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PubMed1993

Effect of Rumalon (glycosaminoglycan-peptide) on the articular tissue.

The effect of glycosaminoglycan-peptide complex (GPC) (Rumalon, made by Robapharm, Switzerland) on cells of the inflammatory periarticular infiltrate and on the articular chondrocytes was studied in experimentally induced papain arthropathy by means of image cytometry and biochemistry. The GPC therapy exhibited some antiinflammatory effect as documented by the reduction of DNA proliferative activity in the inflammatory infiltrate and lowered the activity of hydrolytic enzymes and enzyme inhibitors detected in chondrocytes.

Animals↗

PubMed1993

[Image analysis on isolated colorectal tumor cells].

Image analysis of the nuclear morphology was performed on propidium iodide-stained isolated cells from 9 adenomas and 9 cancers of the colorectum. We analyzed DNA content, seven nuclear geometric features and four shape factors of tumor cells using an image cytometry system that has been developed in our laboratory. Nuclear breadth and the degree of contour irregularity of cancer cells were found to be significantly different from those of adenoma cells. The coefficients of variation (CV) of some nuclear features and the degree of contour irregularity were increased in cancer cells, while CV of the degree of circularity was increased in adenoma cells. Moreover, nuclear in G2 phase were found to be larger and more round than those in G1 phase. These results suggest that cell-cycle-related analysis of nuclear features would be a valid means to assess the nuclear morphology of colorectal tumors.

Adenocarcinoma↗

PubMed1995

[Changes in methylation of tumor cells: a new in situ quantitative approach on interphase nuclei and chromosomes].

DNA methylation is known to be related to the regulation of gene expression. DNA methylation patterns are modified in tumors compared to normal tissue, and in some cases, these variations are linked to cancer progression. Molecular biology techniques are generally used to evaluate DNA methylation status. We describe here a simple and fast immunofluorescent method to quantitate in situ DNA methylation using an image analyser and a CCD camera. Quantification of relative methylation levels in interphase nuclei and metaphase chromosomes was performed on digital images of two types of cells with known methylation levels. The results from image cytometry corresponded to those obtained by molecular studies. Advantages of this approach are that all the DNA of a cell may be examined rather than a limited restriction sequence, and that it may be done on a cell by cell basis rather than on a heterogenous population. In addition, with this method, changes in methylation patterns during tumor progression could be followed and eventually used as a marker for prognosis.

5-Methylcytosine↗

PubMed1995

Multiparameter digital image analysis of biliary, ampullary, and pancreatic adenocarcinomas.

Periampullary adenocarcinomas arise from pancreatic, biliary, or ampullary ductal epithelium. Their origin is difficult or impossible to discern by routine light microscopy of cytologic or small biopsy specimens. We used image analysis to describe the morphologic features of Feulgen-stained nuclei of biliary and ampullary adenocarcinomas and to compare these findings with those of pancreatic ductal adenocarcinomas. Surgically resected cases of ampullary adenocarcinoma (n = 7) and cholangiocarcinoma (n = 26) were selected on the basis of available formalin-fixed, paraffin-embedded tissue and diagnostic clinical data. Disaggregated nuclei were stained by the Feulgen reaction and analyzed using a digital image processor. Data from 15 morphonuclear parameters were assessed and compared with the results from the morphonuclear analysis of 22 pancreatic ductal adenocarcinomas. Multivariate analysis with canonical transformation of the data defined the ampullary adenocarcinomas and cholangiocarcinomas as occupying similar factorial distributions, whereas the pancreatic carcinomas were separate and distinct. Monovariate analysis identified seven parameters distinguishing pancreatic carcinoma from both ampullary carcinoma and cholangiocarcinoma, with P values < or = 0.001 and two others having P values < or = 0.01. Adenocarcinomas of ampullary or bile duct origin possess similar morphonuclear features described by image analysis. Image analysis provides a mechanism to discriminate adenocarcinomas arising from the bile ducts or ampulla from those arising in the pancreas.

Adenocarcinoma↗

PubMed1995

Malignancy associated changes in cervical smears: systematic changes in cytometric features with the grade of dysplasia.

For almost ten years. Malignancy Associated Changes have been consistently found by means of high resolution image analysis of apparently normal uterine cervix cells. The study was performed on normal intermediate cells randomly selected from 1850 cervical smear slides classified as negative, mild dysplasia, moderate dysplasia and severe dysplasia. Size and texture features, performed by the Cyto-Savant software, have shown significant monotonic changes with the grade of dysplasia. These findings indicate that the intensity of Malignancy Associated Changes are directly related to the severity of the adjacent lesions.

