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PubMed2004

Sampling strategy for prostate tissue microarrays for Ki-67 and androgen receptor biomarkers.

OBJECTIVE: To develop an optimal sampling strategy for tissue microarrays using automated digital analysis for androgen receptor (heterogeneous expression) and the cellular proliferation marker Ki-67 (homogeneous expression and evaluated by others using nonautomated methods). STUDY DESIGN: Tissue microarrays were constructed from 23 radical prostatectomy specimens and immunostained for androgen receptor expression and cellular proliferation. Automated digital image analysis was used, and the minimum number of cores necessary to capture variance change <3% was determined. Androgen receptor immunostaining was described by percent positive nuclei (PPN) and mean optical density (MOD). RESULTS: Androgen receptor PPN variance measurements showed that 5 cores should be obtained when a single block of a radical prostatectomy specimen contained cancer. If all of 15 blocks contained cancer, 2 cores should be obtained from each of 6 blocks. An optimal sampling strategy was developed for androgen receptor PPN, androgen receptor MOD and Ki-67 PPN. CONCLUSION: The selection of the number of cores to sample is a tradeoff between the number of cores available that contain cancer and the amount of work involved in the analysis. Sampling no fewer than 5 but no more than 12 cores per radical prostatectomy specimen can capture tissue heterogeneity.

Aged↗
PubMed2004

Digital image analysis and stereology of angiogenesis in polypoid and nonpolypoid colorectal adenomas.

OBJECTIVE: To evaluate 3-dimensional parameters and bidimensional microvascular quantification in the different morphologic presentations of colorectal adenomas. STUDY DESIGN: A study was carried out, including 102 neoplastic colorectal lesions obtained by endoscopy or surgical resection. For the analysis of angiogenesis, immunohistochemistry, digital image analysis, microvascular quantification and stereology were used. RESULTS: Microvascular quantification, volume and microvascular length estimate rose gradually with high grade dysplasia as compared to the low grade ones (P < .001). There was no significant difference in angiogenesis between polypoid and nonpolypoid colorectal adenomas in terms of quantification and microvascular length estimate. CONCLUSION: The use of digital image analysis and stereology added greater objectivity and effectiveness to angiogenic evaluation because they allowed accurate segmentation of hypervascular areas, representation of the characteristic 3-dimensional morphology of the vascular supply and identification of differences in microvascularization in the developmental stages of colorectal cancer. However, no significant relation could be found between macroscopic type and angiogenesis, suggesting that angiogenesis may contribute little to morphogenesis of colorectal adenomas.

Adenoma↗
PubMed1992

Cytomorphologic, cytometric and histomorphologic observations after laser therapy for cervical lesions.

To analyze the healing process after laser therapy for cervical lesions, the clinical, cytologic, histologic and colposcopic features in 109 cases were studied chronologically. The healing process of the cervical epithelium usually began from both the squamous and columnar epithelial borders, starting around the 10th day after laser therapy; the process covered the whole tissue defect with multilayered epithelium within seven weeks. Inflammatory changes also usually abated within that time. Cytomorphologically, laser therapy resulted in the occurrence of (mostly degenerated) "fiber-type" and orangeophilic cells in smears taken during the first two weeks after treatment. Tissue repair cells were seen in smears collected from the first posttherapy day through the fourth week after laser therapy. Using computer-assisted image cytometry, the reparative cells in samples taken shortly after treatment (roughly, the first to fifth days) exhibited more hyperchromatic (3-4N) nuclei than did those in later samples; however, the mean DNA content of the early reparative cells was generally concentrated around that of the 2N reference cells. These findings suggest that follow-up, including cytologic and colposcopic examination, for the early detection of residual or recurrent lesions should start in the eighth week and continue periodically for at least one year.

Biopsy↗
PubMedDate not supplied

Checkpoint effectors CDKN1A and Gadd45 correlate with oxidative DNA damage in human prostate carcinoma.

