Prognostic value of morphometry in low grade papillary urothelial bladder neoplasms

OBJECTIVE: To analyze the prognostic value of morphometry in low grade papillary urothelial bladder neoplasms (LGPUBNs). STUDY DESIGN: The primary (most common) and secondary (second most common) histologic grades were considered in accordance with the 1998 World Health Organization/International Society of Urological Pathology and the 1999 World Health Organization classifications. With the primary grade, 54 cases were papillary urothelial neoplasms of low malignant potential (PUNLMPs) and 66 low grade papillary urothelial carcinomas (LGPUCs), whereas the secondary grade consisted of 45 PUNLMPs and 75 LGPUCs. To assess the proliferative index, an immunohistochemical study was performed. Regarding nuclear morphometry, an image analysis system on Feulgen-stained sections was utilized in different tumor zones (Zs): Z 1, 100-150 cells from the outer layers of the papillae; Z 2, 100-150 cells from the inner layers; and Z 3, 10 largest nuclei. In univariate studies, a t test, and Mann-Whitney U test and Kaplan-Meier curves were applied, whereas a Cox regression model was used for multivariate study of the variables: size, multiplicity, maximum Ki-67 index, mean nuclear area (MNA) and SD, mean nuclear perimeter and SD, and roundness factor. RESULTS: All 120 cases were followed for a mean of 76.6 months (range, 36-168). In univariate studies, many variables showed a significant correlation (p < 0.05) with recurrence prediction, relapse-free interval and histologic grade regardless of adjuvant therapy. Otherwise, only the MNA of the 10 largest nuclei (threshold, 52 microm2) and the maximum proliferative index (threshold, 7.9%) appeared as independent prognostic markers in the multivariate study. CONCLUSION: In LGPUBNs, the independent prognostic value of MNA of the 10 largest nuclei as well as the maximum proliferative index indicates the importance of histologic grade assessment based on the secondary (second most common) grade.     

Nuclear image morphometry and cytologic grade of breast carcinoma

OBJECTIVE: To correlate visual cytologic grade with automated nuclear morphometry of carcinoma of the breast. STUDY DESIGN: We randomly selected 24 histologically proven infiltrating ductal carcinomas of the breast and 10 benign breast lesions (fibroadenoma). Hematoxylin-eosin-stained fine needle aspiration cytology (FNAC) smears were selected for both cytologic grade and automated image morphometry. The same hematoxylin-eosin-stained FNAC smears were studied for area, convex area, standard deviation of nuclear area, diameter, perimeter and convex perimeters of nucleus. At least 100 cells from each case were measured with an image cytometer. RESULTS: Mean nuclear area, standard deviation of nuclear area, nuclear diameter, convex area, convex perimeter and perimeter were significantly increased from benign versus grade 1 carcinomas and grade 1 versus grade 2 and 3 carcinomas (one way ANOVA test). However, there was no significant difference in grade 2 versus grade 3 carcinomas. CONCLUSION: Automated image cytometry rapidly and successfully measures various nuclear parameters. The measurement of various nuclear parameters would be helpful in future applications of automated diagnosis and grading of breast carcinomas from cytologic material.     

Comparison between morphometry and immunostaining of malignant cells in non-small cell lung cancer

OBJECTIVE: To investigate the morphometric properties of tumor cells in combination with the expression of 5 immunomarkers, quantitatively evaluated by image analysis, in a series of 60 non-small cell lung carcinomas (NSCLCs) and to assess their prognostic significance. STUDY DESIGN: Tissue sections from 60 NSCLCs were assessed for the expression of p53, Ki-67, bcl-2, bax and FasL using immunohistochemistry and antibodies. Correlations between morphometric features and clinicopathologic variables, including overall survival and the expression of the above markers, were statistically investigated. RESULTS: Major and minor axis nuclear values, as well as nuclear area and perimeter values, were positively interrelated. No statistically significant correlations were observed between nuclear morphometry and any of the clinicopathologic parameters. p53 Accumulation, when quantitatively estimated, displayed a positive correlation with major axis (P = .008), minor axis (P = .06), nuclear perimeter (P = .006) and mainly nuclear area values (P = .003). Bcl-2- and bax-quantified immunostaining demonstrated a weak negative correlation with shape factor values (P = .039 and P = .025, respectively). Univariate and multivariate analysis showed that stage was the only significant predictor of survival in all patients. In univariate statistical analysis there was a trend between worse survival and shape factor values < 0.8 (P = .0791); this trend almost reached statistical significance in the subgroup of squamous cell carcinomas (P = .0570). CONCLUSION: p53 Accumulation, when quantified, appears to be positively linked with nuclear morphometric values. The prognostic significance of shape factor in NSCLC warrants further investigation.     

