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Automated image analysis DNA cytometry to predict the pathological stage in clinical stage I nonseminomatous testicular germ cell tumors.

OBJECTIVE: Automated image analysis DNA cytometry was used to predict the pathological stage in clinical stage I nonseminomatous testicular germ cell tumor patients. METHODS: Orchiectomy specimens of 74 patients (41 pathological stage I, 33 pathological stage II) were analyzed by modular image analysis computer, and several mathematical indices from the DNA histogram were calculated. RESULTS: The 5c-exceeding rate (rate of hyperpentaploid nuclei) was found to be significantly higher in patients with pathological stage II disease as compared with patients without metastases (p = 0.0174). Tumor tissue from lymph node metastases showed even higher amounts of hyperpentaploid nuclei (p < 0.0005). In this study, all patients with a 5c-exceeding rate above 3.1% had metastatic disease. CONCLUSIONS: Single-cell cytophotometry was able to show significant differences in 5c-exceeding rates between patients with and without tumor metastases. Because of high standard deviations, however, it was not possible to use this parameter for correct staging in all patients.

Analysis of Variance↗

Quantitative AgNOR assessment of cancer cell populations in undifferentiated nasopharyngeal carcinoma.

OBJECTIVE: To evaluate the proliferative activity of cancer cell populations in undifferentiated nasopharyngeal carcinoma (NPC) by quantitative assessment of argyrophilic nucleolar organizer regions (AgNOR) proteins. STUDY DESIGN: Silver-staining of NORs was applied to 18 paraffin sections of undifferentiated NPC containing a vesicular nuclear cancer cell population (VNCC) and spindle-shaped cancer cell population (SSCC). Various features of AgNORs in these two cell populations were analyzed by image cytometry and compared. RESULTS: Mean AgNOR count, mean AgNOR area and AgNOR area-count ratio of VNCC were statistically significantly higher than those of SSCC, while no significant difference was found in AgNOR area/nuclear area ratio between the two cancer cell populations. CONCLUSION: The different cancer cell populations with their distinct morphologic characteristics in undifferentiated NPC have different proliferative activity. The relationship between proliferative heterogeneity of cancer cell populations in undifferentiated NPC and its sensitivity to radiotherapy awaits further study.

Carcinoma↗

Comparison of three different methods for automated classification of cervical cells.

Over 4600 exfoliated squamous cervical cells taken from appropriate Papanicolaou samples were classified as normal, mildly dysplastic, moderately dysplastic and severely dysplastic by an experienced cytopathologist. The slides were de-stained and subsequently re-stained with Feulgen Thionin-SO2 stain. Images of the nuclei were then captured, recorded and processed employing an image cytometry device. Automated classification of the cells was carried out using three different methods--discriminant function analysis, a decision tree classifier and a neutral network classifier. The discriminant function analysis method, which combined all dysplastic cells into an abnormal group, achieved a combined error rate of less than 0.4% for moderate and severe dysplastic cells, and less than 40% for mildly dysplastic cells. All three methods yielded comparable results, which approached those of human performance.

Cell Nucleus↗

Image analysis of nuclear DNA content and morphometric characteristics of the tumor cells in human astrocytomas.

In order to estimate the degree of malignancy of astrocytomas, we applied image analysis to study the nuclear DNA content and morphometric parameters of individual tumor cells in surgically resected specimens of 20 patients. There were nine low grade astrocytomas (LGA) and 11 high grade astrocytomas (HGA). Although the mean DNA index did not indicate a significant difference between LGA and HGA, five (45%) of the 11 HGA had an aneuploid DNA distribution pattern. By contrast, all LGA had a diploid pattern. The ratio of the S-G2M phase (% S-G2M) of the HGA (17.5 +/- 12.3%) was significantly higher than that of the LGA (7.0 +/- 4.6%). From the morphometric analysis of the area and shape of the nucleus of tumor cells, we obtained quantitative estimates of the degree of nuclear pleomorphism in astrocytomas. The nuclei of the tumor cells of HGA had larger sizes, increased anisokaryosis and were more irregular. In contrast to flow cytometry, image analysis can be used for quantitative studies of morphological tumor cell characteristics of relevance in the grading of malignancy.

Adult↗

DNA abnormalities as marker of risk for progression of Barrett's esophagus to adenocarcinoma: image cytometric DNA analysis in formalin-fixed tissues.