BACKGROUND: Although cellular oxidative stress is a major cause of DNA damage, it is still not clear to what degree it affects genetic instability and malignant progression in established prostate carcinoma (PCa). MATERIALS AND METHODS: We examined the expression of CDKN1A and Gadd45 proteins acting on cell cycle checkpoints and DNA repair in PCa relative to the presence of oxidative DNA damage, as measured by the detection of the DNA adduct 8-hydroxy-2-deoxyguanosine (8-OHdG.). Sixteen PCa and 28 benign prostate hyperplasias (BPH) were analyzed. RT-PCR was used to evaluate WAF1 and Gadd45 transcripts. Western blot and ELISA were used to analyze proteins and 8-OHdG adducts. Proliferation was studied by Ki67 image cytometry; telomerase activity was detected by TRAP- ELISA. RESULTS: Multivariate factor analysis provided evidence that, in PCa, DNA checkpoint proteins were associated with 8-OHdG and did not prevent neoplastic cells proliferation. Conversely, in BPH, oxidative DNA damage was inversely correlated with DNA checkpoint proteins and proliferation, suggesting the presence of energy-depleted senescent cells. CONCLUSION: Although in non-malignant tissue extensive oxidative DNA damage drives cells to a metabolic blockage, in PCa neoplastic cells it activates repair mechanisms favoring the escape from senescence and the expansion of DNA-damaged clones.

Aged↗
PubMed2005

Potential of radial basis function neural networks in discriminating benign from malignant lesions of the lower urinary tract.

OBJECTIVE: To investigate the potential value of morphometry and neural network tools for discriminating benign from malignant nuclei and lesions of the lower urinary tract. STUDY DESIGN: The study group consisted of 33 cases of lithiasis, 41 cases of inflammation, 66 cases of benign hyperplasia of the prostate, 4 cases of carcinoma in situ, 48 cases of grade 1 transitional cell carcinoma of the bladder (TCCB) and 123 cases of grade 2 and 3 TCCB. Images of routinely processed voided urine smears stained by the Giemsa technique were analyzed by a custom image analysis system. Analysis of the images gave a data set of features from 31,158 nuclei. A radial basis function (RBF)-type neural network was employed to discriminate benign from malignant nuclei, based on the extracted morphometric and textural features. Subsequently a second RBF classifier was employed to discriminate benign from malignant cases. The nuclei from 156 randomly selected cases (50% of total cases) was used as a training set, and the nuclei from the remaining 159 cases made up the test set. Similarly, in an attempt to discriminate at the patient level, the same 156 cases were used to train an RBF classifier; the remaining 159 cases were used for the test set. The cases used for training and testing the 2 classifiers (nuclear and patient level) were the same for the 2 kinds of classifiers. RESULTS: Application of the RBF classifier permitted the correct classification of 93.64% of benign nuclei and 85.61% of malignant, giving an overall accuracy of 84.45%. At the patient level the RBF classifier permitted an overall accuracy of 94.97%. These results were on the test sets. CONCLUSION: The role of nuclear morphologic features in the cytologic diagnosis of lower urinary tract alterations was confirmed by the results of this study. The observed overlap in feature space indicates that the nuclear characteristics do not form strictly separate clusters; that fact explains the difficulty morphologists have with reproducible identification of nuclei from the lower urinary tract. Application of RBF offers good classification at the nuclear and patient level and promises to become a powerful tool for everyday practice in the cytologic laboratory.

Algorithms↗
PubMed2005

Color image analysis for quantifying renal tumor angiogenesis.