Grading of ductal breast carcinoma by cytomorphology and image morphometry with histologic correlation

OBJECTIVE: To investigate the relevance of image analysis for grading breast carcinoma. STUDY DESIGN: Twenty-five ductal breast carcinoma cases were chosen randomly from routine fine needle aspiration clinics. The results of cytomorphologic grading and image morphometry were correlated with those of histologic grading. The five image morphometric parameters studied were nuclear diameter, nuclear area, nuclear roundness, nuclear perimeter and grey level to compare with chromatin texture. RESULTS: Cytologic grading alone had a high correlation with histologic grading. The lowest correlation was found in grade 2 tumors. When cytologic grading was supplemented with image morphometric parameters, the correlation was higher than that of cytologic grading alone. CONCLUSION: Cytologic grading has a high correlation with histologic grading. The correlation improves further on supplementation with image morphometric parameters.     

Nuclear planimetry in renal-cell tumors

A morphometric study using conventional planimetry was carried out in 73 renal-cell tumors (64 adenocarcinomas and 9 adenomas) in order to correlate the objective nuclear measurements with some histologic and clinical data believed to have prognostic value. The use of a discriminant function to correctly separate adenomas and low-grade adenocarcinomas was also investigated. There was a strong association between the nuclear grade and the following parameters: nuclear area, nuclear major diameter and nuclear elongation. There was also a relationship between an adverse outcome and the major nuclear diameter and nuclear elongation. In a univariate survival analysis, only the clinicopathologic stage, the nuclear grade and the histologic presence of a pseudosarcomatous cell population had predictive value. Multivariate survival analysis using Cox models showed that the clinicopathologic stage, major nuclear diameter, presence of anaplastic cells and nuclear area had predictive value. Although the minor nuclear diameter reached a significant level, a discriminant function that would correctly separate adenomas and low-grade renal-cell carcinomas could not be formulated.     

Value of morphometric nuclear image analysis using the Feulgen reaction in renal cell carcinoma

OBJECTIVE: To evaluate retrospectively the ability of morphometric nuclear image analysis to predict survival in patients with renal cell carcinoma. STUDY DESIGN: The subjects were 40 patients with previously untreated renal cell carcinoma. Pathologic stage was determined using Robson's stage system. Nuclear grade was assigned according to the criteria of Fuhrman et al. We used the Feulgen staining technique, which has been widely used for the histochemical assessment of nuclear DNA content. A minimum of 300 nuclei were analyzed from each subject. Five variables in morphometric nuclear image analysis were measured: nuclear area, nuclear perimeter, nuclear ellipticity, nuclear regularity and DNA content. Cox's proportional hazard model was applied to identify prognostic usefulness with respect to survival time. RESULTS: All nuclear morphometric variables but nuclear regularity correlated with tumor grade. According to univariate survival analyses, Robson stage and nuclear ellipticity revealed a prognosis on survival with statistical significance. After adjustments for age and sex, nuclear ellipticity remained the only significant prognostic factor related to survival (P < .01). The survival rates were relatively high for patients with nuclear ellipticity > 773 as compared to those with nuclear ellipticity < 773 (P < .05). CONCLUSION: These findings indicate that morphometric nuclear image analysis using the Feulgen reaction is a reliable and efficient technique and that nuclear ellipticity is the most discriminating morphometric variable for predicting the prognosis of renal cell carcinoma patients.     

