich assays are performed using antibody-antigen pair combination of biotin-streptavidin on a separated Au film substrate surface with a self-assembled layer. Detection of Alpha Fetoprotein antigens with different concentrations was performed and a minimum detectable concentration of 1 pg/ml was achieved by the bio-sensing system. It is of considerable interest due to its potential application in the biomedical field based on known specific binding of target and labels.     

Amplification of c-myc by fluorescence in situ hybridization in a population-based breast cancer tissue array.

A total of 261 primary breast carcinomas were analyzed for amplification of the c-myc oncogene by fluorescence in situ hybridization performed on tumor tissue array samples. Results were compared with individual clinicopathologic and follow-up data. Thirty-eight (14.6%) of the tumors showed c-myc gene amplification (defined as two or more additional copies of c-myc gene in relation to the number of chromosome 8 centromere). The reproducibility of fluorescence in situ hybridization assay (defined by hybridization with two different myc probes) was good (kappa coefficient 0.402). Statistically significant associations were found between c-myc amplification and DNA aneuploidy (P =.0011), and progesterone receptor negativity (P =.0071), and c-myc amplification also tended to be associated with high histologic grade (P =.064), positive axillary nodal status (P =.080), and a high S-phase fraction (P =.052). c-myc amplification was not significantly associated with overall survival of patients with invasive cancer (P =.32). These data from a population-based tumor material suggest that c-myc amplification is a feature of aggressive breast cancers, but that it is unlikely to be a clinically useful prognostic factor.     

Localization of thymidine phosphorylase in breast cancer tissue

Thymidine phosphorylase levels are higher in some human cancer tissues than in adjacent normal tissue. However, the ultrastructural localization of thymidine phosphorylase in cancer tissue has been demonstrated only in advanced gastric and colorectal cancer. We investigated the localization of thymidine phosphorylase in breast cancer tissue by immunohistochemistry and its ultrastructural localization by immunoelectron microscopy. Surgically resected specimens from 30 cases of breast cancer were analyzed. Immunohistochemical analysis revealed that cancer cells were positive in 13 cases. However, there were 21 cases that showed thymidine phosphorylase-positive inflammatory cells in cancer tissue. Thymidine phosphorylase-positive staining was detected among both cancer cells and inflammatory cells in 11 cases. Thymidine phosphorylase was diffusely positive in the cytoplasm of cancer cells and specifically positive in mitochondria of neutrophils and specific cytoplasmic granules of macrophages in cancer tissue by immunoelectron microscopy. These findings suggest that thymidine phosphorylase is produced by macrophages and is present in mitochondria of neutrophils and cytoplasmic granules of cancer cells.     

Large-scale computations on histology images reveal grade-differentiating parameters for breast cancer

Background  

Tumor classification is inexact and largely dependent on the qualitative pathological examination of the images of the tumor tissue slides. In this study, our aim was to develop an automated computational method to classify Hematoxylin and Eosin (H&E) stained tissue sections based on cancer tissue texture features.     

Relationships between histology, monoclonal antibody reactivity, and prognosis in breast cancer

Infiltrating ductal carcinoma of the breast is histologically heterogeneous and may be subdivided into three main types (A, B, and C) which correlate with the likelihood of long-term survival. Examples of each type were tested against three monoclonal antibodies (H59, H71, and H72) prepared against the breast cancer cell line ZR-75-1. The reaction patterns of type B tumors were significantly different from those of types A and C with antibodies H59 and H71, but not with H72. Thus, the reactivity to these monoclonal antibodies, and presumably the antigen composition of the tumor cells, correlates with the histologic appearances and probability of long-term survival.     

Evaluating tumor heterogeneity in immunohistochemistry-stained breast cancer tissue

Quantitative clinical measurement of heterogeneity in immunohistochemistry staining would be useful in evaluating patient therapeutic response and in identifying underlying issues in histopathology laboratory quality control. A heterogeneity scoring approach (HetMap) was designed to visualize a individual patient's immunohistochemistry heterogeneity in the context of a patient population. HER2 semiquantitative analysis was combined with ecology diversity statistics to evaluate cell-level heterogeneity (consistency of protein expression within neighboring cells in a tumor nest) and tumor-level heterogeneity (differences of protein expression across a tumor as represented by a tissue section). This approach was evaluated on HER2 immunohistochemistry-stained breast cancer samples using 200 specimens across two different laboratories with three pathologists per laboratory, each outlining regions of tumor for scoring by automatic cell-based image analysis. HetMap was evaluated using three different scoring schemes: HER2 scoring according to American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) guidelines, H-score, and a new continuous HER2 score (HER2(cont)). Two definitions of heterogeneity, cell-level and tumor-level, provided useful independent measures of heterogeneity. Cases where pathologists had disagreement over reads in the area of clinical importance (+1 and +2) had statistically significantly higher levels of tumor-level heterogeneity. Cell-level heterogeneity, reported either as an average or the maximum area of heterogeneity across a slide, had low levels of dependency on the pathologist choice of region, while tumor-level heterogeneity measurements had more dependence on the pathologist choice of regions. HetMap is a measure of heterogeneity, by which pathologists, oncologists, and drug development organizations can view cell-level and tumor-level heterogeneity for a patient for a given marker in the context of an entire patient cohort. Heterogeneity analysis can be used to identify tumors with differing degrees of heterogeneity, or to highlight slides that should be rechecked for QC issues. Tumor heterogeneity plays a significant role in disconcordant reads between pathologists.     