OBJECTIVES: To examine DNA content abnormalities in patients with Barrett's esophagus (BE) who progress to esophageal adenocarcinoma, using image cytometric DNA analysis (ICDA) of formalin-fixed tissues. METHODS: Studies were performed on archived biopsies of BE patients' undergoing endoscopic surveillance before developing adenocarcinoma. A comparison group consisted of BE patients' free of cancer during a follow-up period of over 9 yr. Tissue sections were analyzed for the degree of dysplasia and for DNA content abnormalities, using image cytometry. Additional patients were also analyzed in a cross-sectional study of 56 BE cases with and without dysplasia, including 12 cases of adenocarcinoma. RESULTS: Five patients developed adenocarcinoma during follow-up and earlier biopsies obtained before cancer diagnosis showed specialized intestinal metaplasia (SIM) followed by low-grade dysplasia (LGD) in one, SIM followed by high-grade dysplasia (HGD) in one, LGD in two, and HGD in one case. All five showed some DNA abnormality at baseline or in interval biopsies. In the comparison group, five of seven patients showed normal diploid DNA at baseline and on follow-up biopsies. One patient initially had diploid DNA, but developed aneuploidy 11 yr later. Another case initially had aneuploidy, but was diploid on follow-up. Overall, DNA abnormalities were found in 13% of cases with SIM without dysplasia, 60% with LGD, 73% with HGD, and 100% with adenocarcinoma. CONCLUSIONS: (i) Image cytometric DNA analysis is a useful method to examine DNA abnormalities in formalin-fixed tissues and may be more sensitive in predicting progression to adenocarcinoma than HGD. (ii) Histological dysplasia of any grade and DNA abnormalities, help identify BE patients at high risk for adenocarcinoma.

Adenocarcinoma↗

Higher reproducibility of morphometric analysis over the counting method for interphase AgNOR quantification.

In a series of 40 breast carcinomas, the reproducibility of two different methods for interphase AgNOR quantification was evaluated. Two operators independently defined on each slide the interphase AgNOR quantity both by measuring the area of the silver-stained structures using image cytometry and counting the AgNOR number directly at the microscope. The correlation between the values obtained by the two observers was statistically significant, but the correlation coefficient between AgNOR areas (r = 0.79; P < 0.001) was greater than that between AgNOR numbers (r = 0.38; P = 0.014). On the other hand, when interphase AgNOR area and number values obtained by each observer were compared, no significant correlation was found. This study has demonstrated that the two different methods for interphase AgNOR quantification are not comparable, and that morphometric analysis is more objective and reproducible than the counting method.

Humans↗

Application of malignancy-associated changes of the cervical epithelium in a hierarchic classification concept.

For almost 10 years malignancy-associated changes (MAC) have been consistently found by means of high resolution image analysis of apparently normal uterine cervical cells. This study was performed on Feulgen-stained monolayer specimens from 55 healthy women and 19 patients suffering from various stages of cervical neoplasia. About 200 epithelial cells and 30 trout red blood cells in each case were measured by image cytometry at a spatial resolution of 0.27 micron. The philosophy of classification is based on the hierarchic stepwise definition of 'truly normal' specimens by removal of all suspicious specimens, characterized by the presence of atypical cells or subvisually altered cell populations. The suitability of MAC-sensitive classifiers, in combination with classifiers for the recognition of atypical cells, was demonstrated. The demands of automated prescreening were met by several decision tree procedures (95-100% sensitivity, 85-95% specificity). By focussing on the MAC evidence a new risk group from seemingly healthy women may be defined.

Cervix Uteri↗

Cartilaginous tumors: prognostic applications of cytophotometric DNA analysis.