Cell kinetics of human epithelial ovarian cancers

Tumor cellular proliferative activity was evaluated by 3H-thymidine labeling index (LI) determination in 73 lesions from 62 patients with ovarian cancers. The median LI value for the overall series was 5.8% and places this tumor type in a position of intermediate proliferative activity. In this case series, the relationship between proliferative activity and different morphologic and pathologic characteristics of the disease was analyzed. Similar median LI values were found for ascitic effusions and solid tumors and, among these, between primary tumors and metastases. No significant relation was observed between proliferative activity and histologic type, whereas a definite direct correlation was found between the kinetic variable and prognostic features such as pathologic stage and histologic grading. In fact median LI was higher for stage IV for stage I and for grade 3 than for grade 1 tumors. The strong association between tumor proliferative rate and conventional prognostic factors suggests that also on this tumor type the kinetic variable could play a role as a prognostic indicator and modulator of aggressiveness.     

Automated nuclear image morphometry on fine needle aspiration smears of malignant round cell tumors

OBJECTIVE: To analyze nuclear image morphometry in fine needle aspiration cytology smears of different groups of malignant round cell tumors (MRCTs) to evaluate its diagnostic role. STUDY DESIGN: In this study there were 55 cases of MRCT, consisting of 18 Ewing's sarcoma (EW), 10 neuroblastoma (NB), 5 non-Hodgkin's lymphoma (NHL), 6 rhabdomyosarcoma (RMS), 4 peripheral neuroectodermal tumor (PNET), 8 Wilm's tumor (WT), 2 retinoblastoma (RB) and 2 undifferentiated round cell tumor (URCT). A Leica image cytometer with Quantimet 600 software (Leica, Cambridge, U.K) was used to measure nuclear area, nuclear diameter, nuclear perimeter, nuclear convex perimeter (CP), nuclear roundness and nuclear convex area on hematoxylin and eosin-stained cytologic smears. At least 100 cells were studied in each case. RESULTS: The RB group of tumors showed the highest mean nuclear area (NA), convex area (CA), CP, diameter (D), perimeter (P) and roundness (R). RMS had the highest mean CA, and URCT had the highest mean roundness. ANOVA was performed on the tumors and showed significant differences for all the variables in all the groups (P < .000). All the morphometric data (except roundness) were significantly different in RMS versus all other MRCTs except RB. Similarly, morphometric data on WT were also significantly different from that on NHL. Most of the morphometric data (except CA and R) showed significant differences between RB and all other MRCTs except RMS. PNET, EW and NB could not be differentiated with those variables. CONCLUSION: RMS and RB could successfully be differentiated from all other MRCTs with the help of morphometry. It was not possible to differentiate RMS and RB by image cytometry (ICM) since the ICM data overlapped in those two groups. It was possible to differentiate WT and NHL with ICM. Nuclear ICM was not significantly different in the NB, PNET and ES groups, and probably ICM would not be very helpful to differentiate these groups of MRCT.     

Image analysis of DNA content and nuclear morphometry for predicting radiosensitivity of nasopharyngeal carcinoma

OBJECTIVE: To investigate the relationship among nuclear DNA content, nuclear morphology, clinical response, and radiosensitivity in nasopharyngeal carcinoma (NPC) and the suitability of image cytometric analysis of DNA content and nuclear morphology for predicting radiosensitivity of NPC prior to radiotherapy. STUDY DESIGN: Nuclear DNA content and morphology features were detected by image cytometric analysis in 51 biopsy specimens of NPC prior to radiotherapy. The radiotherapeutic effect experienced by the NPC patients was classified as CR (complete response [i.e., complete tumor disappearance]) and PR (partial response [i.e., residual tumor]) according to pathologic analysis of tumor specimens after completion of the scheduled treatment. RESULTS: The mean DNA index; the percentage of cells with the DNA pattern of 2C, 5C, aneuploidy respectively; the mean nuclear area; the mean nuclear perimeter and the mean nuclear diameter in the CR group were significantly higher than they were in the PR group. CONCLUSION: DNA content and nuclear morphometry by image cytometric analysis were significantly correlated with patient outcome and radiosensitivity of NPC. Other measurements of more biomarkers for predicting the radiosensitivity of NPC await further study.     