Cell Transformation, Neoplastic↗

PubMed1995

Applications of neural networks in histopathology.

ANN methods are one of several nonparametric approaches to classification. They are based on an adaptive, connectionist model of information storage and processing and learn by example rather than explicitly programmed classification rules. Their greatest strength probably is the ease with which they capture nonlinear and interactive feature effects during training, though at a higher risk of overtraining then is found with traditional classifiers. ANN can be used to process image data, but so far not to classify complex natural images directly. Instead, image dimensionality is reduced to a set of extracted features for ANN input. ANN finds growing use in time series signal processing and radiology. In anatomic pathology, ANN pilot studies are published for tumor classification. Pap smear analysis, and chromosome identification. Each application requires proper sampling technique, minimising bias in training and testing to assure the ANN classifier will perform prospectively as expected. With careful arrangement of input features, ANN can be used for prognostic as well as diagnostic models.

Humans↗

PubMed1995

Unbiased stereologic techniques for practical use in diagnostic histopathology.

Grading of malignancy by the examination of morphologic and cytologic details in histologic sections from malignant neoplasms is based exclusively on qualitative features, associated with significant subjectivity, and thus rather poor reproducibility. The traditional way of malignancy grading may therefore not be optimal regarding the scientific and clinical value in selecting specific therapy and making prognostic forecast for the individual patient with a solid cancer. A lot of examples in the international literature document the urgent need for replacing morphologically based techniques by introducing quantitative techniques in the histopathologic discipline of malignancy grading. Unbiased stereologic methods, especially based on measurements of nuclear three-dimensional mean size, have during the last decade proved their value in this regard. In this survey, the methods are reviewed regarding the basic technique involved, sampling, efficiency, and reproducibility. Various types of cancers, where stereologic grading of malignancy has been used, are reviewed and discussed with regard to the development of a new objective and reproducible basis for carrying out prognosis-related malignancy grading of solid tumors. This new, unbiased attitude to malignancy grading is associated with excellent virtues, which ultimately may help the clinician in the choice of optimal treatment of the individual patient suffering from cancer. Stereologic methods are not solely applicable to the field of malignancy grading. Surgical pathologists can use a wide range of these techniques in the routine of diagnostic and scientific work. It is not the intention, however, to give an extensive review of the methodology, but two other techniques will be covered: Unbiased estimation of volume of, and total tumor cell number within, solid neoplasms. Three-dimensional tumor size may be of relevance in staging cancer, whereas total tumor cell number may be of interest in the scientific work with malignancies and treatment of cancer.

Diagnosis, Computer-Assisted↗

PubMed1995

3D reconstruction and quantitation of pathological tissues.

3DR techniques are under continuous development, as is the technology which supports them. Systems developed for engineering and other applications are coming within the price range of many users. Recently publications have started to appear using standard high-quality graphics workstations. As these are coming down in price, more applications will be possible. The advent of new computer technology, notably possible. The advent of new computer technology, notably highly parallel systems and chips such as the Intel i860, offer the prospect of very fast reconstructions and the software to make 3DR a routine technique. Most significant however is the continuing increase in type and numbers of scanning systems, both macroscopic and microscopic. Prior to these systems becoming available, the critical limiting factor inhibiting the wide application of 3DR was generating adequately registered, undistorted, complete sets of serial section data for the reconstruction process. The fact that scanning optical microscopes can produce such datasets easily means that the everyday use of 3DR for studies of pathology is now feasible.

Humans↗

PubMed1996

Future diagnostic procedures.

Although the exact proportion of patients with carcinoma in situ in whom disease will progress to invasive lung cancer is not known, and although there have been reports that some individuals may continue to show malignant cells in sputum for several years without symptoms or abnormality on chest radiograph, untreated or suboptimally treated carcinoma in situ has been shown to progress to invasive cancer or metastatic disease. A study by Frost and co-workers showed that cancer developed in approximately 10% of individuals with moderate atypia and 40% of those with severe atypia in sputum cytology. Therefore, the proportion of individuals with carcinoma in situ in whom invasive cancer will develop is likely to be greater than 40%. Lung cancer is almost uniformly fatal when untreated, and, if the disease is allowed to progress to the invasive stage, the results of currently available therapy are poor. A lesson must be learned from cervical cancer screening. As has been shown by Anderson and co-workers, if individuals harboring dysplasia or carcinoma in situ are actively sought for and treated by laser or cryotherapy, the incidence and mortality of invasive cervical cancer can be reduced to extremely low levels. If current work directed toward detecting early lung cancer in sputum cytology specimens in high-risk groups using quantitative image cytometry or molecular markers is successful, the ability to localize small preinvasive lesions with fluorescence bronchoscopy will become even more important for the pulmonologist or thoracic surgeon. Endoscopic ultrasound can be used to determine the depth of tumor infiltration into the bronchial wall or adjacent structures. Biopsy of mediastinal and peribronchial lymph nodes can be performed under sonographic guidance for more accurate staging. By coupling sensitive diagnostic tools to new treatment modalities, such as chemoprevention and various endobronchial therapies, it is hoped that the traditionally poor prognosis for patients with lung cancer can be altered in the near future.