OBJECTIVE: To segment and quantify microvessels in renal tumor angiogenesis based on a color image analysis method and to improve the accuracy and reproducibility of quantifying microvessel density. STUDY DESIGN: The segmentation task was based on a supervised learning scheme. First, 12 color features (RGB, HSI, I1I2I3 and L*a*b*) were extracted from a training set. The feature selection procedure selected I2L*S features as the best color feature vector. Then we segmented microvessels using the discriminant function made using the minimum error rate classification rule of Bayesian decision theory. In the quantification step, after applying a connected component-labeling algorithm, microvessels with discontinuities were connected and touching microvessels separated. We tested the proposed method on 23 images. RESULTS: The results were evaluated by comparing them with manual quantification of the same images. The comparison revealed that our computerized microvessel counting correlated highly with manual counting by an expert (r = 0.95754). The association between the number of microvessels after the initial segmentation and manual quantification was also assessed using Pearson's correlation coefficient (r = 0.71187). The results indicate that our method is better than conventional computerized image analysis methods. CONCLUSION: Our method correlated highly with quantification by an expert and could become a way to improve the accuracy, feasibility and reproducibility of quantifying microvessel density. We anticipate that it will become a useful diagnostic tool for angiogenesis studies.

Algorithms↗
PubMed1992

The DNA profile of breast cancer in situ.

The nuclear DNA content of 46 mammary adenocarcinomas in situ was measured by means of image cytometry in 4-microns-thick Feulgen-stained histological sections. Aneuploidy was found in 23 cases (50%), whereas the other 23 cases exhibited a diploid DNA distribution pattern. Intraductal carcinoma, the most common subtype in the present study, was found to be aneuploid in 19 cases (63%), with the comedo variant showing aneuploidy in as many as 10 of 11 cases (91%). In contrast, lobular and papillary carcinoma in situ exhibited only 17% and 0% aneuploidy, respectively. The percentages of aneuploid and diploid DNA profiles of the in situ lesions in the present study are almost identical to those observed in invasive breast adenocarcinoma. This indicates that the DNA distribution pattern characteristic for an individual breast adenocarcinoma is already established at the in situ stage, i.e., it occurs before invasiveness and does not progress from a euploid to an aneuploid pattern. In clinical routines DNA ploidy measurements may be of help to distinguish between e.g., hyperplasias and in situ lesions and may also indicate in which direction the malignancy potential of in situ lesions will develop.

Adult↗
PubMedDate not supplied

Heterogeneity in prostate cancer: prostate specific antigen (PSA) and DNA cytophotometry.

BACKGROUND: The heterogeneity in prostate cancer is the reason for the difficult diagnosis and prognosis of this tumor. In this study, we looked for a correlation between prostate specific antigen (PSA), tumor staging and DNA cytophotometry. MATERIALS AND METHODS: Twenty-two prostates (pT1-T4) from patients with prostate cancer, who underwent radical prostatectomy, were examined. Preoperative PSA and postoperative DNA image cytometry, after 2-8 needle biopsies out of each organ, were evaluated. RESULTS: The prostate cancer tissues showed, in DNA stemline-interpretation according to Fu, in homogenous diploid tumors an average PSA level of 3.8 ng/ml, and, in homogenous aneuploid tumors, a level of 14.0 ng/ml. Tumors with heterogeneous DNA patterns with a majority of aneuploidy had an average PSA level of 85.6 ng/ml, and heterogeneous tissues with a majority of diploidy a level of 10.9 ng/ml. CONCLUSION: Only the stemline-interpretation of Fu after DNA cytophotometry is efficient for diagnosis of prostate cancer, and allows prognostic statements of the disease.

Cytophotometry↗
PubMed2005

Karyometry in rectal mucosa of patients with previous colorectal adenomas.