Ploidy and nuclear area as a predictive factor of histologic grade in primary breast cancer

OBJECTIVE: To compare ploidy and nuclear area with histologic grade in breast cancer using cytologic samples. STUDY DESIGN: Fine needle aspirates from 85 patients with primary breast cancer were analyzed to identify ploidy and nuclear area. The Feulgen technique was used to stain the material. We used the SAMBA 4000 image analysis system (Grenoble, France) for analyzing ploidy and nuclear area. Each patient underwent a biopsy, and the histologic grade was analyzed. RESULTS: A significant association was found between ploidy and nuclear area, between histologic grade and nuclear area, and between ploidy and histologic grade. As ploidy became aneuploid and polyploid and nuclear area became larger, histologic grade became higher. CONCLUSION: A reliable and rapid evaluation of variables for breast cancer can be achieved using cytologic preparations by measuring ploidy and nuclear area of malignant cells with an image analysis system. Ploidy and nuclear area have a significant association with histologic grade.     

Angiogenesis as an independent prognostic indicator in node-negative breast cancer

OBJECTIVE: To quantitate tumor angiogenesis in carcinoma of the breast (stage T2N0M0) by computerized image analysis of CD-31-stained histologic sections and, keeping in view the heterogeneity of tumors, to assess which areas of neovascularization provide the best prognostic indicator. STUDY DESIGN: Thirty-six cases of infiltrating duct carcinoma of the breast, stage T2N0M0, with follow-up of five years, were analyzed. All cases had received uniform initial treatment in the form of mastectomy with axillary clearance and radiotherapy. Intratumoral microvessel density (IMD) counts were done on "hot spots" and "non-hot spots" on CD-31-stained sections using computerized image analysis. Angiogenesis was correlated with other variables, such as age, menopausal status, histologic grade and proliferative activity by univariate and multivariate analyses. RESULTS: Hot-spot IMD counts were highly significant independent prognostic markers in univariate and multivariate analyses. Background vascularity of a tumor was of no value in prognosticating. CONCLUSION: In patients with node negative breast carcinoma, IMD counts in hot spots are an independent prognostic factor in disease-free and overall survival and can be used to separate out patients with T2N0M0 stage in need of systemic adjuvant therapy.     

Cell kinetics analysis in stage I endometrial carcinoma as determined by image cytometry and immunohistochemistry. Correlation with clinicopathologic characteristics

OBJECTIVE: To test the significance of various proliferative indices in endometrial carcinoma (EC) since previous investigations failed to yield consistent results that would establish them as factors of clinical importance. STUDY DESIGN: Seventy patients with EC were evaluated for various proliferative indices (mitotic index, Ki-67 index, argyrophilic nucleolar organizer (AgNOR) number and area per nucleus, and p53 protein expression) in relation to image cytometry (nuclear area, diameter and roundness) and standard clinicopathologic features (age, histologic type and grade, and depth of invasion). We also tested the proliferation index (PI), which combines the Ki-67 index and AgNOR area in Ki-67-positive nuclei. Slides from each case were double stained for Ki-67 antigen and AgNOR proteins for this purpose. RESULTS: Mitotic counts were significantly higher in papillary-serous (vs. endometrioid) tumors (P = .0001), high grade (vs. low grade) tumors (P = .0001), deeply invasive (P = .017) and p53-positive tumors (P = .017). AgNOR counts correlated only with age (higher in older women, P = .002), while the PI correlated with mitotic counts (P = 0.28) and marginally with depth of invasion (P = .06). Morphometric variables were associated just with histologic type and grade. p53 Protein was expressed exclusively in invasive tumors and was related strongly to histologic type (P = .0029) and grade (P = .0001). CONCLUSION: Our data reestablish the value of classic histopathologic features (mitotic index, histologic type and grade) as the most important tools for EC evaluation. In addition, we suggest that p53 immunostaining may be used for predicting aggressive behavior in EC.     