BACKGROUND: Traditionally, selection of cancer therapy is based on the assessment of the prognosis of the individual patient. The specific type of tumor and the stage of disease have been the most reliable indicators of prognosis. METHODS: Image cytometry to determine DNA content was used in conjunction with clinicopathologic parameters and patient survival to investigate 16 cartilaginous tumors. Histopathologic characteristics, cytometric DNA ploidy status, 2c deviation index (2cDI), DNA malignancy grade (DNA-MG), and 5c-exceeding event (5cEE) were used to learn more about the determination of tumor prognosis. Prognosis was analyzed with a maximum follow-up of 148 months. RESULTS: DNA ploidy status, 2cDI, DNA-MG, and 5cEE are indicators of prognosis. After 148 months of follow-up, patients with aneuploid tumors had a significantly lower overall survival rate compared with those with diploid tumors (P < 0.05). Patients with DNA-MG less than 0.8 or 2cDI less than 1.5 had a significantly longer overall survival rate with respect to the group of patients with a DNA-MG greater than 0.8 or 2cDI greater than 1.5 (P < 0.001). A significant difference was noted in the overall survival rates between patients with tumors with 5cEE less than 3 and 5cEE 3 or greater (P < 0.001). CONCLUSION: Image cytophotometry DNA ploidy status, 2c deviations index, DNA malignancy grade, and 5c exceeding event were investigated and were found to be of prognostic value for patients with cartilaginous tumors.

Adult↗

Effect of focus on cell detection and recognition by the Cell Analyzer.

The effect of defocusing on the quality of signals from live cells detected by an automated image cytometry device, the Cell Analyzer, was examined. The influence of these effects on the ability of this device to automatically locate cells plated into a tissue culture flask was then determined by measuring the performance of cell detection and recognition procedures as a function of focus setting. Acceptable limits for deviation from the optimal focus setting (as determined by microscope objective position) were found to be similar for both these procedures, ranging from 40 microns below to 25 microns above the optimal focus position. These limits were asymmetrical about ideal focus due to a pronounced asymmetry in the effects of positive and negative defocusing on the cell signal.

Animals↗

[Correlation between PMI and DNA degradation of costicartilage and dental pulp cells in human being].

OBJECTIVE: To probe the correlation between the postmortem interval (PMI) and the DNA degradation of costicartilage and dental pulp cells in human being after death, and to seek a new method for estimating PMI. METHODS: The image cytometry was used to measure the DNA degradation under different ambient temperatures (30-35 degrees C, 15-20 degrees C) in 0-15 days after death. RESULTS: The average DNA content of two kinds of tissue was degradated with the prolongation of PMI. But there was a plateau period of 0-4 days for dental pulp cells of human being in 15-20 degrees C. There was a high negative correlativity P<0.01 between the average DNA content and PMI. CONCLUSION: PMI could be estimated accurately according to the DNA degradation of costicartilage and dental pulp cells in human being after death.

Adolescent↗

Differences in quantitative nuclear features between ductal carcinoma in situ (DCIS) with and without accompanying invasive carcinoma in the surrounding breast.

The aim of this study was to demonstrate differences in nuclear morphology between ductal carcinoma in situ (DCIS) without an invasive component and DCIS associated with invasive carcinoma in adjacent breast tissue. DCIS specimens of 60 non-comedo and 21 comedo cases were obtained from two groups of patients with or without invasive carcinoma of the breast. The analysis of DCIS nuclei was performed on formalin fixed deparaffinized thin sections stained with a stoichiometric stain following the Feulgen procedure. Nuclear features, related to nuclear size, shape and DNA distribution, were quantitatively characterized by high resolution image cytometry. Features associated with the presence of invasive carcinoma in the surrounding breast tissue were identified in DCIS nuclei (independent of nuclear grade). Features selected by the stepwise procedure of the discriminant function analysis were typically texture features describing the DNA distribution in the nucleus. A classification function based on the selected nuclear features predicted accurately the presence of invasive carcinoma in all comedo DCIS and in 80% of non-comedo DCIS cases. Our results indicate that quantitative nuclear features of DCIS nuclei are predictive of the accompanying invasion and may be helpful as a new tool in evaluation of DCIS patients.

Breast Neoplasms↗

Biopathologic characteristics of DNA content in crypt cells of transitional mucosa adjacent to carcinomas of the rectum and rectosigmoid.