Bronchoscopes↗

PubMed1996

Methodological considerations on Feulgen-staining applied to cells in primoculture: the model of osteoarthritic synovial cells.

Human synovial cells in primoculture are an interesting model for the study of articular joint diseases and anti-rheumatic drugs. Based on results obtained by image cytometry of Feulgen-stained nuclei, we describe the heterogeneity of synovial cell populations and their progression during culture time in primoculture. Using the hydrolysis properties of the Feulgen reaction and their variations dependent on fixatives, we demonstrate the high acid-lability of the condensed chromatin observed in short term cultured nuclei compared to the acid-resistance of decondensed chromatin in long term cultured nuclei; these variations being probably induced by modifications in the molecular supra-organisation of chromatin during the aging of a culture. Finally, due to the cellular heterogeneity of the biological model and its evolution during culture progression, technical compromises are proposed to obtain optimal Feulgen staining, using Böhm-Sprenger fixative and a 1 h hydrolysis by 6 M HCl at 20 degrees C.

Aged↗

PubMed1996

Prognostic factors in low-stage nonseminomatous testicular cancer.

Whether patients with clinical stage I nonseminomatous testicular germ-cell cancer (NSGCT) should be treated with orchiectomy and retroperitoneal lymph node dissection (RPLND) or orchiectomy and surveillance remains controversial. Proponents of the former approach cite the uncertainty and risks of monitoring young men who may harbor occult metastases, while proponents of the latter strategy contend that surgical staging overtreats 60% to 70% of men. Over the last few years, prognostic factors in the primary testicular tumor have helped clinicians make more rational decisions about whether RPLND or surveillance should follow initial orchiectomy. As of 1996, the most clinically useful prognostic factors are the percentage of embryonal carcinoma and the presence or absence of vascular invasion by tumor cells in the primary tumor. Ongoing work with flow cytometry, image analysis, proliferation markers, and oncogene and tumor-suppressor gene markers may allow us to further stratify patients as to their likelihood of occult metastases and permit rational "risk-adaptive" treatment.

Adult↗

PubMed1995

Predictive value of morphological nuclear parameters and DNA ploidy pattern in precancerous lesions of the uterine cervix.

To evaluate the supportive role of image cytometry and DNA ploidy analysis in the precancerous and cancerous lesions of the uterine cervix, the present study was performed on 45 cervical smears, initially diagnosed as dysplasia and malignant. Twenty normal and inflammatory smears were taken as a control for the study. Morphometric parameters and microphotometric DNA measurements were performed on 50 cells in each case. On the basis of nuclear area dysplastic lesions were categorised into two groups i.e. low grade lesions having nuclear area upto 85 sq. mu m and high grade lesions having nuclear area above 85 sq. mu m. The results were compared with DNA ploidy analysis. It is revealed from the study that 85.7% low grade lesions with diploid and polyploid DNA, value mostly regressed to inflammation and 78.5% high grade lesion with aneuploid DNA value progressed to malignancy. However, initial malignant cases having aneuploid DNA value exhibit invasive cancer during their follow up. It indicates that combination of morphometry and DNA cytometry can be used as an adjunct to cytologic diagnosis to predict the biologic outcome of the lesions.

Aneuploidy↗

PubMed1996

Classification of duct carcinoma in situ (DCIS) with a characterization of high grade lesions: defining cohorts for chemoprevention trials.