OBJECTIVE: To determine whether karyometric measurements taken in biopsies from histologically normal-appearing rectal mucosa could serve as a biomarker for the risk of recurrence of polyps. MATERIALS AND METHODS: Biopsies were taken from the rectal mucosa of cases with a prior history of colonic polyps at the baseline of the study. In 57 cases recurrent polyps occurred (R cases); in 72 cases no recurrent disease was found at the end of the study (NR cases). From each biopsy 100 nuclei were recorded at high resolution. After segmentation, feature extraction and selection of a discriminating subset of features, a number of discriminant functions were derived. Also, measures of nuclear abnormality were computed. RESULTS: The differences in karyometricfeature values for nuclei from biopsies of cases with recurrent or nonrecurrent disease were very small and not notably expressed in the majority of nuclei. It was possible by focusing on nuclei showing clear deviations from normal to derive a discriminant function that exhibited a shift for the NR and R data sets. The distributions of discriminant function scores were then subjected to a second-order discriminant analysis to separate cases according to recurrence status. This function showed a statistically highly significant correlation with recurrence. At one extreme of its score distribution were 11 of 57 cases that had a recurrence, and at the other extreme were 8-10 of 72 cases that had no recurrence. The distributions of nuclear abnormality values for these subsets of cases were drastically different, with an average value of 1.72 for the group that may be at high risk for another recurrence and 1.02 for the group possibly at low risk. All cases with a prior history of colonic polyps showed a nuclear abnormality deviating from normal. CONCLUSION: Measurement of a sample of 100 nuclei from the rectal mucosa will suggest, for approximately 10% of cases, that a high risk for recurrence of adenomatous polyps exists and, for a slightly lower proportion, confirm that the nuclei deviate only slightly from those from individuals with no history of colonic polyps. For the majority of cases with a prior history of adenoma, the nuclei in the biopsy show a notable deviation from normal, but the deviation is practically the same for cases that had a recurrence and those that did not. However, a tentative discriminant function (DF I,3) derived from the characteristics of the extreme cases correctly classified approximately 64% of nonrecurrent and 83% of recurrent cases using a Bayesian decision boundary.

Adenomatous Polyposis Coli↗
PubMed1992

Can continuing medical education prepare the current practitioner for the 21st century?

The traditional approach to continuing medical education (CME) will be inadequate to prepare the practicing pathologist for the 21st century. Seminars at regional or national meetings, audiovisual presentations, and similar CME activities are useful to provide updates or to fill in more detailed information in the basic knowledge that all pathologists must acquire during their training. Different, more imaginative approaches will be necessary for the pathologist wishing to acquire the necessary knowledge and skills to utilize the newly developing techniques in pathology, such as flow cytometry, image analysis, and the myriad diagnostic procedures based on molecular biology. Self-directed learning will continue to be an essential approach to CME, and the availability of computer programs, including videodisks, will be increasingly effective. However, it should be acknowledged that self-directed learning has been available since the invention of the printing press. The current pressure for public accountability of medical practitioners clearly indicates that pathologists must accept the reality that CME will not be recognized unless it is provided by an accredited organization and attendance is documented. Pathologists should anticipate institution of recertification procedures involving peer review, which will require documentable CME. This CME will be based on needs assessment, educational objectives, more effective formats, and evaluation of whether CME, in fact, improved the pathologist's effectiveness in practice. The academicians have their sabbaticals to refresh their knowledge and explore new fields; perhaps minisabbaticals should be arranged for both the academicians and the practicing pathologist who cannot be away from his or her responsibilities for 6 months or 1 year. The medical specialty societies are the most suitable groups for organizing these programs, although the actual programs must be provided in the laboratories that actually perform the procedures.

Certification↗
PubMed2005

Proliferative changes of epidermal cells in lesions of cutaneous leishmaniasis.

In 35 parasitologically proven zoonotic cutaneous leishmaniasis patients, the histopathological and immunohistochemical picture were studied. The haematoxylin and eosin stain, the monoclonal antibodies for T & B lymphocytes, peroxidase anti-peroxidase for P53 protein, and Feulgen staining for DNA imaging cytometry to DNA contents and S-phase (DNA synthesis of cycling cells were evaluated. The out-come results revealed that P53 and S-phase fraction and DNA content must be in mind when dealing with a human cutaneous leishmaniasis. Consequently, the early detection of any nuclear mutation and cellular proliferation in the skin leishmaniasis lesion(s) must be taken into consideration to avoid the miserable formation of the skin cancer.

Adolescent↗
PubMed2005

The evolution of scanning microphotometry: a historical review.

