The recent development of tissue microarray technology has potentiated large-scale retrospective cohort studies using archival formalin-fixed, paraffin-embedded tissues. A major obstacle to broad ...acceptance of microarrays is that they reduce the amount of tissue analyzed from a whole tissue section to a disk, 0.6 mm in diameter, that may not be representative of the protein expression patterns of the entire tumor. In this study, we examine the number to disks required to adequately represent the expression of three common antigens in invasive breast carcinoma--estrogen receptor, progesterone receptor, and the Her2/neu oncogene--in 38 cases of invasive breast carcinoma. We compared the staining of 2 to 10 microarray disks and the whole tissue sections from which they were derived and determined that analysis of two disks is comparable to analysis of a whole tissue section in more than 95% of cases. To evaluate the potential for using archival tissue in such arrays, we created a breast cancer microarray of 8 to 11 cases from each decade beginning in 1932 to the present day and evaluated the antigenicity of these markers and others. This array demonstrates that many proteins retain their antigenicity for more than 60 years, thus validating their study on archival tissues. We conclude that the tissue microarray technique, with 2-fold redundancy, is a valuable and accurate method for analysis of protein expression in large archival cohorts.
Tissue microarrays have facilitated the evaluation of large cohort studies; however, there is little data on the best method for preserving sections once they are cut. We assessed three methods of ...storing precut breast cancer microarray slides: paraffin coating and storage in a nitrogen desiccator, either alone or in combination. We tested the durability of three antigens, cytokeratin, estrogen receptor, and Ki-67 on microarrays stored under these conditions for 3 months at room temperature. Staining was assessed with both manual scoring using traditional brown stain (0–3+) as well as automated scoring using fluorescently stained sections. Staining intensity was compared to that obtained from freshly cut slides. Slides stored under ambient conditions (room temperature and air) for 3 months exhibited marked degradation of all target antigens, in some cases resulting in slides that were virtually unreadable. We found that combined paraffin coating and nitrogen storage resulted in the best preservation of antigenicity, with retention of 72–99% of the antigenicity of a freshly cut slide, depending upon the marker and detection system used. The use of either paraffin coating or nitrogen storage alone protected slides to a lesser degree.
Purpose : β-Catenin is involved in homotypic cell-cell adhesion and the wnt signaling pathway. Deregulation of β-catenin levels, caused in part by mutations of the adenomatous polyposis coli gene,
is ...thought to play a role in the development of colorectal and other cancers. To further elucidate their roles, the expression
pattern of β-catenin and phosphospecific β-catenin was correlated with clinical outcome in a series of patients with colorectal
cancer.
Experimental Design: Immunohistochemical analysis of a tissue microarray with 650 colorectal cancer specimens was performed to study the expression
and subcellular localization of β-catenin and phosphospecific β-catenin. These results were correlated with other clinicopathological
factors and with overall survival.
Results: The majority of cancers retained some degree of β-catenin membranous staining, whereas cytoplasmic or nuclear expression
was seen in 42.5% and 20.4% of specimens, respectively. Phospho-β-catenin showed nuclear staining in 9.5% of specimens, and
there was no apparent membranous or cytoplasmic staining. There was no significant association between β-catenin or phospho-β-catenin
and grade or stage. However, there was a positive correlation between β-catenin and phospho-β-catenin ( P = 0.039), with phospho-β-catenin representing a subset of nuclear β-catenin. Patients with nuclear expression of β-catenin
did not have an altered survival compared with those that did not ( P = 0.5611). Nuclear expression of phospho-β-catenin, however, was associated with an improved survival ( P = 0.0006). In multivariate analysis, only stage and phospho-β-catenin were independently predictive of overall survival ( P < 0.001 and P = 0.0034, respectively).
Conclusions: These findings support a role for β-catenin overexpression in colorectal tumorigenesis and provide initial evidence that
phospho-β-catenin may be a marker for improved overall survival independent of stage and grade.
Tissue microarrays are a method of relocating tissue from conventional histologic paraffin blocks in a manner that tissue from multiple patients or blocks can be seen on the same slide. This is done ...by using a needle to biopsy a standard histologic section and placing the core into an array on a recipient paraffin block. This technique allows maximization of tissue resources by analysis of small core biopsies of blocks, rather than complete sections. Using this technology, a carefully planned array can be constructed using cases from pathology tissue block archives, and a 20-year survival analysis can be done on a cohort of 600 or more patients using only a few microliters of antibody in a single experiment. Furthermore, this cohort can be analyzed thousands of times with different reagents as a result of judicious sectioning of the array block. This review describes this process and discusses the issues of representative sampling in heterogeneous lesions, the issue of antigen preservation, and some technical strategies and methods of array construction. In summary, this technique can provide a highly efficient, high-throughput mechanism for evaluation of protein expression in large cohorts. It has the potential for allowing validation of new genes at a speed comparable to the rapid rate of gene discovery afforded by DNA microarrays.