In the last 6 years a number of non-randomized, predominantly single institutional trials of breast conservation therapy (BCT) with DCIS, have demonstrated that it constitutes a very heterogeneous group of diseases with markedly different risks of local recurrence and invasive transformation. There has been a consensus that DCIS, which exhibits a "comedo" morphology, generally defines a high risk group. Most studies, moreover, have identified the same two features, nuclear grade and necrosis, as contributing most significantly to prognosis. Nuclear grade and necrosis have been identified as independent prognostic variables in several studies. High nuclear grade DCIS which exhibits comedo necrosis defines the majority of all DCIS which will result in local recurrence and invasive transformation after BCT. Studies utilizing image cytometry, to determine ploidy and S-phase fraction and immunohistochemical studies of proliferation and oncogene distribution have shown a significant association with morphologically identified high nuclear grade and aneuploidy, high S-phase fraction or proliferation rate, presence of HER-2/neu and P53 oncogenes and absence of estrogen receptors. Generally the inverse of this association is seen with low nuclear grade DCIS. However, initial hopes that these adjunctive studies would identify subsets within the high nuclear grade group which might be more likely to recur have not been fulfilled.

Anticarcinogenic Agents↗

PubMed1997

Cytologic differentiation grade and malignancy DNA index in prostatic adenocarcinoma.

OBJECTIVE: To compare the predictive value of cytologic differentiation grades traditionally used for adenocarcinoma of the prostate with the malignancy grades obtained by DNA quantification. STUDY DESIGN: Of a total number of 156 cases of cancer of the prostate, 54 were selected for DNA quantification by image cytometry. They were studied cytologically, separating well, moderately and poorly differentiated cases. We obtained Kaplan-Meier survival times based on the cytologic grade. Then DNA quantification was carried out, and cases were selected on the basis of their different malignancy grades. The relative survival curves were obtained. Survival times based on cytologic grade were compared with those from DNA quantification. RESULTS: There was no statistically significant difference in survival times between moderate and poorly differentiated cases, so they were included in the same group. Between well and moderately differentiated cases there were differences related to survival that were significant at P < .01. With respect to DNA quantification, a survival difference between low and high grade malignancy occurred at P < .001. CONCLUSION: DNA quantification clearly allows better prediction of survival than does cytologic grading alone.

Adenocarcinoma↗

PubMed1997

Indeterminate fine-needle aspiration of the breast. Image analysis-assisted diagnosis.

BACKGROUND: Fine-needle aspiration (FNA) of the breast, although effective for the diagnosis of breast carcinoma, has a significant drawback. A minority of cases cannot be classified as benign or malignant. These FNAs are assigned an inconclusive diagnosis, often prompting surgical biopsy. Surgery is justified in some of these cases, but many of these lesions are benign. If these inconclusive FNAs could be accurately diagnosed as benign or malignant, many of these patients might avoid having to undergo surgical biopsy. METHODS: An image analysis and an automated learning system that was developed at the University of Wisconsin (Xcyt) was used to categorize 56 (37 benign and 19 malignant) breast FNAs diagnosed as "indeterminate" and the computer diagnosis compared with the surgical biopsy. For each case, an operator chose a group of cells within a single field on the FNA slide and digitized this image using a video camera. The outline of each nucleus was manually outlined, and the exact border was delineated by the computer. Based on the analysis of three nuclear features (area, texture, and smoothness), the Xcyt system computed a benign or malignant diagnosis and a corresponding probability of malignancy for each case. RESULTS: Probabilities of malignancy for the respective cases ranged from 0.0-1.0. Benign cases were defined as those having probabilities of malignancy < 0.3; those with probabilities above this limit were considered malignant. Using these criteria, the computer identified 33 cases as benign and 23 cases as malignant. When compared with the surgical biopsy, 42 of the cases (75%) were correctly classified with a sensitivity and specificity of 73.7% and 75.7%, respectively. There were only 5 false-negative cases with a false-negative rate of 13.5% and a predictive value of a negative test of 84.8%. CONCLUSIONS: When faced with inconclusive diagnoses of FNAs of breast masses, the authors believe that image analysis may be used as an aid in the further classification of such lesions, thereby providing a more appropriate triage for surgical biopsy.

Adult↗

PubMed1997

Reproducibility of p53 and Ki-67 immunoquantitation in Barrett's esophagus.