This article traces the evolution of scanning microphotometry from its beginning, with an emphasis on the developing technologies that have made feasible the rapid and detailed capture of high-resolution image information. Consideration is given to future directions that may prove fruitful to clinical practice.

History, 20th Century↗
PubMed2006

Angiopoietin-1, 2 and Tie2 expressions in endometrial adenocarcinoma--the Ang2 dominant balance up-regulates tumor angiogenesis in the presence of VEGF.

We investigated Ang1, Ang2 and Tie2 expressions including balance and intratumoral vessels in the role of angiogenesis of endometrial adenocarcinoma. Immunohistochemical staining was performed on 133 patients with endometrial (endometrioid) adenocarcinoma, including 73 with G1, 34 with G2, and 26 with G3. The levels of Ang1, Ang2 and Tie2 expressions were expressed as staining score. Total vessel count (TVC), microvessel count (MVC) and mean vessel diameter (VD) in the CD34-stained tissues were measured in five hot spot areas at x 200 magnification by image cytometry. These results were compared with high and low vascular endothelial growth factor (VEGF) expressions. Ang1, Ang2, Tie2 and CD34 were expressed in the cytoplasm of tumor cells. A significant correlation was found among Ang1, Ang2 and Tie2 expressions. In high VEGF cases, Ang1 expression was correlated negatively with TVC and MVC, but positively with VD, and the Angl < Ang2 group was significantly higher in TVC and MVC and tended to be smaller in VD than the Ang1 > Ang2 group. VD was significantly larger in G3 than in G1. The Ang1 < Ang2 balance may be one of the key factors for angiogenesis of endometrial carcinoma in the presence of high VEGF expression.

Adenocarcinoma↗
PubMed2006

Usefulness of AgNOR technique and CEA expression in atypical metaplastic cells from cervical smears.

OBJECTIVE: To evaluate the proliferating capacity of atypical metaplastic cells in cervical smears by AgNOR technique and image analysis and investigate the probable relation with squamous intraepithelial lesions (SIL) using immunocytochemical assay for carcinoembryonic antigen (CEA). STUDY DESIGN: Eight cervical smears were stained with Papanicolaou stain for diagnosis of atypical metaplastic cells. After removal of the stain the smears were processed with a silver colloidal solution and the AgNOR area determined by image cytometry. Differences in the mean AgNOR protein area between reactive metaplastic cells and controls were tested by Student's t test. The CEA was investigated by immunocytochemical staining in smears with atypical metaplastic cells and smears from high-grade squamous intraepithelial lesions (HSIL). RESULTS: The mean AgNOR areas from atypical metaplastic cells (4.55, 6.66, 4.68, 5.30, 4.97, 6.20, 6.28, and 7.35) were significantly greater those that of intermediate metaplastic cells and cells from low-grade squamous intraepithelial lesions (LSIL) (0.77, 0.99, and 0.82, respectively). The atypical metaplastic cells showed values of mean AgNOR area intermediate between that of basal cells (3.28) and HSIL cells (7.73) or neoplastic cells (16.12). The CEA was strongly expressed by the atypical metaplastic cells. CONCLUSION: The expression of CEA in the atypical metaplastic cells underlies the probable relation to SIL. Although the organizer region areas raised high values, it would be necessary to use a greater number of cases to define whether the AgNOR area is indicative of the proliferative activity of the atypical metaplastic cells.

Biopsy↗
PubMed2006

Digitized pathology: theory and experiences in automated tissue-based virtual diagnosis.