Immunohistochemical localization of transferrin in human breast cancer tissue

The present study was planned to detect the iron binding protein, transferrin (TR) in paraffin sections of the human breast tumors. The distribution of transferrin has been studied in 153 cases (63 benign lesions and 90 malignant tumors). The extent of staining reaction was determined by semiquantitative grading (weak, moderate and consistent). Positivity rate for transferrin was higher (92.2%) in malignant tumors as compared to benign breast lesions (28.5%) with significant p value (P = .0001) for both the groups. The intensity was variable in both the groups, being more intense in the malignant tumors. Tumors with higher grade of malignancy presented consistent positive staining along with the lymph nodes involved. The extent of immunoreactivity revealed a significant positive correlation with axillary lymph node status. However, no significant correlation was found with the age of the patients. Thus the study of transferrin in breast tumors besides being of prognostic significance helps in the further management of malignant lesions of the breast.     

Immunohistochemical localization of transferrin in human breast cancer tissue

The present study was planned to detect the iron binding protein, transferrin (TR) in paraffin sections of the human breast tumors. The distribution of transferrin has been studied in 153 cases (63 benign lesions and 90 malignant tumors). The extent of staining reaction was determined by semiquantitative grading (weak, moderate and consistent). Positivity rate for transferrin was higher (92.2%) in malignant tumors as compared to benign breast lesions (28.5%) with significant p value (p = 0.0001) for both the groups. The intensity was variable in both the groups, being more intense in the malignant tumors. Tumors with higher grade of malignancy presented consistent positive staining along with the lymph nodes involved. The extent of immunoreactivity revealed a significant positive correlation with axillary lymph node status. However, no significant correlation was found with the age of the patients. Thus the study of transferrin in breast tumors besides being of prognostic significance helps in the further management of malignant lesions of the breast.     

A model for determining the optimum histology of sentinel lymph nodes in breast cancer

AIMS: To create and use a geometrical model for sentinel lymph node (SLN) histopathology in breast cancer. METHODS: The model involves a spherical metastasis randomly situated in an SLN. Two extreme situations are taken as the starting points. In one of these, the metastasis is seen in its largest dimension, whereas in the other it is only just visible, approximating 0 mm in size. Intermediate positions are analysed, with different metastasis sizes and different distances between the levels assessed by histology. RESULTS: The findings suggest that sections taken 1 mm apart afford a reasonable means of identifying almost all metastases measuring > 2 mm (referred to as macrometastases here). For nearly all micrometastases to be identified correctly according to the current TNM definitions (that is, metastases > 0.2 mm), a step sectioning protocol with levels of 250 microm or 200 microm would be adequate. CONCLUSIONS: SLNs are the most likely sites of nodal metastasis. Macrometastases are of recognised prognostic relevance so that all should be identified, preferably correctly as macrometastases; an assessment of levels 1 mm apart appears satisfactory and sufficient for this aim. SLNs also offer an ideal method for the study of the significance of micrometastases; for this, step sections separated by 200 or 250 microm are a good choice.     

Preservation of tissue culture cells by deep freezing]

The effect of polyethylene oxide, mol. wght 400 and 4000, the rate of freezing in liquid nitrogen, and thawing in a water bath at 37 degrees C on the survival rate, the growth activity, miotic activity and the activity of some oxidation-reduction enzymes of the most important links of the chain of biological oxidation, SDG, NAD-H2, NADP-H2- diaphorases, electrophoretic motility of continuous human and animal tissue culture cells, RH HEp-2, SPEV were studied. The developed regimen of freezing at a rate of 30 degrees per minute retains 95% viable cells. The miotic activity, enzymatic activity and electrophoretic motility of frozen-thawed cells did not differ from those of the controls.     

Routine assessment of prognostic factors in breast cancer using a multicore tissue microarray procedure

We propose multicore tissue microarray (TMA) as an alternative to whole section for routine assessment of prognostic factors in breast cancer. Since 2004, we introduced the multicore TMA for testing estrogen (ER) and progesterone receptors (PR), proliferation activity by Ki67, and HER2 overexpression and amplification in routine work. At least four tumor foci were selected on the whole section, and a dedicated technician used a stereomicroscope for accurate sampling of the selected areas. To identify a specific case in the TMA, a separate file and a computerized reporting form with the TMA map were created. A preliminary pilot study comparing the TMA results with those obtained on whole sections showed the specificity of the procedure. Moreover, in everyday diagnosis, hormone receptors were repeated on full section when negative in TMA, without significant discrepancy. Retrospective analysis of the 237 breast carcinomas studied by TMA showed the expected correspondence of tumor-grade differentiation with the hormone receptor pattern, the proliferation activity, and HER2 immunohistochemical and FISH values. In conclusion, multicore TMA may be an efficient approach in the routine study of prognostic factors in breast cancer, significantly reducing costs, time, and burden of slides necessary to accomplish these mandatory tests.     

Endobronchial metastatic breast cancer with pagetoid histology mimicking bronchial pagetoid squamous cell carcinoma in situ

We report a case of a 56-year-old woman with endobronchial breast cancer metastasis of unusual histology. The patient presented with persistent cough, and a lesion was noted in the left mainstem bronchus on bronchoscopic examination. Biopsy revealed extensive squamous metaplasia of bronchial epithelium along with large, atypical cells exhibiting pagetoid intraepithelial spread within squamous mucosa. Immunohistochemical stains were compatible with a diagnosis of metastatic breast adenocarcinoma with pagetoid spread. To our knowledge, this is the first reported case of endobronchial breast cancer metastasis with this histologic presentation. In this report, we describe the clinical, radiographic, bronchoscopic, histologic, and immunohistochemical characteristics of this case. We provide a brief review of existing literature on endobronchial breast cancer metastasis. In addition, we discuss the principal differential diagnosis of bronchial pagetoid lesions. This report raises awareness of this uncommon manifestation of metastatic breast cancer.