OBJECTIVE: To test the reproducibility and time effectiveness of two immunoquantitation and sampling methods in Barrett's esophagus (BE) mucosa. STUDY DESIGN: Measurements were performed using image cytometry (CAS 200/486) with "at convenience" sampling and stereology (QPRODIT 5.2) with both at convenience and systematic random sampling. RESULTS: Quantitation of p53 immunohistochemistry (IHC) by CAS was very reproducible by the same observer (r = .99), but its interobserver reproducibility was lower, and the measurement was time consuming (r = .81, 35 minutes). Moreover, CAS also detected a "signal" in the absence of any visually observable brown stain, giving false positive results. Both intraobserver and interobserver reproducibility by QPRODIT 5.2 were good. With QPRODIT, using systematic random sampling, the measurements were more reproducible by different observers and faster (r = .96, 10 minutes) than with QPRODIT with at convenience sampling (r = .91, 20 minutes). Therefore, for assessment of the Ki-67 labelling index (LI) and area percentage (area%) of Ki-67-positive nuclei, we used only QPRODIT with systematic random sampling. While intraobserver reproducibility of both Ki-67 LI and area% was good (r = .96, r = .99), interobserver reproducibility of Ki-67 LI was poorer than area% (r = .72 vs. r = .97). The assessment of Ki-67 LI was time consuming, whereas the measurement of the area% of Ki-67-positive nuclei was very time effective (45 vs. 7 minutes). A strong correlation was found between Ki-67 LI and area% of Ki-67-positive nuclei (r = .92). CONCLUSION: Therefore, we conclude that quantitation of p53 and Ki-67 IHC in BE mucosa can be very reproducible and highly time effective by means of stereology with systematic random sampling.

Barrett Esophagus↗

PubMed1997

Automatic analysis of growth onset, growth rate and colony size of individual bone marrow progenitors.

A method for automatic enumeration of proliferating bone marrow progenitors after single cell sorting is described. The system is based on regular inverse microscopy, recording with a video camera, and image analysis using dedicated software on an Apple computer. Single CD34+ progenitor cells were sorted in 96-well plates. Three times weekly phase-contrast video images of each well were stored and analyzed for the actual number of cells. From the subsequent counts growth curves were plotted for each individual progenitor. Enumeration by image analysis correlated very well with manual cell counting (r = 0.99, P < 0.0001). To show the capability of the method to analyze growth rate and growth delay, more differentiated (CD34+/CD13+/CD33+) progenitors were compared with more primitive (CD34+/CD13+/CD33g-) progenitors. Differences in the timing of colony outgrowth were shown to be based on delay in growth initiation. Initiation of growth was delayed 2.6-3.1 days in CD34+/CD13+/ CD33- fraction of 3 different donors (P < 0.0001). The growth rates of the progenitors in both fractions were not significantly different. The described method seems important to more accurately evaluate subpopulations of progenitors, the effect of growth promoting or inhibiting factors, and effects of cytotoxic drugs and irradiation.

Antigens, CD34↗

PubMed1997

DNA image cytometric measurement as a surrogate end point biomarker in a phase I trial of alpha-difluoromethylornithine for cervical intraepithelial neoplasia.

Cervical intraepithelial neoplasia grade 3 (CIN 3) is considered a high-risk precursor of invasive cervical cancer. alpha-Difluoromethylornithine (DFMO) is a promising antiproliferative chemopreventive agent. The purpose of this study was to evaluate image cytometric measurement of nuclear DNA (ICM-DNA) as a surrogate end point biomarker (SEB) in a Phase I trial of DFMO for CIN. Thirty patients with CIN 3 were treated with DFMO at five doses, ranging from 0.0625 to 1.0 g/m2/day, for 1 month. Half of the patients had histological responses. Twenty-five pre- and posttreatment cervical biopsy specimens (from 11 responders and 14 nonresponders) were available for this analysis. ICM-DNA was performed on 4-micron sections cut from formalin-fixed tissue blocks and stained with a thionin-SO2 Feulgen reaction. ICM-DNAs for each case were expressed as normalized measurements (against the nuclear modal absorbance of lymphocytes) of the absorbance of each cell of interest and were presented in bar histograms. The mean normalized summed absorbance (sigma ODn) was obtained as a mean histogram of the cell population of interest. Nineteen (76%) of 25 patients had a significant decrease in sigma ODn after DFMO treatment. Posttreatment values were significantly lower than pretreatment values in a paired analysis, and responders had significantly lower values than nonresponders. Analyses of different ICM-DNA references, including percentile values of sigma ODn distribution, DNA malignancy grade, and 5c exceeding rate, showed a decrease of mean sigma ODn during DFMO treatment. In addition, the summed posttreatment sigma ODn histograms also showed progressively shorter right shoulders compared with pretreatment histograms in both responders and nonresponders. We concluded that the modulation of sigma ODn reflected the chemoprevention effect of DFMO even before morphological changes appeared, and thus, ICM-DNA may be useful as a SEB in chemoprevention trials of DFMO. Additional reasons for using ICM-DNA as a SEB are the relative simplicity of its use, the high accuracy of the results, the low cost of the reagents, the ability to use small tissue samples, and the objectivity and reproducibility of the procedure.

Adult↗