The transitional mucosa (TM) adjacent to carcinomas of the large bowel shows histologic and mucin histochemical changes that may indicate premalignant change and may be of prognostic value after radical resection. In this study, 10 anterior resection specimens from patients with carcinomas of the rectum and rectosigmoid were used to compare the nuclear DNA content in TM with those in cancer tissue and with those in nontransitional mucosa (N-TM; i.e., uninvolved mucosa remote from tumors showing normal histologic and mucin histochemical features). The nuclear DNA content was assessed using DNA image cytometry on Feulgen-stained sections. As compared with N-TM, crypts in TM contained greater numbers of cells, were elongated, and were more likely to be branched with marked sialomucin secretion, accompanied by a marked reduction in the normal sulfomucin content. The mean nuclear DNA content in the upper, middle, and lower thirds of crypts was significantly higher in TM than in N-TM, and the nuclear DNA content in TM exhibited no correlation to that in tumors. The results suggest that TM adjacent to carcinomas of the rectum and rectosigmoid has higher proliferative activity, providing further evidence that TM may be an unstable premalignant change.

Adenocarcinoma↗

Quantitative pathological study of aggressiveness of giant cell tumor of bone.

Clinical behavior of giant cell tumor of bone (GCT) may vary remarkably from latent to very aggressive. Quantitative pathological methods were used to evaluate the aggressiveness of GCT. Fifteen cytometric parameters were measured and computed on routine sections of 40 GCTs which had been treated with curettage. The surgery factor (with or without additional procedures like bone cement packing or freezing after the curettage) was also taken into account and computed. Nineteen of the 40 GCTs were cured and 21 recurred. While no single one from the 15 parameters measured showed significant differences between the cured and recurrent groups according to the Mann-Whitney's U test or Student's t test, a 4-variable function was established with a stepwise discriminant analysis which could correctly identify 70.8% of the predicted cases as cured or recurrent (jackknife procedure). The function also suggested that in addition to the surgery factor, which no doubt had close relation with the prognosis, the most important risk factor in histological parameters was SA40, i.e. the percent of cells with nucleus larger than 40 square microns. Single cells extracted from paraffin-embedded blocks of 38 GCTs were analyzed by DNA-image cytometry. The 2c deviation index (2cDI) showed wide heterogeneity ranging from those consistent with benign tumors to those with apparent malignant ones, which may account for its diversity in clinical behaviour. Sixteen of the 38 cases had been treated with curettage, 8 of them were cured and followed up for at least 3 years and the remaining 8 recurred. The significant difference of 2cDI between the two groups suggested that these DNA parameters are useful in evaluating the aggressiveness of GCT for the selection of an adequate treatment. Quantitative approaches appeared to be more objective and more sensitive in evaluating the aggressiveness and predicting the prognosis of GCT than the subjective grading system used before.

Bone Neoplasms↗

Diagnostic use of mean nuclear area and nuclear DNA content in preoperative breast cancer cytology.

OBJECTIVE: Fine needle aspiration (FNA) cytology of breast lumps is a routine procedure, and the diagnostic accuracy can be 95%. Occasional discrepancies arise, and it would be valuable to have additional parameters for accurate diagnosis. We evaluated nuclear DNA content and mean nuclear area (MNA) using image cytometry in the diagnosis of preoperative breast cancers by FNA in those with a discrepancy between clinical, radiologic and cytologic diagnoses. STUDY DESIGN: One hundred eighteen consecutive preoperative FNA samples were evaluated for nuclear DNA and MNA and were compared to cytologic and postoperative histologic diagnoses. RESULTS: Sensitivity, accuracy and positive predictive value of routine cytology were 95%, 90%, 95% as compared to nuclear DNA (66%, 66%, 96%) and MNA (61%, 61%, 97%). Combining these 3 parameters gave a sensitivity of 97%, accuracy of 94% and positive predictive value of 99%. CONCLUSION: These results demonstrate that nuclear DNA and MNA combined with routine cytology may be useful adjuncts in preoperative breast cancer cytologic diagnosis when discrepancies arise. This may lead to better and more accurate planning of treatment regimens in preoperative breast cancer patients.

Biopsy, Fine-Needle↗

Evaluation of DNA content in preneoplastic changes of mouse urinary bladder induced by N-butyl-N-(4-hydroxybutyl) nitrosamine.