Mean nuclear area of fine needle aspirates of primary preoperative palpable breast carcinoma using image cytometry

OBJECTIVE: Early, operable breast cancers in appropriate patients are increasingly being treated preoperatively using neoadjuvant chemotherapy. A good response rate is seen with high grade tumors. Nuclear size, which may reflect the grade of the tumor, is also of possible prognostic value in breast cancer. STUDY DESIGN: We measured the mean nuclear area (MNA) of 114 consecutive preoperative fine needle aspirates of palpable, operable breast cancers. We used computerized image cytometry to measure nuclear area to determine tumor biology and possible grade prior to treatment. RESULTS: Histologic grade distribution was as follows: low grade, 15%; moderate grade, 40%; and high grade, 45%. Mann-Whitney test for trend on tumor size and histologic grade between MNA showed a significant relationship between MNA and tumor size (P=.016) but no significance between MNA and histologic grade (P =.22). The chi2 and Fisher Exact Test between MNA and node-positive or -negative patients showed no significance. CONCLUSION: When correlating MNA with tumor size and histologic grade, high MNA is present at a higher frequency as tumor size and histologic grade increase.     

Prognostic significance of mean nuclear volume in pancreatic adenocarcinoma

OBJECTIVE: To evaluate the correlation of stereologically estimated mean nuclear volume of tumor cells with other clinicopathologic prognostic features and survival in pancreatic ductal adenocarcinoma. STUDY DESIGN: The study included 27 patients with primary pancreatic ductal adenocarcinoma. A stereologic method proposed by Gundersen et al was used for the estimation of mean nuclear volume in hematoxylin and eosin-stained histologic sections of each case. Mean nuclear volume values were compared statistically with histopathologic prognostic feature groups and survival. RESULTS: The mean nuclear volume values of tumor cells ranged from 296.83 to 982.79 microns 3 (mean, 633.906 +/- 212.310). Higher values of mean nuclear volume were significantly related to advanced tumor stage and the presence of distant metastasis (Kruskal-Wallis, P = .036; Mann-Whitney U, P = .020). In contrast, nodal stage, tumor grade, perineural invasion, lymphatic and blood vessel invasion, and size of tumor showed no statistical relation to mean nuclear volume of tumor cells. Mean nuclear volume was found to be a significant predictor of survival in univariate analysis (P = .016). CONCLUSION: Estimation of mean nuclear volume may help in predicting the extent of disease and clinical behavior in pancreatic adenocarcinoma.     

Neural network-based segmentation and classification system for automated grading of histologic sections of bladder carcinoma

OBJECTIVE: To develop an image analysis system for automated nuclear segmentation and classification of histologic bladder sections employing quantitative nuclear features. STUDY DESIGN: Ninety-two cases were classified into three classes by experienced pathologists according to the WHO grading system: 18 cases as grade 1, 45 as grade 2, and 29 as grade 3. Nuclear segmentation was performed by means of an artificial neural network (ANN)-based pixel classification algorithm, and each case was represented by 36 nuclei features. Automated grading of bladder tumor histologic sections was performed by an ANN classifier implemented in a two-stage hierarchic tree. RESULTS: On average, 95% of the nuclei were correctly detected. At the first stage of the hierarchic tree, classifier performance in discriminating between cases of grade 1 and 2 and cases of grade 3 was 89%. At the second stage, 79% of grade 1 cases were correctly distinguished from grade 2 cases. CONCLUSION: The proposed image analysis system provides the means to reduce subjectivity in grading bladder tumors and may contribute to more accurate diagnosis and prognosis since it relies on nuclear features, the value of which has been confirmed.     

The enhanced expression of estrogen-related receptor α in human bladder cancer tissues and the effects of estrogen-related receptor α knockdown on bladder cancer cells