Construction of tissue microarrays (TMAs) to efficiently characterize large sets of noninvasive epithelial lesions in the breast by immunohistochemistry is an appealing investigative approach, but ...presents technical challenges. We report methodologic studies performed to optimize methods for building TMAs from noninvasive breast tissues collected in a large case-control study of breast cancer. Using a manual arraying technique with 2.0-mm diameter needles, we constructed TMAs from specimens obtained from 32 women with breast cancer containing the following targets: (1) 28 terminal duct lobular units (TDLUs); (2) 28 ductal carcinomas in situ, and (3) 23 invasive carcinomas. Using careful target selection, we achieved representation of approximately 80% of noninvasive targets with sustained preservation through section 30 of the TMAs. Immunohistochemical staining of TDLU targets demonstrated positive staining for estrogen receptor (ER) in 30.8% of tubules and for progesterone receptor (PR) in 50.0%. To establish an efficient method to evaluate staining results in TDLUs, we created a categorical scoring system to approximate the percentage of tubules containing positive stained cells (<10%, 10% to 50%, >or=50%), and compared the results with those obtained by tubule counting. Comparison between the two methods demonstrated exact agreement for 70.8% of ER and 79.2% of PR stains without two-category discrepancies. ER/PR expression levels in multiple (up to 4) noninvasive targets of the same tissue type (TDLU or DCIS) from a single block showed good correlation. These data suggest that it is feasible to produce TMAs of noninvasive breast structures, albeit with careful selection of targets, and that immunostains of such cores may permit efficient immunohistochemical characterization of peritumoral tissues. Additional exploration of this approach is needed.
Introduction: Automated image analysis provides an objective, quantitative, and reproducible method of measurement of biomarkers. Image quantification is particularly well suited for the analysis of ...tissue microarrays which has played a major pivotal role in the rapid assessment of molecular biomarkers. Data acquired from grinding up bulk tissue samples miss spatial information regarding cellular localization; therefore, methods that allow for spatial cell phenotyping at high resolution have proven to be valuable in many biomarker discovery assays. Here, we focus our attention on breast cancer as an example of a tumor type that has benefited from quantitative biomarker studies using tissue microarray format.
Areas covered: The history of immunofluorescence and immunohistochemistry and the current status of these techniques, including multiplexing technologies (spectral and non-spectral) and image analysis software will be addressed. Finally, we will turn our attention to studies that have provided proof-of-principle evidence that have been impacted from the use of these techniques.
Expert opinion: Assessment of prognostic and predictive biomarkers on tissue sections and TMA using Quantitative immunohistochemistry is an important advancement in the investigation of biologic markers. The challenges in standardization of quantitative technologies for accurate assessment are required for adoption into routine clinical practice.
Central deafness has been linked historically to bihemispheric involvement of the temporal lobe, with more recent findings suggesting that compromise of other cortical and subcortical structures can ...also result in this disorder. The present investigation extends our understanding of the potential anatomical correlates to central deafness by demonstrating that bilateral involvement of an auditory structure within the midbrain can additionally result in this condition. Our subject was a 21-year-old male with a subarachnoid bleed affecting both inferior colliculi. Significant auditory deficits were noted for the middle and late auditory evoked potentials, while electrophysiologic measures of the periphery indicated normal function. The patient was enrolled in a rehabilitation program for approximately 14 weeks. Although initially unresponsive to sounds, the patient regained most of his auditory abilities during the 10 months he was followed. This case documents the range of auditory deficits that may be associated with damage to the inferior colliculi, and it profiles a hierarchical recovery of auditory function consistent with test findings.
Abstract
We sought to determine the test efficiency of the middle latency evoked response for identifying or differentiating subjects with and without central nervous system (CNS) involvement. ...Receiver operating characteristic curves were established for hit and false-positive rates for 26 subjects with CNS lesions and 26 control subjects matched for age and hearing sensitivity. The lesions involved but were not limited to the auditory regions of the CNS. Middle latency evoked response latency and amplitude measurements were made for the N
a
and P
a
waves recorded at C
3
and C
4
electrode sites following stimulation of the left and right ears. Intrasubject comparisons were made for ipsilateral and contralateral stimulation/recording conditions. Amplitude measures were superior to latency measures. For amplitude, percentage differences from contralateral comparisons proved to be the most sensitive and specific measure. The clinical implications of findings are discussed.
Abbreviations:
ABR = auditory brainstem response, CANS = central auditory nervous systems, CNS = central nervous system, MLR = middle latency response, ROC = receiver operating characteristics
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