Using image cytometry analysis we analysed the deoxyribonucleic acid content of preneoplastic lesions induced in C3H/He female mice. Female mice (n = 30) were given 0.05% BBN in their drinking water for 4, 8 and 12 weeks, and were then euthanized. At the 4th week 90% hyperplasias, 90% dysplasias and 10% papillary tumours developed selectively in the bladder, after 8 weeks mice developed 80% hyperplasias and 80% dysplasias and 12 weeks we identified 70% hyperplasias, 70% dysplasias, 10% papillary tumours and 20% squamous metaplasia. Among the ones who developed simple hyperplasia, 58.3% were diploid and 41.7% were aneuploid. In the dysplasia lesions 29.2% were diploid and 70.8% were aneuploid (p = 0.041). All papillary tumours were aneuploid and all epidermoid metaplasias were diploid. These results suggest that aneuploidization, DNA content alteration of preneoplastic lesions, might be considered as a prognostic factor.

Animals↗

Spontaneous malignant transformation of conventional giant cell tumor.

Spontaneous malignant transformation of conventional giant cell tumor (GCT) of bone is exceedingly rare. We report on a case of GCT of the iliac crest in a 35-year-old woman with malignant change into a high-grade osteosarcoma 10 years after the first appearance of GCT on a radiograph. Since the patient refused therapy for personal reasons the tumor remained untreated until sarcomatous transformation occurred. Image cytometry showed DNA aneuploidy and a suspiciously high 2c deviation index (2cDI) in the primary bone lesion. A thorough review of the world literature revealed only seven fully documented cases of secondary malignant GCT which matched the definition of a "sarcomatous growth that occurs at the site of a previously documented benign giant cell tumor" and not treated by radiotherapy. These cases as well as the current one suggest that a spontaneous secondary malignant GCT presents as a frankly sarcomatous tumor in the form of an osteosarcoma or malignant fibrous histiocytoma. It usually appears at sites of typical GCTs-often without any recurrent intermediate state-and is diagnosed 3 or more years after the primary bone lesion. The prognosis is poor.

Adult↗

Validation of the galactose oxidase-Schiff's reagent sequence for early detection and prognosis in human colorectal adenocarcinoma.

Based on the multistage and multifocal nature of colorectal carcinogenesis, it is likely that reduction of cancer mortality through early detection and identification of new prognostic markers is an attainable goal. Well-documented changes occur in mucin glycoconjugates during neoplastic progression in the colon, and the nonneoplastic colonic mucosa in colon cancer patients is morphologically and histochemically abnormal. In this retrospective study, 152 archival colorectal tissues from 49 patients were studied for changes in mucin secretions as detected by the galactose oxidase-Schiff's (GOS) sequence. Intensity of the stain was evaluated in histological sections by semiquantitative analysis, and the area percentage of epithelium stained was quantified by image cytometry. The correlation between gender or tumor size, location and reactivity with peanut agglutinin and quantitative expression of GOS-reactive mucins was determined as well as intratumor and inter individual variability. Reactivity with GOS: (a) decreased during neoplastic progression and malignant conversion in the neoplasm; (b) increased in the normal colonic mucosa of patients with progressively more advanced disease; and (c) was of prognostic significance for patient survival or recurrence both in the normal colon of cancer patients and in invasive neoplasms. These data are consistent with the conclusion that GOS reactivity in the normal colonic mucosa is a dosimeter of exposure to environmental/lifestyle colorectal carcinogens rather than a marker for an oncodevelopmental cancer-associated antigen.

Adenocarcinoma↗

Ploidy-dependent growth and binucleation in cultured rat hepatocytes.

The proliferative activity of rat hepatocytes, cultured in the presence of epidermal growth factor (EGF) and insulin, was examined by immunostaining of S-phase cells labeled with bromodeoxyuridine (BrdU) in culture. Proliferation rates of the different hepatocellular ploidy and nuclearity classes were measured by fluorescence image cytometry or by microscope counting of immunostained cells. Effects of EGF and insulin were largely additive, the binuclear cells being more growth factor-dependent (showing less growth in the absence of factors) than the mononuclear cells. A serial warm-washing procedure was used to remove excess BrdU from the culture medium, allowing the study of hepatocellular binucleation by a BrdU pulse-chase approach. A high rate of binucleation was detected (50%, possibly suggesting a quantal mechanism), indicating that the hormones induce a binucleating (polyploidizing) type of growth similar to that normally observed in the liver of growing rats. The highest proliferative activity (labeling index) in the hepatocyte cultures was found among the diploid cells, independent of the degree of mitogenic stimulation. The labeling index was inversely correlated with ploidy, suggesting that the ability of hepatocytes to proliferate decreases with increasing polyploidization.

Animals↗