AIMS: To describe the theory and develop an automated virtual slide screening system. Theoretical considerations. Tissue-based diagnosis separates into (a) sampling procedure to allocate the slide area containing diagnostic information, and (b) evaluation of diagnosis from the selected area. Nyquist's theorem broadly applied in acoustics, serves to presetting the sampling accuracy. Tissue-based diagnosis relies on two different information systems: (a) texture, and (b) object information. Texture information can be derived by recursive formulas without image segmentation. Object information requires image segmentation and feature extraction. Both algorithms complete another to a "self-learning" classification system. METHODS: Non-overlapping compartments of the original virtual slide (image) are chosen at random with predefined error-rate (Nyquist's theorem). The standardized image compartments are subject for texture and object analysis. The recursive formula of texture analysis computes median gray values and local noise distribution. Object analysis includes automated measurements of immunohistochemically stained slides. The computations performed at different magnifications (x 2, x 4.5, x 10, x 20, x 40) are subject to multivariate statistically analysis and diagnosis classification. RESULTS: A total of 808 lung cancer cases of diagnoses groups: cohort (1) normal lung (318 cases) - cancer (490 cases); cancer subdivided: cohort (2) small cell lung cancer (10 cases) - non-small cell lung cancer (480 cases); non-small cell lung cancer subdivided: cohort (3) squamous cell carcinoma (318 cases) - adenocarcinoma (194 cases) - large cell carcinoma (70 cases) was analyzed. Cohorts (1) and (2) were classified correctly in 100%, cohort (3) in more than 95%. The selected area can be limited to 10% of the original image without increased error rate. A second approach included 233 breast tissue cases (105 normal, 128 breast carcinomas) and 88 lung tissue cases (58 normal, 38 cancer). Texture analysis revealed a correct classification with only 10 training set cases in >92% for both, breast and lung tissue. CONCLUSIONS: The developed system is a fast and reliable procedure to fulfill all requirements for an automated "pre-screening" of virtual slides in tissue-based diagnosis.

Algorithms↗
PubMed1990

Control cells for image cytometric DNA analysis of esophageal tissue and the influence of preoperative treatment.

The nuclear DNA content of 4,960 normal cells in 30 esophagectomy specimens was assessed by image cytometry. In relation to intermediate squamous epithelial cells, the mean transmission values were 25.6% higher for basal squamous epithelial cells, 10.1% higher for fibroblasts and 16.2% lower for lymphocytes. The interslide coefficient of variation (CV) was similar for intermediate and basal squamous epithelial cells, 20.2% and 18.9%, respectively, while fibroblasts and lymphocytes displayed lower interslide CVs, 10.5% and 8.8%, respectively. In spite of their high interslide CVs, the mean DNA values of intermediate and basal squamous epithelial cells had the highest intraslide correlation. This correlation indicates that the preparative and analytical differences between slides that result in alterations in mean DNA values affect squamous epithelial cells of similar origin, size and shape in an analogous manner. Basal squamous epithelial cells replicate their DNA and contain more than the diploid amount; therefore, normal intermediate squamous epithelial cells are suggested as control cells to define the 2c reference value for further DNA studies on squamous epithelial lesions of the esophagus. Preoperative irradiation, with or without chemotherapy, did not alter the DNA values of the investigated control cells.

Adenocarcinoma↗
PubMed1991

DNA ploidy pattern of parathyroid parenchymal cells in renal secondary hyperparathyroidism with relapse.

The nuclei of parathyroid parenchymal cells, analyzed using image cytometry (ICM), in relapsing and non-relapsing secondary hyperparathyroidism due to uremia, showed a DNA-distribution pattern of diploid type. Nevertheless, some differences were observed within the groups, as regards the concept of 'scattered cells' in ICM DNA histograms. The relative incidence of 'scattered cells' was particularly high in the histograms from parathyroid glands with nodular hyperplasia and in those from parathyroid parenchyma grafted into the skeletal musculature. In these two kinds of parathyroid specimens, the 'scattered cells' were both of chief-cell and oxyphil-cell types. In contrast, 'scattered cells' were not so conspicuous when parenchymal cells of glands with diffuse hyperplasia were analyzed. As there is some clinical and histopathological evidence that the cells in both nodular-hyperplastic and autografted parathyroid parenchyma have increased growth potential, it is hypothesized that the relative incidence of the 'scattered cells' in the ICM DNA histograms indicates an increased proliferative activity.

Adult↗