Estrogen-related receptor α (ERRα) belongs to the superfamily of nuclear orphan receptors. However, the role of ERRα in bladder cancer remains unknown. This study examined the expression of ERRα in bladder cancer tissues and explored the molecular mechanisms of ERRα in bladder cancer progression. The expression of ERRα in bladder cancer tissues from 61 patients was determined by immunohistochemistry. We performed quantitative real-time polymerase chain reaction assay to detect the gene expression levels and carried out Western blot assay to measure protein levels. In vitro functional assays, including colony formation, Cell Counting Kit-8, Transwell invasion, and migration assays, were performed to detect bladder cancer cell growth, proliferation, invasion, and migration, respectively. Flow cytometry was used to determine the cell apoptotic rate of bladder cancer cells. Among the 61 detected bladder cancer tissues, 39 bladder cancer tissues showed positive ERRα immunoreactivity. Higher ERRα immunoreactivity score was significantly associated with TNM stage, tumor grade, distant metastasis, and poor survival in patients with bladder cancer. Univariate and multivariate analyses showed that ERRα immunoreactivity was an independent prognostic factor for overall survival in patients with bladder cancer. ERRα was found to be upregulated in bladder cancer cell lines, and knockdown of ERRα suppressed bladder cancer cell growth, proliferation, invasion, and migration; promoted bladder cancer cell apoptosis; and inhibited the epithelial-mesenchymal transition of bladder cancer cells. On the other hand, bladder cancer cell proliferation, invasion, and migration were significantly enhanced after cells were transfected with an ERRα-overexpressing vector. In vivo tumor growth and metastasis assays showed that ERRα knockdown resulted in remarkable inhibition of tumor growth and tumor metastasis in nude mice. Collectively, our results suggest that the enhanced expression of ERRα may play a key role in the development and progression of bladder cancer and ERRα may serve as an important prognostic factor for bladder cancer.     

Proteome-based diagnostics and prognosis of bladder transitional cell carcinoma

More than 90% of bladder tumors are diagnosed as bladder transitional cell carcinoma and the majority of these lesions (70%) are diagnosed as superficial papillary lesions (stage pTa, T1). Recurrences are common to superficial tumors and few lesions will progress to a higher grade and/or stage and muscle invasion. Thus, diagnosing cancer at an early stage, predicting whether a tumor will recur and/or progress and identification of novel targets for cancer intervention, become the main focus of bladder cancer research. The purpose of this article is to briefly review what has been accomplished to date by using proteomic technology in order to develop a new strategy to resolve the problems of early detection, recurrence or therapeutic intervention.     

p53 protein expression and proliferative activity in imprints of superficial transitional cell carcinoma of the bladder

OBJECTIVE: To investigate p53 protein expression and proliferative activity in imprints of tumor biopsies from superficial transitional cell carcinoma of the bladder in relation to the histologic grade of malignancy and recurrence status. STUDY DESIGN: The study group consisted of 70 cases of superficial transitional cell carcinoma of the bladder. In order to investigate p53 protein expression and Ki-67 expression, an immunocytochemical avidin-extravidin complex technique was performed using monoclonal antibodies p53 D0-7 and proliferating cells correspondingly. RESULTS: Thirty-seven percent of superficial transitional cell carcinoma cases showed positive expression of p53 protein. No correlation was found between p53 protein expression and grade of malignancy (P = .45). p53 Protein expression was statistically correlated with a high Ki-67 labeling index (LI) (P < .001) and recurrence status (P < .001). Forty-seven percent of cases showed a Ki-67 LI > 25%. No correlation was found between a high Ki-67 LI and grade of malignancy (P = .703). A significant difference in high Ki-67 LI between recurrent and nonrecurrent tumors of the same grade (P < .001) and between recurrent and nonrecurrent tumors was found independently of grade (P < .001). CONCLUSION: These results on cytologic material could provide useful information on the biologic behavior of superficial transitional cell carcinoma of the bladder at the time of diagnosis.     

Stereologic estimates of volume-weighted mean nuclear volume in aspiration smears of ductal breast carcinoma. Correlation with cytologic grade, tumor size and lymph node status

OBJECTIVE: To estimate cytologic volume-weighted mean nuclear volume and correlate it with other prognostic factors, such as tumor diameter and cytologic grading in relation to nodal infiltration. STUDY DESIGN: The relationships between nodal status and nuclear VV, tumor diameter and cytologic grading, according to the modified Black nuclear grading system, were analyzed on fine needle aspirates of 49 cases of breast cancer by univariate and multivariate logistic regression. RESULTS: Volume-weighted mean nuclear volume (nuclear VV) estimated on fine needle aspiration smears showed a significant correlation with grade of tumor differentiation. CONCLUSION: Stereologic evaluation of nuclear size by nuclear VV is an objective method for the cytologic grading of ductal carcinoma of the breast and has independent prognostic value in relation to nodal status higher than those of tumor diameter and cytologic grade.